BaTiO3 PerOvskiTe CaTalysT in OxidaTive COuPling ...

Oxidation Communications 35, No 3, 525–537 (2012) Gas-phase oxidation reactions

BaTiO3 Perovskite Catalyst in Oxidative Coupling of Methane. Quantum Chemistry Approaches M. Gharibia,b*, M. S. Motallebipoura Petrochemical Research and Technology Company, National Petrochemical Company, 27 Sarv Alley, Shirazi-south, Mollasadra, P.O. Box 1435884711, Tehran, Iran E-mail: [email protected] b Department of Chemistry, Tarbiat Moallem University, 49 Mofateh Avenue, Tehran, Iran a

ABSTRACT The present work tries to show that the quantum mechanics and ab initio calculations can be used as a virtual chemistry lab for studying reactions at surfaces and is divided into three parts. In the first part, the ab initio geometry optimisation has been carried out for the cubic BaTiO3 perovskite structure as a catalyst for oxidative coupling of methane (OCM) reaction by using hybrid DFT (B3PW, B3LYP) is described. The optimised properties and effective Mulliken charges of Ba2+, Ti4+ and O2– for bulk BaTiO3 catalyst are compared with those available from experimental or theoretical data. In the second part, the bond energies for some gas-phase methane conversion are theoretically calculated. The results provide a more accurate approach for describing gas-phase reaction mechanisms. Finally the catalytic cycle responsible for coupling of methane over the catalyst is shown. The results suggest a new mechanism over BaTiO3 that requires only one active site and does not involve the high-energy barrier process of creating lattice vacancies. This mechanism is more consistent with the Ito–Lunsford mechanism on Li/MgO oxide catalyst proposed earlier. Keywords: oxidative coupling of methane (OCM), DFT ab initio modelling, BaTiO3 perovskite catalyst, mechanism. AIMS AND BACKGROUND Methane is the most abundant component of natural gas. The conversion of methane (natural gas) to higher and more valuable hydrocarbons is of significant industrial importance. Among various schemes for methane conversion, heterogeneous oxida*

For correspondence.

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tive coupling, i.e. direct formation of C2+ (ethane, ethylene and higher hydrocarbons) from methane, is a promising route to utilise the vast natural gas resources. After the pioneer works of Keller and Bhasin1, Ito and Lunsford2, and Anderson3, there have been extensive research and development efforts in this area. Such a technology is especially attractive for Iran with its large reserves of the natural gas. Experimental approach. The oxidative coupling of methane exhibits a complex system of homogeneous and heterogeneous reaction steps. Reaction temperature typically varies between 650 and 800°C, and the molar ratio of methane to oxygen is generally 3–10 under atmospheric pressure. A great variety of metal oxides has been tested as potential catalysts. Rare-earth compounds, alkali and alkaline-earth metal compounds, and multivalent metal oxides such as Mn/Na2WO4/SiO2 have been reported as effective catalysts in OCM. Perovskite structure is an important type adopted by numerous compounds with ABX3 stoichiometry, where A and B are cations and X represents an anion such as oxygen or fluorine4. Barium titanate (BaTiO3) perovskite has numerous industrial applications and is an effective catalyst for oxidative coupling of methane (OCM) reaction for petrochemical applications: CH4 + O2 ABO 3

catalyst

C2+ + CO + CO 2 + H2O +…

desired product by-product

ABO3: SrTiO3, BaTiO3, KNbO3 …

The propriety perovskite-type catalyst (BaTiO3+SnCl2) of NPC-RT has shown excellent performance in OCM process with C2+ hydrocarbon yields of up to 30% (Ref. 5). BaTiO3 exhibits the sequence of FE-PT (displace versus order-disorder transition): cubic → tetragonal → orthorombic → rhombohedral. The perfect crystalline structure has been assumed according to the high-symmetry cubic phase at high temperatures6. Extensive experimental studies have shown that BaTiO3 has high activity of converting methane to C2+ compounds in the presence of O2. Figure 1 shows the good catalytic performance of BaTiO3 for OCM process7. In exothermic OCM process the following selective and nonselective overall reactions occur simultaneously: 2CH4 + 0.5O2 → C2H6 + H2O, ∆H*reaction = –174.2 kJ/mol

(1)

C2H6 + 0.5O2 → C2H4 + H2O, ∆H reaction = –103.9 kJ/mol

(2)

*

C2H6 → C2H4 + H2, ∆H*reaction = 114.6 kJ/mol CH4 + 2O2 → CO2 + 2H2O, ∆H reaction = –801.6 kJ/mol *

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(3) (4)

CH4 + 1.5 O2 → CO + 2H2O, ∆H*reaction = –519.1 kJ/mol

(5)

C2H6, C2H4, H2 and O2 → CO, CO2, H2O+ large amount of heat

(6)

Different mechanisms have been suggested for the OCM reaction. One mechanism is based on O– as the active site8. The other is based on O2– with a low coordinate number9. McCarthy and co-workers have tried to make a model for a combination of the homogeneous and heterogeneous reactions. Their attempts have failed until now, because of the lack of accurate information on the elementary heterogeneous steps10. According to the accepted mechanism, O2– and O– species occurring on oxide surface initiate the catalytic process by abstracting a hydrogen atom from methane, leading to the formation of methyl radicals on the catalyst surface; the 2 desorbed radicals are coupled in the gas-phase to form ethane molecules11.

Fig. 1. Activity of methane conversion with various perovskite catalysts7

Theoretical approach. The application of ab initio quantum calculations (ABQC) to catalysis has increased continuously in the recent years. Quantum chemistry methods provide information to model heterogeneous catalytic processes and to investigate unknown mechanisms. The expansion of ABQC in the field of catalysis is mostly due to advances in computing capabilities (large memories, faster CPUs). In recent years, several theoretical studies involving ab initio and semi-empirical methods have been applied to better elucidate the catalytic surfaces and mechanism of OCM process. Ito et al.12 examined chemisorptions of CH4 on MgO clusters. Zhanpeisov et al.13,14 performed quantum chemical MINDO/3 calculation for adsorption of methane on pure and modified MgO, CaO and ZnO cluster surfaces. The energy changes of the OCM process on MgO and Li/MgO catalyst using MNDO-PM3 formalism has been explored by Senkan et al.15 Au et al.8 presented the results of activation of methane on La2O3 catalyst obtained by MP2 method, and in Ref. 16 adsorption of methane on Li/MgO surfaces was investigated. There are still some unsolved problems, such as the relationship between the reactivity and coordination number of 527

active sites, the mechanism for producing C2 hydrocarbons after H abstraction from methane, modelling of catalyst at high temperatures, etc. The first ab initio calculations of BaTiO3 surfaces have been reported by Padilla and Vanderbilt17. The authors considered BaO- and TiO2-terminated surfaces and investigated surface-induced relaxations, defect states and the influence of the surface upon ferroelectricity. Other theoretical efforts on BaTiO3 aimed at an understanding of elastic constants, phase transfer, oxygen vacancies, relaxation, optical band gaps and ferroelectricity, bulk modulus and band structure6,18–26. However, in the present work we attempt to find an optimised bulk model for BaTiO3 catalyst in OCM process by using the hybrid DFT (B3PW, B3LYP) as a new screening method and to suggest a catalytic cycle mechanism over BaTiO3 that is more consistent with the Ito–Lunsford mechanism for Li/MgO catalyst. COMPUTATIONAL DETAILS The strategy employed in this work is first to optimise catalyst geometries by making use of the ab initio density functional theory (DFT) and applying a number of different exchange-correlation functionals which describe the lattice structure of perovskite crystals adequately, including hybrid Lee, Yang and Parr (B3LYP) or Perdew and Wang (B3PW) with the SSD and LANL2DZ basic sets27,28 and then to obtain the bulk properties. The GAUSSIAN 98 program package29 is used for all calculations. The valence electrons of all atoms are described by the standard LANL2DZ (Los Alamos National Laboratory 2-double-z) basic sets. D95V (Dunning/Huzinaga valence double-zeta) is used for the first row such as O (721/41), C (721/41), H (31) and electron core potential for Ba (5s25p66s2) and Ti (3s23p64s23d2). The electron core potential approximation allows one to calculate the effective potentials, to focus on more significant calculations of the valence electron states, and consequently, to save significant amount of computational time. The BaTiO3 perovskite crystal exhibits a cubic structure at high OCM reaction temperatures, with space group Pm3m (Ref. 17). The DFT-B3PW optimised geometry for (1×1×1) cubic BaTiO3 unit cell perovskite-type is depicted in Fig. 2 (Table 1). In this structure, the oxygen atoms located in the face-center positions of a cubic unit cell, from a perfect octahedron with the titanium atom in its center, and barium atom lying outside the oxygen octahedron in the corners of the cube, with a0 representing the cubic lattice constant. In the cubic perovskite structure, the Ti-O, Ba-O, and Ba-Ti bond distances are a0/2, a0(√2/2) and a0(√3/2), respectively. The atomic positions are Ba (0, 0, 0), Ti (0.5, 0.5, 0.5), O1 (0.5, 0.0, 0.5), O2(0.0, 0.5, 0.5), and O3 (0.5, 0.5, 0.0).

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Fig. 2. Optimised geometry for 1×1×1 cubic BaTiO3 unit cell perovskite-type

The bond lengths and lattice parameter are listed in Table 1. Table 1. Comparison of the results for bond lengths and lattice parameter on cubic BaTiO3 structure

Ba–O (1) Ba–O (2) Ba–O (3) Ti–O (1) Ti–O (2) Ti–O (3) Ti–Ba CH3–H a

Bond lengths (Å) this work

expt.a,b

2.8453

2.8365, 2.8415

2.0069

2.0057, 2.0092

3.4767 1.0800

3.4739, 3.4801 1.086f

Lattice parameter (Å) cubic (a=b=c) this work 4.0145 LDAc 3.96 PW91d 3.996 BLYPe 4.08 PBEc 4.03 B3LYPe 4.01 HFc 4.01 expt. 4.0185a, 4.00g, 4.0114b

Ref. 30 ; b Ref. 31; c Ref. 18; d Ref. 20; e Ref. 19; f Ref. 32; g Ref. 33.

For calculations of the bulk crystal, the BaTiO3 catalyst was modelled as a finite lattice BanTinO3n (n=8) molecular cluster. The larger clusters did not significantly change the energetic of the corner or edge atoms. Here, we use the periodic model with a primary BaTiO3 unit cell extended by a factor of 4×2×1 grid which provides a balanced summation in the direct and reciprocal lattices and thereby contains 40 atoms. The surface was fully optimised under the constraint of Cs or C1 symmetry. The optimised lattice constant a0 =4.0145Å used results in the minimum energy for bulk cubic (BaTiO3)8. This cluster size is significantly larger than the molecular sizes of CH4, O2 and OCM products; therefore, the chemisorptions properties of the surface should be minimally altered. As shown in Table 2, the hybrid functional B3LYP/SDD level at 750◦C provides accurate representation of the electronic properties and the HOMO of this model is fully occupied. The OCM reaction is highly exothermic, implying that 2 electrons from the two C–H bonds (methane activation) can be easily transferred to the LUMO and, therefore, the reaction of CH4-surface is 529

feasible. The energy difference between the HOMO and LUMO level is regarded as the band gap energy. Table 2. Calculated results for the optimised BaTiO3 cubic structure at different DFT levels of approximation

Properties Basic set Energy (kcal/mol) Enthalpy (kcal/mol) The Gibbs free energy (kcal/mol) HOMO (kcal/mol) LUMO (kcal/mol)

25°C 750°C 25°C 750°C

DFT/B3LYP SDD –194368.969 –194358.852 –194341.756 –194382.912 –194453.433 –119.992 –36.546

LANL2DZ –194005.396 –193995.198 –193978.118 –194019.087 –194089.181 –95.864 –62.738

DFT/B3PW SDD –194338.341 –194328.165 –194311.107 –194352.152 –194422.460 –120.193 –28.733

RESULTS Catalytic reactions. The model for the (BaTiO3)8 surface includes all possible coordination numbers. This model enables one to investigate the relations between the reactivity and coordination numbers of the reaction sites. Chemical activity of OCM reactions is often connected with O2– and O– species of various surface irregularities such as steps, corners and faces as shown in Fig. 3.

Fig. 3. Chemical activities of catalytic reactions

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Table 1 gives the calculated properties for bulk BaTiO3 crystal and CH4 at the B3PW level, together with experimental data. Standard DFT functional (LDA, BLYP, B3LYP, PW) and uncorrected HF are also added for comparison. The calculated cubic lattice parameters and the bond lengths are quite close to the experimental values obtained by Kim et al.30,31. Our results confirm that B3PW/LANL2DZ can be confidently used, and the agreement with experiments is of the same order as that achieved by others using the more extended theoretical levels. The electronic properties for the equilibrium geometry of BaTiO3 unit cell such as the Mulliken charges and other population analysis are listed in Tables 2 and 3. As shown in Table 3, the calculations reveal that the charge transfer from Ti to O is not complete and the static charges on the Ti and O atoms are smaller than those expected for purely ionic materials. The Mulliken net charges of Ti and O are quite different from those of formal ionic charges of ABO3 perovskite: there is a large overlap between the Ti 3d and O 2p orbitals, resulting in a partly covalent O–Ti chemical bonding. Unfortunately, experimental data for OCM reaction on BaTiO3 catalyst are rather scarce. In contrast, there is practically no bonding of O with Ba atoms and Ba charges remain close to the formal +2e. These results show good agreement with other experimental (or theoretical) values. Table 3. Comparison of effective Mulliken charges Q (e) for bulk BaTiO3 perovskite

Q (e)

Bulk charges Cubic phase (empirical model)b HFa This work Nominal charge a

a

Ba2+ 1.795 1.48 1.855, 1.772 1.7900 +2

O2– –1.386 –1.39 –1.554 –1.1292 –2

Ti4+ 2.364 1.86 2.808 2.2198 +4

Ref. 18; bRef. 34.

Gas phase. As an indication of the accuracy of the method employed, pseudo-potential, and basic sets, the calculated bond energies (kcal/mol) of relevant gas-phase molecules, such as CH4 → CH3 + H, C2H6 → CH3 + CH3, O2 → O + O, with experimental or series of ab inito data at the HF, MP2, BLYP, G2, and G3MP2B3 levels are compared in Table 4. Experimentally, the reaction 4CH4 + O2 → 2C2H6 + 2H2O is exothermic by –84.5 kcal/mol and ∆H25°C = –177 kJ/mol and ∆H800°C = –174.3 kJ/mol. Using DFT/ B3PW level, one obtains –76.23 kcal/mol for reaction energy, which is in a good agreement with experimental data. One should note the significant improvement of DFT/B3PW results as compared to the Hartree–Fock calculations. A rather small discrepancy occurs for equation (2), which could be attributed to the deficiency of the level of this reaction.

531

Table 4. Bond energies (kcal/mol) for some gas-phase molecules and reaction energy for methane conversion to ethane

No Reaction step 1 O2 → O + O 2

H2O → OH + H

3

OH → O + H

4

CH4 → CH3 + H

5

C2H6 → CH3 + CH3

6

4CH4 + O2 → 2C2H6 + 2H2O

a l

Theoretical refs. 22 (HF)a, 113 (MP2) a 121.67c, 106.93d, 113.3 e 89 (HF)a, 119 (MP2) a 123.0 (B3LYP/6-311 + + G(3df, 3pd)) f 113.5 (BLYP/6-31 + G(d)) f 139.4 (SWVN/6-31 + G(d)) f 68 (HF)a, 96 (MP2) a 102.39 ( B3LYP/CC-PVQZ) g 104.5 h 88 (HF)a, 107 (MP2) a 105.79 (G2) i 104.76(G3MP2B3) i 67 (HF)a, 92 (MP2) a 88.65 (G3MP2B3) i 90.81 (G2) i –74 (HF)a, –73 (MP2) a

This work Expt. 116.74 119.106 ± 0.048b, 117.97 ± 0.45 k 98.09 119 ± 1 b, l

100.45

102.3 ± 0.5 b, 101.0 ± 0.2 k

103.7

104 ± 1 b , 104.9 ± 0.09 j

90.65

88 ± 2 b, 90.2 ± 0.2 j

–76.23

–84.5 b

Ref. 9; b Ref. 35; cRef. 36; dRef. 37; e Ref 38; f Ref. 39, gRef. 40; hRef. 41; i Ref. 42; jRef. 43; k Ref. 44, Ref. 45.

Modelling the catalytic cycle reaction. As pointed out by Olah et al.46, many of commonly used oxides as catalysts for OCM reaction give remarkably similar conversion-selectivity results, suggesting a common active site and reaction mechanism, accordingly, alternative cycles in OCM process on BaTiO3 catalyst similar to the Ito–Lunsford mechanism2 is proposed in Fig. 4. In this figure, we use notation in which brackets enclose the surface defect, and species indicated outside the brackets are adsorbents at the defect site. There is a relation between the net atomic charge and the coordination number. The results show that net atomic charge decreases with an increase in the coordination number. As shown in Fig. 4, the lower channel involves proton migration between defects. The upper loop shows an analogous reaction with a mobile hydrogen atom.

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mobile hydrogen atom

proton migration

Fig. 4. Alternative cycles in OCM process by BaTiO3 catalyst similar to the Ito-Lunsford mechanism. 2[Ba2+O2–] + 2CH4 (g) 2[Ba2+O2–] H + 2CH3 °(g). The lower channel involves proton migration between defects. The upper loop shows an analogous reaction with a mobile hydrogen atom (Va: an anion vacancy)

Developed cycle. A more comprehensive and accurate cycle for the oxidative coupling of methane over BaTiO3 is composed of reactions (8)–(12) in Table 5 and indicated by bold arrows in Fig. 5. This cycle begins with adsorption of molecular oxygen on the [Ba2+O2–] defect, resulting in the formation of a surface ion (equation (8) of Table 5). This species abstracts hydrogen from methane forming another stable intermediate [Ba2+O2–]O2H, which, in turn, reacts further with another methane molecule to form the [Ba2+O2–]O defect, as well as a CH3 radical, and a water molecule (equation (10) of Table 5). As discussed in the previous section, [Ba2+O2–] O has a significant hydrogen affinity; therefore, it may abstract hydrogen from methane (equation (11) of Table 5) and then participate in a second reaction with methane to form water and restore the active site (equation (12) of Table 5). All other assumed surface reactions shown in Fig. 5 with thin arrows are listed in the lower portion of Table 5.

Fig. 5. Predicted possible catalytic cycles that involve only one [Ba2+O2–] reactive site The reactions are given in Table 5

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Table 5. All predicted catalytic cycle reactions related to Fig. 5

Eq No Reaction The main catalytic reaction for OCM process indicated by bold arrows in Fig. 5 (8) (9) (10) (11) (12)

(7) (13) (14) (15) (16) (17)

[Ba2+O2–] + O2(g) → [Ba2+O2–]O2 [Ba2+O2–]O2 + CH4(g) → [Ba2+O2–]O2H + CH3(g) [Ba2+O2–]O2H + CH4(g) → [Ba2+O2–]O + H2O(g) + CH3(g) [Ba2+O2–]O + CH4(g) → [Ba2+O2–]OH + CH3(g) [Ba2+O2–]OH + CH4(g) → [Ba2+O2–]+ H2O(g) + CH3(g) All other surface reactions with thin arrows in Fig. 5 We assumed that the atoms are released into the gas phase [Ba2+O2–]+ CH4(g) → [Ba2+O2–]H + CH3(g) [Ba2+O2–]+ O2(g) → [Ba2+O2–]O + O(g) [Ba2+O2–]O + O2(g) → [Ba2+O2–]O2 + O(g) [Ba2+O2–]H + O2(g) → [Ba2+O2–]OH + O(g) [Ba2+O2–]H + O2(g) → [Ba2+O2–]O2H [Ba2+O2–]O2H + CH4(g) → [Ba2+O2–]OH + OH(g) + CH3(g)

Fig. 6. HOMO and LUMO topologies of the optimised cubic BaTiO3 model

In Fig. 6 the HOMO and LUMO topologies of the cubic BaTiO3 model are compared. The character of the HOMO or LUMO of the model plays an important role in activation of CH4. The HOMO consists of oxygen electrons and LUMO is mainly composed of Ti and Ba orbitals for activation of CH4. CONCLUSIONS The homogeneous-heterogeneous natural gas conversion processes such as OCM reaction has considerable significance in the utilisation of the natural gas as petrochemical feedstock. There are, however, some problems such as the low performance of current catalytic processes for direct conversion of natural gas to value-added higher hydrocarbons. Quantum chemical calculations offer the opportunity to probe details of catalytic chemistry that are difficult to obtain from experiment. The quantum mechanics and

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ab initio calculations can be used to the virtual chemistry lab for studying many reactions at surfaces. Using hybrid DFT (B3PW, B3LYP) as a new screening method, we suggested a novel catalytic cycle mechanism for OCM reaction over BaTiO3 and showed that the mechanism on optimised (BaTiO3)8 model catalysts is more consistent with the Ito–Lunsford mechanism on Li/MgO oxide catalyst. In general, our calculations show good agreement with experimental results. As mentioned by Reynolds47 ’Many industrial problems are simply too large for timely solutions using large-scale ab initio quantum calculations…industrial chemists are often more interested in trends than absolute numbers’. Therefore, the results of the present study could be used in ongoing works to determine the model of perovskite surfaces, multi-layered structures, interfaces between perovskites and gas-phase ions, defects in perovskite crystals, and investigation of active sites by making use of various ab inito calculations on OCM catalytic. There is every reason to believe that the theoretical study of processes on surfaces based on ab initio electronic structure methods will also prosper in the future. It will provide further exciting insights into fundamental mechanisms but it will also become a very valuable tool in the research and development process48. ACKNOWLEDGEMENT This research was supported by the Petrochemical Research and Technology Company of National Petrochemical Company (NPC-RT). One of authors, M. Gharibi, would like to thank Dr. S. Sahebdelfar for helpful comments on the improvement of the text. REFERENCES   1. G. E. KELLER, M. M. BHASIN: Synthesis of Ethylene via Oxidative Coupling of Methane: I. Determination of Active Catalysts. J. Catal., 73, 9 (1982).   2. T. ITO, J. H. WANG, C. H. LIN, J. H. LUNSFORD: Oxidative Dimerization of Methane over a Lithium-promoted Magnesium Oxide Catalyst. J. Am. Chem. Soc., 107, 5062 (1985).   3. J. R. ANDERSON: Methane to Higher Hydrocarbons. Appl. Catal., 47, 177 (1989).   4. F. F. FAVAT, Ph. D’ACRO, R. ORLANDO, R. DOVESI: A Quantum Mechanical Investigation of the Electronic and Magnetic Properties of CaMnO3 Perovskite. J. Phys. Condens. Matter., 9, 489 (1997).   5. A. HASSAN, E. BAGHERZADEH: WO Pat.,005042 A1, NPC-RT Co., 2005.   6. H. DONNERBERG: Atomic Simulation of Electrooptic and Magnetooptic Oxide Materials. Springer Tracts in Modern Physics, Vol. 151, 1999.   7. X. LI, K. TOMISHIGE, K. FUJIMOTO: Oxidative Coupling of Methane by Water as the Oxidant on Perovskite Oxide Catalysts. Catalysis Letters, 36, 21 (1996).   8. C.-T. AU , T.-J. ZHOU, W.-J. LAI, C.-F. NG: An ab initio Study of Methane Activation on Lanthanide Oxide. Catalysis Letters, 49, 53 (1997).   9. M. A. JOHNSON, E. V. STEFANOVICH, T. N. TRUONG: An ab initio Study on the Oxidative Coupling of Methane over a Lithium-doped MgO Catalyst: Surface Defects and Mechanism. J. Phys. Chem., B 101, 3196 (1997). 10. E. E. Wolf: Methane Conversion by Oxidative Processes. VNR Catalysis Series, 1992, p. 274.

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M. CHALLACOMBE, P. M. W. GILL, B. G. JOHNSON, W. CHEN, M. W. WONG, J. L. ANDRES, M. HEAD-GORDON, E. S. REPLOGLE, J. A. POPLE: Gaussian 98. revision A.7; Gaussian, Inc., Pittsburgh, PA, 1995. 30. Y.-I. KIM, K.-S. RYU, S.-H. NAHM, J.-S. PARK: Structure and Electron Charge-density Analysis of Nano-sized BaTiO3 Powder Prepared by Solvothermal Method. Current Applied Physics, 6, e266 (2006). 31. Y.-I. KIM, J. K. JUNG, K.-S. RYU: Structural Study of Nano BaTiO3 Powder by Rietveld Refinement. Materials Research Bulletin, 39, 1045 (2004). 32. B. G. JOHNSON, P. M. W. GILL, J. A. POPLE: The Performance of a Family of Density Functional Methods. J. Chem. Phys., 98, 5612 (1993). 33. K. H. HELLWEGE, A. M. HELLWEGE (Eds): Ferroelectrics and Related Substances. LandoltBornstein, Vol. 3, Springer Verlag, 1969. 34. H. B. SHU, G. C. ZHOU, X. L. ZHONG, L. Z. SUN, J. B. WANG, X. S. CHEN, Y. C. ZHOU: Effects of Lattice Strain and Ion Displacement on the Bonding Mechanism of the Ferroelectric Perovskite Material BaTiO3: First-principles Study. J. Phys. Condens. Matter, 19, 276213 (2007). 35. R. C. WEAST, M. J. ASTLE (Eds): CRC Handbook of Chemistry and Physics. 62nd ed. CRC Press, Boca Raton, FL, 1981–1982. 36. T. MÜLLER, M. DALLOS, H. LISCHKA, Z. DUBROVAY, P. G. SZALAY: A Systematic Theoretical Investigation of the Valence Excited States of the Diatomic Molecules B2, C2, N2 and O2. Theor. Chem. Acc., 105, 227 (2001). 37. C. W. BAUSCHLICHER, S. R. LANGHOFF: Full CI Benchmark Calculations on N2, NO, and O2: A Comparison of Methods for Describing Multiple Bonds. J. Chem. Phys., 86, 5595 (1987). 38. J. M. MARTELL, J. D. GODDARD, L. A. ERIKSSON: Assessment of Basis Set and Functional Dependencies in Density Functional Theory: Studies of Atomization and Reaction Energies. J. Phys. Chem., A 101, 1927 (1997). 39. B. JURSIC, R. MARTIN: Calculation of Bond Dissociation Energies for Oxygen Containing Molecules by ab initio and Density Functional Theory Method. Int. J. Quant. Chem., 59, 495 (1996). 40. R. JANOSCHEK: Quantum Chemical B3LYP/cc-pvqz Computation of Ground-state Structures and Properties of Small Molecules aith Atoms of Z ≤ 18 (Hydrogen to Argon). Pure Appl. Chem., 73, 1521 (2001). 41. R. NEUMANN, N. C. HANDY: Investigations Using the Becke-Roussel Exchange Functional. Chem. Phys. Lett., 246, 381 (1995). 42. Handbook of Chemistry and Physics. 79th ed. CRC Press, NY, 1998, 9–80. 43. Y. R. LUO: Handbook of Bond Dissociation Energies in Organic Compounds. CRC Press, Boca Raton, 2003. 44. B. ROSEN: Spectroscopic Data Relative to Diatomic Molecules. Pergamon Press, Oxford, 1970. 45. D. F. McMILLEN, D. M. GOLDEN: Hydrocarbon Bond Dissociation Energies. Ann. Rev. Phys. Chem., 33, 493 (1982). 46. G. A. OLAH, Á. MOLNAR: Hydrocarbon Chemistry. 2nd ed. Wiley-Interscience, New York, 2003, 109–113. 47. C. H. REYNOLDS: Semiempirical MO Methods: The Middle Ground in Molecular Modeling. J. Mol. Struct. (Theochem.), 401, 267 (1997). 48. A. GROSS: The Virtual Chemistry Lab for Reactions at Surfaces: Is It Possible? Will It Be Useful? Surface Science, 500, 347 (2002). Received 10 October 2009 Revised 31 November 2009

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Oxidation Communications 35, No 3, 538–544 (2012) Gas-phase oxidation reactions

Direct Kinetic Study of the OH-radical Initiated Oxidation of Pivalaldehyde, (CH3)3CC(O)H, in the Gas Phase E. Szabó, G. L. Zügner, M. Farkas, I. Szilágyi, S. Dóbé* Institute of Materials and Environmental Chemistry, Chemical Research Center of the Hungarian Academy of Sciences, Pusztaszeri út 59–67, H-1025 Budapest, Hungary E-mail: [email protected] ABSTRACT The low-pressure fast discharge flow method was used to study the kinetics of the oxidation reaction system OH + (CH3)3CC(O)H + O2 at room temperature. OH radicals were monitored by resonance florescence detection; helium was the carrier gas. The rate constant for the overall reaction between OH radicals and pivalaldehyde, (CH3)3CC(O)H was determined to be k1 (298 K) = (2.65 ± 0.34) × 10–11 cm3 mol–1 s–1, given with 2σ statistical uncertainty. The OH yields of ГOH = 0.26 ± 0.10 and ГOH = 0.15 ± 0.06 were measured for the pivaloyl radical, (CH3)3CC(O), + O2 reaction at P = 1.81 and 3.46 mbar reaction pressures, respectively. Comparison with literature and the atmospheric implications have been discussed. Keywords: atmospheric kinetics, pivalaldehyde, OH reaction rate constant, OH product yield. AIMS AND BACKGROUND Aldehydes, among them pivalaldehyde (PVA), 2,2-dimethyl-propanal ((CH3)3CC(O)H), are important trace constituents of the atmosphere. They are emitted both from biogenic and anthropogenic sources, and are reaction intermediates formed in the atmospheric degradation of most atmospheric organics1. The oxidative degradation of aldehydes contributes significantly to the HOx and O3 budget of the troposphere and they are precursors for peroxyacylnitrates, which are components of urban smog and efficient reservoirs of NOx (Ref. 2). Similarly to other aldehydes, a major atmospheric loss process of pivalaldehyde is the reaction with OH radicals (equation (1)). The reaction takes place predominantly via the abstraction of the aldehydic H atom3,4 *

For correspondence.

538

leading to the pivaloyl (PVL), 2,2-dimethyl-propionyl ((CH3)3CC(O)), radical. PVL reacts exclusively with O2 in the atmosphere and is known to form the pivaloylperoxyl radical, (CH3)3CC(O)O2 (equation (2a)) (Ref. 3). By analogy with the acetyl (CH3CO) + O2 (Refs 5 and 6) and propionyl (C2H5CO) + O2 (Refs 7 and 8) reactions, OH formation is also an expected reaction channel in the reaction of PVL radicals with O2 (equation (2b)). OH + (CH3)3CC(O)H → (CH3)3CC(O) + H2O (CH3)3CC(O) + O2 → products

(1) (2)

(CH3)3CC(O) + O2 + M → (CH3)3CC(O)O2 + M

(2a)

(CH3)3CC(O) + O2 → OH + other products

(2b)

Objective of our study was to determine rate constant for the overall reaction between OH radicals and pivalaldehyde, k1, and to determine OH yields (branching ratios), ГOH = k2b/k2, at room temperature and a few mbar pressure of helium. Peroxy radicals (step (2a)) and the co-product to OH (step (2b)) have not been determined in the present work. EXPERIMENTAL The low-pressure fast discharge flow method (DF) was applied to investigate the oxidation kinetics of pivalaldehyde initiated by OH radicals. OH was detected directly by A2Σ(v=0)←X2Π(v=0) resonance fluorescence (RF) using a microwave-powered resonance lamp for excitation. Details of the kinetic apparatus and RF detection have been presented previously9,10. The 60.0-cm long, 4.01-cm i.d. flow tube reactor was constructed of Pyrex and was coaxially equipped with a movable quartz injector. The inner surface of the reactor was coated with a thin layer of halocarbon wax to reduce heterogeneous wall effects. OH radicals were reacted with (CH3)3CC(O)H in the flow reactor in the presence and absence of O2. OH was produced in the reaction of H atoms with a slight excess of NO2 inside the movable injector: H + NO2 → OH + NO. Hydrogen atoms were obtained by microwave-discharge dissociation of H2, in large excess of He flow. Helium was the main carrier gas which was regulated by calibrated mass flow controllers. The smaller flows of reactants and radical-source molecules were regulated by needle valves and determined from the pressure rise over time in known volumes. The reaction pressure was measured with a calibrated capacitance manometer. The flow tube was connected downstream to a detection block where the induced resonance fluorescence of OH was viewed through an interference filter centred at 307 nm and detected by a photomultiplier. The multiplier output was fed into a purpose-built data acquisition PC-board for signal averaging and further analysis. The detection limit for OH was ~2 × 109 mol cm–3 (at S/N = 1 signal-to-noise ratio).

539

The experimental procedure involved monitoring of the concentration of OH radicals at different positions of the movable injector (at varied reaction times) in the presence and absence of O2, i.e. for OH + (CH3)3CC(O)H and OH + (CH3)3CC(O)H + O2. Both PVA and O2 were used in large excess over OH and they entered the reactor upstream through side arms. The initial OH concentration was [OH]0 ≈ 3 × 1011 mol cm–3, along with [(CH3)3CC(O)H] ≈ 4 × 1012 and [O2] ≈ 1 × 1015 mol cm–3. The ‘O2 flow on’ and ‘O2 flow off’ runs, or vice versa, were conducted in a back-to-back manner. The linear flow velocity was vlin ≈ 15 m s–1 allowing kinetic measurements to be performed on the millisecond time scale. Materials used in the experiments have been listed in Table 1. Table 1. Materials used in the experiments

Name He H2 Ar O2 NO2 (CH3 )3CC(O)H

Supplier Messer-Griesheim Messer Griesheim Linde Messer Griesheim Messer Griesheim Aldrich

Purity (%) 99.996 99.995 99.9990 99.995 98 > 97

Notes a b b

c d

As a carrier gas, it was passed through liquid-nitrogen-cooled silica-gel traps before entering the flow system; b used as 5% H2 + 10% Ar mixture in He; c purified by low-temperature trap-to-trap distillation in vacuum and used as 1% mixture in He; d degassed by freeze-pump-thaw cycles prior to use. It was metered in the flow tube either directly or premixed in 10–15% with He. a 

RESULTS The experiments were carried-out at room temperature, T = 298 ± 3 K, and P = 1.82 and 3.46 mbar pressures of He. OH signals were found to decrease with increasing reaction time both in the absence and presence of O2, but the depletion was slower and the OH signals were bigger when oxygen was added to the system indicating the reformation of OH via reaction (2b). OH obeyed first-order kinetics both for OH + PVA and OH + PVA + O2. Figure 1 presents representative OH kinetic plots, where the decay curves drawn are non-linear least squares (LSQ) fit to the experimental data.

540

Fig. 1. Typical OH decay plots determined for OH + (CH3)3CC(O)H and OH + (CH3)3CC(O)H + O2 in back-to-back experiments (P = 1.82 mbar, T = 299 K, [OH]0 ≈ 3.4 × 1011, [PVA] = 3.6 × 1012 and [O2] = 1.9 × 1015 mol cm–3)

The decay constant measured in the absence of O2 is the pseudo-first order rate constant for the reaction OH + PVA (1), k1′ = k1 [PVA] + const; a plot of the k1′ values versus the pivalaldehyde concentration is shown in Fig. 2. Data plotted include all k1′ both from the 1.82 and 3.46 mbar experiments and show no discernible pressure dependence. The experimental data have defined a reasonable good straight line with a small intercept. A linear LSQ analysis has provided the rate constant value of k1(298 K) = (2.65 ± 0.34) × 10–11 cm3 mol–1 s–1.

(The errors above and throughout this Communication designate 2σ statistical uncertainty.)

Fig. 2. Plot of the pseudo-first order decay constant versus the pivalaldehyde concentration in the absence of O2. The slope of the straight line provides the rate constant for the reaction OH + (CH3)3CC(O)H

541

Under our experimental conditions only reactions (1), (2a), (2b) and the heterogeneous loss of OH took place, OH + wall → products (w). As we have shown in a previous publication5, the system of differential equations of reactions (1)–(w) can be solved analytically, providing equation (I) for the OH yield: ГOH = (k1′ – k*) / (k1′ – kw)

(I)

where k1′ is the OH decay constant with (CH3)3CC(O)H (see also above); k* – the OH decay constant with (CH3)3CC(O)H + O2, and kw – the ‘wall’ rate constant of OH. The depletion of OH radicals on the wall of the reactor was determined in separate experiments to be kw = 8 ± 3 s–1. The experimental conditions and kinetic results are summarised in Table 2. The OH yield has been found significant at the low pressures of the investigations; the more accurate result was obtained at 1.81 mbar where the OH reformation was more significant and the reproducibility was better: ГOH(1.81 mbar) = 0.26 ± 0.10. Table 2. Experimental conditions and kinetic results for the OH-initiated oxidation of pivalaldehyde (T = 298 ± 3 K)

P (He) (mbar) 1.81 3.46

[PVA] k′1 a (1012 mol cm–3) (s–1) 0.77–4.37 22.8–223.3 1.70–6.49 63.6–177.6

k* b (s–1) 18.1–192.1 58.2–172.2

ГOH ± 2σ c

Runsd

0.26 ± 0.10 0.15 ± 0.06

13  5

OH decay constant in the absence of O2; b OH decay constant in the presence of O2; the O2 concentration was ~2 × 1015 mol cm–3; c OH yield (branching ratio), ГOH = k2b/k2; d number of back-to-back determinations of k1′ and k*. a

DISCUSSION The rate constant we have determined for the overall reaction between OH radicals and pivalaldehyde, k1(300 K) = (2.65 ± 0.34) × 10–11 cm3 mol–1 s–1, agrees well with most of the data reported in literature. As for instance, in a recent study by D’Anna et al.4 the rate constant value of k1(298 K) = (2.86 ± 0.13) × 10–11 cm3 mol–1 s–1 has been determined by relative-rate kinetic experiments (for further references see also the paper of these authors). In an early work from our own laboratory, a significantly higher rate constant was determined for reaction (1) using also the DF-RF method9. The reason of the disparity is not known; the only apparent difference is a somewhat higher, but still not unusually high, wall consumption of OH (kw = 20 s–1) in the previous experiments9. Le Crane and co-workers3 have carried out a detailed laboratory study on the atmospheric chemistry of pivalaldehyde using flash photolysis–UV absorption and continuous photolysis – FTIR absorption methods in 930 mbar synthetic air at 296 K. Pivaloyl (PVL), (CH3)3CC(O), radicals were produced by reacting pivalaldehyde with Cl atoms. The authors have shown that the atmospheric fate of PVL radicals is the 542

addition to O2 to give pivalylperoxyl radical, (CH3)3CC(O)O2, (reaction (2a)) with a yield of >0.98. The OH yields of 0.26 and 0.15 we have determined do not contradict with the results of Le Crane et al., since we have performed the experiments at much lower pressures. In two of our recent studies, OH formation was shown to be the dominating reaction product for the CH3CO + O2 (Ref. 5) and C2H5CO+O2 (Ref. 7) reactions in the low pressure regime of the DF technique. At ~1 mbar pressure of He, the OH yields were found close to unity, but they decreased quickly with increasing pressure as understood by the efficient competition between the pressure dependent O2-addition and the pressure-independent OH elimination5,7. In our current study, we have observed also significant, although smaller OH yields for the reaction (CH3)3CC(O) + O2. Some pressure dependence is also evident by the presented data, but measurements were done only at two pressures and no OH yields could be determined above ~4 mbar because of the small OH signals at higher pressures. The OH yields are compared in Table 3. Table 3. Comparison of OH yields (ГOH) for the acyl + O2 reactions in the low pressure regime (T = 300 K)

Reaction (CH3)3CC(O) + O2 CH3CO + O2 C2H5CO + O2 (CH3)3CC(O) + O2 CH3CO + O2 C2H5CO + O2

P(He) (mbar) 1.81 2.00 2.00 3.46 3.50 3.59

ГOH 0.26 ± 0.10 0.86 0.86 ± 0.14 0.15 ± 0.06 0.57 0.68 ± 0.10

Reference this work Kovács et al.5 Zügner et al.7 this work Kovács et al.5 Zügner et al.7

The OH yields presented in Table 3 are seen to be close to each other for the CH3CO + O2 and C2H5CO + O2 reactions, while they are more than 3-times smaller for the (CH3)3CC(O) + O2 reaction at comparable pressures. One possible explanation for the apparent small OH yields is that the pivaloyl radical is much less stable thermally compared with its acetyl and propionyl counterparts3,11 and so the decomposition reaction of PVL, (CH3)3CC(O) + M → (CH3)3C + CO + M, could compete efficiently with its reaction with the oxygen molecule. In this case, estimation of ГOH is no longer viable by using the simple equation (I). The high rate constant we have determined for reaction (1) implies a short tropospheric lifetime of pivalaldehyde. Taking an average global OH concentration of [OH]global = 1 × 106 mol cm–3 (Ref. 12), one obtains τOH ≈ 1/(k1(298 K) × [OH]global) = 11 h estimate for the lifetime of PVA in the troposphere with respect to its reaction with OH radicals. Photolysis may be of comparable importance for the atmospheric removal of PVA, but no photochemical study of pivalaldehyde has been reported in the gas phase. Based on our current experimental findings, and analogies with the acetyl and propionyl radical reactions with O2 (Refs 5 and 7), the OH yield for the PVA 543

+ O2 reaction must be very small in atmospheric pressure air. Thus, the atmospheric photooxidation of pivalaldehyde is likely to occur via the OH initiation step, followed by the formation of the peroxyl radical (CH3)3CC(O)O2 which undergoes subsequent reactions with NO, NO2 and other peroxyl radicals. ACKNOWLEDGEMENTS This work has been supported by the European Atmospheric Chemistry Project SCOUT-O3 (contract GOCE-CT2004-505390) and the Hungarian Research Fund (contract OMFB-00992/2009). REFERENCES   1. A. MELLOUKI, G. le BRAS, H. SIDEBOTTOM: Kinetics and Mechanisms of the Oxidation of Oxygenated Organic Compounds in the Gas Phase. Chemical Reviews, 103, 5077 (2003).   2. B. J. FINLAYSON-PITTS, J. N. PITTS: Chemistry of the Upper and Lower Atmosphere. Theory, Experiments, and Applications. Academic Press, San Diego, San Francisco, New York, 2003.   3. J. P. le CRANE, E. VILLENAVE, M. D. HURLEY, T. J. WALLINGTON, S. NISHIDA, K. TAKAHASHI, Y. MATSUMI: Atmospheric Chemistry of Pivalaldehyde and Isobutyraldehyde: Kinetics and Mechanisms of Reactions with Cl Atoms, Fate of (CH3)3CC(O) and (CH3)2CHC(O) Radicals, and Self-reaction Kinetics of (CH3)3CC(O)O2 and (CH3)2CHC(O)O2 Radicals. J. of Physical Chemistry A, 108, 795 (2004).   4. B. D’ANNA, W. ANDRESEN, Z. GEFEN, C. NIELSEN: Kinetic Study of OH and NO3 Radical Reactions with 14 Aliphatic Aldehydes. Physical Chemistry Chemical Physics, 3, 3057 (2001).   5. G. KOVÁCS, J. ZÁDOR, E. FARKAS, R. NÁDASDI, I. SZILÁGYI, S. DÓBÉ, T. BÉRCES, F. MÁRTA, G. LENDVAY: Kinetics and Mechanism of the Reactions of CH3CO and CH3C(O)CH2 Radicals with O2. Low-pressure Discharge Flow Experiments and Quantum Chemical Computations. Physical Chemistry Chemical Physics, 9, 4142 (2007).   6. M. A. BLITZ, D. E. HEARD, M. J. PILLING: OH formation from CH3CO+O2: A Convenient Experimental Marker for the Acetyl Radical. Chemical Physics Letters, 365, 374 (2002).   7. G. L. ZÜGNER, I. SZILÁGYI, J. ZÁDOR, E. SZABÓ, S. DÓBÉ, X. L. SONG, B. S. WANG: OH Yields for C2H5CO + O2 at Low Pressure: Experiment and Theory. Chemical Physics Letters, 495, 179 (2010).   8. M. T. BAEZA-ROMERO, M. A. BLITZ, D. E. HEARD, M. J. PILLING, B. PRICE, P. W. SEAKINS: OH Formation from the C2H5CO+O2 Reaction: An Experimental Marker for the Propionyl Radical. Chemical Physics Letter, 408, 232 (2005).   9. S. DÓBÉ L. KHACHATRYAN, T. BÉRCES: Kinetics of Reactions of Hydroxyl Radicals with a Series of Aliphatic Aldehydes. Berichte der Bunsen-Gesellschaft für physikalische Chemie, 93, 847 (1989). 10. K. IMRIK, E. FARKAS, G. VASVÁRI, I. SZILÁGYI, D. SARZYNSKI, T. BÉRCES, F. MÁRTA: Laser Spectrometry and Kinetics of Selected Elementary Reactions of the Acetonyl Radical. Physical Chemistry Chemical Physics, 6, 3958 (2004). 11. S. JAGIELLA, H. G. LIBUDA, F. ZABEL: Thermal Stability of Carbonyl Radicals. Part I. Straightchain and Branched C-4 and C-5 Acyl Radicals. Physical Chemistry Chemical Physics, 2, 1175 (2000). 12. D. E. HEARD, M. J. PILLING: Measurement of OH and HO2 in the Troposphere. Chemical Reviews, 103, 5163 (2003). Received 13 May 2011 Revised 16 June 2011

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Oxidation Communications 35, No 3, 545–559 (2012) Oxidation in the presence of Cu-containing compounds

Kinetic and Mechanistic Study of Oxidation of Succinamide by Diperiodatocuprate(III) in Aqueous Alkaline Medium K. M. Naik, S. T. Nandibewoor* P. G. Department of Studies in Chemistry, Karnatak University, 580 003 Dharwad, India E-mail: [email protected] ABSTRACT The kinetics of oxidation of succinamide (SUC) by diperiodatocuprate (III) (DPC) in aqueous alkaline medium at a constant ionic strength of 0.50 mol dm−3 was studied spectrophotometrically. The reaction between SUC and DPC in alkaline medium exhibits 1:4 stoichiometry (SUC: DPC). The reaction is of first order in [DPC] and has less than unit order both in [SUC] and [alkali]. However, the order in [SUC] and [alkali] changes from first order to zero order as their concentration increase. Intervention of free radicals was observed in the reaction. Increase in periodate concentration decreased the rate. The oxidation reaction in alkaline medium has been shown to proceed via a monoperiodatocuprate (III)–SUC complex, which decomposed slowly in a rate-determining step followed by other fast steps to give the products. The main oxidative products were identified by spot test and GC-MS. The reaction constants involved in the different steps of the mechanism were calculated. Keywords: succinamide, diperiodatocuprate (III), oxidation, kinetics, mechanism. AIMS AND BACKGROUND Succinamide is a well-known compound, which is synthetically important to the chemists. Succinamides are a sub-class of anticonvulsants, indicated for the treatment of seizures associated with epilepsy1. Although there is a known cure seizures associted with the disorder, succinamides are most often used in conjuction with other anticonvulsant medications to control other types of seizures (such as other generalised tonic-clonic or grand mal seizures) as part of a comprehensive course of treatment for epilepsy and other disorders. Succinamides are sold under several names, including ethosuximide (Zarontin) and celontin. Zarontin is the only succinamide that is *

For correspondence.

545

regularly used in the United States today, as celontin has a higher rate of side effects. Zarontin effectively controls partial seizures, but in some individuals may actually increase the likelihood of generalised seizures. Use of one of products, succinic acid, ranges from scientific applications such as radiation dosimetry and standard buffer solutions to applications in argiculture, food, medicine, plastics, cosmetics, textiles, plating and waste gas scrubbing. In recent years, the study of the highest oxidation state of transition metals has intrigued many researchers. Transition metals in a higher oxidation state can be stabilised by chelating with suitable polydentate ligands. Metal chelates such as diperiodatocuprate(III) (Ref. 2), diperiodatoargentate(III) (Ref. 3), and diperiodatonickelate(IV) (Ref. 4) are good oxidants in a medium with an appropriate pH value. Copper(III) is shown to be an intermediate in the copper(II)-catalysed oxidation of amino acids by peroxydisulphate5. The oxidation reaction usually involves the copper(II)–copper(I) couple, and such aspects are detailed in different reviews6. The use of diperiodatocuprate(III) (DPC) as an oxidant in alkaline medium is new and restricted to a few cases because of its limited solubility and stability in aqueous medium. Copper complexes have occupied a major place in oxidation chemistry because of their abundance and relevance in biological chemistry7. Copper(III) is involved in many biological electron-transfer reactions8. When the copper(III) periodate complex is oxidant and multiple equilibria between different copper(III) species9 are involved, it would be interesting to know which of the species is the active oxidant. The kinetics of oxidation of SUC by DPC has not been reported in literature. In view of the medicinal value and potential pharmaceutical importance of SUC and lack of literature on the oxidation mechanism of this drug by DPC, there was need for understanding the oxidation mechanism of this bioactive compound. Hence the title reaction was investigated in details. EXPERIMENTAL Materials and methods. All chemicals used were of reagent grade, and doubly distilled water was used throughout the work. A solution of SUC was prepared by dissolving an appropriate amount of SUC (S. D. fine) in doubly distilled water. The required concentration of SUC was prepared from its stock solution. The copper(III) periodate complex was prepared by standard procedure10. Existence of copper(III) complex was verified by its UV-vis. spectrum, which showed an absorption band with maximum absorption at 415 nm. The aqueous solution of copper(III) was standardised by iodometric titration and gravimetrically by the thiocyanate11 method. The copper(II) solution was prepared by dissolving the known amount of copper sulphate (BDH) in distilled water. Periodate solution was prepared by weighing the required amount of sample in hot water and used after keeping it for 24 h. Its concentration was ascertained iodometrically12 at neutral pH by phosphate buffer. KOH and KNO3 (BDH, AR)

546

were employed to maintain the required alkalinity and ionic strength, respectively, in reaction solutions. Instruments used. (i) For kinetic measurements, a Peltier Accessory (temperature control) attached Varian CARY 50 Bio UV-vis. spectrophotometer (Varian, Victoria3170, Australia) was used; (ii) for product analysis, a QP-2010S Shimadzu gas chromatograph mass spectrometer, Nicolet 5700-FT-IR spectrometer (Thermo, USA) and for pH measurements, an Elico pH-meter model LI120 were used. Kinetic measurements. The kinetic measurements were performed on a Hitachi 15020 UV-vis. spectrophotometer. The kinetics was followed under pseudo-first order conditions, where [SUC]>[DPC] at 25±0.1°C, unless specified. The reaction was initiated by mixing the DPC with SUC solution, which also contained the required concentration of KNO3, KOH, and KIO4. The progress of the reaction was followed spectrophotometrically at 415 nm by monitoring the decrease in absorbance due to DPC with the molar absorbency index, ε to be 6230 ± 100 dm3 mol−1 cm−1. It was verified that there is a negligible interference from other species present in the reaction mixture at this wavelength. The pseudo-first order rate constants, kobs, were determined from the lg (absorbance) versus time plots and were the average of duplicate runs (Table 1). The plots were linear up to 80% completion of reaction under the range of [OH−] used and the rate constants were reproducible to within ±5%. The orders for various species were determined from the slopes of plots of log kobs versus lg (respective concentration) of species except for [DPC] in which no variation of kobs was observed as expected to the reaction condition. During the kinetics, a constant concentration, viz. 5.0 × 10−5 mol dm−3 of KIO4 was used throughout the study unless otherwise stated. Since periodate is present in excess in DPC, the possibility of oxidation of SUC by periodate in alkaline medium at 25°C was tested. The progress of the reaction was followed iodometrically. However, it was found that there was no significant reaction under the experimental conditions employed compared to the DPC oxidation of SUC. The total concentrations of periodate and OH− were calculated by considering the amount present in the DPC solution and that additionally added. Kinetic runs were also carried out in N2 atmosphere in order to understand the effect of dissolved oxygen on the rate of reaction. No significant difference in the results was obtained under a N2 atmosphere and in the presence of air. In view of the ubiquitous contamination of carbonate in the basic medium, the effect of carbonate was also studied. Added carbonate had no effect on the reaction rates. The spectral changes during the reaction are shown in Fig. 1. It is evident from the figure that the concentration of DPC decreases by observing the absorbance at 415 nm.

547

Table 1. Effect of [DPC], [SUC], [IO4–] and [OH–] on the oxidation of succinamide by diperiodatocuprate(III) in alkaline medium at 25oC, I = 0.5 mol dm–3

[DPC] × 105 (mol dm–3) 1.0 3.0 5.0 8.0 10.0

[SUC] × 104 (mol dm–3) 4.0 4.0 4.0 4.0 4.0

[IO4–] ×105 (mol dm–3) 5.0 5.0 5.0 5.0 5.0

[OH–] (mol dm–3) 0.4 0.4 0.4 0.4 0.4

kobs × 103 (s–1) kcal ×103 (s–1) 4.06 4.77 4.71 4.72 4.98

4.83 4.83 4.83 4.83 4.83

5.0 5.0 5.0 5.0 5.0 5.0

1.0 2.0 4.0 6.0 8.0 10.0

5.0 5.0 5.0 5.0 5.0 5.0

0.4 0.4 0.4 0.4 0.4 0.4

1.99 3.18 4.71 5.61 6.39 6.97

2.00 3.28 4.83 5.72 6.31 6.72

5.0 5.0 5.0 5.0 5.0

4.0 4.0 4.0 4.0 4.0

1.0 3.0 5.0 8.0 10.0

0.4 0.4 0.4 0.4 0.4

6.75 5.46 4.71 3.65 3.21

6.97 5.70 4.83 3.92 3.48

5.0 5.0 5.0 5.0 5.0

4.0 4.0 4.0 4.0 4.0

5.0 5.0 5.0 5.0 5.0

0.04 0.08 0.1 0.2 0.4

1.42 2.35 2.73 3.83 4.71

1.42 2.34 2.68 3.81 4.83

Fig. 1. UV-vis. spectra changes during the oxidation of succinamide by alkaline diperiodatocuprate(III) at 298 K, [DPC] – 5.0 × 10–5, [SUC] – 5.0 10–4, [OH–] – 0.4 and I = 0.5 mol dm–3 with scanning time of: 1.0 min (1), 2.0 min (2), 3.0 min (3), 4.0 min (4), 5.0 min (5), 6.0 min (6) and 7.0 min (7)

548

Regression analysis of experimental data to obtain the regression coefficient r and standard deviation S of points from the regression line was performed using Microsoft 2003 Excel program. Stoichiometry and product analysis. Different sets of reaction mixtures containing various ratios of DPC to SUC in the presence of constant amounts of OH− and KNO3 were kept for 4 h in a closed vessel under nitrogen atmosphere. The remaining concentration of DPC was estimated by spectrophotometrically. The results indicated 1:4 stoichiometry as given in equation (1). O H2C

C

NH2

H2C

C

NH2

+ 4 [Cu(H2IO6)(H2O)2] + 4 OH–

O

(1)

O H2C H2C

C C O

OH OH

+ 2 NH2OH + 4 Cu2+ + 4 H2IO63– + 8 H2O



The reaction product was extracted with ether and recrystallised from aqueous alcohol. Only one product was obtained as evidenced by a single spot on thin layer chromatography, which was identified as succinic acid by spot test13(a) and was characterised by GC-MS spectral studies. GC-MS data was obtained on a QP-2010S Shimadzu gas chromatograph mass spectrometer. The mass spectral data showed a molecular ion peak at 102 m/z confirming the presence of succinic acid (Fig. 2). All other peaks observed in GC-MS can be interpreted in accordance with the observed structure of succinic acid. Another product, hydroxylamine (NH2OH), was identified by CHN data analysis, H = 0.8.98%, N = 41.83% and O = 41.12% and further it was identified by spot test13(b). Another product of Cu(II) was identified by UV-vis. spectra. The reaction products do not undergo further oxidation under the present kinetic conditions.

549

40

45

55

74 73

%

20 10

100

46

63

53 0

40.0

45.0

50.0

61 55.0

60.0

78

69 65.0

70.0

75.0

83 80.0

85.0

90.0

95.0

100.0

Fig. 2. Mass spectrum of reaction product, succinic acid with its molecular ion peak at 102 m/z

RESULTS Reaction orders. The reaction orders were determined from the slope of lg kobs versus lg (concentration) plots by varying the concentrations of SUC, alkali, and periodate in turn, while keeping all other concentrations and conditions constant, except for DPC concentration. Effect of [DPC]. The oxidant, DPC concentration was varied in the range 1.0×10−5 to 1.0×10−4 mol dm−3, and the fairly constant kobs values indicate that order with respect to [DPC] was unity (Table 1). This was also confirmed by linearity of the plots of lg [absorbance] versus time (r ≥ 0.9986, S ≤ 0.013) up to 80% completion of the reaction. Effect of [SUC]. The effect of [SUC] on the rate of reaction was studied at constant concentrations of alkali, DPC, and periodate at a constant ionic strength of 0.5 mol dm−3. The substrate, SUC, was varied in the range 1.0 × 10−4 to 1.0 × 10−3 mol dm−3. The kobs values increased with increase in concentration of SUC. The order with respect to [SUC] was found to be less than unity (Table 1) (r ≥ 0.9995, S ≤ 0.007) under the experimental concentrations. This less than unit order in the SUC was also confirmed by the linear plot of kobs versus [SUC]0.54. The plots of kobs versus [SUC] were nonlinear (Fig. 3). However, at lower concentrations of SUC, the reaction was first order in [SUC] and at high concentration of SUC, the reaction was independent of [SUC]. The order in [SUC] changes from first order to zero order as [SUC] increases. 0

2

[SUC]0.54 × 104 (mol dm–3) 4 6 8 10

12

1.4

0.8

1.0

0.6

0.8 0.6

0.4

0.4

0.2

0.2 0.0

0

1

2 3 4 5 [SUC] × 104 (mol dm–3)

6

0.0

Fig. 3. Plots of kobs versus [SUC]0.54 and kobs versus [SUC] (from Table 1)

550

kobs × 102 (s–1)

kobs × 102 (s–1)

1.2

1.0

Effect of [alkali]. The effect of increase in concentration of alkali on the reaction was studied at constant concentrations of SUC, DPC, and periodate at a constant ionic strength of 0.5 mol dm−3 at 25◦C. The rate constants increased with increase in alkali concentration (Table 1), indicating apparently less than unit order dependence of rate on alkali concentration (r ≥ 0.9994, S ≤ 0.006). Similar to as in the case of SUC, the order in alkali changes from first order to zero order as [OH–] increases. Effect of [periodate]. The effect of increasing concentration of periodate was studied by varying the periodate concentration from 1.0 × 10−5 to1.0 × 10−4 mol dm−3 keeping all other reactants concentrations constant. It was found that the added periodate had a retarding effect on the rate of reaction, the order with respect to periodate concentration being negative less than unity (Table 1). Effect of ionic strength (I) and dielectric constant of the medium (D). The addition of KNO3 at constant [DPC], [SUC], [OH−], and [IO4−] was found that increasing ionic strength had no significance on the rate of the reaction. Dielectric constant of the medium, D, was varied by varying t-butyl alcohol and water percentage. The D values were calculated from the equation D = DwVw + DBVB, where Dw and DB are dielectric constants of pure water and t-butyl alcohol, respectively, and Vw and VB – the volume fractions of components water and t-butyl alcohol, respectively, in the total mixture. There was no effect of dielectric constant on the rate of reaction. Thus, from the observed experimental results, the rate law for reaction is given as follows: rate = kobs [SUC ]0.54 [DPC]1.0 [OH–]0.52 [IO4–]–0.32.

Effect of added products. The externally added products, succinic acid and copper(II) (CuSO4) in the range 1.0×10−4 to 1.0×10−3 mol dm−3 did not have any significant effect on the rate of the reaction. Polymerisation study (Test for free radicals). DPC is a single equivalent oxidant. Hence, intervention of a free radical, generated from the organic compound, was expected. In view of this, for the reaction the possibility of formation of free radicals was detected as follows. The reaction mixture, to which a known quantity of acrylonitrile (scavenger) had been added initially, was kept in an inert atmosphere for 2 h. On diluting the reaction mixture with methanol, a white precipitate was formed, indicating the intervention of free radicals in the reaction. The blank experiments of either DPC or SUC alone with acrylonitrile did not induce any polymerisation under the same condition as those induced for reaction mixture. Initially, added acrylonitrile decreases the rate of reaction indicating free radical intervention, which is the case in earlier work14. Effect of temperature. The rate of reaction was measured at 4 different temperatures under standard condition. The rate constants, kobs, of the reaction were obtained from 551

the slopes and intercepts of 1/kobs versus 1/[SUC], 1/kobs versus [H3IO62−], and 1/kobs versus 1/[OH−]. The data are subjected to the least square analysis which is given in Table 2. From the plot of lg kobs versus 1/T the activation parameters have been calculated and tabulated in Table 2. Table 2. Effect of temperature on kobs, for the oxidation of succinamide by diperiodatocuprate(III) in aqueous alkaline medium

Temperature (K)

kobs × 103 (s–1)

293 298 303 308

  2.96   4.71   7.86 13.02

DISCUSSION The water-soluble copper(III) periodate complex is reported15 to be [Cu(HIO6)2]5−. However, in an aqueous alkaline medium and at a high pH range as employed in the study, periodate is unlikely to exist as HIO64− (as present in the complex) as is evident from its involvement in the multiple equilibria16 (equations (2)–(4)) depending on the pH of the solution. H5IO6

H4IO6– + H+, K1′ = 5.1 × 10–4

(2)

H4IO6

H3IO6 + H , K2′ = 4.9 × 10

(3)

H3IO62–

H2IO63– + H+, K3′ = 2.5 × 10–12

(4)

–

2–

+

–9

Periodic acid exists in acid medium as H5IO6 and as H4IO6− around pH 7. Thus, under the conditions employed in alkaline medium, the main species are expected to be H3IO62− and H2IO63−. At higher concentrations, periodate also tends to dimerise17. However, formation of this species is negligible under conditions employed for kinetic study. Hence, at the pH employed in this study, the soluble copper(III) periodate complex exists as diperiodatocuprate(III), [Cu(H3IO6)(H2IO6)]2−, a conclusion also supported by earlier work2. MECHANISM The reaction between the diperiodatocuprate(III) complex and succinamide in alkaline medium has the stoichiometry 1:4 (SUC: DPC) with a first order dependence on [DPC] and an apparent order of less than unity in [substrate], [alkali] and negative fractional order dependence on the periodate. Based on the experimental results, a mechanism is proposed for which all the observed orders in each constituent such as [oxidant], [reductant], [OH−], and [IO4−] may be well accommodated. Lister18 proposed 3 forms of copper(III) periodate in alkaline medium as diperiodatocuprate(III) 552

(DPC), monoperiodatocuprate(III) (MPC) and tetrahydroxocuprate(III). The tetra­ hyd­roxocuprate(III) is ruled out, as its equilibrium constant is 8.0 × 10−11 at 40°C. Hence, in the present study, DPC and MPC are considered to be as active forms of copper(III) periodate complex. The result of increase in rate of reaction with increase in alkalinity (Table 1) can be explained in terms of prevailing equilibrium of formation of [Cu(H­3IO6)(H2IO6)]2– from [Cu(H3IO6)2)] – as given below. [Cu(H3IO6)2]– + OH–

K1

[Cu(H2IO6)(H3IO6)]2– + H2O

(5)

Also the decrease in the rate with increase in periodate concentration suggests that the displacement of a ligand periodate takes place to give a free periodate and monoperiodatocuprate(III) (MPC) species from [Cu(H2IO6)(H3IO6)]2– as given in equation (6): [Cu(H2IO6)(H3IO6)]2–

K2

[Cu(H2IO6)(H2I)2] + [H3IO6]2–

(6)

Such a type of equilibria has been well noticed in literature19. It may be expected that a lower periodate complex such as monoperiodatocuprate(III) (MPC) is more important in the reaction than the DPC in view of the observed inverse fractional order in periodate. With the known equilibrium constants19, the individual concentrations of [DPC]f, DPC and MPC were calculated. It was found that MPC was in higher concentration and nearly paralleled the rate variation with different [OH–]. Furthermore, the spectra of Cu (III) periodate complex was dependent on [OH–] and the absorption becomes almost constant, indicating the predominance of one species, presumably [Cu(H2IO6)(H2O)2]. Because of this and the fact that rate is a function of [OH–] (less than unit order), the main oxidation species is likely to be [Cu(H2IO6)(H2O)2] and its formation equilibrium (6) is important for the reaction. The less than unit order in [SUC] presumably results from formation of a complex (C) between the MPC species and succinamide prior to the formation of the products. K3 is the composite equilibrium constant comprising the equilibrium to bind active species, MPC to SUC species to form a complex (C). This complex(C) undergoes decomposition in a slow step to give the free radical species of SUC, periodate, NH2+ and Cu(II). This free radical species of SUC reacts with 1 mol of MPC species in a fast step to form 3-carbamoylpropanoic acid, Cu(II), and periodate. This 3-carbamoylpropanoic acid further reacts with 1 more mol of MPC in a fast step to form a free radical species of 3-carbamoylpropanoic acid, Cu(II), NH2+ and periodate. Further this free radical reacts with 1 more mol of MPC to form the final products, i.e. succinic acid, Cu (II) and periodate. NH2+ ion reacts with hydroxyl ion in a fast step to form the hydroxylamine. So, the detailed mechanistic pathways for the oxidation of 6-MP by diperiodatocuprate (III) is presented in Scheme 1.

553

Scheme1 Detailed mechanistic pathways for the oxidation of succinamide by alkaline diperiodatocuprate(III)

Since Scheme 1 is in accordance with the generally well-accepted principle of non-complementary oxidations taking place in sequence of 1-electron steps, the reaction between the substrate and oxidant would afford a radical intermediate. A free-radical scavenging experiment revealed such a possibility. This type of radical 554

intermediate has also been observed in earlier work20. Spectroscopic evidence for the complex formation between oxidant and substrate was obtained from UV-vis. spectra of SUC (5.0 × 10−4 mol dm–3), DPC (5.0 × 10−5 mol dm–3), [OH−] (0.4 mol dm−3) and mixture of both. A hypsochromic shift of about 5 nm from 306 to 301 nm in the spectra of DPC was observed. The Michaelis–Menten plot also proved the complex formation between DPC and SUC, which explains the less than unit order dependence on [SUC]. Such a complex between a substrate and an oxidant has been observed in other studies21. The diamagnetic (dsp2) square planar structure of Cu (III) periodate in the form of DPC, MPC and the paramagnetic (sp3) complex of Cu (III) and SUC can be formulated as shown below.

Scheme 1 leads to rate law (7) (see appendix). rate =

kK1K2K3[DPC][SUC][OH–] [H3IO6 ] + K1[OH ][H3IO62–] + K1K2[OH–] + K1K2K3[SUC][OH–] –

2–

(7)

which explains all the observed kinetic orders of different species. kobs =

rate [DPC]

=

kK1K2K3[SUC][OH–] [H3IO62–] + K1[OH–][H3IO62–] + K1K2[OH–] + K1K2K3[SUC][OH–]

. (8)

Rate law (8) can be re-arranged into the following form, which is suitable for verification: 1 kobs

=

[H3IO62–] kK1K2K3[OH ][SUC] –

+

[HIO62–] kK2K3[SUC]

+

1 kK3[SUC]

+

1 k

.

(9)

According to equation (9), other conditions being constant, plots of 1/kobs versus 1/[OH−] (r ≥ 0.999, S ≤ 0.029), 1/kobs versus 1/[SUC] (r ≥ 0.997, S ≤ 0.016), and 1/kobs versus [H3IO62−] (r ≥ 0.998, S ≤ 0.011) should be linear and are found to be so (Fig. 4). The slopes and intercepts of such plots lead to the values of K1, K2, K3, and k as 1.92 555

dm3 mol–1, 2.09 ×10−5 mol dm−3, 1.55 × 104 dm3 mol−1 and 9.10 × 10−3 s−1, respectively at 298 K. The value of K1 is in a good agreement with earlier work19. Using these K1, K2, K3, and k values, the rate constants under different experimental conditions were calculated by equation (8) and compared with experimental data. There is a good agreement between them (Table 1), which fortifies Scheme 1. 0.45

0.6

a 1/kobs × 10–3 (s)

1/kobs × 10–3 (s)

0.35

0.4

298 K

0.3 0.2

298 K

0.30 0.25 0.20 0.15 0.10

0.1 0

b

0.40

0.5

0.05 0

2

4 6 8 10 1/[SUC] × 10–3 (dm3 mol–1) 0.8

0.00

0.50 1.00 1.50 1/[H3IO62–] × 10–4 (mol dm–3)

2.00

c

0.7 1/kobs × 10–3 (s)

0.00

12

0.6

298 K

0.5 0.4 0.3 0.2 0.1 0

0

5

10

15

20

25

30

1/[OH–] × 10–3 (dm3 mol–1)

Fig. 4. Verification of rate law (8) in the form of equation (9) for the oxidation of succinamide by diperiodatocuprate(III): a – plot of 1/kobs versus 1/[SUC], b – plot of 1/kobs versus 1/[H3IO62–] and c – plot of 1/kobs versus 1/[OH–] at 298 K

The negligible effect of ionic strength and dielectric constant of the medium on the rate qualitatively explains the reaction between neutral charged species, as seen in Scheme 1. The values of ΔH* and ΔS* were both favourable for electron transfer processes. The negative value of ΔS* indicates that the complex (C) is more ordered than the reactants22. The value of ΔS* within the range for radical reaction has been ascribed23 to the nature of electron pairing and unpairing processes and to the loss of degrees of freedom formerly available to the reactants upon the formation of rigid transition state. The observed modest enthalpy of activation and a relatively low value of the entropy of activation as well as a higher rate constant of the slow step indicate that the oxidation presumably occurs via inner-sphere mechanism. This conclusion is supported by earlier observation24. 556

Table 3. Thermodynamic activation parameters for the oxidation of succinamide by diperiodatocuprate(III) in aqueous alkaline medium

Parameters Ea (kJ mol–1) ∆H* (kJ mol–1) ∆S* (J K–1 mol–1) ∆G*298 K (kJ mol–1) lg A

Values   74   72 –48   86   10.6

APPENDIX Derivation of rate law for the reaction

According to Scheme 1 rate = – d[DPC]/dt = k[C] =

(i)

kK1K2K3[DPC][OH–][SUC] [H3IO6]2–

.

(ii)

Total concentration of DPC is given by [DPC]T = [DPC]f + [Cu(H2IO6)(H3IO6)]2– + [Cu(H2IO6)(H2O)2] + [C]

(

= [DPC]f 1 + K1[OH–] +

K1K2[OH–] [H3IO62–]

+

K1K2K3[OH–][SUC] [H3IO62–]

)

(iii)

,

where T and f refer to total and free concentrations. Therefore, [DPC]f =

[DPC]T[H3IO62–] [H3IO62–] + K1[H3IO62–][OH–] + K1K2[OH–] + K1K2K3[OH–][SUC]

.

(iv)

Similarly, [SUC]T = [SUC]f + [C] [SUF]f =

[SUC]T[H3IO62–] [H3IO62–] + K1K2K3[OH–][Cu(H3IO6)2]–

(v) .

In view of the low concentration of DPC used, the term K1K2K3[OH–][Cu(H3IO6)2]– compared to [H3IO62–] can be neglected. Hence, [SUC]f = [SUC]T

(vi)

[OH–]f = [OH–]T .

(vii)

Similarly,

557

Substituting equations (iv), (vi), and (vii) in (ii) and omitting the subscripts T and f, we get rate =

kK1K2K3[DPC][SUC][OH–] [H3IO62–] + K1[OH–][H3IO62–] + K1K2[OH–] + K1K2K3[SUC][OH–]

which explains all the observed kinetic orders of different species. kobs =

rate [DPC]

=

kK1K2K3[SUC][OH–] [H3IO62–] + K1[OH–][H3IO62–] + K1K2[OH–] + K1K2K3[SUC][OH–]

. (ix)

CONCLUSIONS Among the various species of DPC in aqueous alkaline medium, [Cu(H2IO6)(H2O)2] is considered as active species for the title reaction. The results indicated that in carrying out this reaction, the role of pH in the reaction medium is crucial. Rate constant of slow step and other equilibrium constants involved in the mechanism are evaluated. The overall mechanistic sequence described here is consistent with all the experimental evidences including the product, spectral, mechanistic and kinetic studies. REFERENCES   1. WEAVER, F. DONALD: Epilepsy and Seizures: Everything You Need to Know. Firefly Books, Toronto, 2001.   2. K. B. REDDY, B. SETHURAM, T. NAVANEETH RAO: Kinetics of Oxidation of Benzaldehydes by Copper(III) in t-Butanol-Water Medium. Indian J. Chem., 23A, 593 (1984).   3. A. KUMAR, VAISHALI, P. RAMAMURTHY: Kinetics and Mechanism of Oxidation of Ethylene­ diaimine and Related Compounds by Diperiodatoargentate (III) Ion. Int. J. Chem. Kinet., 32, 286 (2000).   4. R. S. SHETTAR, S. T. NANDIBEWOOR: Kinetic, Mechanistic and Spectral Investigations of Ruthenium (III) Catalysed Oxidation of 4-hydroxycoumarin by Alkaline Diperiodatonickelate(IV) (Stopped Flow Technique). J. Mol. Cat. A: Chem., 234, 137 (2005).   5. M. G. RAMREDDY, B. SETHURAM, T. NAVANEETH RAO: Effect of Copper(II) on Kinetics and Mechanism of Silver(I) Catalysed Oxidation of Some Amino Acids by Peroxydisulfate Ion in Aqueous Medium. Indian J. Chem., 16A, 313 (1978).   6. K. D. KARLIN, Y. GULTNEH: Progress in Inorganic Chemistry (Ed. S. J. Lippard). Vol. 35, Wiley, New York, 1997, p. 220.   7. N. KITAJIMA, Y. MORO-KA: Copper-dioxygen Complexes Inorganic and Bioinorganic Perspectives. Chem. Rev., 94, 737 (1994).   8. J. PEISACH, P. ALSEN, W. E. BLOOMBERG: The Biochemistry of Copper. Academic Press, New York, 1966, p. 49.   9. N. SRIDEVI, J. PADMAVATHI, K. K. M. YUSUFF: Determination of the Nature of the Diperiodatocuprate(III) Species in Aqueous Alkaline Medium through a Kinetic and Mechanistic Study on the Oxidation of Iodide Ion. Transition Met. Chem., 26, 315 (2001). 10. C. P. MURTHY, B. SETHURAM, T. NAVANEETH RAO: Kinetics of Oxidation of Some Alcohols by Diperiodatocuprate(III) in Alkaline Medium. Z. Phys. Chem., 262, 336 (1981). 11. G. H. JEFFERY, J. BASSETT, J. MENDHAM, R. C. DENNY: Vogel’s Textbook of Quantitative Chemical Analysis. 5th ed. ELBS, Longman, Essex, UK, 1966, p. 455.

558

12. G. P. PANIGRAHI, P. K MISRO: Kinetics and Mechanism of Os(VIII) Catalysed Oxidation of Unsaturated Acids by Sodium Periodate. Indian J. Chem., 16A, 201 (1978). 13. (a) F. FEIGL: Spot Tests on Organic Analysis. Elsevier, New York, 1975, p. 579; (b) F. FEIGL: Spot Tests on Organic Analysis. Elsevier, New York, 1975, p. 288. 14. G. C. HIREMATH, R. M. MULLA, S. T. NANDIBEWOOR: Mechanistic Study of the Oxidation of Isonicotinate Ion by Diperiodatocuprate(III) in Aqueous Alkaline Medium. J. Chem. Res., 197 (2005). 15. K. B. REDDY, B. SETHURAM, T. NAVANEETH RAO: Photon Cross Sections in Copper, Platinum and Gold at 81 keV. Z. Phys. Chem., 268, 706 (1987). 16. T. S. KIRAN, C. V. HIREMATH, S. T. NANDIBEWOOR: Kinetic, Mechanistic and Spectral Investigations of Ruthenium(III)/Osmium(VIII) Catalysed Oxidation of Paracetamol by Alkaline Diperiodatoargentate(III) (Stopped Flow Technique). Appl. Cat. A: Gen., 305, 79 (2006). 17. B. SETHURAM: Some Aspects of Electron Transfer Reactions Involving Organic Molecules. Allied Publishers, New Delhi, 2003, p. 78. 18. M. W. LISTER: The Stability of Some Complexes of Trivalent Copper. Can. J. Chem., 31, 638 (1953). 19. R. N. HEGDE, N. P. SHETTI, S. T. NANDIBEWOOR: Oxidative Degradation and Deamination of Atenolol by Diperiodatocuprate(III) in Aqueous Alkaline Medium: A Mechanistic Study. Polyhedron, 28, 3499 (2009). 20. R. R. HOSAMANI, R. N. HEDGE, S. T. NANDIBEWOOR: Mechanistic Study on the Oxidation of L-phenylalanine by Copper(III) in Aqueous Alkaline Medium: A Kinetic Approach. Monatsh Chem., 141, 1069 (2010). 21. P. N. NAIK, S. D. KULKARNI, S. A. CHIMATADAR, S. T. NANDIBEWOOR: Mechanistic Study of Oxidation of Sulfacetamide by Diperiodatocuprate(III) in Aqueous Alkaline Medium. Indian J. Chem., 47A, 1666 (2008). 22. A. WEISSBERGER: Investigations of Rates and Mechanism of Reactions (Ed. E. S. Lewis). Techniques of Chemistry. Vol. 4. Wiley, New York, 1974, p. 421. 23. C. WALLING: Free Radicals in Solution. Academic Press, New York, 1957, p. 38. 24. S. A. FAROKHI, S. T. NANDIBEWOOR: Kinetic, Mechanistic and Spectral Studies for the Oxidation of Sulfanilic Acid by Alkaline Hexacyanoferrate(III). Tetrahedron, 59, 7595 (2003). Received 9 February 2011 Revised 20 June 2011

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Oxidation Communications 35, No 3, 560–568 (2012) Oxidation in the presence of Cr-containing compounds

Kinetics and Mechanism of Oxidation of Benzaldehyde and 4-Nitrobenzaldehyde by Pyridinium Dichromate in Aquo–Acetic Acid Medium Bhupendra Amba*, B. L. Hiranb Chemical Kinetic Laboratory, Department of Chemistry, Government P. G. College, 458 441 Neemuch (M.P.), India E-mail: bk_amb2009@ yahoo.com b Chemical Kinetics Laboratory, Department of Chemistry, College of Science M. L. Sukhadia University, 313 001 Udaipur, India a

ABSTRACT Oxidation of benzaldehyde and 4-nirtobenzaldehyde by pyridinium dichromate (PDC) leading to corresponding benzoic acids is first order with respect to PDC and benzaldehydes and second order with respect to [H+]. The rate of oxidation decreases with increase in dielectric constant of solvent suggesting cation–dipole interaction. Electron-deficient reaction centre in the transition state is suggested. Formation of instant cyclic chromic ester between hydrated benzaldeyde and protonated pyridinium dichromate, followed by C–H bond fission in rate-determining step, has been suggested. Activation parameters have been evaluated. A mechanism consistent with experimental observations has been proposed. Keywords: kinetics, oxidation, 4-nitrobenzaldehyde, benzaldehyde, PDC. AIMS AND BACKGROUND A lot of work has been initiated in the area of development of new Cr(VI) reagents1–4 for the effective and selective oxidation of organic substrates. Mahanti and Banerji5 have reviewed synthetic, kinetic and mechanistic aspects of reactions of complexed Cr(VI) compounds. Very effective and interesting oxidising agent of Cr(VI), pyridinium dichromate2 (PDC), has been recently reported6,7. Only a few reports about the kinetics and mechanistic aspects of oxidation by PDC are available in literature5–8. The kinetics of oxidation of aromatic aldehydes by PDC has not been investigated. We report here the kinetics of oxidation of benzaldehyde and 4-nitrobenzaldehyde in acetic acid–perchloric acid medium with this oxidant. *

For correspondence.

560

EXPERIMENTAL All the aldehydes and other chemicals used were of AnalaR grade (E. Merck) and purity was checked by m.p. or b.p. Acetic acid (E. Merck, India) was purified by reffuxing with CrO3 and followed by distillation acid. The standard solutions of the purified benzaldehydes were prepared in purified acetic. PDC solutions were prepared in purified acetic acid. Solid PDC was prepared by reported method2 and crystallised from acetonitrile solution. Its purity was checked iodometrically and by m.p. The products of oxidation of benzaldehyde by PDC were confirmed as corresponding benzoic acid by m.p., tlc and spectral analysis. Stoichiometry investigation revealed that 3 mol of benzaldehyde consume 1 mol of PDC. 3ArCHO + 3H2O + PDC {2Cr(VI)} → 3ArCOOH + 2Cr(III) + 6H+

(1)

Kinetic measurements. The reactions were carried out under pseudo-first order conditions. A known volume of substrate, perchloric acid and acetic acid were mixed in reaction flask and kept in thermostat maintained at constant temperature (± 0.1K). The reaction was initiated by adding rapidly pre-determined volume of PDC solution into the above reaction mixture. Aliquots of (5.0 ml) were withdrawn at regular time intervals and added to 10 ml of 10% potassium iodide solution. This solution was titrated against previously standardised sodium thiosulphate (hypo) using starch as an indicator. The rate constants kobs were computed from the linear plots of lg [hypo] versus time by least square method. The results were reproducible to ±3%. Orders with respect to different reactants were determined by the Oswald isolation method. RESULTS AND DISCUSSION Effect of oxidant concentration on rate. The rate coefficients are independent of the initial concentration of PDC in the concentration range of 0.00333–0.00125 M, indicating first order of the reaction in PDC. Plots of lg [PDC] versus time are linear with correlation coefficient r >0.99. The results are summarised in Table 1. Effect of substrate concentration on rate. The rate of oxidation increases on increasing the concentration of benzaldehyde (Table 1). The plot of 1/kobs versus 1/[aldehyde] gave linear line but it did not have positive intercepts as required by the Michaelis– Menten kinetics9. This shows that either the complex formed is very unstable such that it can not be detected by kinetics study or it is not formed. Plot of lg kobs versus lg [substrate] is also a straight line in each case with a slope of ca. 1 (for benzaldehyde, 4-nitrobenzaldehyde are 0.76, 1.098, respectively). The order with respect to substrate is one.

561

Table 1. Variation of rate with oxidant concentration, substrate concentration, solvent composition, added perchloric acid, added Mn(II)/Ce(III), added pyridine and ionic strength temperature 308 K

[Sub- AcOH [H+] [PDC] [NaSO4] [NaClO4] strate] (%) ×10 ×103 ×103 ×103 ×102 (v/v) (mol (mol (mol (mol (mol dm–3) dm–3) dm–3) dm–3) –3 dm 1 2 3 4 5 6 2.0 30 15 3.33 0 0 2.0 30 15 2.00 0 0 2.0 30 15 1.75 0 0 2.0 30 15 1.50 0 0 2.0 30 15 1.25 0 0 2.0 30 15 2.00 0 0

Mn(II) Ce(III) Pyri­dine kobs × 105 (s–1) ×103 ×103 ×104 –H 4-NO2 (mol (mol (mol dm–3) dm–3) dm–3) 7 0 0 0 0 0 0

8 0 0 0 0 0 0

9 0 0 0 0 0 0

10 13.80 13.70 12.97 13.26 13.70 13.70

11 47.50 47.48 46.25 47.26 47.48 47.48

2.5 3.0 3.6 4.0 4.6

30 30 30 30 30

15 15 15 15 15

2.00 2.00 2.00 2.00 2.00

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

17.08 61.45 20.17 77.75 22.51 94.30 25.23 104.56 27.13 124.30

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

30 30 30 30 30 30 30 30 30 30 30 30 30

01 02 03 04 05 06 08 10 11 12 13 14 15

2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

00.05 00.37 00.23 01.12 00.52 02.90 01.07 04.66 01.43 07.18 02.14 09.55 03.86 16.12 05.75 22.82 07.09 28.76 08.53 33.69 09.40 38.95 11.52 44.58 13.70 47.48

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

10 15 20 25 30 35 40 45 50 55

15 15 15 15 15 15 15 15 15 15

2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

07.74 08.69 09.60 11.30 13.68 13.96 15.59 18.45 21.51 27.72

22.88 28.15 31.01 38.02 47.48 53.16 64.04 76.97 99.61 131.50

to be continued

562

Continuation of Table 1

1

2

3

5

6

15 15 15 15 15 15

4 2.00 2.00 2.00 2.00 2.00 2.00

2.0 2.0 2.0 2.0 2.0 2.0

30 30 30 30 30 30

2.0 2.0 2.0 2.0 2.0 2.0

8

9

10

11

0 0 0 0 0 0

7 00 02 04 06 08 10

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

13.70 12.90 12.10 11.60 10.80 09.60

47.48 43.70 42.01 37.99 34.62 31.82

30 30 30 30 30 30

15 15 15 15 15 15

2.00 2.00 2.00 2.00 2.00 2.00

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

00 02 04 06 08 10

0 0 0 0 0 0

13.70 12.92 12.11 11.65 10.75 09.62

47.48 43.70 42.01 37.99 34.62 31.82

2.0 2.0 2.0 2.0 2.0 2.0

30 30 30 30 30 30

15 15 15 15 15 15

2.00 2.00 2.00 2.00 2.00 2.00

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

00 01 02 03 04 05

13.70 13.70 12.90 13.70 13.32 13.15

47.48 47.25 47.48 46.88 47.55 47.50

2.0 2.0 2.0 2.0

30 30 30 30

15 15 15 15

2.00 2.00 2.00 2.00

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

13.50 13.70 14.05 13.93

47.60 47.84 46.25 47.15

2.0 2.0 2.0 2.0

30 30 30 30

15 15 15 15

2.00 2.00 2.00 2.00

0 0 0 0

0 0 0 0

0 0 0 0

13.75 12.90 13.35 13.70

47.56 47.74 46.25 47.15

5.61 10.0 12.8 20.0 0 0 0 0

5.61 10.0 12.8 20.0

Stability of pyridinium dichromate (oxidant) in solution. The rate of oxidation does not change with increase in pyridine concentration. This shows that PDC is not hydrolysed. There was no change in optical density and spectra of PDC solution in acetic acid–water–perchloric acid mixture on long standing or at heating up to 60oC. These two experiments show that PDC is quite stable compound under aqueous kinetic conditions. Polymerisation test for free radical. The oxidation of benzaldehydes by PDC in an atmosphere of nitrogen failed to induce the polymerisation of inhibitor-free acrylonitrile (contained 0.001 mol dm–3 acrylonitrile). Further, the addition of a radical scavenger, acrylonitrile had no effect on the rate; suggesting the absence of free radical intermediate in the reaction. Effect of solvent composition on rate. The rate of oxidation of benzaldehydes is affected considerably by changing solvent composition of acetic acid–water mixture and increases with increase in volume percentage of acetic acid. The rate of oxida563

tion depends on the polarity of the medium, when the polarity (dielectric constant) of the solvent is increased, the rate of oxidation decreased (Table 1). The plot of lg kobs versus D+1/2D–1 is a straight line with negative slopes10, indicating the presence of dipole–dipole type of interaction in rate-determining step10. The plots of lg kobs versus 1/D(dielectric constant) with positive slopes > 20 (for benzaldehyde, 4-nitrobenzaldehyde are +111, +156, respectively) suggest cationdipole type of interaction11. Results are contrary to earlier observation in oxidation of substituted benzaldehyde by acid bromate12 and similar to observation in oxidation of substituted benzaldehyde by QBC (Ref. 13). Effect of perchloric acid concentration on rate. Reaction was carried out by changing the perchloric acid concentration in the reaction medium. Increase in [HClO4] was found to increase the rate of oxidation indicating that the oxidation of aldehyde is catalysed by acid. A plot of lg kobs versus lg [H+] was linear in the range of [H+] =0.1– 1.5 mol dm–3 with a slope of ca. 2 (for benzaldehyde, 4-nitrobenzaldehyde – 2.0067, 2.0093, respectively), i.e. the order with respect to [H+] is two. It is more probable that one H+ is taken by PDC and the other attacks the aldehyde to give RC+HOH. The second order with respect to [H+] indicates an interaction between hydrated benzaldehyde and protonated PDC forming a cyclic ester, which then decomposes in a slow step forming the corresponding the benzoic acids. Similar effects have been observed in the oxidation of benzaldehyde by Hiran et al.13 with QBC, Ramkrishanan and Chockalingam14 with PDC and Rocek and Chivsheung15 with Cr(VI). These results are contrary to ealier observed by Aruna et al.16, Elango and Karunakaran17, Pillay and Jameel18. They observed a first order rate dependence in [H+] concentration in the oxidation of benzaldehydes by QDC, QFC, PFC, respectively. The protonated PDC may function as the effective oxidant species similar to the chromium trioxide oxidation. A protonated Cr(VI) species is likely to be a better electrophile and oxidant compared to the neutral one. The Zucker–Hammett19, Bunnett hypothesis20 and Bunnett–Olsen criterion21 were also applied but the slopes of the plots do not fit in the criterion. This indicates that the water molecule is not acting as proton abstractor in the rate-determining step. The rate law of the oxidation process can be expressed as follows: O O || || K1 O==Cr O  Cr == O + H+ Ň | O– PyH+ O– PyH+

O O || || HO  Cr+ O  Cr == O Ň | – + O– PyH+ O PyH

(2)

[PDC]

–d[PDC]/dt = k1[PDC][aldehyde][H+]2

(3)

Effect of ionic strength on rate. There was no effect of sodium sulphate, sodium nitrate and sodium perchlorate concentration on the rate of the reaction (Table 1). 564

This proves that opposite or similar charge species are not interacting22 in the ratedetermining step. Effect of Mn(II) and Ce(III) ion on rate. The rate of oxidation decreases gradually on the addition of Ce(III) and Mn(II) ions (Table 1). This effect suggests that Cr(VI) acts as a 2-electron oxidant23. Thermodynamic parameters. To calculate various thermodynamic parameters the rate constants were measured at 303 to 328 K. The rate of oxidation increases with increase in temperature (Table 2A). The activation parameters evaluated on the basis of the Arrhenius plot are given in Table 2, A and B. The plot of lg k1 against 1/T is a straight line. The entropy of activation is: –208 and –188 J K–1 mol–1 (Table 2B) for benzaldehyde and 4-nitrobenzaldehyde, respectively, indicating that the oxidation involves the rupture of C–H bond in the rate-determining step. Glasstone24 has suggested that if entropy of activation is large and positive, the reaction will be normal and fast but if it is negative, the reaction is slow. In our case, the value of entropy of activation falls in the category of slow reaction. High negative values of entropy of activation also shows bimolecular reaction in the rate-determining step in the presence of water as a solvent and the involvement of a proton transfer agent during the rate-determining step. The high negative entropy of activation indicates the following possibilities: – formation of an aldehyde hydrate, which proceeds in the rate-determining steps25; – formation of cyclic transition state; – if esterification, than this step would also exhibit a negative entropy26,27; – formation of more polar activated state and hence aggregation of solvent molecule around activated state. Table 2A. Variation of rate with temperature

Temperature [PDC]×103 [Substrate] (K) (mol dm–3) ×102 (mol dm–3) 303 2.0 2.0 308 2.0 2.0 313 2.0 2.0 318 2.0 2.0 323 2.0 2.0 328 2.0 2.0

AcOH% (v/v)

[H+]× 10 (mol dm–3)

30 30 30 30 30 30

15 15 15 15 15 15

kobs× 105 (s–1) –H

4-NO2

11.45 13.70 16.25 18.54 22.60 26.28

  39.41   47.48   59.17   71.90   82.66 101.10

565

Table 2B. Thermodynamic parameters

Aldehyde Benzaldehyde 4-Nitrobenzaldehyde

Average Energy of pZ=A activation (dm–3 s–1 mol–1) ∆Ea* (kJ mol–1) 1.33×102 27.38 ± 1.1 1.38×103 30.13 ± 1.4

Entropy of activation ∆S* (J K–1 mol–1) –208.22 ± 5.7 –188.74 ± 5.2

Free energy ∆G* (kJ mol–1) 91.51 ± 2.0 88.26 ± 1.9

The specific rates of reaction of benzaldehyde and 4-nitrobenzaldehyde in identical conditions are 30.15 and 47.48 ×10–5 s–1, respectively. From the relative rates, it can be observed that the benzaldehyde, which has electron-withdrawing group has higher rate and 4-nitrobenzaldehyde with electron-donating group has lower rate. Electron-withdrawing group will increase the positive charge on carbonyl carbon of the aldehyde and making hydration more probable. The higher rate of oxidation of 4-nitrobenzaldehyde could be due to combined –I and –R effects, indicating more positive charge on carbonyl carbon atom. Similar effects has been observed by Lucchi28 in Cr(VI); Aruna et al.16 in QDC and Rama­ krishnan and Chokalingam14 in PFC. Considering all these experimental data, the following reaction scheme may be suggested: O O || || O==Cr O  Cr == O + H+ | | – – + O PyH+ O PyH [PDC] Ph  C == O + H+ | H H | Ph  C+ + H2O | OH

K1

K2

K3 fast

O O || || HO  Cr+ O  Cr == O | | – + O PyH O– PyH+ (C1) Ph  C+ O  H | H (C2) OH | Ph  C  H + H+ | OH

(i)

(ii)

(iii)

The interaction of C1 with C2 can be ruled out as there was no effect of ionic strength on rate while solvent effect indicates cation–dipole type of interaction11. (iv)

566

(v)



(vi)

(vii)

Overall reaction is: 3 Ph-CHO + PDC → 3 Ph-COOH +2 Cr(III)

(4)

which is consistent with observed stoichiometric equation and product study. Based on the above mechanism, the rate law can be derived as follows: rate = –d[PDC]/dt = k [C] = kk′ [Ph-CHOH(OH)] [H+PDC] = kk′ K1K2 [Ald] [H+] [H+] [PDC] = kk′ K1K2[Ald] [PDC] [H+]2 = k1 [PDC] = kobs [PDC]



kobs = k1 = kk′ K1K2 [Ald] [H+]2.

This rate law is consistent with all the observed experimental results. Acknowledgement The authors are thankful to Prof. S. N. Joshi (Retired professor, M.L.Sukhadia University, Udaipur) for his valuable suggestions. Authors are also thankful to U.G.C., New Delhi, India, for providing financial assistance. Abbreviation QBC – quinolinium bromochromate; PFC – pyridinium fluorochromate; QFC – quinolinium fluorochromate; PDC – pyridiniumdichromate; QDC – quinolinium dichromate; PCC – pyridinium chlorochromate.

REFERENCES 1. E. J. COREY, W. J. SUGGS: Synthetic and Kinetic Aspects of PCC. Tetrahedron Letters, 2461 (1978). 2. E. J. COREY, G. SCHMIDT: Synthetic and Kinetic Aspects of PDC. Tetrahedron Letters, 399 (1979).

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3. F. S. GUZICE, F. A. LUZZIO: Kinetics of Oxidation of Benzaldehyde by 2,2 BPCC. Synthesis, 691 (1980).   4. N. NARAYANA, T. R. BALASUBRAMANIAN: Kinetics of Oxidation of Benzaldehyde by PBC. Indian J. Chem., 25B, (1986).   5. M. K. MAHANTI, K. K. BANERJI: Synthetic and Kinetic Aspects of Cr(VI). J. Indian Chem. Soc., 79, 31 (2002).   6. G. MANGALAM, S. M. SUNDARAM: Kinetics of Oxidation of Aryl Methyl Sulphide by PDC. J. Indian Chem. Soc., 68, 77 (1991).   7. K. SUGANYA, G. BABURAO, S. KABIAN: Kinetics of Oxidation of Benzyl Alcohol and Substituted Benzyl Alcohol by PDC. Oxid. Commun., 26, 373 (2003).   8. A. N. PALNIAPPAN, K. BHARATHI, K. G. SEKER: Kinetics of Oxidation of 8-hydroxy Quinoline (Oxime) by PDC. Polish J. Chem., 72 (1), 122 (1998).   9. S. AGRAWAL, K. CHAUDHARY, K. K. BANERJI: Kinetics of Oxidation of Substituted Benzaldehyde by PFC. J. Org. Chem., 56, 5111 (1991). 10. K. J. LAIDLER: Chemical Kinetics. 3rd ed. Harper and Row, 1987, p.183. 11. E. S. AMIS: Solvent Effect on Reaction Rates and Mechaniam. Academic Press, New York, 1966, p. 45. 12. G. V. BAKORE, K. K. BANERJI, R. SHANKER: Kinetics of Chromic Acid Oxidation of Benzyl Alcohol in Aqueous Acetic Acid. Z. Phys. Chem. (Frankfurt), 45, 129 (1965). 13. B. L. HIRAN, R. K. MALKANI, P. CHAUDHARY, P. VERMA, N. SHORGER: Kinetics of Oxidation of Para-substituted Benzaldehyde by QBC. Asian J. Chemistry, 18 (4), 3081 (2006). 14. P. S. RAMAKRISHAN, P. CHOKALINGAM: Kinetics of Oxidation of Substituted Benzaldehydes by PFC. J. Indian Chem. Soc., 70, 581 (1993). 15. J. ROCEK, Ng. CHIU-SHEUNG: Kinetics of Oxidation of Benzaldehydes by Cr(VI). J. Am. Chem. Soc., 96, 1522 (1974). 16. K. ARUNA, P. MANIKYMBA, V. SUNDRAM: Kinetics of Oxidation of Benzaldehydes by QDC. Asian J. Chem., 6, 542 (1994). 17. K. P. ELANGO, K. KARUNAKARAN: Kinetics of Oxidation of Benzaldehydes by QFC. Oxid. Commun., 9, 59 (1996). 18. M. K. PILLAY, A. A. JAMEEL: Kinetics of Oxidation of Benzaldehydes by PFC. Indian J. Chem., 31, 46 (1992). 19. L. ZUCKER, L. P. HAMMETT: The Effect of Nuclear and Side Chain Substitution on the Oxonium Ion Catalyzed Iodination of Acetophenone Derivatives. J. Am. Chem. Soc., 61, 2779 (1939). 20. J. F. BUNNETT: Kinetics of Reaction in Moderately Concentrated Aqueous Acids. II. An Emprical Criterion of Mechanism. J. Am. Chem. Soc., 83, 4968 (1961). 21. J. F. BUNNETT, F. P. OLSEN: Bunnett-Olsen’s Criterion. Can. J. Chem., 44, 1917 (1966). 22. A. FROST, RALPHG PEARSON: Kinetics and Mechanism. John Wiley & Sons, Inc., Japan, 1961 p. 150. 23. A. THANERAJAN, R. GOPALAN: Kinetics of Oxidation of 2-propanol with Cr(VI). J. Indian Chem. Soc., 67, 453 (1990). 24. S. GLASSTONE, K. J. LAIDER, H. EYRING: Theory of Rate Processes. McGraw-Hill, New York, 1941. 25. R. P. BELL: Adv. Phys. Org. Chem., 4, 1 (1966). 26. E. K. EURANTO: The Chemistry of Carboxylic Acids and Esters (Ed. S. Patai). Interscience, 1969, p. 505. 27. H. A. SMITH, F. P BYRNE: The Kinetics of Acids-catalyzed Esterification of Cyclohexanedicarboxylic Acids. J. Am. Chem. Soc., 72, 4406 (1950). 28. E. LUCCHI: Kinetics of Oxidation of Benzaldehydes by Cr(VI). Boll. Sci. Fac. Chem. Ind. Bologna, 333 (1940). Received 8 March 2010 Revised 29March 2010

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Oxidation Communications 35, No 3, 569–576 (2012) Oxidation in the presence of Cr-containing compounds

Kinetic and Mechanistic Studies of the Oxidation of Crotonaldehyde by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium Dipti, A. Tomar*, A. Kumar Department of Chemistry, Meerut College, 250 001 Meerut, (U.P.) India E-mail: [email protected] ABSTRACT The kinetics of oxidation of crotonaldehyde by a new Cr(VI) reagent tetraethylammonium chlorochromate (TEACC) has been studied in acetic acid–water medium (50% v/v). The reaction shows first order dependence each in [TEACC], [crotonaldehyde] and [H+] ions. Increase in acetic acid content of the solvent medium increases the rate of the reaction. Ionic strength of the medium has no influence on oxidation rate. The reaction rates have been determined at different temperatures and the activation parameters have been computed. Stoichiometery shows that 1 mol of crotonaldehyde reacts with 1 mol of TEACC. A mechanism in conformity with kinetic data is proposed. Keywords: kinetics, mechanism, oxidation, crotonaldehyde, tetraethylammonium chlorochromate. AIMS AND BACKGROUND The mechanism of oxidation of organic substrates by chromium (VI) has been studied extensively by a large number of invstigators1–6. Tetraethylammonium chlorochromate has been used as a selective and effective oxidant7. The compound is capable of acting both as electron transfer and oxygen atom transfer agent. There seems to be only few studies on the oxidation reaction of TEACC (Refs 8–13). EXPERIMENTAL Materials. Tetraethylammonium chlorochromate (TEACC) was prepared by literature method7. Chromium(VI) oxide (Lancaster) was dissolved in 6 M HCl and stirred at 0°C for 5 min. A solution of tetraethylammonium hydroxide (20% in H2O) (Lancaster) was added to it and the resulting orange yellow solid was washed and dried under *

For correspondence.

569

reduced pressure. Chromium content was determined iodometrically. Solution of crotonaldehyde was prepared by dissolving requisite volume in 50% (v/v) aqueous acetic acid. The ionic strength was maintained with the use of concentrated solution of NaClO4 (C.D.H.). Perchloric acid (C.D.H.) and all other chemicals were used as such without further purification. Kinetic measurments. The reaction was carried out under pseudo-first order conditions by keeping a large excess of [crotonaldehyde] with respect to [TEACC]. The medium of the reaction was always 1:1 (v/v) acetic acid–water in the presence of perchloric acid. Kinetic measurements were performed in a Shimadzu UV 160 A spectrophotometer at 350 nm. The optical density was measured at various intervals of time. Computation of rate constant was made from the plot of lg [TEACC] against time. Stoichiometry and product analysis. The stoichiometry of the reaction was determined by allowing excess of oxidant to react with crotonaldehyde under kinetic conditions. The disappearance of Cr(VI) was monitored until a contancy in the absorbance was found; it indicated a 1:1 stoichiometry.

The epoxide formed in the reaction mixture was identified by periodate test for epoxide14. RESULTS The pseudo-first order rate constants were determined at initial concentrations of reactants. The results obtained are given in Table 1. The plots for different concentrations of the tetraethylammonium chlorochromate versus time were linear and the rate constants were independent of initial concentration of tetraethylammonium chlorochromate showing first order dependence of the rate on [TEACC]. The reaction is first order with respect to [crotonaldehyde], too. The plot of lg k1 against lg [crotonaldehyde] was linear with a slope of unity thus confirming first order dependence in [crotonaldehyde]. The second order rate constants k2= k1 ⁄substrate give the concordant value. Rate of oxidation were found to increase with increase in [H+] and the slopes of the plots of lg k1 versus lg [HClO4] were approximately unity showing that the reaction is acid-catalysed and follows first order dependence in [HClO4]. Similar observation was made for QFC (Ref. 15) oxidation reaction. Concequently the empirical rate law is described as follows: –

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d[TEACC] dt

= kobs[crotonaldehyde][TEACC][HClO4].

Table 1. Rate constants for the oxidation of crotonaldehyde by TEACC at 25°C Solvent – acetic acid–water (50–50% v/v)

[TEACC] × 103 (mol dm–3) 0.66 0.88 1.11 1.33 1.55 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11

[Crotonal­dehyde] ×102 (mol dm–3) 5.00 5.00 5.00 5.00 5.00 2.00 3.00 4.00 5.00 6.00 5.00 5.00 5.00 5.00 5.00

[H+] × 10 (mol dm–3)

k1 × 104 (s–1)

k2 × 102 (mol–1 dm3 s–1)

0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.77 0.96 1.16 1.35 1.55

4.57 4.42 4.45 4.39 4.51 1.72 2.50 3.64 4.45 5.05 2.05 4.45 7.10 9.36 11.82

0.86 0.83 0.91 0.89 0.84

Effect of ionic strength. Increase in the ionic strength of the medium by adding sodium perchlorate has no appreciable effect on the reaction rate16. Effect of solvent composition. The reaction has been studied under various composition of acetic acid–water mixture. The rate of oxidation of crotonaldehyde decreases with increases in the dielectric constant of the medium (Table 2). A linear plot between lg k1and 1/D (inverse of dielectric constant, the unit of D is Debye) has a positive slope (Fig. 1) is obtained, suggesting an interaction between ion and dipole17.

Fig. 1. Plot of lg k1 versus 1/D [crotonaldehyde] – 5.00 × 10–2 mol dm–3; [TEACC] – 1.11 × 10–3 mol dm–3; [HClO4] – 7.73 × 10–1 mol dm–3; [NaClO4] – 1.66 × 10–1 mol dm–3; temperature 25°C

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Table 2. Dependence of rate on solvent composition [Crotonaldehyde] – 5.00 × 10–2 mol dm–3; [TEACC] – 1.11 × 10–3 mol dm–3; [HClO4] – 7.73 × 10–1 mol dm–3; [NaClO4] – 1.66 × 10–1 mol dm–3; temperature 25°C

k1 × 10–4(s–1) 4.18 4.45 5.01 5.85 7.59 13.10

CH3COOH : H2O 40:60 50:50 60:40 70:30 80:20 90:10

1/D 0.020 0.023 0.028 0.035 0.048 0.074

Effect of radical forming agent. When the reaction was initiated by adding acrylonitrile into a solution containing crotonaldehyde and TEACC, no retardation in the rate was observed. No turbity due to polymerisation of acrylonitrile was observed. Thus the formation of radical intermediate may be ruled out in the course of the reaction. Effect of temperature. The rate of reaction was studied at different temperatures (293–313 K). The values of the activation energy (Ea) were evaluated from the Arrhenius plot, i.e lg k1 versus 1/T plot (Fig. 2). From this value, the thermodynamic parameters ∆H*, ∆S* and ∆G* were evaluated (Table 3).

Fig. 2. Plot of lg k1 versus 1/T [crotonaldehyde] – 5.00 × 10–2 mol dm–3; [TEACC] – 1.11 × 10–3 mol dm–3; [HClO4] – 7.73 × 10–1 mol dm–3; [NaClO4] – 1.66 × 10–1 mol dm–3; temperature 25°C Table 3. Temperature dependence and activation parameters of oxidation of crotonaldehyde by TEACC

Temperature (K) 293 298 303 308 313

k1 × 104 (s–1) 3.24 4.45 6.35 8.71 12.38

Temperature Ea coefficient (kJ mol–1)

Note: mean Ea = 50.97 kJ mol–1

572

1.96 1.96 1.95

46.11 53.39 49.04 55.34

∆H* (kJ mol–1) 48.53 48.49 48.45 48.41 48.36

∆G* -∆S* –1 (kJ mol ) (J K–1 mol–1) 87.73 133.80 88.45 134.09 89.03 133.94 89.69 134.03 90.20 133.69

DISCUSSION It was found that the epoxide is the only product of oxidation of crotonaldehyde by TEACC. On the basis of the above findings, the following mechanism has been proposed. Scheme 1

The rate-determining step may be the formation of unstable complex between the protonated TEACC and crotonaldehyde. An electrophile attack of Cr(VI) being positively charged in the protonated TEACC on the double bond leads to a cyclic 4-member intermediate which rearranges to give the epoxide in the final step. However, since in the proposed mechanism (Scheme 1) direct chromium to carbon bond does not account for the insensitivity to sterric effect very often observed in the oxidation of olefins18 by Cr(VI) the most favourable reaction path may be via 3-member type addition intermediate as depicted in Scheme 2. Both schemes envisage an oxygen atom transfer from the oxidant. This is in accordance with the earlier observation made for the QFC oxidation of unsaturated aldehyde19. Furthermore, UV-vis. spectral studies did not show any evidence for the formation of TEACC-substrate complex (Fig. 3).

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Scheme 2

Fig. 3. UV-spectra of TEACC in aqueous acetic acid 50% (v/v) (A) and TEACC in aqueous acetic acid 50% (v/v) with crotonaldehyde (B)

Rate law. Based on the proposed mechanism, the rate law for the TEACC oxidation of crotonaldehyde (S) may be derived as follows: TEACC + H+

S + TEACCH+

K1 K2 K3

TEACCH+

(1)

intermediate

(2)

slow (r.d.s.)

574

fast

intermediate ––––→ product

(3)

rate = – d[TEACC]/dt ≈ [TEACCH ][S] +

d[TEACC]/dt = k1[TEACCH+][S].

(4)

By applying law of mass action in equation (1): k2 =

[TEACCH+] [H+][TEACC]

[TEACC+] = k2[TEACC][H+].

(5)

Substituting the value of from [TEACC+] equation (5) into equation (4) it gives: –d[TEACC]/dt = k1k2[S][TEACC][H+] –d[TEACC]/dt = kobs[S][TEACC][H+],

(6)

where k1k2 = kobs. CONCLUSIONS The method developed in this investigation has advantages of simplicity, accuracy and precision applicability. The titrimetric procedure does not require a screening indicator unlike many other procedures proposed earlier. REFERENCES 1. B. H. OZGUINS, D. NEBAHAT: Kinetics of Oxidation of Substituted Benzyl Alcohols by Quinolinium Chlorochromate. J. Chem. Res. Synop., (1), 32 (1997). 2. I. DAVE, V. SHARMA, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Some α-hydroxy Acids by Quinolinium Fluorochromate. Indian J. Chem., 39A, 728 (2000). 3. N. NARAYANAN, T. R. BALSUBRAMANIAN: Kinetics and Mechanism of the Oxidation of Benzhydrol by Pyridinium Bromochromate. J. Chem. Res., (S), 336 (1991). 4. K. MISHRA, J. SINGH, G. L. AGARWAL, A. PANDEY: Oxidation of Crotonaldehyde by Quinolinium Chlorochromate. A Kinetic Study. Oxid Commun, 26 (1), 52 (2003). 5. S. AGARWAL, K. CHOWDHARY, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Thioacids by Pyridinium Fluorochromate. Transition Met. Chem., 16, 661 (1991). 6. P. APARNA, S. KOTHARI, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Primary Aliphatic Alcohols by Pyridinium Bromochromate. Proc. Indian Acad. Sci., 107, 213 (1995). 7. A. PANDURANGAN, V. MURUGESAN: Kinetics and Mechanism of the Oxidation of Benzyl Alcohol by Tetraethylammonium Chlorochromate. J. Ind. Chem. Soc., 73, 484 (1996). 8. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of D-Galactose by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. Asian J. Chem., 18 (4), 3053 (2006). 9. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of D-Mannose by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. Asian J. Chem., 18 (4), 3073 (2006).

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10. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of D-Fructose by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. J. Indian Chem. Soc., 83, 1153 (2006). 11. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of D-Glucose by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. Oxid Commun, 30 (1), 88 (2007). 12. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of L-Sorbose by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. J. Indian Chem. Soc., 84, 1162 (2007). 13. A. TOMAR, A. KUMAR: Kinetic and Mechanistic Study of the Oxidation of Acrylic Acid by Tetraethylammonium Chlorochromate in Aqueous Acetic Acid Medium. Oxid Commun, 31 (1), 211 (2008). 14. D. J. PASTO, C. R. JOHNSON: Organic Structure Determination. Prentice Hall, New Jersey, 1969, p. 376. 15. G. L. AGARWAL, R. JAIN: Kinetics and Mechanism of the Oxidation of Allyl Alcohol by Quinolinium Fluorochromate. Oxid Commun, 20, 273 (1997). 16. G. L. AGARWAL, S. JHA: Kinetics and Mechanism of the Oxidation of Diols by Pyridinium Chlorochromate. Revue Roumaine de Chemie, 34, 1969 (1989). 17. E. S. AMIS: Solvent Effects on Reaction Rates and Mechanism. Academic Press, New York, 1966, p. 42. 18. J. ROCEK, A. DROZD: Nature of the Transition State in the Oxidation of Olefins by Chromium(VI). J. Am. Chem. Soc., 91, 991 (1969). 19. G. L. AGARWAL, R. JAIN: Epoxidation of Crotonaldehyde by Quinolinium Fluorochromate. A Kinetic Study. Oxid Commun, 22, 514 (1999). Received 2 May 2010 Revised 19 June 2010

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Oxidation Communications 35, No 3, 577–582 (2012) Oxidation in the presence of Cr-containing compounds

Oxidation of Substituted S-phenylmercaptoacetic Acids by Quinoxalinium Dichromate K. G. Sekar*, G. Manikandan Department of Chemistry, National College, 620 001Tiruchirappalli, Tamilnadu, India E-mail: [email protected]; [email protected] ABSTRACT The conversion of S-phenylmercaptoacetic acid to the corresponding sulphoxide was performed in 50% (v/v) water–acetic acid mixture in the presence of perchloric acid medium. The order with respect to S-phenylmercaptoacetic acid and quinoxalinium dichromate were both one and inverse first order with respect to hydrogen ion concentration. Decrease in dielectric constant of the medium increased the rate of reaction. Ionic strength had a considerable influence on a reaction rate, indicating the involvement of a dipole in the rate-limiting step. In general, the electron-withdrawing substituents enhance the reaction rate and electron-releasing substituents retard the reaction rate. A suitable mechanism and rate law in consonance with the observed facts is proposed. Keywords: kinetics, oxidation, phenylmercaptoacetic acid, quinoxalinium dichromate. AIMS AND BACKGROUND Quinoxalinium dichromate (C8H6N2H2)Cr2O7 (QxDC) has been used as a mild, efficient and selective oxidising reagent in synthetic organic chemistry1. H N Cr2O72– N H quinoxalinium dichromate *

For correspondence.

577

However, there are not many reports on the characteristic aspects of reactions of QxDC studies reported so far on the kinetics of oxidation of S-phenylmercaptoacetic acid2–7 to give diverse products, involving different intermediates in aqueous medium. The use of an insulated acid substrate contains groups or atoms between the reaction site and the bulk of the molecule in a similar study is rare. Now, we report the oxidation of S-phenylmercaptoacetic acid by quinoxalinium dichromate. EXPERIMENTAL Reagents. S-phenylmercaptoacetic acids were prepared and purified by literature method8. QxDC was prepared by a known procedure1 and its purity was determined by iodometric assay. Acetic acid was refluxed over chromium trioxide for 6 h and then fractionated9. All other chemicals were of Analar grade. The reaction mixture was homogeneous throughout the course of the reaction. Kinetic measurements. The reactions were followed under pseudo-first order conditions by maintaining always the substrate concentration in excess over that of QxDC. The reactions were carried by monitoring the decrease in the concentrations of QxDC and were followed spectrophotometrically at 470 nm for up to 80% of the reaction. The rate constants were evaluated from the linear plot of log absorbance against time by the least square method and were reproducible within ± 3%. Stoichiometry. The stoichiometric runs were carried out in the presence of excess QxDC which reveals that 1 mol of oxidant consumes 1 mol of substrate confirming the stoichiometry of the reaction as 1:1. Product analysis. The kinetic reaction mixture was left to stand for 24 h under kinetic conditions. It was extracted with ether and the residue that separated during solvent evaporation was analysed by IR spectroscopy. The following frequencies corresponding to the sulphoxide were observed: 1024 cm–1 (=S=O group), 1713 cm–1 (–C=O group) and 3434 cm–1 (–COOH group). The product was further confirmed by TLC. The yield of sulphoxide was 90% as determined by weight measurement of the reactant and product. RESULTS AND DISCUSSION The reaction was studied under different experimental conditions in the presence of acetic acid–water (50% v/v) as solvent medium. At a constant temperature, the rate increased steadily on increasing the concentration of the substrate as shown in Table 1. The linear plot of lg k against lg [substrate] with a slope of unity clearly indicates that the reaction has unit order dependence on the concentration of the substrate. The specific reaction rate constant of k2 = k1/[S] confirms the first order in the S-phe­ nylmercaptoacetic acid.

578

Table 1. Rate data on the oxidation of S-phenylmercaptoacetic acid by quinoxalinium dichromate at 313 K

[PMA]×103 (mol dm–3) 2.5–12.5 5.0 5.0 5.0 5.0

[QxDC]×103 (mol dm–3) 2.0 1.5–3.5 2.0 2.0 2.0

[H+]×10 (mol dm–3) 3.5 3.5 3.5–17.5 3.5 3.5

AcOH:H2O [NaClO4]×102 (%–v/v) (mol dm–3) 50:50 – 50:50 – 50:50 – 40:60–60:40 – 50:50 0.00–20.20

k1×104 (s–1) 1.27–8.52 3.99–1.62 3.26–0.78 2.72–3.71 3.26–3.13

The reaction was found to be first order with respect to the oxidant as evidenced by the good linearity in the plot of lg absorbance versus time (r=0.990). Increase in ionic strength of the medium by adding sodium perchlorate has no effect on the reaction rate indicating the involvement of charged species in the rate-determining step (Table 1). The kinetic runs were performed at different concentrations of perchloric acid which acted as the catalyst. The rate decreased with an increase in the concentration of hydrogen ion, this suggests that H+ ions react with S-phenylmercaptoacetic acid and forms a non-reactive species. Plots of k versus 1/[H+] and lg k versus lg [H+] are also straight lines with unit slope indicating an inverse first order dependence on hydrogen ion concentration. The acetic acid composition in the solvent mixture was varied while maintaining the other variables constant, as shown in Table 1. The rate was found to increase considerably on increasing the acetic acid content of the medium. It is due to the fact that the reaction is facilitated by an increase in polarity or nucleophilicity. The addition of acrylonitrile, which is a very good trapper of free radicals did not have any retarding effect on the reaction. It indicates that no free radicals participation in the reaction. The addition of Mn2+ retard the rate of the oxidation considerably showing that the rate-determining step involves a 2-electron transfer in the mechanism. The reaction was performed at different temperatures, viz. 303, 313, 323 and 333 K while maintaining the concentrations of substrate, oxidant and H+ constant (Table 2) and from the Eyring plot10 of ln (k2/T) versus 1/T, the thermodynamic parameters were calculated.

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Table. 2. Thermodynamic parameters for the oxidation of para- and meta- S-phenylmercaptoacetic acids by quinoxalinium dichromate

S. Substitu- Order No ents with respect to substrate 1 H 1.14 2 p-OMe 0.88 3 p-Me 0.90 4 p-Br 1.11 5 p-Cl 1.14 6 p-NO2 0.87 7 m-OMe 0.79 8 m-Me 0.82 9 m-Br 1.08 10 m-Cl 1.18 11 m-NO2 1.28

k1 ×104 (s–1) 303 K 313 K 323 K 333 K 2.54 0.72 0.94 5.98 5.04 30.12 3.01 1.89 10.21 9.07 28.21

3.26 1.12 1.55 9.43 6.76 45.71 4.84 2.98 14.45 12.88 38.90

5.58 1.85 2.38 13.89 8.87 64.94 6.52 5.81 19.21 16.62 50.09

8.21 2.71 3.56 19.98 10.98 87.22 9.27 8.94 25.09 21.08 65.24

∆H* (kJ mol–1)

–∆S* (J K–1 mol–1)

∆G* (kJ mol–1) at 313 K

r

13.60 15.19 15.02 13.48 8.37 11.79 12.37 18.28 9.73 9.03 8.95

184.32 183.41 182.91 181.33 196.29 180.99 187.40 169.86 191.72 194.47 190.64

71.29 72.60 72.27 70.24 69.81 68.44 71.03 71.45 69.74 69.90 68.62

0.990 0.999 0.999 0.999 0.998 0.998 0.996 0.996 0.999 0.997 0.999

MECHANISM AND RATE LAW

The oxidation of S-phenylmercaptoacetic acid with quinoxalinium dichromate was catalysed by perchloric acid. It is first order with respect to the concentrations of each of the oxidant and substrate and inverse first order with respect to H+. Product analysis clearly indicates that the obtaining of the corresponding sulphoxide. From these observations, the following mechanism and rate law were proposed. C6H5SCH2COOH + H+ K2

(C8H7N2)Cr2O7H+

K1

C6H5SC+H3COOH

(C8H7N2)Cr2O7 + H+

(C8H7N2)Cr2O7H+ + C6H5SCH2COOH

K3

complex

O || complex C6H5–S– –––––→ CH2COOH + H+ + Cr(IV) k4



Rate law:

rate = k4 [complex] = k4 K3 [QxDCH+] [PMA] = k4 K3 K2 [QxDC] [PMA] [H+]/K2 [H+] –d(QxDC)/dt = k4K1 K2 K3 [QxDC] [PMA] [H+]/K2 [H+]

The proposed mechanism and the rate law support all the observations made including the effect of solvent polarity and the negative entropy of activation. 580

EFFECT OF SUBSTITUENTS

The rate constant k1 was estimated for the substituted S-phenylmercaptoacetic acids at 4 different temperatures, viz. 303, 313, 323 and 333 K. The thermodynamic parameters have been computed from a plot of ln k2/T versus 1/T using the Eyring equation. The negative values of the entropies of activation (∆S*) suggested that the transition state formed was considerable rigid, resulting in a reduction in the degree of freedom of the molecules. The constancy of the (∆G*) values indicated a common mechanism for the oxidation of all the substrates. As ∆H* and ∆S* do not vary linearly, no isokinetic relationship is observed. This indicated the absence of enthalpy – entropy compensation effect11. Exner12 criticised the validity of such a linear correlation between ∆H* and ∆S* as the quantities are dependent on each other, when measurements at 2 different temperatures have been made. The rate data can be analysed by the following equation13: lg k1 (T2) = a + b lg k1 (T1),

where a and b are intercept and slope and T2>T1. The plot of lg k1 (303 K) against lg k1 (313 K) gave a straight line with r = 0.997 – such a good correlation indicates that the oxidation of the substrates with different substituents follows a common mechanism. To have an idea about the order with respect to each of the substrate the oxidation has been studied at 313 K and the results are given in Table 2. It is interesting to note that all the substituted S-phenylmercaptoacetic acids show a unit order dependence on the reaction rate. The rate data for the oxidation of para- and meta-substituted S-phenylmercaptoacetic acids give a good correlation for the plot of lg k versus σ (Fig. 1) (r = 0.994, ρ = +1.54) with the Hammett value at 313 K. Similar phenomenon has been observed in the oxidation of substituted benzaldehydes by PFC (Ref. 14) and IDC (Ref. 15). The positive ‘ρ’ value indicates that electron-withdrawing substituents enhance the rate of oxidation and electron-releasing substituents decrease the rate of the reaction.

Fig. 1. The Hammett plot of lg k313 K versus σ for the oxidation of S-phenylmercaptoacetic acids by QxDC (numbers as given in Table 2)

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CONCLUSIONS The oxidation of S-phenylmercaptoacetic acid by quinoxalinium dichromate was studied in full depth and a mechanism involving the substrate, oxidant and H+ is proposed. In the slow rate-determining step, the substrate reacts with the positively charged species. The product is the corresponding sulphoxide. The orders with respect to the concentrations of substrate and oxidant are one. The perchloric acid reacts with substrate to form a non-reactive species. The negative sign of the entropy change suggests that the transition state is more orderly when compared with the reactants. REFERENCES   1. N. DEGRIMENBASI, B. OZGUN: Quinoxalinium Dichromate: A New and Efficient Reagent for the Oxidation of Organic Substrate. Monatshefte fur Chemie, 133, 1417 (2002).   2. S. KABILAN, M. UMA, K. KRISHNASAMY, P. SANKAR: Oxidation of S-phenylmercaptoacetic Acid by Pyridinium Dichromate. J. Indian Council Chem., 10 (1), 21 (1994).   3. S. KABILAN, K. KRISHNASAMY, P. SANKAR: Oxidative Cleavage of Phenylthioacetic Acid by Pyridinium Dichromate in Acetonitrile Medium: Kinetic and Correlation Study. Oxid Commun, 18 (3), 288 (1995).   4. S. KABILAN, R. GIRIJA, V. RAJAGOPAL: Oxidative Cleavage of S-phenylmercaptoacetic Acid by Pyridinium Chlorochromate: Kinetic and Correlation Analysis. Int. J. Chem. Kinet., 31 (10), 109 (1999).   5. K. SATHIYANARAYANAN, R. SUSEELA, CHANG WOO LEE: Oxidation of S-phenylmercaptoacetic Acid by N-chloronicotinimide: A Kinetic Study. J. Ind. Eng. Chem., 12 (2), 280 (2006).   6. K. SATHIYANARAYANAN, C. PAVITHRA, CHANG WOO LEE: Kinetics and Mechanism of S-phenylmercaptoacetic Acid by Chromium(VI). J. Ind. Eng. Chem., 12 (5), 727 (2006).   7. N. M. I. ALHAJI, A. M. UDUMAN MOHIDEEN, K. KALAIMATHI: Mechanism of Oxidation of p-substituted Phenylthio Acetic Acids with N-bromophthalimide, E. J. Chem., 8 (1), 1 (2011).   8. S. GABRIEL: Oxidation of Some Sulphur Compounds. Ber., 12, 1879 (1939).   9. K. S. P. ORTON, A. E. BRADFIELD: The Purification of Acetic Acid. The Estimation of Acetic Anhydride in Acetic Acid. J. Chem. Soc., 983 (1927). 10. H. EYRING: The Activated Complex in Chemical Reactions. J. Chem. Phys., 3, 107 (1935). 11. M. G. ALDER, J. E. LEFFLER: The Role of the Solvent in Radical Composition Reactions: Phenyl Azotriphenyl Methane. J. Am. Chem. Soc., 76, 1425 (1954). 12. O. EXNER: Concerning the Isokinetic Relationship. Nature, 201, 488 (1964). 13. M. J. MALAWSKI: The Linear Relation between Enthalpy and Entropy of Activation. Roczniki Chem., 38, 1129 (1964). 14. P. S. RAMAKRISHNAN, P. CHOCKALINGAM: Kinetics of Oxidation of Substituted Benzaldehydes by Pyridinium Fluorochromate in Acetic Acid – Perchloric Acid Medium. J. Indian Chem. Soc., 70, 581 (1993). 15. K. BALASUBRAMANIAN, K. LAKSHMANAN, K. G. SEKAR: Kinetics and Mechanism of Oxidation of Aromatic Aldehydes by Imidazolium Dichromate. Asian J. Chem., 11 (4), 1451 (1999). Received 14 April 2012 Revised 17 May 2012

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Oxidation Communications 35, No 3, 583–590 (2012) Oxidation in the presence of Ni-containing compounds

Kinetics and Mechanism of Oxidation of L-lysine and L-ornithine by Dihydroxydiperiodatonickelate(IV) In Alkaline Liquids Jinhuan Shan*, Haixia Shen, Heye Wang, Xiaoqian Wang College of Chemistry and Environmental Science, Hebei University, 071 002 Baoding, China E-mail: [email protected] ABSTRACT The kinetics of oxidation of L-lysine and L-ornithine by diperiodatonickelate(IV) (DPN) in alkaline liquids at a constant ionic strength has been studied spectrophotometrically in the temperature range of 288.2–308.2 K. The reaction shows first order dependence on [DPN], fractional order dependence on each reductant. [IO4–] and [OH–] have retarding effect on the rate of reaction. When reductant was oxidised by Ni(IV), the reaction rate decreases with the increase of ionic strength. A mechanism involving the diperiodatonickelate(IV) (DPN) as the reactive species of the oxidant has been proposed. The rate constants of the rate-determining step and the activation parameters were calculated. Keywords: L-lysine, L-ornithine, dihydroxydiperiodatonickelate(IV), kinetics, mechanism. AIMS AND BACKGROUND In recent years, the study of the highest oxidation state of transition metals has intrigued many researchers. This can provide new and valuable information in some fields. Transition metals in a higher oxidation state can generally be stabilised by chelation with suitable polydentate ligands. Metal chelates such as diperiodatocuprate(III) (Ref. 1), diperiodatoargentate(III) (Ref. 2) and diperiodatonickelate(IV) (Ref. 3) are good oxidants in a medium with an appropriate pH value. Ni(IV) complexes have been employed as oxidising agents for the investigation of some organic compounds such as tetrahydrofurfuryl alcohol4, L-leucine5, 4-hydroxycoumarin6, gabapentin7, etc. Amino acids act not only as the building blocks in protein synthesis but they also play a significant role in metabolism and have been oxidised by a variety of *

For correspondence.

583

oxidising agents8. The study of the oxidation of amino acids is of interest because of their biological significance and selectivity towards the oxidant to yield the different products9. L-lysine and L-ornithine are essential dietary amino acids. In the present investigation, we have obtained the evidence for the reactive species for DPN in alkaline medium. A literature survey reveals that there are no reports on the kinetics of oxidation of L-lysine and L-ornithine by DPN. Hence, in order to understand the active species of oxidant and to propose a suitable mechanism based on experimental results, the title reaction was investigated. EXPERIMENTAL Materials. All the reagents used were of A.R. grade. All solutions were prepared with doubly distilled water. The solution of oxidation was prepared10 and standardised11 by the method reported earlier. Its UV spectrum was found to be consistent with that reported in literature. The concentration of DPN was derived from its absorption at 410 nm. The solution of oxidation was always freshly prepared before use. The ionic strength μ was maintained by adding KNO3 solution and the pH of the reaction mixture was adjusted with a KOH solution. The kinetic measurements were performed on a UV-vis. spectrophotometer (TU-1900, Beijing Puxi Inc., China), which had a cell holder kept at constant temperature (± 0.1ºC) by circulating water from a thermostat (BG-chiller E10, Beijing Biotech Inc., Beijing). Apparatus and kinetics measurements. All kinetics measurements were carried out under pseudo-first order conditions. 2 ml of the oxidation solution containing a definite concentration of Ni(IV), OH–, IO4– were transferred to upper branch of the λ-type tube and 2 ml of reductant solution with an appropriate concentration was transferred separately to the lower branch of this tube. After thermal equilibration at the desired temperature in a thermostat, the two solutions were mixed well and immediately transferred into a 1-cm thick rectangular quartz cell in a constant temperature cell holder (±0.1ºC). The reaction process was monitored automatically by recording with a TU-1900 spectrophotometer. All other species did not absorb significantly at this wavelength. Details of the determinations are described elsewhere12. The oxidation product was identified as the corresponding aldehyde and Ni(II) by spot test13. RESULTS EVALUATION OF PSEUDO-FIRST ORDER RATE CONSTANTS

Under the conditions of [reductant]0>>[ Ni(IV)]0, the plots of ln(At–A∞) versus time were straight lines, details of the evaluation are described in our previous work14. Dependence of rate on the concentration of reductant. At constant temperature, kobs values increase by increasing the concentration of reductant while keeping the [Ni(IV)], [OH–], [IO4–], and μ constant. The order with respect to reductant was fractional. The 584

plots of 1/kobs versus 1/[reductant] were straight lines with a positive intercept (Figs 1 and 2). 288.2 K

320

1/kobs (s)

240 293.2 K 160 298.2 K 80

303.2 K 308.2 K

0

70

140

210

280

350

1/[L-lysine] (l/mol)

Fig. 1. Plots of 1/kobs versus 1/[L-lysine] at different temperatures L-lysine: [Ni(IV)] = 7.83×10–6 mol/l; [OH–]= 0.0155 mol/l; [IO4–] = 2.0×10–3 mol/l; μ= 0.0325 mol/l (r ≥ 0.998) 288.2 K 100

75 1/kobs (s)

293.2 K 50 298.2 K 25

0

303.2 K 308.2 K 65

130

195

260

325

1/[L-ornithine] (l/mol)

Fig. 2. Plots of 1/ kobs versus 1/[L-ornithine] at different temperatures L-ornithine: [Ni(IV)] = 7.83×10–6 mol/l; [OH–] = 0.0155 mol/l; [IO4–] = 4.0×10–3 mol/l; μ= 0.1095 mol/l (r ≥ 0.995)

Dependence of rate on the concentration of IO4–. At constant [Ni(IV)], [reductant], [OH–], μ and temperature, the experimental results indicate that kobs decreases while increasing the [IO4−]. The order with respect to [IO4−] was negative fractional and the plot of 1/kobs versus [IO4−] was linear (Figs 3 and 4).

585

64

1/kobs (s)

56 48 40 32

0

9

18

27

36

45

[IO4–] × 104 (mol/l)

Fig. 3. Plot of 1/kobs versus [IO4−] at temperature 298.2 K L-lysine: [Ni(IV)] = 7.83×10–6 mol/l; [L-lysine] = 9.0×10–3 mol/l; [OH–] = 0.0155 mol/l; μ= 0.0385 mol/l (r = 0.999) 30

1/kobs (s)

25

20

15

10

0

9

18 27 [IO4–] × 104 (mol/l)

36

45

Fig. 4. Plot of 1/kobs versus [IO4−] at temperature 298.2 K L-ornithine: [Ni(IV)] = 7.83×10–6 mol/l; [L-ornithine] = 6.0×10–3 mol/l; [OH–] = 0.0155 mol/l; μ = 0.1015 mol/l (r = 0.999)

Dependence of rate on the concentration of OH−. At constant [Ni(IV)], [reductant], [IO4−], μ and temperature, kobs values decreased with the increase in [OH–] (Table 1). Table 1. Rate dependence on [OH–] at temperature 298.2 K

[OH–] ×102 (mol/l) kobs ×103(s–1)

L-lysine L-ornithine

0.55 44.00 63.90

1.05 26.81 42.80

1.55 20.52 35.08

2.05 16.65 31.13

3.05 12.78 23.30

L-lysine: [Ni(IV)] = 7.83×10–6 mol/l, [IO4–] = 2.00×10–3 mol/l, [L-lysine] = 9.0×10–3 mol/l, μ = 0.0415 mol/l; L-ornithine: [Ni(IV)] = 7.83×10–6 mol/l, [IO4–] = 4.00×10–3 mol/l, [L-ornithine] = 6.00×10–3 mol/l, μ = 0.1065 mol/l.

586

Dependence of rate on the ionic strength. With other conditions fixed, the reaction rate decreased with increase in ionic strength when reductant was oxidised by Ni(IV), indicating that there was negative salt effect to reductant (Table 2). Table 2. Rate dependence on ionic strength μ at temperature 298.2 K

µ (mol/l) kobs ×103 (s–1) µ (mol l ) kobs ×103 (s–1) –1

  0.0265 22.35

L-lysine   0.0515   0.0765 18.97 16.26

  0.1015 15.18

  0.1265 14.30

  0.0315 41.42

L-ornithine   0.0565   0.0815 35.98 33.48

  0.1215 29.37

  0.2315 25.45

L-lysine: [Ni(IV)] = 7.83×10–6 mol/l, [IO4–]=4.00×10–3 mol/l, [L-lysine] = 9.0×10–3 mol/l, [OH–] =1.55×10–2 mol/l; L-ornithine: [Ni(IV)] = 7.83×10–6 mol/l, [IO4–] =4.00×10–3 mol/l, [L-ornithine]=6.00×10–3 mol/l, [OH–] =1.55×10–2 mol/l.

DISCUSSION In alkaline solution, equilibria (1)–(3) were observed and the corresponding equilibrium constants at 298 K were determined by Aveston15.     2IO4–+2OH–

H2I2O104–, lgβ1 =15.05

(1)

IO4– + OH– +H2O

H3IO62–, lgβ2 = 6.21

(2)

  

H2IO6 , lgβ3 = 8.67

(3)

IO4 + 2OH –

–

3–

The distribution of all species of periodate in alkaline solution can be calculated from the equilibria (1)–(3). The dimer H2I2O104– and IO4– species can be neglected, the main iodic acid species are H3IO62– and H2IO63–. According to literature, the main existent form of oxidant was [Ni(OH)2(H2IO6)2]4– over the experimental concentration range of OH–. Neglecting the concentration of ligand dissociated from Ni(IV), the main species of periodate are H2IO63– and H3IO62–, where [IO4–]t ≅ [H3IO62–]+[H2IO63–]

(4)

Equations (5) and (6) can be obtained from equations (2), (3) and (4): [H2IO63–] =

[H3IO62–] =

β3[OH–] β2 + β3[OH–] β2 β2 + β3[OH–]

[IO4–]t = f([OH–]) [IO4–]t,

(5)

[IO4–]t = φ([OH–]) [IO4–]t,

(6)

Here, [IO4–]t represents the concentration of original over all periodate ions which is approximately equal to the sum of [H2IO63–] and [H3IO62–]. In the [OH–] range used in 587

this work, the main species of periodate is H2IO63–. Based on the discussion, the formula of the Ni(IV) periodate complex may be represented either by [Ni(OH)2( H3IO6)2]2– or the less protonated ionic species [Ni(OH)2 (H2IO6)2]4–. We preferred to use the latter to represent DPN because it is close to that suggested by Mukherjee16. Based on the above discussion, the following reaction mechanism was proposed:     K1



[Ni(OH)2(H2IO6)2]4– + OH– [Ni(OH)2(HIO6)]2– + H2IO63– + H2O   (7) (DPN)      (MPN) K2

R(NH2)CHCOO– + H2O R(NH3+)CHCOO– + OH–     (A)   (B)

(8)

[Ni(OH)2(HIO6)]2– + R(NH3+)CHCOO–    (MPN)      (B)

(9)

K3

complex

k

complex ––––→ Ni(II) + product

(10)

R = –CH2CH2CH2CH2NH2 or R = –CH2CH2NH2.

Reaction (10) was the rate-determining step. The oxidation products of L-lysine and L-ornithine are the corresponding aldehydes. [Ni(IV)]t = [DPN]e + [MPN]e + [complex]e.

Subscripts t and e stand for total concentration and concentration at equilibrium, respectively. As the rate of the disappearance of [Ni(IV)]t was monitored, the rate of the reaction can be derived as follows: –

d[Ni(IV)t] dt

=

kobs = 1 kobs 1 kobs

=

=

1 k

kK1K2K3[A] K1K2K3[A] + K1[OH–] + [H2IO63–]

[Ni(IV)t

kK1K2K3[A] K1K2K3[A] + K1[OH–] + [H2IO63–] +

K1[OH–] + f([OH–])[IO4–]

1

kK1K2K3

[A]

K1K2K3[A] + K1[OH–] K1K2K3[A]

+

f([OH–]) K1K2K3[A]

[IO4–]

(11)

(12)

(13)

(14)

In this report, equation (13) shows that the order in L-lysine and L-ornithine should be of fractional order and 1/kobs versus 1/[reductant] should be linear, equation (14) shows that the plot of 1/kobs versus [IO4–] should also be linear, and equation (12) shows that kobs values decreased with the increase in [OH–]. The rate equations 588

derived from the reaction mechanisms are consistent with our experimental results. The activation energy and the thermodynamic parameters (298.2 K) were evaluated by the previously published method17 (Table 3). Table 3. Rate constants (k) and the activation parameters for the rate-determining step

k ×102 (mol–1 l s–1)

T (K)

Thermodynamic activation parameters (298.2 K)

L-lysine L-ornithine L-lysine L-ornithine

288.2 293.2 298.2 303.2 308.2 2.68 4.21 6.33   9.99 17.25 3.12 4.38 6.68 11.39 18.73 –1 * Ea=67.63 kJ mol , ΔH = 65.15 kJ mol–1, ΔS* = –48.96 J K–1 mol–1 Ea = 66.88 kJ mol–1, ΔH* = 64.41 kJ mol–1, ΔS* = –50.70 J K–1 mol–1

The plots of lnk versus 1/T have the following intercept (a) slope (b) and relative coefficient (r): L-lysine: a = 24.57, b = –8134.94, r = 0.998; L-ornithine: a = 24.374, b = –8045.2, r= 0.995.

CONCLUSIONS Among various species of Ni(IV) in alkaline liquids, monoperiodatonickelate is considered as the active species for the title reaction. The rate constant of slow step and other equilibrium constants involved in the mechanism are evaluated and activation parameters with respect to the slow step of the reaction were computed. The overall mechanistic sequence described here is consistent with product studies, mechanistic studies and kinetic studies. Based on the above discussion and results, it was found that the rate constants of the rate-determining step and the activation parameters for L-ornithine and L-lysine are very contiguous. In the reaction system, both the L-ornithine and L-lysine formed hexa-cyclic intermediate compounds with Ni(IV). At the same time we also observed that the rate of the rate-determining step of L-ornithine is a little quicker than that of L-lysine because L-lysine embodies a longer carbon chain, which has the special steric hindrance. So the formation of intermediate adduct between Ni(IV) complex and L-ornithine is more stable than that of L-lysine. Therefore, the rate constants of the rate-determining step for L-ornithine are larger than those for L-lysine, which is consistent with experimental phenomena. REFERENCES 1. W. J. NIU, Y. ZHU, K. C. HU, C. L. TONG, H. S. YANG: Kinetics of Oxidation of SCN– by Diperiodatocuprate(III) (DPC) in Alkaline Medium. Int. J. Chem. Kinet., 28, 899 (1996). 2. T. S. SHI: Studies of Unusual Oxidation States of Transition Metals. (I) Kinetics and Mechanism of Oxidation of Potassium Thiocyanate by Diperiodatoargentate(III) Ion in Alkaline Medium. Sci. China, Ser. B Chem., 12, 471 (1990). 3. U. CHANDRAIAH, C. P. MURTHY, K. SUSHAMA: Kinetics of Oxidation of Lactic, Mandelic and Glycollic Acids by Diperiodatonickelate(IV) in Alkaline Medium. Indian J. Chem., 28(A), 162 (1989).

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  4. Z. T. LI, F. L. WANG, A. Z. WANG: Kinetics and Mechanism of Oxidation of Tetrahydrofuryl Alcohol by Dihydroxydiperiodatonickelate(IV) Complex in Aqueous Alkaline Medium. Int. J. Chem. Kinet., 24, 933 (1992).   5. R. T. MAHESH, P. D. POL, S. T. NANDIBEWOOR: Kinetics and Mechanism of Oxidation of L-leucine by Alkaline Diperiodatonickelate(IV): A Free Radical Intervention, Deamination, and Decarboxylation. Monatsh. Chem., 134, 1341 (2003).   6. R. S. SHETTAR, S. T. NANDIBEWOOR: Kinetic, Mechanistic and Spectral Investigations of Ruthenium(III)-catalysed Oxidation of 4-hydroxycoumarin by Alkaline Diperiodatonickelate(IV) (Stopped Flow Technique). J. Mol. Catal. A: Chem., 234, 137 (2005).   7. C. V. HIREMATH, D. C. HIREMATH, S. T. NANDIBEWOOR: Ruthenium(III) Catalysed Oxidation of Gabapentin (Neurontin) by Diperiodatonickelate(IV) in Aqueous Alkaline Medium: A Kinetic and Mechanistic Study. J. Mol. Catal. A: Chem., 269, 246 (2007).   8. D. S. MAHADEVAPPA, K. S. RANGAPPA, N. M. M. GOWDA, B. THIMME GOWDA: Kinetic and Mechanistic Studies of Oxidation of Arginine, Histidine, and Threonine in Alkaline Medium by N-chloro-N-sodio-p-toluenesulfonamide. Int. J. Chem. Kinet., 14, 1183(1982).   9. (a) D. LALOO, M. K. MAHANTI: Kinetics of Oxidation of Amino Acids by Alkaline Hexacyanoferrate(III). J. Phys. Org. Chem., 3, 799 (1990); (b) R. M. KULKARNI, D. C. BILEHAL, S. T. NANDIBEWOOR: Deamination and Decarboxylation in the Chromium(III)-catalysed Oxidation of L-valine by Alkaline Permanganate and Analysis of Chromium(III) in Microscopic Amounts by a Kinetic Method. Transition Met. Chem., 28, 199 (2003). 10. L. C. W. BAKER, H. G. MUKERJEE, S. B. SARKAR, B. K. CHOUDHURY: Synthesis and Characterisation of Lithium Bexaorth Periodatonickelate(IV). Indian J. Chem., 21 (A), 618 (1982). 11. C. P. MURTHY, B. SETHURAM, T. N. RAO: Oxidation by Tetravalent Nickel Part 1: Kinetics of Electron Transfer from Some Aliphatic Alcoholos to Ni (IV) in Aqueous Alkaline Media. Z. Phys. Chem. (Leizig), 287, 1212 (1986). 12. J. H. SHAN, L. P. WANG, S. G. SHEN, H. W. SUN: Kinetics and Mechanism of Oxidation of Some Hydroxy Butyric Acid Salts by Ditelluratocuprate(III) in Alkaline Medium. Turk. J. Chem., 27, 265 (2003). 13. F. FEIGL: Spot Tests in Organic Analysis. Elsevier Publishing Co., New York, 1956, p. 208. 14. J. QIAN, M. Z. GAO, J. H. SHAN, S. G. SHEN, H. W. SUN: Kinetics of Oxidation of Ethanolamine by Dihydroxydiperiodatonickelate(IV) in Alkaline Media. J. Guangxi Normal Univ., 21 (1), 248 (2003). 15. J. AVESTON: Hydrolysis of Potassium Periodate: Ultracentrifugation Potentiometric Titration and Raman Spectra. J. Chem. Soc. A, 273 (1969). 16. H. G. MUKHERJEE, B. MANDAL, S. DE: Preparation and Studies of the Complex Periodatoferrate(III) Hexahydrate and Periodatonickelate(IV) Monohydrate. Indian J. Chem., 23(A), 481 (1984). 17. J. H. SHAN, T. Y. LIU: Kinetics and Mechanism of Substitution Reactions of Bis(N,N-diethyldithio­ carbamato)alkylxanthatocobalt(III) with Dipropylamine and Di-n-butylamine in Methanol. Acta Chim. Sinica, 52, 1140 (1994). Received 11 May 2009 Revised 23 August 2009

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Oxidation Communications 35, No 3, 591–598 (2012) Analytical section

An Approach for Biodiesel Characterisation I. Sharafutdinova, А. Pavlovab*, D. Stratievc, P. Petkovd, I. Shishkovab, R. Dinkovc Lukoil Neftochim Bourgas JSC, 8104 Bourgas, Bulgaria Research Laboratory, Lukoil Neftochim Bourgas JSC, 8104 Bourgas, Bulgaria E-mail: [email protected] c Process Engineer Department, Lukoil Neftochim Bourgas JSC, 8104 Bourgas, Bulgaria d ‘Prof. Assen Zlatarov’ University, 8010 Bourgas, Bulgaria a

b

ABSTRACT The determination of fatty acid methyl esters (FAMEs) by gas chromatography is one of the most common analyses performed in biodiesel research. By using the potential of the capillary gas chromatography, the flame ionisation detector and the non-polar column was developed fast, efficient and reproducible method for determination of fatty acid methyl esters (FAMEs) content in the biodiesel. Based on the quantitative results of the biodiesel component composition was carried out evaluation degree of unsaturation. The analytical procedure has been successfully applied for determination of fatty acid ester contents in 7 samples of biodiesel, produced by several regional producers of bio-products in Bulgaria. Keywords: gas chromatography, biodiesel, fatty acid methyl esters, degree of unsaturation. AIMS AND BACKGROUND The environmental pollution, the diminishing oil supplies, the overproduction of agricultural crops, the energy dependence of a number of countries account for the still increasing use of motor fuels with bio-components. To this effect, the most common and future-oriented is considered to be the biodiesel. The biodiesel is a blend of FAMEs, the content of which varies depending on the feedstock source. From chemical point of view, the various vegetable and animal oils have different fatty acid content. The fatty acids differ in terms of the carbon chain length and the acids degree of unsaturation. The properties of the biodiesel are strongly *

For correspondence.

591

affected by the content of the individual fatty acid esters. The presence of high levels of linolenate acid (С18:3) above 8%, significantly facilitates the oxygenation processes in the bio-product1–3. Moreover, the presence of fatty acid esters in biodiesel of a higher degree of unsaturation undeniably has a strong influence on a number of physical properties when mixing the biodiesel with petroleum diesel fuel (e.g. cetane number, viscosity, oxidation stability, etc.)4,5. In this context, the determination of biodiesel content prior its mixing with petroleum diesel is a serious challenge in front of the refinery research laboratories. There is a wide variety of analytical techniques of detection, characterisation and quantification of the fatty acid ester content in the biodiesel6–12. Generally, the methods of analysis of the fatty acid ester content are two: gas chromatography and high efficiency liquid chromatography13,14. Of course, the quest for new solutions is continuous not only for determination of the individual fatty acid ester content in the biodiesel, but also some new approaches are being developed dedicated to the processing of analytical information in view of the product specificity, the assortment expansion and the quality of the fuels. No doubt, the problem remains open. Lukoil Neftochim Bourgas is in the process of active determination of the general targets and requirements in terms of the introduction of the biodiesel to the produced diesel fuel. As part of this ever developing effort, the tasks for production of transportation fuels containing biodiesel are clearly outlined. The present study offers a method for determination of FAME content in the biodiesel based on the capillary gas chromatography combined with flame ionisation detector. The derived quantitative data are used for assessment of the biodiesel unsaturation in order to characterise its quality before it may be used as a blend component. EXPERIMENTAL Reagent. n-Heptane was supplied from Мerck, Germany. The Certified Reference Materials (CRM), containing methyl myristate/C14:0, methyl palmitate/C16:0, methyl stearate/C18:0, cis-9-oleic methyl ester/C18:1, methyl linoleate/C18:2, methyl linolenate/C18:3, methyl arachidate/C20:0, methyl eicosenoate/C20:1, methyl behenate/C22:0, methyl erucate/C22:1 and methyl lignocerate/C24:0 were purchased from Supelco, USA. Apparatus. The gas chromatographic quantification of FAME content was performed by 5890 series GC equipped with a flame ionisation detector (FID) (Agilent Technologies, Inc., USA). A fused-silica capillary column PONA (50 m length, 199 μm i.d., film thickness 0.5 μm) is used. Hydrogen was used as a carrier gas at a constant flow rate 1.0 ml/min. For the analysis of FAME, the column temperature was programmed from 170°С (held for 1 min) to 280°С at a rate of 6°С/min (held 30 min). Split/splitless injector temperature is 250°С and detector temperature was 280°С. Injection volume was 0.2 µl and split ratio 1:100. 592

The gas chromatography-mass spectrometry analysis was performed using a 7890A series GC device with 5975C series mass selective detector (MSD) Inert XL EI/CI (Agilent Technologies, Inc., USA). The chromatographic column with non-polar cross-linked methyl-silicone phase and the chromatographic analysis conditions were similar to those used with the GC FID analysis. The temperature of transfer line, ion source and quadrupole mass spectrometry analyser was held at 250, 230 and 150°С, respectively. Mass spectrum was acquired in electron impact mode at 70 еV by the full scan mode. Sample preparation. About 0.8 g of biodiesel sample were weighed in a 5-ml volumetric flask, after that the flask was filled up to the marking with n-heptane and the ready mixture was weighed. The GC FID calibration was carried out by a series of appropriate n-heptane prepared CRM solutions, prepared in the same way as described above. The correction factors for each methyl ester of the respective fatty acid were determined. The analysis was performed on 7 biodiesel samples of vegetable origin, produced by several regional bio-product producers in Bulgaria. RESULTS AND DISCUSSION By means of the analysis performed is generated a method for determination of the FAME content in the biodiesel samples. In order to achieve a maximum good separation of the respective acid methyl esters in the biodiesel the chromatographic analysis conditions systematically are changed. Also the quantity of the solvent used for preparation of the calibration mixtures was optimised. By using mixtures of certified reference materials and by means of the mass spectrometry analysis are identified and determined the retention times of the methyl esters of the acids present in the tested samples. By means of the chromatographic column used there are 15 compounds separated and identified, and the respective retention times and coefficient of variation are illustrated in Table 1. The table data demonstrate very good repeatability of the optimum conditions selected for analysis of the FAMEs with the used non-polar stationary cross-linked methyl silicone phase. Quantity optimisation of the solvent used for preparation of calibration mixtures and for biodiesel samples testing is the next parameter tested in the course of methodology development of the biodiesel analysis. For this purpose in our tests are assessed n-heptane and ultra-low-sulphur diesel fuel as solvents. The quantitative results derived from the conducted tests are summarised in Table 2. The data show that the absolute differences in the contents of acid methyl esters are insignificant for both tested levels of 5 and 30% when mixing biodiesel with n-heptane. It is established that a blend of biodiesel and low-sulphur diesel fuel is not suitable for the purposes of our testing because of the significant lowering of the methyl ester content in the tested acids.

593

Table 1. Retention time and coefficient of variation of FAMEs

Compounds Caprylic acid methyl ester Capric acid methyl ester Lauric acid methyl ester Myristic acid methyl ester Palmitic acid methyl ester Palmitoleic acid methyl ester Linoleic acid methyl ester Linolenic acid methyl ester cis-9-Oleic acid methyl ester Stearic acid methyl ester Gondanoic acid methyl ester Arachidic acid methyl ester Erucic acid methyl ester Behenic acid methyl ester Lignoceric acid methyl ester

Retention time (min)   4.697   6.590   7.812 12.708 15.830 16.175 19.042 19.102 19.285 19.525 22.730 23.223 27.497 28.178 35.320

Coefficient of variation (%) 0.012 0.014 0.020 0.015 0.019 0.009 0.014 0.019 0.017 0.026 0.003 0.004 0.018 0.012 0.025

Table 2. Chromatographic data from analysis on blends of biodiesel with n-heptane and with diesel fuel

Compounds

С16:0 С18:0 С18:1 С18:2

Sunflower oil as methyl esters (RM) (%)   6.23   3.99 27.90 61.92

30% RM in nheptane (%)

5% RM in nheptane (%)

5% RM in diesel fuel (%)

  6.07   3.96 27.10 61.74

  6.00   3.60 27.80 59.98

  6.40   4.40 26.20 57.80

Table 3 shows the results of a soybean oil-type certified reference material analysis with known compounds composition, carried out at specified chromatographic testing conditions. The test sample preparation was carried out just before the analysis performance. Thus the results in the table include the variations caused by the sample preparation and the chromatographic analysis. The derived average values for individual FAME content in the soybean oil correspond to the input quantities in the reference material – the analytical yield varies from 92 to 99%. The relative standard deviations are less than 5.2%.

594

Table 3. Precision of the chromatographic method

Compounds Myristic acid methyl ester Palmitic acid methyl ester Linoleic acid methyl ester Linolenic acid methyl ester cis-9-Oleic acid methyl ester Stearic acid methyl ester Gondanoic acid methyl ester Arachidic acid methyl ester Erucic acid methyl ester Behenic acid methyl ester Lignoceric acid methyl ester

FAME mix rapeseed oil (%) 1.000 3.998 12.006 5.027 59.950 2.999 1.018 3.000 5.003 2.999 3.000

Average Recovery sd (n=5) (%) (%) (%) 1.02 4.11 11.96 4.89 60.10 3.09 1.10 3.12 4.99 2.87 2.95

98 97 99 97 99 97 92 96 99 96 98

0.02 0.01 0.02 0.11 0.32 0.11 0.04 0.03 0.01 0.15 0.11

RSD (%) 2.0 0.2 0.2 2.2 0.5 3.6 3.6 1.0 0.2 5.2 3.7

The physical and chemical properties of the biodiesel are defined by its composition. The distribution of the fatty acid methyl esters and the biodiesel degree of unsaturation have strong impact on the overall behaviour and suitability of the biodiesel as a component blended with petroleum diesel. The changes advanced in the quality of biodiesel produced from various row materials may to a high degree be explained with these 2 characteristics5,15–17. The distribution of the fatty acid methyl esters in the tested samples is summarised in Table 4. There are 2 main types of fatty acid methyl esters – methyl esters of saturated fatty acids, and methyl esters of unsaturated fatty acids, respectively with 1, 2 and 3 double bonds in the carbon chain. The tests conducted show that the analysed samples contain large quantities of methyl esters of unsaturated fatty acids, where prevailing are the methyl esters of mono-unsaturated cis-9-oleinic (C18:1) and di-unsaturated linoleate (C18:2) acids. The data for quantitative distribution of mono-, di- and poly-unsaturated fatty acid methyl esters in the analysed samples are presented in Table 5. This quantitative information is used for determination of the total unsaturation of the tested biodiesel samples. The degree of unsaturation of every biodiesel sample is calculated by summing the individual contributions of the methyl esters of mono-, di- and poly-unsaturated acids present in the samples by multiplying their mass quantity by factors 0.8, 1.7 and 2.6. These factors are calculated based on the molecular weights of the mono-, di- and poly-unsaturated acid methyl esters, the number of double bonds and atomic weight of the iodine atom which theoretically impacts the double bond in the acids17. This approach for evaluation of the biodiesel unsaturation is suitable because no toxic solvents are used and/or it is not required preliminary preparation of the sample for determination of the total unsaturation of the biodiesel compared to the standard approaches18. The calculated degree of unsaturation of the tested samples is presented in Table 5. The data derived for the biodiesel unsaturation are correlated to their experimentally established iodine numbers (Table 5). With increasing the biodiesel unsaturation degree increases also 595

the iodine number. However, more detailed and reliable structural information on the origin of the overall unsaturation of the biodiesel is obtained by the chromatographic analysis and the relevant quantitative data interpretation applied. The physical factor ‘iodine number’ is a rather general quantitative indicator for measurement of the bioproduct unsaturation, as very often this parameter is strongly affected by the presence of compounds naturally occurring in the biodiesel (for example, carotenes and squalene). Table 4. Compound composition (wt.%) of biodiesel produced by regional producers

Compounds Caprylic acid methyl ester Capric acid methyl ester Lauric acid methyl ester Myristic acid methyl ester Palmitic acid methyl ester Palmitoleic acid methyl ester Stearic acid methyl ester cis-9-Oleic acid methyl ester Linoleic acid methyl ester Linolenic acid methyl ester Arachidic acid methyl ester Gondanoic acid methyl ester Behenic acid methyl ester Erucic acid methyl ester Lignoceric acid methyl ester Total content (%)

C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0

1 nd   0.04 nd   0.15   7.76   0.15   3.18 36.23 48.26   4.23 nd nd nd nd nd 100

2 nd nd nd   0.06   5.57 nd   3.74 30.30 54.54 nd   0.27   1.89   0.69 nd   0.49 97.55

3 nd nd nd nd   5.48 nd   2.54 45.41 39.29   4.14   0.48   0.98   0.58   0.71   0.09 99.70

Sample 4   0.05   0.04 nd   0.07   5.38   0.18   2.57 43.73 38.92   3.81   0.54   0.94   0.50   0.65   0.05 97.43

5 nd nd nd   0.12 10.21   0.28   2.77 41.13 37.10   5.62   0.25   0.43   0.14 nd nd 98.05

6   0.11   0.02   0.06   0.37 12.06   0.38   4.05 29.55 49.97   0.39   0.30   0.35   0.60   0.11   0.22 98.54

7   0.12 nd nd   0.20   8.36   0.23   3.97 27.96 57.20   0.10   0.30   0.24   0.66 nd   0.23 99.57

Sample 1 – Astra Bioplant, Slivo pole; sample 2 – Green oil, Silistra; sample 3 – Sun rays, Provadia; sample 4 – Astra Bioplant, Slivo pole, 17.04.09; sample 5 – Brevo, Sofia; sample 6 – TIT Kameno, No10; sample 7 – TIT Kameno; nd – not determined. Table 5. Fatty acid methyl esters content

Test name

Sample 1 2 3 4 5 6 7 Saturated acid methyl esters (%)   11.13   10.82    8.82    9.20   13.49   13.84   17.79 Mono-unsaturated acid methyl   36.26   32.19   36.73   45.50   42.84   28.43   30.39 esters (%) Di-unsaturated acid methyl   48.26   54.54   48.10   38.92   37.10   57.20   49.97 esters (%) Poly-unsaturated acid methyl    4.23    nd    2.21    3.81    5.62    0.10    0.39 esters (%) Degree of unsaturation (%) 122.1 118.5 116.9 112.5 112.0 143.0 131.1 Iodine number (g J2/100 g) 117 117.6 119 117 108 127 118

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CONCLUSIONS The presented study represents a research effort to harness the potential of our laboratory equipment and develop a method for identification and quantification of the fatty acid methyl esters content in the biodiesel, produced by the regional producers in Bulgaria. Their quantification is achieved by using capillary GC with FID. The fast and efficient separation of the biodiesel main compounds is carried out on a non-polar chromatography column. The main compounds of the tested biodiesel samples are methyl esters of saturated and unsaturated fatty acids ranging in number of carbon atoms from C8 to C24. The concentrations of C18:1 and C18:2 unsaturated fatty acids methyl esters are highest. The determined mono-, di- and poly-unsaturated fatty acid contents are used for quantification of the total unsaturation of the tested biodiesel samples. The degree of unsaturation results are correlated to their established iodine numbers. The applied approach for interpretation of the quantitative information finds application in the development of new batches of petroleum diesel blended with various quantities of biodiesel. REFERENCES   1. M. J. RAMOS, C. M. FERNANDEZ, A. CASSA, L. RODRIGUEZ, A. PEREZ: Influence of Fatty Acid Composition of Raw Materials on Biodiesel Properties. Bioresource Technology, 100, 261 (2009).   2. L. C. MEHER, D. VIDYA SAGAR, S. N. NAIK: Technical Aspects of Biodiesel Production by Transesterification. A Review. Renew. Sust. Energ. Rev., 10, 248 (2006).   3. A. K. DOMINGOS, E. B. SAAD, W. W. D. VECHIATTO, H. M. WILLHELM, L. P. RAMOS: The Influence of BHA, BHT and TBHQ on the Oxidation Stability of Soybean Oil Ethyl Esters (Biodiesel). J. Braz. Chem. Soc., 18 (2), 416 (2007).   4. G. KNOTHE: Depenence of Biodiesel Fuel Properties on the Structure of Fatty Acid Alkyl Ester. Fuel Processing Technology, 86, 1059 (2005).   5. G. A. PEREYRA-IRUJO, N. G. IZQUIERDO, M. COVI, S. M. NOLASCO, F. QUIROZ, L. A. N. AGUIRREZABAL: Variability in Sunflower Oil Quality for Biodiesel Production: A Simulation Study. Biomass and Bioenergy, 33 (3), 459 (2009).   6. A. C. PINTO, L. L. N. GUARIERO, M. J. C. REZENDE, N. M. RIBEIRO, E. A. TORRES, W. A. LOPES, P. A. de P. PEREIRA, J. B. de ANDRADE: Biodiesel: An Overview. J. Braz. Chem. Soc., 16 (6B), 1313 (2005).   7. B. DIEHL, G. RANDEL: Analysis of Biodiesel, Diesel and Gasoline by NMR Spectroscopy – A Quick and Robust Alternative to NIR and GC. Lipid Technology, 19 (11), 258 (2007).   8. S. SCHOBER, I. SEIDL, M. MITTELBACH: Ester Content Evaluation in Biodiesel from Animal Fats and Lauric Oils. Eur. J. Lipid. Sci. Technol., 108, 309 (2006).   9. S.-H. CHEN, K.-C. CHEN, H.-M. LIEN: Determination of Fatty Acids in Vegetable Oil by Reversed Phase Liquid Chromatography with Fluorescence Detection. J. Chromatogr. A, 849 (2), 357 (1999). 10. L. MONDELLO, P. Q. TRANCHIDA, R. COSTA, A. CASILLI, P. DUGO, A. COTRONEO, G. DUGO: Fast GC for the Analysis of Fats and Oils. J. Sep. Sci., 26, 1467 (2003). 11. Z. XU, K. HARVEY, T. PAVLINA, G. DUTOT, G. ZALOGA, R. SIDIQUID: An Improved Method for Determining Medium- and Long-chain FAMEs Using Gas Chromatography. Lipids, 45, 199 (2010).

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12. C. HÄRTIG: Rapid Identification of Fatty Acid Methyl Esters Using a Multidimensional Gas Chromatography–Mass Spectrometry Database. J. Chromatogr. A, 1177 (1), 159 (2008). 13. EN 14331:2004: Liquid Petroleum Products–Separation and Characterisation of Fatty Acid Methyl Esters (FAME) from Middle Distillates. Liquid Chromatography (LC)/Gas Chromatography (GC) Method. 14. EN 14103:2011: Fat and Oil Derivatives-Fatty Acid Methyl Esters (FAME). Determination of Ester and Linolenic Acid Methyl Ester Contents. 15. S. K. HOEKMAN, A. BROCH, C. ROBBINS, E. CENICEROS, M. NATARAJAN: Review of Biodiesel Composition, Properties, and Specifications. Renewable and Sustainable Energy Reviews, doi: 10.1016/j.rser.2011.07.143 16. G. KNOTHE, R. O. DUNN, M. O. BAGBY: Biodiesel: The Use of Vegetable Oils and Their Derivatives as Alternative Diesel Fuels. In: Fuels and Chemicals from Biomass (Eds B. C. Saha, J. Woodward). ACS Symposium Series, 666, 172 (1997). 17. ЕN 14214:2003: Automotive Fuels. Fatty Acid Methyl Esters (FAME) for Diesel Engines. Requirements and Test Methods. 18. ЕN 14111:2003: Fat and Oil Derivatives. Fatty Acid Methyl Esters (FAME). Determination of Iodine Value. Received 5 January 2012 Revised 12 February 2012

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Oxidation Communications 35, No 3, 599–610 (2012) Analytical section

Synthesis and Spectroscopic Characterisation of Hydroxy Ketones Using Green Chemical Approaches R. Saharana*, P. S. Vermab, I. K. Sharmab, J. Joshia Department of Chemistry, MNIT, 302 017 Jaipur, India Department of Chemistry, University of Rajasthan, 302 004 Jaipur, India E-mail: [email protected]; [email protected]

a

b

ABSTRACT The synthesis and characterisation of hydroxy ketones such as 3-hydroxy-1-phenylbutanone and 3-hydroxy-2-pentanone employing microbial transformation (using whole cells of baker yeast in their free as well as immobilised form in mixtures of glycerol and water) and using electrochemical technique are reported. In electrochemical technique effect of scan rate and pH on the reduction peaks have been analysed. The kinetic parameters were also calculated and the process was found to be diffusion controlled. The products obtained were purified and then characterised by spectroscopic techniques. These green methodologies were found to be more effective, safe, economical, easy to handle and environmental friendly and provide improved synthetic routes to many valuable compounds. Keywords: hydroxy ketones, biocatalysts (baker yeast), cyclic voltammetry (glassy carbon electrode), constant current electrolysis. AIMS AND BACKGROUND Chiral alcohols are versatile and convenient building blocks in the synthesis of biologically important compounds, therefore asymmetric reduction of ketones mediated by microbes becomes a promising route to synthesise chiral alcohols in an excellent stereoselectivity1. The asymmetric reduction of prochiral ketones is one of the most important reactions in organic synthesis. Enantiopure alcohols, which can be prepared by chemical or biocatalytic methods, are significant chiral building blocks of industrial special and fine chemistry. Disadvantages of chemical processes are often extreme reaction conditions and insufficient optical purity yields2. Among the various possible biocatalysts, baker yeast (BY, Saccharomyces cerevisiae) mediated reduction of synthetic substrate is a useful method for preparing chiral intermediate in synthesis *

For correspondence.

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chemistry because it is emerged as one of the most frequently employed microorganisms in whole cell conditions due to its high bioavailability, easiness of treatment inexpensive and mild reaction conditions3,4. Regio- and enantioselective reduction of diketones can be achieved readily by using a biocatalyst. As a result, optically active hydroxyketones and diols have been successfully synthesised5. The electrochemical reduction is one of the greener approaches because it is pollution free as electrons are regarded as one of the reagents, therefore it reduces the use of at least one hazardous chemical reagent6,7. These reactions can take place in a low-temperature environment, reducing the local consumption of energy, the risk of corrosion, material failure, and accidental release8. Electrochemical techniques are also very useful to investigate kinetics and mechanisms of the reactions, hence electro-organic synthesis provides alternative synthetic route9. Chiral α-hydroxy ketones constitute a significant structural block in many biologically active natural products. They are also remarkable synthons for the asymmetric synthesis of natural products10,11. Chiral α-keto alcohols are important intermediates for the production of pharmaceuticals, flavours and fragrances12. Chiral α-hydroxy ketones are found in antidepressants, in selective inhibitors of amyloid-β protein production (used in the treatment of the Alzheimer disease), in farnesyl transferase inhibitors (kurasoin A and B), and in antitumor antibiotics (olivomycin A and chromomycin A3). α-Hydroxy ketones are as fine chemicals are used as building blocks for the production of larger molecules. They can also be used in synthesis of many other important products, such as amino alcohols, diols. Chemical synthetic approaches are not attractive and are economically not profitable because of the lack of selectivity and large number of chemical steps required13. 3-Hydroxy-2-pentanone is a natural identical flavour ingredient according to the European legislation and is used as aromas by flavour industry. β-Hydroxy ketones are key intermediates in the construction of numerous natural products14. β-Hydroxy ketones belong to an extremely important class of biological compounds and can serve as versatile building block for the asymmetric synthesis of carbohydrates, amino acids and many other biomolecules. They also provide privileged structural functionalities that exist in many important natural products. For example, the β-hydroxy ketones functionalities exist in macrolide classes of antibiotics such as telithromycin and cethromycin which are targeted primarily against gram-positive bacterial strains including Streptococcus pneumoniae and S. pyogenes, fastidious gramnegative strains including Haemophilus influenza and Moraxella catarrhalis, atypical Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophilia. They were also found in conagenin that can improve the antitumor efficacy of adriamycin and mitomycin C against murine leukemias, which suggest its potential utility for cancer chemotherapy15. In view of the above applications, the present work relates to the reduction of 2, 3-pentanedione and phenyl-1,3-butanedione to the corresponding hydroxy ketones such as 3-hydroxy-2-pentanone16 and 3-hydroxy-1-phenyl-butanone17 employing 600

electrochemical technique to evaluate electrode reaction using cyclic voltammetry and constant current electrolysis and microbial transformation using baker yeast (in a mixture of glycerol and water). These 2 strategies provide an useful and environmental friendly synthetic toolbox. EXPERIMENTAL All chemicals used in the present investigation were of analytical grade. All the solvents were dried and then distilled out. Doubly distilled water was used to prepare the required solutions. 1H NMR spectra were recorded using a Joel (Japan) 300MHZ spectrophotometer. FT-IR spectra were recorded by a Nicolet (USA) FT-IR spectrophotometer. MICROBIAL TRANSFORMATION

Reduction using free baker yeast. In a 500-ml flat bottom flask, a mixture of water and glycerol (50:50), 10 g fresh baker yeast, 10 g sucrose were placed and the suspension was stirred for 30 min. Chosen carbonyl compound (2 mM) dissolved in minimum amount of absolute alcohol was then poured into the suspension. The resulting mixture was magnetically stirred for appropriate time (Table 1). After completion of the reaction, the product was filtered using celite (filter aid powder), extraction was done with diethyl ether (30 ml) and the procedure was repeated 3 times. The ether was first evaporated from ether extract and then dried over calcium chloride to yield the product which was then characterised by boiling/melting point measurement and spectral techniques, viz. IR, NMR (Table 1). Table 1. Physical and spectral data from free baker yeast mediated reduction

Product name

Reac­tion Boiling/ Yield time (h) melting (%) point (oC)

IR data (cm–1)

H NMR (δ-value)

1

3-Hydroxy-1phenylbutanone

96

294

76

3360 (–OH), 3045 (Ar–CH str.) 2950 (CH str.) 1670,1590 (C=C ring str.), 1105, 1200 (C–O str. sec. alcohol)

2.0 (OH), 7.8 (ortho-CH), 2.7 (–CH2), 1.21 (–CH3)

3-Hydroxy-2pentanone

96

105

74

3420 (–OH), 2960 (–CH str.) 1465, 1380 (CH-ben), 1130 (C–O)

2.0 (OH), 1.76 (–CH2) 0.96 (–CH2–CH3) 2.09 (–CH3)

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Reduction using immobilised baker yeast. The experiment was performed under similar conditions with immobilised baker yeast, obtained by immobilisation of baker yeast (5 g) in polyacrylamide gel. The details of immobilisation are given below. Immobilisation of baker yeast (BY) in polyacrylamide gel: the gel was prepared using the following solutions: Solution A – 10 g acrylamide and 2.5 g N,N-methylenebisacrylamide in 100 ml doubly distilled water; Solution B – 5.98 g trihydroxy methyl amino methane, 0.46 ml N,N,N′,N″tetramethyl ethylenediamine and 48 ml 1N HCl solution to 100 ml solution; Solution C – 560 mg ammonium persulphate in 100 ml doubly distilled water; Solution D – 34.2 g sucrose in 100 ml doubly distilled water. The solutions were then mixed in the following sequence – solution A (10 ml) + solution B (5 ml) + solution D (20 ml) + baker yeast (5 g) + solution C (5 ml). The resulting solution was then deareated and allowed to polymerise for nearly 1 h. The resulting gel was cut into small pieces. The resulting final products obtained were characterised by boiling point measurement and spectral analysis, viz. IR and NMR (Table 2). Table 2. Physical and spectral data from immobilised baker yeast mediated reduction

Product name

Reac- Boiling/ Yield IR data (cm–1) tion melting (%) time (h) point (oC) 3-Hydroxy-196 294 83 3365 (–OH), phenyl3040 (Ar–CH str.) butanone 2970 (CH str.) 1670, 1590 (C=C ring str.), 1120, 1200 (C–O str. sec. alcohol) 3-Hydroxy-296 105 80 3430 (–OH), pentanone 2965 (–CH str.) 1465, 1385 (CH-ben), 1120 (C–O)

H NMR (δ-value)

1

2.0 (OH), 7.8 (ortho-CH), 2.65 (–CH2), 1.20 (–CH3) 2.1 (OH), 1.76 (–CH2) 0.96 (–CH2–CH3) 2.0 (–CH3)

Reduction using electrochemical technique. The completely computer-controlled basic electrochemistry system model ECDA-001 was used for recording cyclic voltammograms of selected compounds at different pH and scan rates in aqueous methanol using potassium chloride as supporting electrolyte at glassy carbon electrode. Cyclic voltammetric studies were carried out using a glassy carbon working electrode (A = 0.1 mm2), Ag/AgCl reference electrode and a platinum auxiliary electrode. All the measurements were carried out at room temperature. The working electrode was polished intensively with aluminium oxide (0.4 μm) on a polishing cloth and degreased in methanol prior to each electrochemical measurement. 602

The solutions were purged with purified dry nitrogen for 5 min prior to the experiments in order to remove dissolved oxygen from the media and blank cyclic voltammograms were recorded. Solution of 1mM of reactant was added to blank solution, then initial potential, final potential, scan rate and current sensitivity were provided and the resulting current was measured as a function of applied potential. Constant current electrolysis. Carbonyl compounds were subjected to constant current electrolysis at constant current at 1.0 A for 8 h in aqueous methanol. Galvanostat supplied by OMEGA type ICVD 60/2 was used to perform the experiment. A Remi hot plate cum magnetic stirrer (2 M LH model) was used to stir the solution throughout the electrolysis. A two-compartment H-shaped glass cell provided with a fritz glass disc (G-4) was used for electrolysis. Rectangular plates of stainless steel (SS-316) each of size (4 cm × 6 cm) were used as cathode as well as anode. The Britton Robinson buffer of appropriate pH and the supporting electrolyte (CH3COONa) was filled in both the limbs of H-shaped glass cell. Carbonyl compound was dissolved in minimum amount of methanol and placed in the cathodic compartment and electrolysed at constant current (1.0 A). After the completion of reaction, extraction was done with diethyl ether (30 ml) and the procedure was repeated 3 times. The ether extract was dried over calcium chloride and then characterised by combined application of boiling/melting point measurement, chromatographic and spectral techniques (Table 3). Table 3. Physical and spectral data from electrochemical reduction

Product name

Reac- Boiling/ Yield IR data (cm–1) tion melting (%) time (h) point (oC) 3-Hydroxy-16 294 84 3370 (–OH), phenyl3045 (Ar–CH str.) butanone 29550 (CH str.) 1675, 1590 (C=C ring str.) 1100, 1200 (C–O str. sec. alcohol) 3-Hydroxy-28 105 82 3420 (–OH), pentanone 2965 (–CH str.) 1470, 1385 (CH-ben), 1125 (C–O)

H NMR (δ-value) 1

2.0 (OH) 7.8 (ortho-CH) 2.6 (–CH2) 1.22 (–CH3) 2.1 (OH), 1.75 (–CH2) 0.96 (–CH2–CH3) 2.0 (–CH3)

RESULTS AND DISCUSSION Microbial transformation. Microbial reduction of phenyl-1,3-butanedione and 2,3pentanedione has been carried out as shown by the schemes below.

603

Scheme 1

Scheme 2

Asymmetric reduction of carbonyl compounds using whole cells of baker yeast as biocatalysts involves 2 enzyme systems. One of them is the enzyme catalysing the asymmetric reduction, and other is the cofactor regeneration system, which supplies NADPH from NADP+ through the oxidation of the energy source such as carbohydrates. Saccharomyces cerevisiae cells has an extra cellular invertase (β-Dfructosidase), that hydrolyses sucrose into glucose and fructose, which are transported into the cell by hexose transporters and metabolised through glycolysis. Addition of sucrose to the reaction mixture increases the bioreduction. It is due to enhanced regeneration of the co-factor in baker yeast in the presence of glucose that uses as electron donor. Although water is the most suitable and natural solvent for biocatalysis from the viability and activity point of view, glycerol is an alternative green solvent. It has the advantage with respect to substrate solubility and product separation. Therefore asymmetric reduction in a mixture of water and glycerol has advantages of both the solvents while carrying out the reduction using either free or immobilised whole cells. The reduction carried out using whole cells of immobilised baker yeast gave high yield as compared to free whole cells due to enhanced operational stability of free baker yeast (FBY), isolation of the products and repeated reused. Electrochemical reduction. Electrochemical reduction of phenyl-1,3-butanedione and 2,3-pentanedione has been carried out as shown by the schemes below.

604

Scheme 3

Scheme 4

Scheme 5

In the cyclic voltammogram of 2,3-pentanedione in acidic medium only one irreversible cathodic peak was observed while in neutral and basic media (pH 7 and 9, respectively) 2 cathodic peaks were observed which were irreversible in nature. At pH 7 and 9, the first cathodic peak is due to the reduction of 2,3-pentanedione to 3-hydroxy-2-pentanone via uptake of 2e– and 2H+ process. The reduction process does not stop at 3-hydroxy-2-pentanone and it is further reduced via 2e–/2H+ process yielding 2,3-pentanediol as final product. In acidic medium 3-hydroxy-2-pentanone was formed as final reduction product via 2e–/2H+ irreversible process. In the cyclic voltammograms of phenyl-1,3-butanedione at pH 5, 7 and 9 single irreversible cathodic peak was observed due to the reduction of >C=O moiety to the corresponding secondary alcohol yielding 3-hydroxy-1-phenyl butanone as final product. Kinetic parameters evaluated from the cyclic voltammograms are given in Table 4. Effect of pH. The influence of pH on reduction process was examined. On increasing pH the reduction peak shifts towards more negative values as shown in Figs 1 and 2. The observed shift in E½ with decreasing pH to more positive values can be explained by protonation of the carbonyl group, thus easing the reduction. This dependence indicates that there is a proton transfer in the electrode reactions.

605

Table 4. Voltammetric data evaluated from cyclic voltammograms of phenyl-1,3-butanedione (at pH 7.0) and 2,3-pentanedione (at pH 5.0)

S. No

Compound

Scan rate ν (mV/s)

Cathodic peak Cathodic peak Peak current / potential current square root of Epc (mV) Ipc (μA) scan rate (Ip/√ν)

1 phenyl-1,3-butanedione

100 200 300 400 500

–533 –561 –583 –577 –591

205 300 375 448 508

20.5 21.21 21.65 22.4 21.77

2 2,3-pentanedione

100 200 300 400 500

–690 –702 –709 –713 –722

166 239 296 355 410

16.6 16.90 17.09 16.2 17.44

Fig. 1. Cyclic voltammogram of phenyl-1,3-butanedione at different pH: 5.0 (1), 7.0 (2), 9.0 (3), where IP – initial potential = 900, FP – final potential = –1000, SR – scan rate = 500, CS – current sensitivity = 0.01

606

Fig. 2. Cyclic voltammogram of 2,3-pentanedione at different pH: 5.0 (1), 7.0 (2), 9.0 (3), where IP – initial potential = 1200, FP – final potential = –1200, SR – scan rate = 500, CS – current sensitivity = 0.01

Effect of scan rate. The effect of scan rate on cathodic peak potential (Epc) was also studied. Figures 3–5 show the effect of scan rate on cathodic peak potential (Epc). On increasing the scan rate Epc is shifted towards more negative potentials indicating an irreversible electron transfer process. The dependence of the voltammetric peak current (Ipc) of the wave on the square root of scan rate (v1/2) is linear with correlation coefficients close to unity (Graph 1) at all the pH. Under these conditions the current process was diffusion-controlled. Thus 2, 3-pentanedione and phenyl-1,3-butanedione were reduced electrochemically in a diffusion-controlled irreversible cyclic voltammetry wave.

Fig. 3. Cyclic voltammogram of phenyl-1,3-butanedione at various scan rate at pH 7.0: 100 (1), 200 (2), 300 (3), 400 (4) and 500 (5), where IP – initial potential = 1000, FP – final potential = –1000, CS – current sensitivity = 0.01

607

Fig. 4. Cyclic voltammogram of 2,3-pentanedione at various scan rates at pH 5.0: 100 (1), 200 (2), 300 (3), 400 (4), 500 (5), where IP – initial potential = 1000, FP – final potential = –1200, CS – current sensitivity = 0.01

Fig. 5. Cyclic voltammogram of 2,3-pentanedione at various scan rates at pH 7.0: 100 (1), 200 (2), 300 (3), 400 (4) and 500 (5), where IP – initial potential = 1000, FP – final potential = –1300, CS – current sensitivity = 0.01

608

Graph 1.Variation of the cathodic peak currents (Ip) with square root of scan rate (v1/2) for phenyl-1, 3-butandione (at pH 7.0) and 2, 3-pentanedione (at pH 5.0)

CONCLUSIONS Hydroxy ketones were prepared and characterised on the basis of analytical and spectral data. These alcohols such as 3-hydroxy-2-petanone, 2,3-pentanediol, 3-hydroxy-1phenyl butanone were prepared using microbial transformation and electrochemical procedures. Biocatalysis provides an alternative opportunity to prepare pharmaceutically important chiral compounds useful in the development of drugs. These two strategies follow green methodology over conventional chemical methods in terms of effectiveness, safety, economical, ecofriendly, easy to handle and provide new and improved synthetic routes to many valuable compounds in fields of pharmaceutical, flavour, fragrances, perfume industry and as fine chemicals are used as building blocks for the production of larger molecules. ACKNOWLEDGEMENTS Authors thank the Director, Malaviya National Institute of Technology, Jaipur (India) and Head, Department of Chemistry, University of Rajasthan, Jaipur (India) for providing necessary research facilities. One of the author (Ritu Saharan) wishes to thanks University Grant Commission New Delhi (India) for providing Junior Research fellowship. REFERENCES 1. K. NAKAMURA, Y. KAWAI, S. OKA, A.OHNO: Stereochemical Control in Microbial Reduction. 8. Stereochemical Control in Microbial Reduction of β-keto Ester. Bull. Chem. Soc. Jpn., 62, 875 (1989). 2. T. DREPPER, T. EGGERT, W. HUMMEL, C. LEGGEWIE, M. POHL, F. ROSENAU, S. WILHELM, K. E. JAEGER: Novel Biocatalysts for White Biotechnology. Biotechnol. J., 1, 777 (2006).

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  3. X. L. GAO, G. ZHOU, Y. K. GUAN, W. D. LI, Y. L. LI: Baker’s Yeast Mediated Reduction of Optically Active Diketone. Chinese Chemical Letters, 12 (4 ), 291 (2001).   4. X. Y. WANG, J. N. CUI, W. M. REN, F. LI, C. L. LU, X. H. QIAN: Baker’s Yeast Mediated Reduction of Substituted Acenaphthenequinones: Regio- and Enantioselective Preparation of Monohydroxyacenaphthenones. Chinese Chemical Letters, 18, 681 (2007).   5. K. NAKAMURA, R. YAMANAKA, T. MATSUDAB, T. HARADAB: Recent Developments in Asymmetric Reduction of Ketones with Biocatalysts. Tetrahedron: Asymmetry, 14, 2659 (2003).   6. G. P. MAMATHA, B. S. SHERIGARA, K. M. MAHADEVAN: Electrochemical Reduction of 2-acetyl Benzofuran and Its Derivatives at Glassy Carbon Electrode. Indian J. of Chemical Technology, 14, 566 (2007).   7. H. JAYADEVAPPA, Y. SHIVRAJ, K. M. MAHADEVAN, B. E. KUMARASWAMY, A. K. SATHPATHI, B. S. SHERIGARA: Electrochemical Behaviour of Some Industrially Important Azonaphthol Derivatives at Glassy Carbon Electrode. Indian J. of Chemical Technology, 13, 269 (2006).   8. M. A. MATTHEWS: Green Electrochemistry. Examples and Challenges. Pure Appl. Chem., 73, 1305 (2001).   9. R. YUAN, S. WATANABE, S. KUWABATA, H. YONEYAMA: Asymmetric Electroreduction of Ketone and Aldehyde Derivatives to the Corresponding Alcohols Using Alcohol Dehydrogenase as an Electrocatalyst. J. Org. Chem., 62, 2494 (1997). 10. N. O. MAHMOODI, H. G. MOHAMMADI: Enantio-, Regio-, and Chemoselective Reduction of Aromatic α-diketones by Baker’s Yeast. Monatshefte fur Chemie, 134, 1283 (2003). 11. R. CHENEVERT, S. THIBOUTOT: Baker’s Yeast Reduction of 1,2-diketones. Preparation of Pure (S)-(–)-2-hydroxy-1-phenyl-1-propanone. Chemistry Letters, 1191 (1988). 12. N. O. MAHMOODI, M. N. NAVROOD: Enantio-, Regio-, and Chemoselective Reduction of Aromatic α-diketones by Baker’s Yeast in Diverse Organic–Water Solvent Systems. ARKIVOC, (iii) 37 (2007). 13. P. HOYOS, J. V. SINISTERRA, F. MOLINARI, A. R. ALCANTARA, D. D. MARIA: Biocatalytic Strategies for the Asymmetric Synthesis of α-Hydroxy Ketones. P. Acc. Chem. Res., 43 (2), 288 (2010). 14. J. ZHANG, M.-Z. JIN, W. ZHANG, L. Y. LIU, Z. L. LIU: Photoinduced Transformation of α,βepoxyketones to β-hydroxyketones by Hantzsch 1,4-dihydropyridine. Tetrahedron Letters, 43, 9687 (2002). 15. Z. LU, H. MEI, J. HAN, YI PAN: The Mimic of Type II Aldolases Chemistry: Asymmetric Synthesis of β-hydroxy Ketones by Direct Aldol Reaction. Chem. Biol. Drug Res., 76, 181 (2010). 16. C. HÖCKELMANN, F. JÜTTNER: Off-flavours in Water: Hydroxyketones and Ionone Derivatives as New Odour Compounds of Freshwater Cyanobacteria. Flavour Fragr. J., 20, 387 (2005). 17. K. AHMAD, S. KOUL, S. C. TANEJA A. P. SINGH, M. KAPOOR, RIYAZ-ul-HASSAN, V. VERMA, G. N. QAZI: Enzyme Directed Diastereoselectivity in Chemical Reductions: Studies towards the Preparation of All Four Isomers of 1-phenyl-1,3-butanediol. Tetrahedron: Asymmetry, 15 (7), 1685 (2004). Received 18 March 2012 Revised 21 April 2012

610

Oxidation Communications 35, No 3, 611–618 (2012) Analytical section

Comparative Study of Chitin and Chitosan D. T. Zvezdovaa*, V. G. Georgievab, L. T. Vlaevb Department of Organic Chemistry, ‘Prof. Dr. Assen Zlatarov’ University, 8010 Bourgas, Bulgaria E-mail: [email protected] b Department of Physical Chemistry, ‘Prof. Dr. Assen Zlatarov’ University, 8010 Bourgas, Bulgaria a

ABSTRACT Chitin and chitosan are natural aminopolysaccharides with unique structures, multidimensional properties, complex structure–activity relationships and wide-ranging applications in biomedical and other industrial areas. Beside cellulose, chitin is the second, most popular polysaccharide. In the present work, a comparison between the X-ray diffraction patterns, FT-IR spectra and the thermal behaviour as expressed by TG-DTG-DTA curves of chitin and chitosan (commercial products of SIGMAAldrich) is made. The knowledge of these physicochemical characteristics is believed to contribute to a better understanding of the chemistry of these biopolymers and their utilisation as starting materials in different areas of science and technology. Keywords: chitin, chitosan, FT-IR spectra, thermal analysis, X-ray diffraction patterns. AIMS AND BACKGROUND Chitin and chitosan are natural biopolymers (aminopolysaccharides) with complex structures, interesting properties and wide applicability. It is estimated that, annually, 1011 t of these products are obtained1–3. Chitin is a linear biopolymer in the form of highly-crystalline microfibres and possesses higher degree of polymerisation than cellulose. As polysaccharide, chitin consists of β-(1-4)-linked 2-acetamido-2-deoxyD-glucopyranose fragments4–6 and is structurally similar to cellulose. Therefore, it can be characterised as aminopolysaccharide with acetamido functionalities at C-2 positions instead of the hydroxyl groups, typical for cellulose (Scheme 1).

*

For correspondence.

611

Scheme 1 Structures of cellulose, chitin and chitosan

cellulose (C6H10O5)n             chitin (C8H13O5N)n

chitosan (C6H11O4)n

Chitin exists as a reinforcing element in the cuticles of arthropods, cell walls of most fungi, shells of crustaceans such as crabs and shrimps, exoskeletons of krill, cuticles of insects as well as specific component of many other living organisms. It is a white, solid, non-elastic polymeric material and represents one of the major sources of surface pollution in coastal areas. In its native state, chitin is crystalline. Chitin has been reported to exist in different crystalline forms. For instance, α-chitin, which is the most abundant biopolymer, is also thermodynamically the most stable one. It occurs in the cuticles of insects, crabs and shrimps, in the exoskeletons of krill, as well as in a number of other systems. β-Chitin, however, scarcely exists in Nature; one example in this respect is its occurrence in the extracellular spines of the euryhaline diatoms4. Chitin can also been found as a waste product of the crab-meat canning industry and it can be extracted in large quantities from crab and shrimp shells7. Due to their special chemical and biological properties and widespread availability, chitins and their derivatives have extensive applications in many industrial, agricultural, food and medical fields4. Recently, chitin has been considered as biomaterial in some important scientific and industrial areas such as biomedicine, pharmacology and biotechnology. This is due to its biocompatibility, biodegradability and biological activities6. In addition, chitin has been widely used as adsorbent in some adsorption studies7,8. Chitosan, namely poly-β(1-4)-2-amino-2-deoxy-D-glucopyranose is a natural high-molecular mass biopolymer, which occurs as a component of the cell wall of some fungi. It is generally prepared by extensive deacetylation of chitin9. There are 612

no pronounced nomenclature differences between chitin and chitosan with respect to the degree of N-deacetylation. The major procedure for preparation of chitosan is based on the deacetylation of chitin in the presence of strongly-alkaline medium. When deacetylation is incomplete, however, a polymer with mixed functionalities (acetamido- and amino groups) is obtained, and the material (named as chitosans) has different properties. Degrees of deacetylation of 70 – 80% are very common. The degrees of deacetylation and crystallinity of chtosans are their principal characteristics, affecting both the solubility in aqueous medium and capacity of forming complexes with some metals10. Chitosan is well-known as an excellent biosorbent for dyes, pigments and heavymetal ions removal in near-neutral solutions, mainly, because of the high content of amino (–NH2) functional groups in the biopolymer11–13. These excellent adsorption characteristics can be attributed to the high hydrophilicity, due also to the high content of hydroxyl groups in the glucoside units. Another reasons for this good hydrophilicity is the presence of a large number of acetamido functional groups. The high chemical reactivity of these groups and the flexible structure of the polymer chain are other important characteristics1,13,14. Chitosan appears to be more useful as compared to chitin, since the abundance of polar and hydrophilic functional groups determines its properties as chelating agent and allows for chemical modification15. Using various methods of physical and/or chemical modification may give rise to changes of some structural properties of chitin and chitosan, and the generation of novel functional groups opens room for new areas in the application of these valuable biopolymers1,3,13,16. The structures of cellulose, chitin and chitosan are shown in Fig.1. The aim of the present study was to compare the structural characteristics of chitin and chitosan by employing instrumental methods such as X-ray diffraction, FT-IR spectroscopy and thermogravimetric analysis. The knowledge of the physicochemical characteristics of these materials will contribute to their applicability as materials in different areas of the applied organic and polymer chemistry. EXPERIMENTAL Materials. Chitin derived from crab shells was commercially obtained from SIGMAAldrich (Cat. No C9752) and was used without further purification. Before use, chitin was vigorously grounded in agate mortar and dried in air at 60oC for 4 h. Chitosan derived obtained from crab shells was commercial product of SIGMAAldrich (Cat. No C3646), with degree of deacetylation ≥75% and was also used without further purification. Before use, chitosan was vigorously grounded in agate mortar and dried in air at 60oC for 4 h. X-ray diffraction. The degree of crystallinity and the size of crystallites were determined by means of X-ray diffraction method. Diffraction patterns were recorded in a symmetrical reflection mode using an URD-6 Seifert diffractometer and a copper target X-ray tube (λ=0.154 nm) operated at 40 kV and 30 mA. CuKα radiation was 613

monochromised with a graphite monochromiser and a Ni filter. WAXS curves were recorded in the 2θ range 4–40o, with a step size of 0.1o. Samples were powdered and pressed into a sample holder. Samples with the radius of 2 cm and the thickness of 1 mm thick were prepared. FT-IR spectrometry. The FT-IR spectra of chitin and chitosan were recorded using KBr pellets. The samples were prepared as follows: 2 mg of the samples were grounded together with 200 mg KBr into the fine powder with the particles size below 5 µm and compressed to form clear disk. The FTIR spectra were recorded using a Bruker Tensor-27 spectrometer at ambient temperature in the wavenumber range between 4000–400 cm–1. Thermal analysis. The thermal analysis measurements (TG-DTG-DTA) were carried out on a NETZSCH STA 449F3 apparatus by increasing temperature from ambient to 800oC. The measurements were performed in dry air atmosphere, at a heating rate of 12oC min–1. The sample mass was 7.0–9.0 mg and it was placed in an aluminum crucible without pressing. Calcined α-Al2O3 powder was used as reference sample. RESULTS AND DISCUSSION X-ray diffraction patterns of chitin and chitosan are shown in Fig. 1.

Fig. 1. X-ray diffraction patterns of chitin (1) and chitosan (2)

In the case of α-chitin, the maximum intensity of the peaks was located at 2θ = 9.7; 19.8; 21.1 and 26.6o. According to literature data4, the unit cell of α-chitin is orthorhombic with dimensions a=0.474 nm, b=1.886 nm and c=1.032 nm. Chitosan possesses the same type of crystal lattice and showed 3 crystalline reflections at 2θ = 614

9.5; 19.8 and 21.6o, respectively. The degree of crystallinity of the chitosan was smaller as compared to chitin. The analysis of the XRD curves and the calculations of the degree of crystallinity of chitin and chitosan was performed, using the Sherrer equation4: Dhkl =

λ w cos θ

,

(1)

where w is the half-width of the peak, related to (hkl) lattice planes. The degree of crystallinity was calculated as the ratio of the total, integral intensity of crystalline peaks to the total, integral intensity scattered by a sample over the whole range of measurements. Data concerning the crystallinity and size of crystallites of studied materials are summarised in Table 1. Table 1. XRD results for α-chitin and chitosan sample

Sample α-Chitin Chitosan

Degree of crystallinity 0.51 0.34

D020 12.6   7.1

Size of crystallites Dhkl (nm) D110 D021 D013 8.0 5.0 16.3 5.2 2.9 –

Dave 10.5   5.1

*The size Dhkl of crystallites in the direction, perpendicular to the lattice planes, related to the most pronounced crystalline peaks.

The FT-IR spectra of chitin and chitosan are shown in Fig. 2.

Fig. 2. FT-IR spectrum of chitin (1) and chitosan (2)

There were strong and broad absorption bands at ca. 3443 cm–1 in the spectrum of chitin, due to the axial stretching of O–H bonds. The bands at 3280 cm–1 were assigned to the –NH-groups. The very strong band within 3000–2800 cm–1 was assigned to the –CH, –CH2 and –CH3 groups present in chitin3. Strong absorption bands (doublets) 615

appeared at ca. 1696–1607 cm–1, and were assigned to amide I; the single band at ca. 1554 cm–1, corresponded to amide II absorption. The band2, corresponding to the axial stretching of C–H bonds was centred at 2910 cm–1. The bands at 1653 and 1580 cm–1 corresponded to the amide I and amide II vibrations, respectively. The bands at 1417 and 1377 cm–1 resulted from coupling of the C–N axial stretchings and N–H angular deformations; and the bands within 1153–897 cm–1 were assigned to the polysaccharide skeleton, including the glycoside functionalities (C–O and C–O–C stretching vibrations)2. Bands appearing at 1646, 1096 and 1590 cm–1 corresponded to the νC=O, νC–O stretching and δNH2 bending modes of vibration, respectively. These bands indicated that chitosan was not completely deacetylated1. The assumed assignments of the absorption bands are shown in Table 2. Table 2. Observed absorption bands of chitin and chitosan and their assignments

Chitin wavenumber assignment (cm–1) 1 2 3500 stretching vibration of isolated OH group 3474 and stretching vibration of 3434 OH group 3280 3000–2800

1724 1656

1626

1557

stretching vibration of NH group mainly due to CH–, –CH2– and –CH3 groups

Chitosan Refs wavenumber assignment (cm–1) 3 4 5 7 3500 stretching vibration of free OH group 3, 7, 3450 stretching vibration of 10 H-bonded OH group 3

3360

Refs 6 7 1, 3, 5, 6, 8, 10, 11 1,5

stretching vibration of NH group 2920, 2880, symmetric or asym1, 2,5 1430, 1320, metric CH2 stretching 1275, vibration attributed to 1245 pyranose ring carbonyl group 4 1730 sarbonyl group 4, 5, 8 amide stretching of 3, 7 1660 stretching in C=O (νC=O) 2, 5, 8 C=O (νC=O) in vicinity with amide group 1638 H–O–H bending vibra1 tion stretching of C–N vibra- 3, 7, 1590 angular deformation of 1, 2, 4, tion of superimposed 10 N–H bonds of the amino 5, 8 C=O group, linked to group (δ–NH2) OH group by H bonding stretching or N–H defor- 3 1560 NH-bending vibration in 5 mation of amine group amide group 1472 vibrational frequency of –NH-group

to be continued

616

Continuation of Table 2

1377 1345

1

2 symmetrical deformation or rocking of CH3 group CO–NH deformation and CH2 group

3 4 7, 10 1415,1320 7

5 vibrations of OH, CH in the ring

6 1,5

1380

bending vibration in 1, 5, 6, –CH3 in vicinity with 11 amide group 1255–1249 C–O group 1,5 1150–1040 C–O and –C–O–C– 1, 2, 4, stretching in glicosidic 5, 11 linkage 1096, 1030 stretching of C–O group 1, 6 in amide group 897, 850, 838 CH3COH group 1, 4, 5

TG, DTG and DTA curves of samples of 10 mg chitin and chitosan obtained at a heating rate of 6oC min–1 are shown in Fig. 3.

Fig. 3. TG, DTG and DTA curves of chitin (1) and chitosan (2) obtained at a heating rate of 6oC min–1

The 1st endothermic peak was observed between 50 and 120oC, with a minimum at 86oC. A mass loss of 8.9% was associated with this effect and it can be attributed to the evaporation of absorbed water in the internal polymer. The 2nd thermal effect was observed within the range of 268–312oC, with an exothermic peak at 292oC. It was associated also with the mass loss (51.6%), which corresponded to thermal degradation of the polymeric chain with vaporisation of volatile compounds. The onset of the pyrolysis of polysaccharide structure was associated with random splits of the glycoside bonds, followed by a further decomposition with the formation of acetic and butyric acids as well as other low-molecular fatty acids, with the predomination of C2, C3 and C6 homologues1. 617

CONCLUSIONS The present work is associated with comparative studies on the structures and physicochemical characteristics of chitin and chitosan by using X-ray diffraction, FT-IR and thermal analysis instrumental methods. This has been achieved by comparison of their X-ray diffraction patterns, FT-IR spectra and TG-DTG-DTA scans. The data obtained are considered to serve as useful tool for the identification of chitin and chitosan obtained from various sources, and by employing different preparation methods. REFERENCES   1. S. TOKURA, H. TAMURA: Chitin and Chitosan. Comprehensive Glycoscience, 2, 449 (2007).   2. K. V. H. PRASHANTH, R. N. THARANATHAN: Chitin/Chitosan: Modifications and Their Unlimited Application Potential. An Overview. Trends in Food Science & Technology, 18, 117 (2007).   3. M. RINAUDO: Chitin and Chitosan: Properties and Applications. Prog. Polym. Sci., 31, 603 (2006).   4. D. STAWSKI, S. RABIEJ, L. HERCZYNSKI, Z. DRACZYNSKI: Thermo­gravimetric Analysis of Chitins of Different Origin. J. Thermal. Anal. Calorim., 93 (2), 489 (2008).   5. T. WANJUN, W. CUNXIN, C. DONGHUA: Kinetic Studies on the Pyrolysis of Chitin and Chitosan. Polymer Degrad. Stabil., 87, 389 (2005).   6. G. AKKAYA, I. UZUN, F. GÜZEL: Kinetics of the Adsorption of Reactive Dyes by Chitin. Dyes and Pigments, 73, 168 (2007).   7. A. Y. DURSUN, C. S. KALAYCI: Equilibrium, Kinetic and Thermodynamic Studies on the Adsorption of Phenol onto Chitin. J. Hazard. Mater., B123, 151 (2005).   8. B. BENGUELLA, H. BENAISSA: Cadmium Removal from Aqueous Solution by Chitin: Kinetic and Equilibrium Studies. Water Res., 36, 2463 (2002).   9. C. PENICHE-COVAS, W. ARGÜELLES-MONAL, J. S. ROMAN: A Kinetic Study of the Thermal Degradation of Chitosan and Mercaptan Derivative of Chitosan. Polym. Degrad. Stab., 39, 21 (1993). 10. F. A. LOPEZ, A. L. R. MERCE, F. J. ALGUACIL, A. LOPEZ-DELGADO: A Kinetic Study on the Thermal Behaviour of Chitosan. J. Therm. Anal. Calorim., 91 (2), 633 (2008). 11. G. CRINI: Non-conventional Low-cost Adsorbents for Dye Removal: A Review. Bioresource Technol., 97, 1061 (2006). 12. N. SAKKAYAWONG, P. THIRAVETYAN, W. NAKBANPOTE: Adsorption Mechanism of Synthetic Reactive Dye Wastewater by Chitosan. J. Coll. Int. Sci., 286, 36 (2005). 13. F. SHAHIDI, R. ABUZAYTOUN: Chitin, Chitosan, and Co-products: Chemistry, Productions, and Health Effects. Advances in Food and Nutrition Research, 49, 93 (2005). 14. P. MIRETZKY, A. F. CIRELLI: Hg(II) Removal from Water by Chitosan and Chitosan Derivatives: A Review. J. Hazard. Mater., 167 (1–3), 10 (2009). 15. A. KAMARI, W. S. WAN NGAH, L. K. LIVE: Chitosan and Chemically Modified Chitosan Beads for Acid Dyes Sorption. J. Environ. Sci., 21, 296 (2009). 16. E. GUIBAL: Heterogeneous Catalysis on Chitosan-based Materials: A Review. Prog. Polym. Sci., 30, 71 (2005). Received 13 September 2011 Revised 28 October 2011

618

Oxidation Communications 35, No 3, 619–626 (2012) Analytical section

Complex Formation and Thermodynamics In [Cd–L-amino Acid–Vitamin-PP] Complexes at Dropping Mercury Electrode K. Rai, F. Khan* Electrochemical Laboratory, Department of Chemistry, Dr. H. S. Gour University, 470 003 Sagar, Madhya Pradesh, India E-mail: [email protected]; farid.fk@ rediffmail.com; [email protected] ABSTRACT This paper describes the ternary complexes formation of Cd(II) with L-glutamine, L-asparagine, L-valine, L-leucine, α-alanine and L-glycine as primary ligands and vitamin-PP (nicotinamide) as secondary ligand by polarographic technique at pH = 7.30 ± 0.01, µ = 1.0 M KNO3 at 25 and 35 ºC. Cd(II) formed 1:1:1, 1:1:2, and 1:2:1 complexes with these ligands, as confirmed by the Schaap and McMaster method. The sequence of the stability constant of complexes was L-glutamine < L-asparagine < L-valine < L-leucine < α-alanine < L-glycine. The cathodic reduction of the complexes involves 2 electrons and the waves were reversible and diffusion-controlled in nature. The changes in thermodynamic parameters such as enthalpy ∆H, free energy ∆G and entropy ∆S were also determined that showed that the complexes are lesser stable at higher temperature. Keywords: vitamin-PP, stability constant, L-amino acids, cadmium. AIMS AND BACKGROUND Metal complexes of amino acids and vitamins are subject of active research and there are some publications related to the coordination chemistry of both groups of ligands1–3. In addition to the naturally occurring amino acids, some synthetic analogues and derivatives of amino acids and vitamins have been considered due to their biological or theoretical significance as well as important applications in chemistry or medicines4. The coordination behaviour of amino acids to the metal ions are well known with strong binding to ions via a chelate binding mode involving both carboxylate and amine group5. *

For correspondence.

619

Nicotinamide (3-pyridine carboxylic acid amide, commonly known as vitaminPP) is a reactive moiety of the coenzyme nicotinamide adenine dinucleotide6,7. The vitamin-PP is a biologically important ligand and its metal complexes are more effective than the free ligands8. Studies on metal complexes of nitrogen-containing ligands, especially heteroaromatic nitrogen bases with amino acids are of much interest9. A metal complex plays an essential role in agriculture, pharmaceutical and industrial chemistry10. Cadmium is a soft, light-coloured metal with high vapour pressure causing it to be rapidly oxidised to cadmium oxide in air11. Cadmium is a modern toxic metal which was discovered in 1817, and its industrial use was minor until about 50 years ago. But now it is a very important metal with many applications12. Cadmium also affects several genes involved in the stress response to pollutants or toxic agents. Metal centers, being positively charged, are favoured to bind to negatively charged bio-molecules, i.e. amino acids and vitamins offer excellent ligands for binding to metal ions. The pharmaceutical use of metal complexes therefore has excellent potential. A broad array of medicinal applications of metal complexes has been investigated, and several recent reviews summarise advances in these fields13–17. Yet there is large work on metal toxicity and study of first aim to sites of toxic metal effects in general, or specially Cd(II), remains to be known18. Such interaction of transition-metal ions with amino acids and vitamins is of immense biological importance19–21. EXPERIMENTAL All the chemicals were of reagent grades and used without further purification and their solutions were prepared in doubly distilled water. The concentrations of Cd(II), L-amino acids and vitamin-PP (nicotinamide) were taken in a ratio of 1:40:40 and the pH of the analyte was fixed at 7.30 ± 0.01 which was adjusted with the required value of perchloric acid or sodium hydroxide as needed. The pH of the analyte was stabilised by the addition of potassium dihydrogen phosphate buffer. The current-voltage curves were obtained on a manual polarograph using polyflex galvanometer (PL-50). The polarographic cell was of Laitinin and Lingane22 type in which capillary of 5.0 cm in length with 0.04 mm in diameter was used. The m2/3 t1/6 value was 2.40 mg2/3 s1/2 at 60.02 cm effective height of mercury. The pH of the analyte was measured by the digital pH meter (Model l11-101 E). In complex formation of [Cd–L-amino acids–vitamin-PP] system, the concentrations of primary ligands, i.e. L-amino acids varied from 0.5 to 30.0 mM at 0.025 and 0.050 M (fixed) concentration of secondary ligand (vitamin-PP), respectively. Cd(II) showed a well-defined 2-electron reversible reduction wave with half-wave potential –0.586 V versus S. C. E. at pH = 7.30 ± 0.01 and µ = 1.0 M KNO3 at 25 and 35º C (Ref. 23). The nature of the current-voltage curves for complexes was also reversible. The concentrations of Cd(II), KNO3 and Triton X-100 (as suppressor) in the analyte were 0.50 mM, 1.0 M and 0.001%, respectively. The value of E1/2 became more negative with the addition of secondary ligand (vitamin-PP) to binary complexes 620

of the [Cd–L-amino acidate] system that showed ternary complex formation of 1:1:1, 1:1:2 and 1:2:1 complexes. RESULTS AND DISCUSSION The values of stability constant are given in Table 1 and the data and plots of Fij [X, Y] against [X] (where Fij is a Schaap and McMaster24 function to evaluate the stability constant βij, X – L-glycine, Y – vitamin-PP and i and j – their stoichiometric numbers, respectively) for [Cd–L-glycine–vitamin-PP] system are given in Table 2 and Fig. 1, respectively, to determine the values of function F00, F10, F20 and F30. 500 450

F00 [X, Y]

400

F10 [X, Y] × 103

Fij[X, Y]

350

F20 [X, Y] × 106

300

F30 [X, Y] × 108

250 200 150 100 50 0

0

500

10 20 [glycine] × 103 [vitamin-PP] = 0.025 M

30

450 F00 [X, Y]

400

F10 [X, Y] × 103

Fij[X, Y]

350

F20 [X, Y] × 106

300

F30 [X, Y] × 108

250 200 150 100 50 0

0

10 [glycine] × 103 [vitamin-PP] = 0.050 M

20

Fig. 1. Plots of Fij[X, Y] versus [X] for [Cd–L-amino acids–vitamin-PP] system Table 1. Stability constant of [Cd-L-amino acid-vitamin-PP] system

Primary ligands L-Glycine α-Alanine L-Leucine L-Valine L-Asparagine L-Glutamine Vitamin-PP [nicotinamide]

lg β01 lg β02 lg β03 lg β10 lg β20 lg β30 – – – 4.30 7.60 9.64 – – – 4.23 7.46 9.43 – – – 4.17 7.35 9.34 – – – 4.11 7.21 9.16 – – – 4.07 7.18 9.10 – – – 4.00 7.04 8.91 1.85 2.73 – – – –

lg β11 4.60 4.53 4.36 4.28 – 4.15 –

lg β12 lg β10 7.96 9.68 – 9.56 7.40 9.41 7.35 9.36 7.25 9.25 7.16 9.13 – –

621

622

0.00 0.50 1.00 2.00 3.00 4.00 5.00 6.00 8.00 10.00 20.00 30.00

[gly]× 10–3­ (M)

F00[X,Y] F10[X,Y] F20[X,Y] F30[X,Y] E1/2 r–V lgIm/Ic F00[X,Y] F10[X,Y] F20[X,Y] F30[X,Y] lgIm/Ic E1/2 r–V ×103 ×106 ×108 versus ×103 ×106 ×108 versus SCE SCE 0.586 –       3.1055 78.095 159.47 43.65 0.586 – 5.88 250.34 279.00 43.65 0.647 0.0072     121.61 237.02 161.65 43.65 0.660 0.0072 326.55 641.34 281.31 43.66 0.656 0.0147     245.03 241.93 163.83 43.65 0.670 0.0147 539.72 533.83 283.49 43.65 0.672 0.0220     832.10 414.49 168.20 43.66 0.680 0.0222 1657.99 826.06 287.86 43.64 0.681 0.0299    1790.50 595.79 172.57 43.66 0.689 0.0299 3386.98 1127.03 292.23 43.67 0.688 0.0378    3146.49 785.85 176.94 43.67 0.696 0.0378 5752.81 1436.73 296.59 43.66 0.694 0.0457    4926.33 984.64 181.31 43.68 0.701 0.0457 8781.46 1755.11 300.95 43.65 0.698 0.0539    7155.22 1192.02 185.67 43.67 0.705 0.0539 12499.66 2082.29 305.33 43.66 0.706 0.0539   13069.34 1633.28 194.39 43.66 0.712 0.0621 22107.80 2762.74 314.05 43.65 0.712 0.0539   21095.05 2109.49 203.14 43.67 0.718 0.0621 34787.28 3478.14 322.78 43.66 0.732 0.0621 100281.00 5013.89 246.79 43.66 0.740 0.0621 151584.68 7578.94 366.43 43.65 0.745 0.0621 263723.95 8790.69 290.42 43.65 0.750 0.0621 376615.08 12553.64 410.11 43.66 lg A = 0.49, lg B = 4.89, lg C = 8.20, lg D = 9.64 lg A = 0.77, lg B = 5.39, lg C = 8.45, lg D = 9.64

Table 2. Polarographic characteristics and Fij[X, Y] values for the [Cd–glycinate–vitamin-PP] system

The stability of binary and ternary complexes compared by the values of lg Km were calculated by the following equation25: lg Km = lg β11 – 1/2 [lg β02 + lg β20 ].

The values of lg Km were calculated as 0.635, 0.628, 0.611, 0.602, –0.277 and 0.588 for [Cd–L-glycine–vitamin-PP], [Cd–α-alanine–vitamin-PP], [Cd–L-leucine– vitamin-PP], [Cd–L-valine–vitamin-PP], [Cd–L-asparagine–vitamin-PP] and [Cd–Lglutamine–vitamin-PP] system, respectively. The positive values of lg Km confirmed that the ternary complexes are more stable than their corresponding binary complexes and the negative values of lg Km showed that the binary complexes are more stable than the corresponding ternary complexes. The trend of stability constant of the complexes was: L-glutamine < L-asparagine < L-valine < L-leucine < α-alanine < L-glycine. The increase of stability constants of metal complexes with amino acids can be explained on the basis of side chain26. The amino acids act as bidentate ligands which bound with the metal ions through COOH and amino groups making 5-member chelate ring systems27. Vitamin-PP coordinates through the nitrogen of the pyridine ring28. The L-glycine formed the complexes of maximum stability whereas the L-glutamine formed complexes of minimum stability with Cd(II). The values of stability constants (lg β) varied from 1.85 to 9.68 (Ref. 29). It is clear from the values of stability constants of complexes that these values are quite reasonable values, therefore, either amino acids or in combination with metals could be used in the treatment of metal toxicity. The values of thermodynamic parameters such as ∆H, ∆G and ∆S of the complexes are given in Table 3. The values showed that these complexes were not stable at higher temperature20. The negative values of ∆H indicated that the complexes were formed with the evolution of heat. The thermodynamic parameters of the complexes have been calculated by the following equations30:

∆H = 2.303 RT1 T2 (lg β2 – lg β1)/T2 – T1

(i)



∆G = –2.303 RT lg β

(ii)



∆G = ∆H – T∆S

(iii)

623

624

[Cd–L-gln–vit.-PP]

[Cd–L-asn–vit.-PP]

[Cd–L-val–vit.-PP]

[Cd–L-leu–vit.-PP]

[Cd–α-ala–vit.-PP]

[Cd–gly–vit.-PP]

System

Stability constant lg β11 lg β12 lg β21 25◦C/ 25◦C/ 25◦C/ 35◦C 35◦C 35◦C 4.60 7.96 9.68 4.15 7.52 9.22 4.53 – 9.56 4.11 – 9.13 4.36 7.40 9.41 4.01 7.04 9.00 4.28 7.35 9.36 3.87 7.03 8.96 – 7.25 9.25 – 6.90 8.88 4.15 7.16 9.13 3.82 6.82 8.77 –

18.0604

14.7003 15.5403

13.86.03 14.2803 15.1204

–

17.2204 13.4403 16.8004

14.7003 15.4204 17.2204

17.6404

18.9004 18.4804 19.3204

–∆H lg β11 lg β12 lg β21 (35–25ºC) for difference of 10ºC lg β11 25◦C/ 35◦C 6.2728 5.8490 6.1774 5.7926 5.9455 5.6517 5.8365 5.4544 – – 5.6592 5.3839

Table 3. Thermodynamic parameters of [Cd–L-amino acids–vitamin-PP] ternary system

–∆G lg β12 25◦C/ 35◦C 10.8547 10.5987 – – 10.0910   9.2221 10.0229   9.9081   9.8865   9.7249   9.7638   9.6121 lg β21 25◦C/ 35◦C 13.2002 12.9947 13.0365 12.7678 12.8320 12.6846 12.7638 12.6282 12.6138 12.5155 12.4502 12.3604

lg β11 25◦C/ 35◦C 18.8794 18.8881 17.6197 17.6216 14.6803 14.6820 17.2008 17.2027 – – 13.8413 13.8428

–∆S lg β12 25◦C/ 35◦C 18.4440 18.4460 – – 15.0865 15.0881 13.4067 13.4081 14.6672 14.6688 14.2476 14.2491

lg β21 25◦C/ 35◦C 19.2762 19.2783 18.0166 18.0186 17.1773 17.1792 16.7576 16.7594 15.4980 15.4997 15.0786 15.0802

CONCLUSIONS The aim of the present investigation is to study the coordination chemistry of Cd(II) with simple biologically relevant molecules like amino acids and vitamins by the polarographic technique. Presently, EDTA and its derivative have been used against the toxicity of Cd but initially they work effectively but simultaneously they increase the nephrotoxicity28. Hence, the use of such drugs against cadmium is questionable. Our results (stability constants) showed that amino acids or their complexes could be used against Cd toxicity ACKNOWLEDGEMENT The authors are thankful to University Grant Commission, New Delhi, for providing the Rajeev Gandhi fellowship to KR and the Head, Department of the Chemistry, Dr. H. S. Gour University, Sagar, for providing the laboratory facilities. REFERENCES   1. O. SZAZUCHIN, S. M. NAVARIN: Synthesis of Complex Combination of Zn (II) and Ni (II) with Amino Acids: D-penicilamine and L-cystein. Antibiotiki, 6, 56 (1965).   2. A. AHMED El-SHERIF: Mixed Ligand Complex Formation Reactions and Equilibrium Studies of Cu(II) with Bidentate Heterocyclic Alcohol (N,O) and Some Bio Relevant Ligands. J. Solution Chem., 39, 131 (2010).   3. ZAKIYAN, IFFET, GUNDUZ, N. TURGUL: Synthesis of Mn (III) Complexes of Schiff Bases. Mercel-Dekker Inc., 31 (7), 1175 (2001).   4. M. K. SINGH, M. N. SHRIVASTAVA: Stepwise Formation of Beryllium (II) and Alluminium (II) Chelates with Aspartic and Glutamic Acid. J. Inorg. Nucl. Chem., 35, 2433 (1972).   5. M. AGHAIE, F. KESHAVARZ REZAIE, K. ZARE, H. AGHAIE: Thermodynamic Study on the Interaction between Tl(I) Ion and L-alanine. Asian J. of Chemistry, 22, 959 (2010).   6. Y. MIWA, T. MIZUNO, K. TSUCHIDA, T. TAGA, Y. IWATA: Experimental Charge Density and Electrostatic Potential in Nicotinamide. Acta Crystallogr., Sect. B, 55, 78 (1999).   7. J. W. PARK, Y. H. PAIK: Interaction of Tris (2,2-bypyridine) – Ruthenium (II) with PSS. Bull. Korean Chem. Soc., 6, 23 (1985).   8. J. R. J. SORENSEN, H. SIGEL, MARCEL DEKKER: Metal Ions in Biological Systems. Marcel Dekker, New York, 1982, p. 14, 77.   9. K. HUSSAIN REDDY: Nuclear Activity of Transition Metal Complex. A Review. J. Indian Chem. Soc., 80, 67 (2003). 10. D. N. DHAR, T. L TAPLOO: Schiff Bases and Their Application. Sci. Ind. Res., 41, 501 (1982). 11. WHO: Air Quality Guidelines for Europe. WHO Regional Publications, European Series No 23, 1987. 12. A. NAVAS-ACIEN, E. K. SILBERGELD, R. SHARRETT, E. CALDERON- ARANDA, E. SELVIN, E. GUALLAR: Metals in Urine and Peripheral Arterial Disease. Environ. Health Perspect., 113, 164 (2005). 13. H. SAKURAI, Y. KOJIMA, Y. YOSHIKAWA, K. KAWABE, H. YASUI: Zinc Complexes as Candidates for Insulin-mimetic Drugs. Coord. Chem., 226, 187 (2002). 14. P. J. SADLER, H. LI, H. SUN: Coordination Chemistry of Metals in Medicine: Target Sites for Bismuth. Coord. Chem. Rev., 185, 689 (1999). 15. H. ALI, J. E. van LIER: Metal Complexes as Photo- and Radio-sensitizers. Chem. Rev., 99, 2379 (1999).

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16. A. Y. LOUIE, T. J. MEADE: Metal Complexes as Enzyme Inhibitors. Chem. Rev., 99, 2711 (1999). 17. W. A. VOLKERT, T. J. HOFFMAN: Therapeutic Radiopharmaceuticallly. Chem. Rev., 99, 2269 (1999). 18. S. CLEMENS: Toxic Metal Accumulation Responses to Exposure and Mechanism of Tolerance in Plants. Biochim., 88, 1707 (2006). 19. A. D. JONES, D. R. WILLIAMS: Thermodynamic Considerations in Co-ordination. Part IX. Heat Capacity Investigations into Complex Formation between Some Lanthanide(III) Ions and Histidine. J. Chem. Soc.A, 3159 (1971). 20. J. M. PRATT: Inorganic Chemistry of Vitamin B12. Academic Press, London, 1972. 21. S. K. SHAPIRO, F. SCHLENK: Transmethylation and Methionine Biosynthesis. University of Chicago Press, 1965. 22. L. MEITES: Polarographic Techniques. Interscience Pub., New York, 1965, p. 1. 23. F. KHAN, A. KHANAM: Polarographic Study of Ternary Complexes of [Cd–L-amino acidate– vitamin-PP] System. J. Ind. Chem. Soc., 85, 89 (2008). 24. W. B. SCHAAP, D. L. McMASTER: A Polarographic Study of Mixed Ligand Complex Formation; Complexes of Cu and Cd with Oxalate Ion and Etylenediamine. J. Am. Chem. Soc., 83, 4699 (1961). 25. R. TAMAMUSHI, H. TANAKA: Polarographic Study on the Electrode Reaction of Zinc Ion. Z. Phys. Chem., 39, 117 (1963). 26. M. TANAKA, M. TABATA: An Attempt to Discriminate between Hydrophobic and Aromatic π-π Interaction in the Copper (II) Ternary Complexes, CuLA with L = 1,10-phenanthroline or 2,2bypyridyl and A = pairs-X-subsitituted Phenylalaninates. Inorg. Chem., 46, 9975 (2007). 27. I. SAKYAN, E. LOGOGLU, S. ARSLAN, N. SARI, N. AKIYAN: Antimicrobial Activities of N-(2hydroxy-1-naphthalidene)-amino Acid (Glycine, Alanine, Phenylalanine, Histidine, Tryptophane) Schiff Bases and Their Manganese(III) Complexes. Biometals, 17 (2), 115 (2004). 28. M. D. WALKER D. R. WILLIAMS: Thermodynamic Consideration in Coordination Part XDIII. Formation Constant for Cadmium–Amino Acids Complexes as Determined by Glass and Solid-state Cadmium-Electrode Potentiometry. J. Chem. Soc., Daltons Trans., 1186 (1974). 29. K. PISREWICZ, D. MORA, F. PFLUEGER, G. FIELDS, F. MARI: Polypeptide Chains Containing D-γ-hydroxyvaline. J. Am. Chem. Soc., 127, 6207 (2005). 30. J. C. F. ROSOTTI, H. ROSOTTI: The Determination of Stability Constant. McGraw Hill Book Co., London, 1961. Received 30 October 2010 Revised 18 December 2010

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Oxidation Communications 35, No 3, 627–632 (2012) Photooxidation in biological systems

Photooxidation of Caffeic Acid in the Presence of Peroxydisulphate in Aqueous Solution M. S. Swaragaa*, M. Adinarayanab Department of Chemistry, Osmania University, 500 007 Hyderabad, India Department of Chemistry, PG College of Science, Saifabad, Osmania University, 500 004 Hyderabad, India E-mail: [email protected]; [email protected]

a

b

ABSTRACT The photooxidation of caffeic acid in the presence of peroxydisulphate (PDS) in aqueous solution has been carried out in a quantum yield reactor using a high-pressure mercury vapour lamp. The rate of photooxidation was found to increase with increase in [PDS] and light intensity and independent of [caffeic acid]. The quantum yield of oxidation of caffeic acid was found to increase with increasing [caffeic acid] and light intensity and independent of [PDS]. The plots of lg (rate) versus lg [PDS] are linear with a slope of one indicating first order dependence of rate on [PDS]. Caffeic acid ortho-quinone was found to be the product of oxidation of caffeic acid. On the basis of experimental results and product analysis a probable mechanism is suggested. Keywords: oxidation of caffeic acid, sulphate radical anion. AIMS AND BACKGROUND The consumption of fruits and vegetables has been shown to decrease the risk of cardiovascular diseases1 and cancer2. There are strong evidences that phenolic antioxidants present in plants are responsible for their protective effects3. Hydroxycinnamic acids are widely distributed in the plant kingdom and are important sources of antioxidants due to their free radical scavenging properties4. It has been shown that these antioxidants protect low-density lipoproteins against oxidation induced by metmyoglobin5, Cu+2 ions and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)6. These antioxidants can also acts as scavenger of hypochlorite7, peroxy­ nitrite8 and 2,2′-diphenyl-1-picrylhydrazyl radical9. In particular, caffeic acid and chlorogenic acid have been proved to be highly active in most of these studies. Both electrochemical10–13 and chemical oxidation of these compounds have been studied *

For correspondence.

627

by many authors10,14–16 especially in aqueous medium at different pH, but mechanisms involved in their oxidative process are not yet well understood. Keeping in view the efficient antioxidant property of caffeic acid, we have undertaken a systematic study of oxidation of caffeic acid by SO4•– radical to understand the mechanism of its oxidation by SO4•– radicals. SO4•– radicals are generated by photolysis of peroxydisulphate. Peroxydisulphate is activated at 254 nm to give SO4•– by homolytic fission of –O–O– bond. In the present communication we report the kinetic results on the oxidation of caffeic acid by SO4•– radicals. EXPERIMENTAL Caffeic acid was supplied from Sigma and was used as received. The solution of caffeic acid and peroxydisulphate was prepared using doubly distilled water. The peroxydisulphate (PDS) solution was standardised cerimetrically using ferroin indicator. The concentration of caffeic acid is determined by measuring the absorbance at 310 nm and from the known molar absorption coefficient value at this wavelength. Irradiations were carried out in a quantum yield reactor model QYR-20 using highpressure mercury vapour lamp. In general, intensity measurements were carried out using ferrioxalate actinometry. In a typical reaction, caffeic acid and PDS solutions were mixed in a specially designed 1-cm path length cuvette, which is suitable, both for irradiations in the reactor and absorbance measurements. The absorbance measurements were carried out on a Hitachi UV-spectrophotometer model 3410. The progress of the reaction was followed by measuring the absorbance at the λmax of caffeic acid by interrupting irradiations at regular intervals of time (Fig. 1, absorption spectra at various times). The reaction rates have been calculated from the plots of absorbance versus time (Fig. 2) using a computer program. The quantum yields have been calculated from the initial rates of oxidation of caffeic acid and light intensity at 254 nm. This is the wavelength at which peroxydisulphate is activated to radical reactions. The light intensity at 254 nm was measured by peroxydisulphate actinometry.

Fig. 1. Absorption spectra of photooxidation of caffeic acid in the presence of peroxydisulphate at different irradiation times [caffeic acid] – 5.00 × 10–5 mol dm–3, [PDS] – 6.00 × 10–4 mol dm–3, light intensity – 1.01 × 1015 quanta s–1

628

Fig. 2. Plot of absorbance versus time [caffeic acid] – 5.00 ×10–5 mol dm–3, [PDS] – 8.00 × 10–4 mol dm–3, light intensity – 1.01 × 1015 quanta s–1, λmax = 310 nm

RESULTS AND DISCUSSION The photooxidation of caffeic acid in the presence of peroxydisulphate (PDS) in aqueous solution has been carried out in a quantum yield reactor using a high-pressure mercury vapour lamp. The initial rates of oxidation of caffeic acid are measured under different experimental conditions. The reaction rates of photooxidation of caffeic acid by PDS were found to increase with increase in [PDS] and light intensity and independent of [caffeic acid] (Table 1). The order in [PDS] has been found to be one (Fig. 3). The quantum yields are calculated from the initial rates of oxidation of caffeic acid and light intensity absorbed by PDS at 254 nm. The quantum yields of the reaction have been found to depend on [caffeic acid] and light intensity and independent of [PDS]. Table 1. Effect of [PDS], [caffeic acid] and light intensity on the rate and quantum yields of oxidation of caffeic acid by SO4•– in a aqueous solution

[PDS] × 104 (mol dm–3) 2.00 4.00 6.00 8.00 2.00 2.00 2.00 2.00 2.00

[Caffeic acid] × 105 [Intensity] × 1015 (mol dm–3) (quanta s–1) 5.00 1.01 5.00 1.01 5.00 1.01 5.00 1.01 1.00 1.01 2.00 1.01 5.00 1.01 1.00 0.506 1.00 1.01

Rate × 108 (mol dm–3 s–1) 1.5 3.0 5.3 6.1 1.3 1.4 1.5 0.350 1.3

φ 2.10 2.13 2.55 2.22 0.384 0.802 2.10 0.210 0.384

629

Fig. 3. Effect of [PDS] on the photooxidation of caffeic acid in the presence of PDS in aqueous solution

In both electrochemical10–13 and chemical oxidation10,14–16 of caffeic acid in aqueous solution at different pH formation of phenoxyl radical as intermediate has been suggested15,17–18. It is reported that caffeic acid o-quinone is the main product in the anodic oxidation of caffeic acid and it is sufficiently stable only in acid solution. Caffeic acid o-quinone has characteristic absorption maxima at 248 and 394 nm (Ref. 19). It is reported that carbon-centered alkyl radical generally adds to the phenoxyl oxygen while oxygen centered radicals prefer to add to ortho- or para-position of the phenoxyl radicals20,21. Hayon22 studied the photolysis of peroxydisulphate in aqueous solution and reported the formation of sulphate radical anion. It is an important source of free radicals and is used as an initiator in free radical induced oxidations. Sulphate radical anion, which is produced on photolysis of peroxydisulphate in the initiation step might react with caffeic acid to produce phenoxyl radicals. The effect of light intensity on the quantum yield of oxidation of caffeic acid indicates that the role of light is restricted to initiation step in the activation of PDS to sulphate radical anions. The increase in quantum yield with increase in [caffeic acid] suggests that excited state of caffeic acid might be acting as a sensitiser to transfer its energy to PDS to generate sulphate radical anions. The rate of oxidation of caffeic acid is independent of [caffeic acid] suggesting that sulphate radical anion might react with caffeic acid in a fast step to give phenoxyl radicals. Increase of reaction rate with increase in [PDS], suggests that PDS is involved in the rate-controlling step of generating radicals by the absorption of light. The UV spectrum of caffeic acid product showed characteristic absorptions at 226 and 384 nm, respectively, indicating the formation of caffeic acid ortho-quinone as product. Based on the experimental results and the above discussions the following mechanism has been proposed for the oxidation of caffeic acid by SO4•– radicals. 630

hν

S2O82–

hν

caffeic acid caffeic acid*

HO HO

+

S2O82–

H

H

C

C COOH + SO4

2SO4 – caffeic acid* 2SO4 –

HO O

caffeic acid O O

caffeic acid

H

H

C

C COOH + HSO4–

S2O82–

H H

+

HO

H 2–

C

C COOH + SO4

H

H

C

C COOH

O

H

H

– C COOH + SO4 C – SO4

–H O O

caffeic acid ortho-quinone

CONCLUSIONS Photochemical oxidation studies of caffeic acid in the presence of peroxydisulphate have been carried out under different experimental conditions. From kinetic results on the oxidation of caffeic acid by SO4•–, and formation of ortho-quinone as the product of oxidation a probable mechanism has been proposed. REFERENCES 1. L. A. BAZZANO, J. HE, L. G. OGDEN, C. M. LORIA, S. VUPPUTURI, L. MYERS, P. K. WHELTON: Fruit and Vegetable Intake and Risk of Cardiovascular Disease in US Adults. The First National Health and Nutrition Examination Survey. Epidemiologic Follow up Study. Am. J. Clin. Nutr., 76, 93 (2002). 2. G. BLOCK, B. PATTERSON, A. SUBAR: Fruit, Vegetables and Cancer Prevention. A Review of the Epidemiological Evidence. Nutr. Cancer., 18, 1 (1992). 3. C. S. YANG, J. M. LANDAU, M. T. HUANG, H. L. NEWMARK: Inhibition of Carcinogenesis by Dietary Polyphenolic Compounds. Ann. Rev. Nutr., 21, 381 (2001). 4. C. A. RICE-EVANS, N. J. MILLER, G. PAGANGA: Structure – Antioxidant Activity Relationships of Flavonoids and Phenolic Acids. Free. Radic. Bio. Med., 20, 933 (1996).

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  5. C. CASTELLUCCIO, G. PAGANGA, N. MELIKIAN, G. P. BOLWELL, J. PRIDHAM, J. SAMPSON, C. RICE-EVANS: Antioxidant Potential of Intermediates in Phenylpropanoid Metabolisms in Higher Plants. FEBS Lett., 368, 188 (1995).   6. M. NARDINI, M. D’AQUINO, G. TOMASSI, V. GENTILI, M. di FELICE, C. SCACCINI: Inhibition of Human Low Density Lipoprotein Oxidation by Caffeic Acid and Other Hydroxycinnamic Acid Derivatives. Free. Radic. Biol. Med., 19, 541 (1995).   7. O. FIRUZI, L. GIANSANTI, R. VENTO, C. SEIBERT, R. PETRUCCI, G. MARROSU, R. AGOSTINO, L. SASO: Hypochlorite Scavenging Activity of Hydroxycinnamic Acids Evaluated by a Rapid Microplate Method Based on the Measurement of Chloramines. J. Pharm. Pharmacol., 55, 1021 (2003).   8. A. S. PANNALA, R. RAZAQ, B. HALLIWELL, S. SINGH C. A. RICE-EVANS: Inhibition of Peroxynitrite Dependent Tyrosine Nitration by Hydroxycinnamate: Nitration or Electron Donation. Free. Radic. Biol. Med., 24, 594 (1998).   9. J. H. CHEN, C. HO: Antioxidant Activities of Caffeic Acid and Its Related Hydroxycinnamic Acid Compounds. J. Agric. Food. Chem., 45, 2374 (1997). 10. C. GIACOMELLI, K. CKLESS, D. GALATO, F. S. MIRANDA, A. SPINELLI: Electrochemistry of Caffeic Acid Aqueous Solutions with pH 2.0 to 8.5. J. Braz. Chem. Soc., 13, 332 (2002). 11. S. KALLEL TRABELSI, N. BELHADJ TAHAR, R. ABDELHEDI: Electrochemical Behaviour of Caffeic Acid. Elecrochim. Acta, 49, 1647 (2004). 12. P. HAPIOT, A. NEUDECK, J. PINSON, H. FULCRAND, P. NETA, C. ROLANDO: Oxidation of Caffeic Acid and Related Hydroxycinnamic Acids. J. Electroanal. Chem., 405, 169 (1996). 13. H. HOTTA, H. SAKAMOTO, S. NAGANO, T. OSAKAI, Y. TSUJINO: Unusually Large Number of Electrons for the Oxidation of Polyphenolic Antioxidants. Biochim. Biophys. Acta, 1526, 159 (2001). 14. H. FULCRAND, A. CHEMINAT, R. BROUILLARD, V. CHEYNIER: Characterization of Compounds Obtained by Chemical Oxidation of Caffeic Acid in Acidic Conditions. Phytochemistry, 35, 499 (1994). 15. J. L. CILLIERS, V. L. SINGLETON: Characterization of the Products of Non-enzymic Auto Oxidative Phenolic Reactions in a Caffeic Acid Model Systems. J. Agric. Food. Chem., 39, 1298 (1991). 16. S. FOLEY, S. NAVARATNAM, D. J. MVGARVEY, E. J. LAND, T. G. TRUSCOTT, C. A. RICEEVANS: Singlet Oxygen Quenching and the Redox Properties of Hydroxycinnamic Acids. Free Radic. Biol. Med., 26, 1202 (1999). 17. W. A. WATERS: Comments on the Mechanism of One Electron Oxidation of Phenols: A Fresh Interpretation of Oxidative Coupling Reactions of Plant Phenols. J. Chem. Soc. B, 2026 (1971). 18. L. PAPOUCHADO, R. W. SANDFORD, G. PETRIE, R. N. ADAMS: Anodic Oxidation Pathways of Phenolic Compounds: Part 2. Stepwise Electron Transfers and Coupled Hydroxylations. J. Electroanal. Chem., 65, 275 (1975). 19. R. PETRUCCI, P. ASTOLFI, L. GRECI, F. L. SASO, G. MARROSU: A Spectroelectrochemical and Chemical Study on Oxidation of Hydroxycinnamic Acids in Aprotic Medium. Electrochim Acta, 52, 2461 (2007). 20. H. W. GARDNER, K. ESKIN, G. W. GRAMS, G. E. INGLETT: Radical Addition of Linoleic Hydroperoxides to Alpha Tocopherol or the Analogous Hydroxychroman. Lipids, 7, 324 (1972). 21. R. YAMAUCHI, K. KATO, Y. VENO: Free Radical Scavenging Reactions of Alpha Tocopherol during the Autooxidation of Methyl Linoleate in Bulk Phase. J. Agric. Food. Chem., 43, 1455 (1995). 22. L. DOGLITTI, E. HAYON: Flash Photolysis of Peroxidisulfate Ions in Aqueous Solutions: The Sulfate and Ozonide Radical Anions. J. Phys. Chem., 71, 2511 (1967). Received 10 June 2009 Revised 26 July 2009

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Oxidation Communications 35, No 3, 633–650 (2012) Synthesis of bioactive compounds

Bioactive Complex Compounds Based on d-Metals and Some Nitrogen-containing Ligands: Synthesis, Structure and Properties M. Samkharadzea, M. Rusiab, N. Lekishvilib*, Kh. Barbakadzeb, N. Kakhidzea, Z. Pachuliac, K. Giorgadzeb, R. Gigaurib Department of Chemistry, ‘Akaki Tsereteli’ Kutaisi State University, Kutaisi, Georgia E-mail: [email protected] b Department of Chemistry, Faculty of Exact and Natural Sciences, Institute of Inorganic and Organic Hybrid Compounds and Non-traditional Materials, ‘Ivane Javakhishvili’ Tbilisi State University,Tbilisi, Georgia E-mail: [email protected] c Sukhumi State University, Sukhumi, Georgia E-mail: [email protected] a

ABSTRACT Novel nitrogen-containing coordination compounds some of d-metals – Ag(I), Hg(II), Zn, Fe(II), Cu(II), Mn(II), Cd(II), Ni(II) and Co(II) have been synthesised and studied. The optimum conditions of the synthesis have been established. The evaluation of relative complex forming-ability of the selected organic ligands and study of their electronic structure their quantum-chemical investigation were carried out. It was established that the obtained coordination compounds are finely dispersed substances, insoluble in water and ethanol. The composition and structure of synthesised coordination compounds have been established by elemental analyses. The study of IR spectra of these compounds showed that SbS43– group in studied substances represents exteriorly spherical tetrathiostibiate(V) ion. Analysis of the synthesised compounds thermographs shows that thermal destruction of studied samples begins at ∼150°C, runs stage-by-stage and deletion of the ligands is completely finished approximately at 400–500°C. Bioscreening of the obtained compounds have been carried out. Their antimicrobial, antiviral and parasitic activity has been determined. The area of their application has been established. Keywords: industrial waste, stibium, coordination compound, bioscreening, composite, application. *

For correspondence.

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AIMS AND BACKGROUND Transformation of stibium-containing industrial waste into stable coordination compound with specific properties with the purpose of their further application belongs to a variety of topical issues of applied and coordination chemistry. Successful solution of this problem will not only create new raw materials resource base, but also will solve important ecological problem – will protect environment from pollution by stibium-containing waste. The presented work is devoted to this problem, in which reaction-responsive stibium compounds extracted from arsenic production waste are considered, on the basis of which new nitrogen-containing bioactive coordination compounds are received. 2,2′-dipyridyl as a ligand, for a great while is studied during the synthesis of coordination compounds1. The reason of this study is non-uniform and contains both pure chemical and applied aspects. The ample opportunities of 2,2′-dipyridyl for creation of coordination com­pounds of different types and behaviour should be sought in its structure:

N

N

Because of the fact that nitrogen atoms hold in molecule such position that optimum conditions are created for formation of 5-member cycle, 2,2′-dipyridyl from the very begin­ning became the focus of scientists interest, and due to this fact coordination compounds with metal halogenides, nitrates, sulphates and almost every other soluble salt, containing it, are studied in details. Sodium tetra-thioarsenates(V) water solutions action with the products of interaction of d-metals soluble salts with bidentate ligand 2,2′-dipyridyl leads us to the formation of appro­priate coordination compound2. Having used the same method we implemented the synthesis of coordination compounds tetrathiostibiates(V) with 2,2′-dipyridyl. Salts dissolved in d-metals water are used as basic substances: Ag(I) and Hg(II) nitrates, Zn, Fe(II) and Cu(II) sulphates, Mn(II), Cd(II), Ni(II) and Co(II) chlorides; sodium tetra­thio­stibiate is used as precipitator, while 2,2-dipyridyl (C5H4N)2 (shortly dipy) is used as nitrogen containing ligand. We also used ethylenediamine (H2N–CH2– CH2–NH2), one of the best bidentate ligand, for obtaining of complex compounds of d-metals(II). EXPERIMENTAL Spectral analysis. IR spectra were obtained with a spectrophotometer FT-IR ‘Thermo Nicolet’ in Ge plates and UR-20 (400–4000 cm–1) in liquid paraffin. Thermogravimetric analyses carried out on F. Paulik, I. Paulik and L. Erdey systems Q 1500 type thermal 634

analyser by Hungarian firm MOM, heating rate 10oC/min, sensibility DTA 1/10. Melting points were determined by a melting point meter apparatus MPM-HV2, Germany; metal content – on an atomic absorptive spectro­photometer Perkin Elmer 603. X-ray pattern of 4 samples Fe(II), Ni(II), Ag(I) and Hg(II) given here was studied by use of the method of homology. For that purpose table data of American cardcatalogue ASTM-20-1917 and ASTM-20-1918 have been used, those are basically in good correlation with X-ray reflection of complexes received. Theoretical (virtual) bioscreening was carried out by using of internet-system program PASS C&T. Standard microbiological methods for study of antibacterial activity of synthesised compounds have been used. RESULTS AND DISCUSSION For preliminary estimation of relative complex forming-ability of the selected organic ligands and study of their electronic structure we have carried out their quantumchemical investigation. Quantum-chemical calculations were performed on PC with an AMD processor with the built-in coprocessor by using Mopac 2000 and CS Chem3D Ultra, v8 (Ref. 3). We gave the following key-words to guide each computation: EF GNORM = 0.100 MMOK GEO-OK AM1 MULLIK LET DDMIN=0.0 GNORM=0.1 GEO-OK. We have calculated energetical and geometrical parameters, effective charges on atoms and electron occupation of atomic orbitals (electronic density) in 2,2′-dipyridyl and ethylene­diamine molecules.

Fig. 1. 3D model of 2,2′-dipyridyl

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Fig. 2. 3D model of ethylene­diamine

In the molecule of 2,2′-dipyridyl (Fig. 1) the bond lengths and valence bond angles of nitrogen atoms N1 and N12 with neighbour atoms (∠C2–N1–C6 = 118.1º and ∠C7–N12–C11 = 118.1º) indicate mainly their sp2 hybridised position. Analysis of values of effective charges on atoms (Table 1) shows that potentially electron-donor atoms are N1 (q1 = –0.105052), C3 (q3 = –0.177231), C5 (q5 = –0.167740), C8 (q8 = –0.167764), C10 (q10 = –0.177228) and N12 (q12 = –0.104952). But electron occupation of atomic orbitals (Table 2) shows that in spite of comparatively high negative relative charge of carbon atoms (C3, C5, C8, C10) by comparison with nitrogen atoms, they are unable to form σ-bond with metal atom by donor-acceptor mechanism as they have not an electron pair on the second energetical level. The electron pairs of nitrogen atoms are located on 2s orbital (electron occupation 1.71732 (N1) and 1.71738 (N12)) and only they have capacity to form σ-bond with metal atom by donor-acceptor mechanism. Table 1. Relative charges and electronic density on the dipyridyl atoms

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Atom (i)

Relative charges on atoms (qi)

Electronic density (qi (d))

Atom (i)

Relative charges on atoms (qi)

Electronic density (qi (d))

N1 C2 C3 C4 C5 C6 C7 C8 C9 C10

–0.105052 –0.071475 –0.177231 –0.095118 –0.167740   0.025459   0.025459 –0.167764 –0.095095 –0.177228

5.1051 4.0715 4.1772 4.0951 4.1677 3.9745 3.9745 4.1678 4.0951 4.1772

C11 N12 H13 H14 H15 H16 H17 H18 H19 H20

–0.071521 –0.104952   0.161749   0.144233   0.139053   0.146123   0.146118   0.139062   0.144225   0.161696

4.0715 5.1050 0.8383 0.8558 0.8609 0.8539 0.8539 0.8609 0.8558 0.8383

Table 2. Electron occupation of atomic orbitals

Atom N1 C3 C5 C8 C10 N12

2s 1.71732 1.22757 1.22712 1.22715 1.22752 1.71738

2py 1.22193 0.92718 0.97761 1.00769 0.99355 1.13945

2px 1.06728 1.00124 0.94049 0.94466 1.00113 1.05008

2pz 1.09853 1.02123 1.02252 0.98827 0.95502 1.19804

Thus, the molecule contains 2 potentially electron-donor atoms – N1 and N12 because of that it represents as a bidentate ligand and is capable to form coordination compounds with d-metals in the form of 5-member cycle:

N

N Mn+

In the molecule of ethylenediamine (Fig. 2) the bond lengths and valence bond angles of nitrogen atoms N3 and N4 with neighbour atoms (∠C1–N3–H9 = 109.5º, ∠C1–N3–H10 = 109.5º, ∠H9–N3–H10 = 110.6º, ∠C2–N4–H11 = 110.3º, ∠C2–N4–H12 = 111.3º and ∠H11–N4–H12 = 109.3º) indicate mainly theirs sp3 hybridised position. Analysis of values of effective charges on atoms (Table 3) shows that potentially electron-donor atoms are N3 (q3 = –0.349295) and N4 (q4 = –0.349288). Electron occupation of atomic orbital (Table 4) shows that the electron pairs of nitrogen atoms are located on 2s and 2pz orbitals (electron occupation for N3 atom is 1.58211 (2s) and 1.49979 (2pz), but for N4 atom is 1.58220 (2s) and 1.52119 (2pz)). Nitrogen atoms – N3 and N4 have capacity to form σ-bond with metal atom by donor-acceptor mechanism. Table 3. Relative charges and electronic density on the ethylenediamine atoms

Atom Relative charges Electronic density Atom (i) on atoms (qi) (qi (d)) (i) C1 C2 N3 N4 H5 H6

–0.080091 –0.080043 –0.349295 –0.349288   0.035061   0.105054

4.0801 4.0800 5.3493 5.3493 0.9649 0.8949

H7 H8 H9 H10 H11 H12

Relative charges on atoms (qi) 0.105135 0.035121 0.149137 0.140076 0.149103 0.140031

Electronic density (qi (d)) 0.8949 0.9649 0.8509 0.8599 0.8509 0.8600

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Table 4. Electron occupation of atomic orbitals

Atom N3 N4

2s 1.58211 1.58220

2py 1.14530 1.12511

2px 1.12209 1.12078

2pz 1.49979 1.52119

Thus, the molecule contains 2 potentially electron-donor atoms – N3 and N4 because of that it represents as a bidentate ligand and is capable to form coordination compounds with d-metal ion in the form of 5-member cycle: H2C

CH2

H2N

NH2 M n+

For comparison of the complex formation ability for selected ligands we calculated their complexes with Cu2+. The Ni–Cu2+ bond orders for dipyridyl PCu1–N2 = 0.333011 and PCu1–N3 = 0.335304 the correspond charges – q1 = 0.633404, q2 = –0.184639 and q13 = –0.184586. For ethylenediamine PCu1–N2 = 0.566363 and PCu1–N3 = 0.566054, the charges – q1 = 0.775783, q2 = –0.191420 and q3 = –0.190861. The above-mentioned allowed us to conclude that the complex of Cu+-ion with ethylenediamine is more stable than with dipyridyl. CH2

H2C N

N

NH2

H2N 2+

Cu2+

Cu

Synthesis of d-metals tetrathiostibiate coordination compound with 2,2′-dipyridyl was carried out by exchange reaction, as result of which tetrathiostibiate complexes of corresponding d-metals are precipitated, formation of which can be explained by the unity of the following consecutive reactions: 638

AgNO3 + dipy → [Ag(dipy)]NO3

(a)

3[Ag(dipy)]NO3 + Na3SbS4.9H2O → [Ag(dipy)]3SbS4↓ + 3NaNO3 + 9H2O   (b)

or in total: 3AgNO3 + 3dipy + Na3SbS4.9H2O → [Ag(dipy)]3SbS4↓ + 3NaNO3 + 9H2O

As to other metals(II) dipyridylates, in particular when M = Fe, Co, Ni, Zn, Cd, Hg, Cu and Mn: MX2 + ndipy → [M(dipy)n]X2

(a)

3[M(dipy)n]X2 + 2Na3SbS4.9H2O → 6NaX + [M(dipy)n]3(SbS4)2↓ + 18H2O   (b)

or in total: 3MX2 + 3ndipy + 2Na3SbS4.9H2O → [M(dipy)n]3(SbS4)2↓ + 6NaX + 18H2O

The essence of this method comprises in the fact that nitrates, chlorides and sulphates of the mentioned metals form stable, but water-soluble coordination compounds with 2,2′-dipyridyl. That is why their extraction from mother solution in chemically pure form for further transformation is not necessary: corresponding d-metals tetrathiostibiate(V) dipyridy­la­tes and ethylenediamine complexes are instantaneously precipitated during treatment by these solutions precipitator – sodium tetrathiostibiate. The obtained coordination compounds represent finely dispersed substances, insoluble in differently coloured water and ethanol (Tables 5 and 6). All of them are extracted without crystallisation water, except of Fe(II) dipyridylates, which adds to 3 molecules of water. They have no certain melting temperature, since they resolve before melting. The composition of the synthesised complexes was established by elemental analysis: stibium was determined by the Evins method, sulphur – by gravimetric method, metal – by volumetric method, and nitrogen – by the Duma micromethod. The composition and structure of the synthesised complexes, except of elemental analyses, was determined by physicochemical research methods. Study of IR spectra of these comp­le­x­es adsorption shows that SbS43– group in the studied substances repre­sents exteriorly spherical tetrathiostibiate(V) ion. In the long-wave 380 and 384 cm–1 spec­tral region absorption band are observed, that correspond to ν3 oscillation of SbS43– ion (Ref. 4). The obtained dipyridyl coordination compounds are finely divided various colour compounds dissolved in water, ethanol and other usual organic solvents. All these compounds, besides dipyridylates of Fe(II), separated without crystallisation water. They have distinct melting temperature because they are dismissed until melting.

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Table 5. Some basic characteristics of the synthesised dipyridyl complexes of tetrathiostibiates(V) of some d-metals

No

Compound

1 2 3 4 5 6 7

[Ag(dipy)]3SbS4 [Zn(dipy)2]3(SbS4)2 [Cd(dipy)2]3(SbS4)2 [Hg(dipy)2]3(SbS4)2 [Cu(dipy)2]3(SbS4)2 [Mn(dipy)3]3(SbS4)2 [Fe(dipy)3]3(SbS4)2• 3H2O 8 [Co(dipy)]3SbS4 9 [Ni(dipy)3]3(SbS4)2

Yield Colour (%) 88.7 black 93.3 yellow 86.3 orange 96.6 brown 95.8 black 95.6 brown 89.8 red

Elemental analysis data (%) found calc. M Sb N S H2O M Sb N S H2O 31.07 11.69   8.06 12.31 – 30.83 11.92   8.03 12.46 – 12.02 14.92 10.29 15.71 – 11.77 15.05 10.36 15.87 – 19.02 13.73   9.47 14.46 – 18.81 13.49   9.27 14.30 – 29.53 11.95   8.24 12.59 – 29.71 12.07   8.02 12.44 – 11.72 14.97 10.33 15.77 – 11.85 15.01 10.11 15.65 –   7.97 11.77 12.18 12.40 –   8.02 11.98 12.01 12.27 –   7.88 11.46 11.86 12.07 2.54   7.66 11.37 11.73 11.91 2.59

90.2 black   8.50 11.70 12.11 12.33 –   8.43 11.53 11.86 12.14 – 85.7 black   8.47 11.71 12.12 12.33 –   8.32 11.84 12.32 12.18 –

Comparison of free (incoordinate) ligand – 2,2′-dipyridyl spectrum with d-metals (Fe, Zn, Mn) tetra­thio­stibiate(V) dipyridylates spectra shows displacement of high frequency absorption bands (Fig. 3). Free ligands absorption band is at 1584 cm–1, while in the complexes it is displaced to 1600–1610 cm–1, that is related to heterocyclic nitrogen coordination with metals atoms5–7. Table 6. Some basic characteristics of the synthesised ethylenediamine complexes of tetrathiostibiates(V) of some d-metals

No 1 2 3 4 5 6 7

Compound [Ag(en)]3SbS4 [Zn(en)2]3(SbS4)2 [Cd(en)2]3(SbS4)2 [Hg(en)2]3(SbS4)2 [Cu(en)2]3(SbS4)2 [Co(en)3]3(SbS4)2 [Ni(en)3]3(SbS4)2

Yield Colour (%) 94.1 88.7 93.9 95.2 94.6 89.7 91.5

black yellow orange brown grey black black

M 42.94 18.57 28.17 41.17 18.14 14.53 14.51

Elemental analysis data (%) found calc. Sb N S M Sb N 16.16 11.15 17.02 43.07 16.21 10.90 23.06 15.91 24.29 18.68 22.99 15.79 20.34 14.03 21.42 28.09 20.40 14.15 16.66 11.49 17.55 41.21 16.54 11.70 23.18 15.99 24.41 18.07 23.27 15.82 20.01 20.71 21.08 14.67 20.16 20.48 20.02 20.72 21.08 14.59 19.96 20.69

S 16.81 24.19 21.25 17.34 24.69 21.45 20.84

By taking into account the above-mentioned we come to a conclusion that the formulas of the synthesised coordination compounds can be presented as follows:

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For Fe(II) complex, as for crystalline hydrate, absorption bands are observed in 1630 cm–1 region (Fig. 3) that points to the presence of crystallisation water in the compounds. Individuality of investigated substance is testified by the results of X-ray phase studies (Table 7).

wavenumber (cm–1)

Fig. 3. IR spectra of the synthesised compounds a – [Fe(dipy)3]3(SbS4)2.3H2O; b – [Zn(dipy)2]3(SbS4)2; c – [Mn(dipy)3]3(SbS4)2

Thermal studies of the synthesised compounds assured us that dipyridyl complexes do not contain crystallisation water, except of dipyridylate of Fe(II) tetrathiostibiates, that is testified by the IR spectroscopic data (Fig. 3), too. The thermal behaviour of these compounds is almost similar. Thermolysis of Fe(II) tetra­thiostibiate(V) dipyridylates [Fe(dipy)3]3(SbS4)2.3H2O was considered as an example (Fig. 4a). The destructive process began at 80oC; in the temperature interval 80–150oC of the corresponding DTA curve one can observe the exothermic peak with 641

maximum. The mass loss equal to 3.57% corresponds to 3 mol crystallisation water (theor. – 2.54%). The next stage of the thermolysis process in the temperature interval 150–380oC proceeds with difficultly. On the DTA one can observe 3 exothermic peaks with maxima at 210, 250 and 360oC. The mass loss at this moment is about 45.00% that corresponds to the removal of 6 mol crystallisation water (theor. – 44.03%). In the tem­pe­ra­tu­re in­ter­val­380–530oC one can observe an exothermal effect with maximum at 480oC with mass loss of 27.57%. Based on the theoretical evaluation (25.03%) the corresponding peak may belong to the loss of 3 mol ligands and 2 mol sulphur. After this process the mass loss of the investigated sample comprises 1%, which is equal to the removal of 1 mol sulphur (theor. – 1.5%). Foreseen from the above-mentioned, the thermolysis of dipyridylate of Fe(II) tetra­thiostibiate may be represented by the following approximate scheme:    80–150oC

150–380oC    380–530oC

>530oC

[Fe(dipy)3]3(SbS4)2.3H2O –→ [Fe(dipy)3]3(SbS4)2 –→ [Fe(dipy)]3(SbS4)2–→ Fe3Sb2S6 –→ …

    –3H2O   –6dipy    –3dipy; –2S  

Fig. 4. DTA, DTG and TG curves of the synthesised coordination compounds a – [Fe(dipy)3]3(SbS4)2.3H2O; b – [Fe(dipy)2]3(SbS4)2

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–S

Table 7. Results of X-ray analysis of the synthesised dipyridyl complexes of tetrathiostibiates(V) of some d-metals (F, Ni, Hg and Ag)

[Fe(dipy)3]3(SbS4)2.3H2O I/I0 100 5 5 4 5 5 5 10 4 6 4 5 4

da/n 11.05   9.31   7.69   4.79   4.28   4.18   3.708   3.562   3.36   3.10   2.54   2.227   2.156

[Ni(dipy)3]3(SbS4)2 I/I0 10 2 2 2 1 1 2 2 3 2 1 1 1

da/n 11.0   7.08   6.44   5.55   5.07   4.28   3.95   3.86    3.74   3.63   2.69   2.51   2.44

[Ag(dipy)]3SbS4 I/I0 5 15 32 25 100 35 5 15 15 5 28 10 8 40 10 30 30

da/n 9.15 6.18 3.63 3.56 3.34 3.13 2.978 2.90 2.80 2.675 2.564 2.368 2.22 2.054 1.898 1.758 1.744

[Hg(dipy)2]3(SbS4)2 I/I0 3 33 6 43 100 10 8 10 5 16 5

da/n 9.8 6.33 3.83 3.36 3.186 2.915 2.765 2.529 2.127 2.057 1.953

Only exception is presented by Hg(II) complex – thermolysis begins on relatively low (100°C) temperature and completes by total decay (Fig. 5a). This fact is caused by instability of Hg(II)-containing compounds at high temperatures. Thus, the obtained results allow to make a conclusion that molecules of 2,2′dipyridyl are coordinated with d-metals atoms by means of nitrogen atoms, while SbS43– group is located in the external (second) sphere of the complex. Since ethylenediamine (H2N–CH2–CH2–NH2) is one of the best bidentate ligand, we have set as a goal the reception of complex compounds of d-metals tetrathiostibiates in the system M2+–SbS43––en–H2O. Sodium tetrathiostibiate Na3SbS4.9H2O, ethylenediamine (50% water solution) and salts of water-soluble d-metals have been used by us as a mother (basic) substances. Coordination compounds of d-metal tetrathiostibiates with ethylenediamine have been received by means of exchange reaction – by the action of sodium tetrathiostibiate with the products of interaction of 50% ethylenediamine and d-metals salts, without extraction of the latter in individual status. Ag(I), Cd, Zn, Hg(II), Cu, Co and Ni(II) complexes with ethylene­diamine have been synthesised according to the following reactions: AgNO3 + en → [Ag(en)]NO3 3[Ag(en)]NO3 + Na3SbSu.9H2O → [Ag(en)]3SbS4↓ + 3NaNO3 + 9H2O    (a)

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Fig. 5. DTA, TGA and TG curves of the synthesised coordination compounds a – [Hg(dipy)2]3(SbS4)2; b – [Ni(dipy)3]3(SbS4)2

MX2 + nen → [M(en)n]X2 3[M(en)n]X2 + 2Na3SbS4.9H2O → [M(en)n]3(SbS4)4↓ + 6NaX + 18H2O    (b)

where M = Zn, Cd, Hg, Cu, Ag, Co, Ni; X = Cl–, 1/2SO42– or NO3–. Since the Fe(II) ethylenediamine complex is extracted from water solution in the form of precipitate, while received precipitate is insoluble in the sodium tetrathiostibiates, we were not able to receive Fe(II) tetrathiostibiate complex by means of exchange reaction with ethylenediamine, as it was achieved during synthesis of other d-metals aminates. The synthesised complexes are finely crystalline compounds of various colouring, they are insoluble in the water, spirit, and other ordinary organic solvents. Synthesised compounds, except of elemental analysis, have been studied by IR spectro­scopy, X-ray graphical studies and thermo-gravimetric analysis. 644

wavenumber (cm–1)

Fig. 6. IR spectra of the synthesised compounds a – [Zn(en)2]3(SbS4)2; b – [Co(en)3]3(SbS4)2

Study of the IR spectra of the synthesised compounds (Fig. 6) shows that the NH2 group absorption bands are significantly shifted in comparison with uncoordinated ligand. For free, uncoor­di­na­ted ethylenediamine absorption bands in 1595 and 3510 cm–1 regions are characteristic, while in coordination compound under investigation the absorption bands of this group are located in 1620 and 3370 cm–1 region that is characteristic for valence vibration of H2N→M+ bond. And since silver(I) coordination number is equal to 2, while in case of other d-metals, proceeding from the quality of their oxidation, this number increases up to 4, we can draw a conclusion that ethylenediamine plays the role of cyclic bidentate ligand, and the synthesised complexes have the following structure:

Individuality of obtained products has been checked by means of X-ray phase ana­ly­sis. Cu(II), Ni(II) and Ag(I) compounds show sufficiently defined X-ray pattern, while as to Hg(II), Zn(II) and Co(II) tetrathiostibiates of ethylenediamine (Table 8), they turn out to be X-ray amorphous (Ref. 8). X-ray diagram of the American card-catalogue ASTM 20-1692 (Ref. 9) (C5H8N2.2HCl pure ligand) has been used with the purpose of study of received X-ray diagrams. It turns out that in our case correlation takes place, but the certain amount of X-ray reflections is not deciphered (decoded), and for this purpose the method of homology has been used. More perfect X-ray diagram existing in the American 645

card-catalogue ASTM 24-1670 partially filled the gap and gave us the picture that is almost similar to Ni(II) and Cu(II) tetrathiostibiate complexes with ethylenediamine. It may be noted that the samples are virtually similar to the compared references and represent ethylenediamine complexes. Below it is also considered the thermolysis of [Co(en)3]3(SbS4)2 as a sample (Fig. 7a). Removal of 9 mol of ligand occurs at 100–280°C temperature range in 3 stages: at 100–130, 130–180 and 180–280°C. At the 1st stage the mass decreases by 15.00% (theoretically 14.79%), at the 2nd – by 16.42%, and at the 3rd – by 15.01%. It may be noted that the removal of 3 mol of ligand corresponds with each of these stages. After 280°С, as it was mentioned, the process of thermolysis proceeds as well as in case of normal (neutral) salts. In 280–560°C temperature range the mass decreases by 7.14% that corresponds to the removal of 3 mol of sulphur (theor. – 7.89%). At DTA curve 2 exothermic effects are observed within this range, with peaks at 340 and 520°C.

Fig. 7. DTA, TGA and TG curves of the synthesised coordination compounds a – [Co(en)3]3(SbS4)2; b – [Zn(en)2]3(SbS4)2

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Further decay of the samples continues above 520°C, endothermic effect is observed at DTA curve with peak at 820°C, the mass loss comprises of 24.28% that should be caused by removal of stibium sulphide form (theor. – 25.27%). Table 8. Results of X-ray analysis of the synthesised ethylenediamine tetrathiostibiates(V) of some d-metals (Cu, Ni and Ag)

[Cu(en)2]3(SbS4)2

I/I0 3 4 1 2 10 1 5 5 1 1 3 2 4 4 2 1 2 1 2 2

da/n 5.67 5.41 5.10 4.796 4.70 4.23 3.95 3.70 3.59 3.70 3.528 3.30 3.028 2.80 2.734 2.64 2.584 2.413 2.127 2.02

[Ni(en)3]3(SbS4)2

I/I0 5 5 4 4 10 5 6 9 9 6 10 5 4 3

da/n 8.29 7.69 5.69 5.38 5.10 4.74 4.04 3.91 3.75 3.53 3.36 3.186 2.529 2.449

[Ag(en)]3SbS4

I/I0 2 1 2 6 10 3 2 1 2

da/n 9.50 8.23 3.18 2.82 2.58 2.42 2.37 2.20 2.07

On the assumption of above-mentioned, the probable scheme of thermolysis of Co(II) tetrathiostibiates(V) of ethylenediamine can be presented as follows:   100–130oC     180–280oC    280–560oC   560–860oC

[Co(en)3]3(SbS4)2 –→ [Co(en)2]3(SbS4)2 –→ Co3(SbS4)2 –→ 2CoS.Sb2S2 –→ 3CoS

   –3en      –3en     –3S    –Sb2S2

As well as in other cases, here the process of [Hg(en)2]3(SbS4)2 thermolysis (Fig. 8a) differs from the corresponding processes of other ethylenediamine complexes that during heating experiences total decay without any residue. Decay of [Hg(en)2]3(SbS4)2 sample begins from 120°C and becomes especially intense in the 210–270°C temperature range. At that time the mass loss is equal to 9.33% (theor. – 8.21%) that corresponds to the removal of 2 mol of ethylenediamine.

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Fig. 8. DTA, TGA and TG curves of the synthesised coordination compounds a – [Hg(en)2]3(SbS4)2; b – [Cu(en)2]3(SbS4)2

In the 270–450°C temperature range the exothermic peak has several maxima at DTA curve that points out that complex process runs on. During this process total loss of remained mass takes place. Study of the synthesised complexes [Cu(II), Cd and Ag(I) (ethylenediaminate tetrathiostibiates] thermographs shows that removal of ethylenediamine in all compounds occurs in 2 or 3 stages. For these complexes with ethylenediamine (as well as in case of normal salts) mass increase is observed in the process of thermolysis. Thus, the investigations show that in case of d-metal ethylenediamine complexes treatment by sodium tetrathiostibiates water solution corresponding compounds [Ag(en)]3SbS4 and [M(en)n]3(SbS4)2, are received in the form of precipitate (where n = 2, 3). We have carried out preliminary virtual (theoretical) bioscreening of obtained structures by using of internet-system program PASS C&T (Ref. 10). The estimation of probability of acti­vity of compounds is carried out via parameters Pa (active) and Pi (inactive); when Pa > 0.5–0.7, the compound also will show activity experimentally and probably will be analog of known pharmaceutical agents, too. Evaluated relative 648

bioactivities of some synthesised compounds are given in Table 9. Microbiological study of the investigated compounds confirmed the evaluated virtual concepts.

Antibacterial activity enhancer

Antineurotoxic

Cytoprotectant

Antineoplastic (brain cancer)

Neurotoxin

Antiviral (Arbovirus)

Antihelmintic (Nematodes)

Antiseborrheic

Compound

Urethanase inhibitor

Table 9. Virtual relative bioactivity of some synthesised coordination compounds

0.695/ 0.010 0.657/ 0.025 0.695/ 0.010 0.640/ 0.034

0.672/ 0.041 0.581/ 0.071 0.672/ 0.041 0.636/ 0.052

0.661/ 0.035 0.615/ 0.063 0.661/ 0.035 0.650/ 0.041

0.637/ 0.047 0.522/ 0.084 0.637/ 0.047 –

Pa/Pi [Fe(dipy)3]3(SbS4)2 3H2O 0.865/ 0.021 [Ag(dipy)]3(SbS4)2 0.824/ 0.041 [Mn(dipy)3]3(SbS4)2 0.865/ 0.021 [Ag(en)]3(SbS4)2 0.768/ 0.066 .

0.820/ 0.005 0.766/ 0.011 0.820/ 0.005 0.651/ 0.034

0.586/ 0.053 0.496/ 0.105 0.586/ 0.053 0.543/ 0.075

0.687/ 0.046 0.636/ 0.089 0.687/ 0.046 0.681/ 0.049

0.719/ 0.029 0.670/ 0.065 0.719/ 0.029 0.663/ 0.070

CONCLUSIONS Novel nitrogen-containing coordination compounds of some d-metals such as Ag(I), Hg(II), Zn, Fe(II), Cu(II), Mn(II), Cd(II), Ni(II) and Co(II) have been synthesised and studied. The optimum conditions of the synthesis have been established. It was established that their extraction from mother solution in chemically pure form for further transformations is not necessary: the corresponding d-metals tetrathiostibiate(V) dipyridylates and ethylenediamine complexes are instantaneously precipitated during treatment by these solutions precipitator – sodium tetrathiostibiate. It was shown that the obtained coordination compounds are finely dispersed substances, insoluble in water, ethanol and usual organic solvents. The composition and structure of synthesised coordination have been established by elemental analyses. The study of the IR spectra of these compounds showed that SbS43– group in the studied substances represents exteriorly spherical tetrathio­stibiate(V) ion. Analysis of thermographs of the synthesised compounds shows that thermal destruction of studied samples begins at ∼150°C, runs stage-by-stage and deletion of the ligand is completely finished approximately at 400–500°C. Bioscreening of obtained compounds was carried out. Their antimicrobial, antiviral and parasitic activity has been established. The area of their application has been established.

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REFERENCES   1. A. A. GRINBERG: Introduction in Chemistry of Complex Compounds. IL Press, Moscow, 1960.   2. I. DIDBARIDZE, G. KHELASHVILI, M. RUSIA, M. INJIA, G. GIGAURI: Coordination Compounds of Tetrathioarsenates of d-metals(II) with 2,2′-dipyridyl. Georgian Engineerig News, 4, 97 (1997).   3. M. J. S. DEWAR, E. G. ZOEBISCH, E. F. HEALY, J. J. P. STEWART: Development and Use of Quantum Mechanical Molecular Models. 76. AM1: A New General Purpose Quantum Mechanical Molecular Model. J. Am. Chem. Soc., 107, 3902 (1985).   4. K. NAKAMOTO: IR and Combination Scattering Spectrum of Inorganic and Coordination Compounds. Mir, Moscow, 1991.   5. A. IREMADZE, I. CHILOGIDZE, Iu. KHARITONOV: Oscillatory Spectra of Complexes of Nickel with Oxymethylnikatinamides. Coord. Chem., 8 (4), 1239 (1978).   6. M. TSINTSADZE, T. MACHALADZE, M. KERESELIDZE, L. SKHIRTLADZE, R. KURTANIDZE, V. VARAZASHVILI, T. PALAVANDISHVILI, M. TSARAKHOV: Coordination Compounds of Zinc Sulphats with ortho-amino-4 and 5-methylpirydines. Proc. of the Academic Sciences of Georgia, 25 (1–2), 33 (1999).   7. A. TSIBADZE, Iu. KHARITONOV, G. TSINTSADZE, Zh. PETRIASHVILI: Investigation of Oscillatory Spectra of Cyanates Complexes of Metals with Hydrazides of Isonicotinic Acid. Coord. Chem., 1 (4), 525 (1975).   8. G. LIPSON, G. STIPL: Interpretation of Powder X-ray Pictures. Vol. II. Mir, Moscow, 1972.   9. The American Card File ASTM (American Society for Testing and Materials), 1977. 10. A. SADIM, A. LAGUNIN, D. FILIMINOV, V. POROIKOV: Internet-System of Prog­no­ses of the Spectrum of Bioactivity of Chemical Compounds. Chem.-Farm. J., 36 (10), 21 (2002). Received 5 January 2012 Revised 12 February 2012

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Oxidation Communications 35, No 3, 651–661 (2012) Oxidative stress in biological systems

Oxidative Stress Provoked by Low and High Temperatures in Wild Type and Ethylene-insensitive Mutant Eti5 of Arabidopsis thaliana (L.) H e y n h D. Todorovaa*, I. Sergieva, I. Moskovaa, V. Alexievaa, M. Hallb Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Street., Bl. 21, 1113 Sofia, Bulgaria b Institute of Biological Sciences, University of Wales, Cledwyn Building, Aberystwyth, Ceredigion, SY23 3DD, UK E-mail: [email protected] a

Abstract Arabidopsis thaliana (L.) H e y n h wild type (wt) and ethylene-insensitive (eti5) type plants were used in this study. The plants were grown in growth chamber and 38 days after sowing the plants were subjected to low temperature (LT) 4°C or high temperature (HT) 38°C for 24 h in darkness. The content of stress markers and enzyme activities were measured at 0, 24, 48 and 120 h after the temperature treatment. The aim of our investigation was to compare the effect of low and high temperature on hydrogen peroxide (H2O2), malondialdehyde (MDA), free proline and carbonyl group content, ascorbate/dehydroascorbate content as well as catalase, guaiacol peroxidase (POD), and superoxide dismutase (SOD) activities in both types of Arabidopsis. Data obtained showed higher levels of the stress-markers MDA and carbonyl groups as well as decreased catalase activity (detoxifying H2O2) and increased SOD activity (producing H2O2) at the end of the measuring period (120 h) in the wt than in the mutant, which indicates that the wt is more sensitive to temperature stress than the mutant. On the other hand, the observed higher levels of stress markers (carbonyl groups, MDA) in both genotypes at 0 h after HT treatment as compared to LT is indicative that Arabidopsis is more sensitive to HT stress probably due to the fact that this plant species is cold-tolerant. Keywords: Arabidopsis thaliana (L.) H e y n h, ethylene-insensitive mutant (eti5), temperature stress, stress markers.

*

For correspondence.

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Aims and Background The small size of Arabidopsis thaliana (L.) H e y n h, its short life cycle and a small genome have made it popular model system for genetic analyses of plant responses to abiotic stresses. Arabidopsis mutants with defects in growth responses to light or low temperatures, in detoxification of active oxygen species (AOS), in absorption of UV-B radiation, etc. have all provided insights into the molecular mechanisms of the stress tolerance phenomenon1. According to Mattoo and Suttle2, and Raz and Fluhr3, a plethora of abiotic and biotic environmental stresses exert their impact on plants via the gaseous hormone ethylene. In this sense, similarly to plant senescence, the sensitivity to environmental changes may be mediated by specific ethylene receptor(s) whose activation signal is then transduced via a cascade pathway to elicit a response4,5. Plants demonstrate a rapid physiological and biochemical response to changing environmental conditions6. Biotic and abiotic stresses provoke a sharp accumulation of active oxygen species, known as the ‘oxidative burst’. The steady-state level of AOS in the cells is determined by the activity of antioxidant systems7,8. In all situations the balance between AOS formation and consumption is precisely controlled. When AOS production becomes far greater than the capacity of the tissues to scavenge them, the oxidative stress is favoured. Therefore, the augmentation of antioxidant defences plays a pivotal role in preventing oxidative stress in plants9. The activities of most antioxidant enzymes under stressful conditions generally increase and correlate well with enhanced tolerance. Catalase (CAT, EC 1.11.1.6), superoxide dismutase(s) (SOD, EC 1.15.1.1), various peroxidases such as guaiacol peroxidase (POA, EC 1.11.1.7) are all usually increased as a result of stress, but the degree of augmentation is clearly limited by the relatively poor capacity for induction of the various enzymes of the antioxidant defence system9. Little information is available on signal transduction sequences and the exact mechanisms underlying the physiological response both of stressed and acclimated plants. Hydrogen peroxide also makes an important contribution in plant defence since it is a diffusible signal for acclimation, stress tolerance and other defence responses. Moreover, hydrogen peroxide functions as a part of the triggering mechanism for senescence which then becomes autocatalytic10. Temperature is one of the major environmental factors affecting nutrient distribution, growth and development of plants. It is well known that Arabidopsis is a cold-tolerant plant11–13. It was established that Arabidopsis is among the so-called ‘hardy’ species which, when exposed to low non-freezing temperatures are able to develop a tolerance to sub-zero temperatures11 – an adaptive phenomenon known as cold hardening. In Arabidopsis, the observed responses to cold include the induction of number of genes and proteins, increases in soluble carbohydrates, adjustments in the unsaturation of cellular lipids, accumulation of anthocyanins, etc.14 However, little information is available about the effects of high temperatures on Arabidopsis. Although the biological significance of the phytohormones in overall plant growth and development is well established, the plant hormone mutants appear to 652

be a promising experimental system to investigate the role of phytohormones in plant stress responses. It was established that the ethylene-insensitive mutant (eti5) (Ref. 15) of Arabidopsis had characteristics of delayed senescence accompanied by increased amounts of leaf pigments and soluble proteins16 as well as elevated endogenous polyamine17,18 and cytokinin19 levels as compared to the wt. Because of the fact that the extreme temperatures may induce senescence, we were interested to determine the changes in the oxygen-related enzymatic and non-enzymatic defence systems as well as the accumulation of organic solutes after low or high temperature treatments during recovery period in wt and eti5 mutant of Arabidopsis thaliana, differing in their sensitivity to ethylene, as well as to assess the sensitivity of both types to extreme temperatures. Experimental Wild type and ethylene-insensitive mutant (eti5) plants of Arabidopsis thaliana (L.) H e y n h were grown in a growth chamber on soil/perlite mixture (3:1) under the following conditions: 16/8 h day/night photoperiod; 70 µmol m–2 s–1 photon flux density; 26/22°C day/night temperature; 60% air humidity. 38-day-old plants were subjected to low 4°C (LT) or high 38°C (HT) temperatures for 24 h in darkness. After the temperature treatments normal growth conditions were restored and samples were collected at every subsequent stage of recovery period – 0, 24, 48 and 120 h. Measurements were performed using material derived from leaf nodes 3–7. Malondialdehyde (MDA) content was measured as a parameter reflecting bio­ membrane integrity deterioration20. The amount of carbonyl groups as products of protein breakdown was determined according to Levine21. Ascorbate/dehydroascorbate levels were quantified using the method of Law et al.22 Free proline content was quantified using the method of Bates et al.23 Hydrogen peroxide content was determined by a reaction with 1M potassium iodide24. Soluble protein content was determined according to Bradford25. The activities of the enzymes catalase10, POD (Ref. 26), and SOD (Ref. 27) measured were determined in 100 mM phosphate buffer (pH 7.6), containing 5 mM EDTA and 1% w/v polyvinylpyrrolidone. All experiments were repeated 3 times with 3 replicates each. The data reported are mean values ±SE. Results and discussion Comparative studies on the effects of temperature treatments (4°C, 24 h or 38°C, 24 h in darkness) on wt and eti 5 of Arabidopsis thaliana (L.) H e y n h were performed and the factors and mechanisms involved in the temperature stress in both Arabidopsis genotypes were studied. Our particular working hypothesis was that the extreme temperatures provoke an oxidative burst, and antioxidants and organic solutes play an important role in the possible tolerance, since a crucial factor to overcome oxidative stress is the speed of activation of plant antioxidant reserves. Correlative studies have 653

indicated that this response is an important aspect of the tolerance to a particular stress. Moreover, we were interested to determine whether the low- and high temperature stresses, both generating active oxygen species, would cause similar changes in AOS scavenging systems. Content of stress markers. Evidence has accumulated suggesting that AOS (superoxide, hydroxyl free radicals, hydrogen peroxide, and singlet oxygen), are the major deleterious factors in plants exposed to different environmental stresses, such as extreme temperatures, herbicides, salt and other xenobiotic treatments, water shortage, nutrient deficiency and toxicity. AOS caused deteriorations both in cell integrity and membrane functions by lipid peroxidation and oxidation of proteins. According to Suzuki and Mittler28, the role of AOS is not only in plant sensing but also in signalling mechanisms in relation to temperature stress. We did not measure the actual changes in the levels of free radicals (the reason for lipid peroxidation) and singlet oxygen (provoking various oxidative modifications of amino acids in proteins which lead to carbonyl formation) but some changes in the amounts of carbonyl groups and MDA content (Fig. 1) were detected. A sharp increase in the amount of carbonyl groups in the wt of Arabidopsis 0–24 h after both temperature treatments was observed, although the effect of HT was more dramatic. During the recovery period the amounts of carbonyl groups in treated plants tended to decrease, but did not reach the control level. In the eti5 mutant the carbonyl group content in controls was much higher than in wt and, although there were some changes in amounts in response to the treatments, these were relatively small. In relation to MDA (the final product of lipid peroxidation), the temperature stresses also acted in a different manner in wt and eti 5. In the wt a significant increase in MDA content was found at 0–24 h after the stress, and the effect of HT was more than 2-fold higher than that of LT, but by the end of the experiment the MDA content in HT-treated plants decreased to the control level, while in LT-treated plants it remained higher than the control. In eti5 where the content of MDA was comparable to that in wt the effects of both temperatures were again relatively small. Thus, on the basis of the overall net changes in the amounts of carbonyl groups and MDA, these data suggest that wt of Arabidopsis was more sensitive to both temperature treatments than the eti5 mutant. Similar data were reported by other authors, but in pea plants subjected to high salinity. According to them, the accumulation of stress-markers is higher in the susceptible variety (Lincoln) than the tolerant (Puget)29. A considerable increase in the malondialdehyde content was also established in broccoli plants subjected to LT (Ref. 30), which is related to oxidative processes. Similar effects were detected after LT treatment of Ixora coccinea31,32.

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Fig. 1. Changes in the content of carbonyl groups, malondialdehyde and free proline in rosette leaves of wild type (wt) and ethylene insensitive mutant (eti5) of Arabidopsis thaliana subjected to high (38°C) and low (4°C) temperature during the recovery period of 120 h

Usually most of the stresses provoke an increase in the amount of osmolytes – proline, soluble sugars and quaternary ammonium compounds. The increased proline concentration is supposed to play a role as a part of the endogenous defense system33, but is also discussed as a stress marker34. The amount of free proline in eti5 mutant plants was similar to wt in the controls, however, although some increase was observed in response to the treatments with more at LT than at HT the changes were small. On the other hand, in wt the free proline content increased markedly in HT and especially in LT-treated plants 24 h after treatments. However, at 48 h the amount of free proline decreased significantly and, 120 h after temperature stresses reached levels below control values. It is considered that in tolerant cells and plants the endogenous concentrations of this compound are higher. According to Tantau and Dörffling35, during the first day of a stress the increase in proline and soluble sugars caused by LT was more pronounced in chilling resistant Euphorbia pulcherima plants in comparison with the chilling sensitive ones. Ascorbate/dehydroascorbate levels. It was found that both temperature treatments considerably increased ascorbate+dehydroascorbate levels in wt (Fig.  2), which 655

remained high till the end of the experimental period as compared to the control. In eti5 ascorbate+dehydroascorbate levels were lower than in wt and did not change as markedly in response to stress or in the recovery period. Most probably the changes in wt are а prerequisite for the increased capability for detoxification of AOS by involvement of the ascorbate-glutathione cycle36,37. It is well known that the ascorbate plays a pivotal role in the plant antioxidant system by reducing the superoxide radical to hydrogen peroxide similarly to the superoxide dismutase, as well as by a rapid reaction with the singlet oxygen38. It may be the increased amount of ascorbate that indicates that this non-enzymatic antioxidant assists in the detoxification of AOS formed after low- and high-temperature treatment.

Fig. 2. Changes in the content of ascorbate+dehydroascorbate in rosette leaves of wild type (wt) and ethylene insensitive mutant (eti5) of Arabidopsis thaliana subjected to high (38°C) and low (4°C) temperature during the recovery period of 120 h

Defence enzyme system activities and hydrogen peroxide. Hydrogen peroxide concentration decreased markedly both in wt and eti5 control plants during the experimental period (Fig. 3). Hydrogen peroxide concentrations in treated wt plants were somewhat higher than the control, but whereas in HT-treated plants there was a drop towards control values during the recovery period, in LT-treated plants the concentration remained high. In eti5, on the other hand, both treatments resulted in decreased levels, which then rose during the recovery period. Okuda et al.39 also observed that the levels of H2O2 rise in leaves of cold-treated winter wheat. A similar situation was also registered in chilled maize seedlings40, and chilled Arabidopsis thaliana callus41 as well as in chilling-induced senescence of broccoli30. Heat shock was also found to increase the H2O2 content. When tobacco seedlings were subjected to 40°C for 1h a 50% increase of H2O2 was observed8.

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Fig. 3. Changes in the content of hydrogen peroxide and the activities of catalase, guaiacol peroxidase and superoxide dismutase in rosette leaves of wild type (wt) and ethylene insensitive mutant (eti5) of Arabidopsis thaliana subjected to high (38°C) and low (4°C) temperature during the recovery period of 120 h

Since the endogenous concentration per se of H2O2 (similar to other AOS) depends on the balance between the rate of its generation (as a metabolite of enzyme reactions and during photosynthesis) versus its decomposition and utilisation (enzymatic and non-enzymatic) the activities of SOD, catalase and POD were also evaluated. Catalase and peroxidase can destroy H2O2 while SOD catalyses the disproportionation of superoxide radicals into H2O2. Although SOD removes superoxide anions (O2–), it does so by converting a toxic derivative of oxygen (O2–) into another one (H2O2). Taken together, all these reactions result in the minimisation of the formation of hydroxyl radicals and singlet oxygen which both lead to lipid peroxidation and to protein oxidation. A number of data are available regarding the changes in the activity of catalase and peroxidase as a result of stress factors – high temperature42, water deficit43, herbicides44, etc.

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Catalase activity (Fig. 3) in both genotypes was similar. However, while levels rose somewhat in controls during the recovery period this was more marked in wt than in eti5. In wt the temperature treatments had little effect and levels changed little up to 120 h. In eti5, on the other hand, there was a marked increase in both treatments although the levels eventually fell back to control levels in the HT-treatment. Peroxidase activity (Fig. 3) was initially higher in wt than in eti5, but rose in both genotypes during the recovery period. However, whereas in eti5 both treatments evoke an increase in enzyme activity, in wt there is a decrease in response to HT and an increase in response to LT as compared to the control. In both genotypes there is a decreasing trend during recovery followed by a return closer to control values. The temperature treatment enhanced POD activity in eti5 immediately after the stress rather than the other antioxidant enzymes like catalase and SOD. Probably the observed significant decrease of H2O2 content in the treated mutant plants in the beginning of the recovery period is mainly due to its utilisation by peroxidases. Enhanced peroxidase activity after extreme temperature treatment, accompanied by an increased secondary metabolite production was reported by Zobayed et al.45 SOD activity increased somewhat immediately after both stresses in wt plants in relation to the control (Fig. 3), and it tended to decrease, including in the control, during the subsequent recovery period. At 120 h, SOD activity in both treatments was higher than in the control, but the effect of LT was more pronounced. In eti5, SOD activity was somewhat lower than in wt but changed little in the control and in LT-treatment over the recovery period. High temperature elicited an increase in this parameter, but returned to control values by the end of the recovery period. According to some authors, increased SOD activity is attributable to a higher tolerance to LT (Ref. 46). Comparing SOD activities in chilling-tolerant Zea diplo­ perennis and cold-sensitive Zea mays Jahnke et al.47 observed that SOD from chillingtolerant plants was double than that of cold-sensitive ones. However, contradictory results also exist29. Conclusions To conclude, the correlative investigation on the effects of LT- and HT-treatments showed that there were higher levels of the stress-markers MDA and carbonyl groups during the recovery period in the wt, which provides an indication that the wt is more sensitive to temperature stress than the mutant. Moreover, the observed decreased catalase activity (detoxifying H2O2) and increased SOD activity (producing H2O2) at the end of the experimental period in wt as compared to eti5 is also indicative that the wt is more susceptible to temperature treatments. Additionaly, the same temperature treatments caused significant alterations in the chlorophyllase activity of wt, while eti5 was not affected considerably48. On the other hand, the observed higher levels of stress markers in both genotypes after cessation of the HT-treatment as compared to the LT is indicative that the HT stress is more severe to Arabidopsis plants. This 658

is also supported by the fact that the HT-treatment provoked more significant alterations in polyamine18 and cytokinin19 levels, in chlorophyll content and chlorophyllase activity48, as well as in the parameters of the fast fluorescence reflecting the efficiency of photosystem II (unpublished data). This is probably due to the fact that this plant species is cold-tolerant. Acknowledgements This work was supported by the Copernicus Programme ERBIC15CT960914. References   1. E. L. Fiscus, R. Philbeck, A. B. Britt, F. L. Booker: Growth of Arabidopsis Flavonoid Mutants under Solar Radiation and UV Filters. Environ. Exp. Bot., 41, 231 (1999).   2. A. K. Mattoo, J. C. Suttle (Eds): The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, 1991.   3. V. Raz, R. Fluhr: Ethylene Signal Is Transduced via Protein Phosphorylation Events in Plants. Plant Cell, 5, 523 (1993).   4. R. Solano, J. Ecker: Ethylene Gas: Perception, Signaling and Response. Curr. Opinion in Plant Biol., 1, 393 (1998).   5. A. Stepanova, J. Ecker: Ethylene Signaling: from Mutants to Molecules. Curr. Opinion in Plant Biol., 3, 353 (2000).   6. H. K. Lichtenthaler: An Introduction to the Stress Concept in Plants. J. Plant Physiol., 148, 4 (1996).   7. K. Asada: Ascorbate Peroxidase – a Hydrogen Peroxide Scavenging Enzyme in Plants. Physiol. Plant., 85, 235 (1992).   8. C. Foyer, H. Lopez-Delgado, J. Dat, I. Scott: Hydrogen Peroxide- and Glutathioneassociated Mechanisms of Acclimatory Stress Tolerance and Signalling. Physiol. Plant., 100, 241 (1997).   9. C. Foyer, P. Descourvieres, K.-J. Kunert: Protection against Oxygen Radicals: an Important Defence Mechanisms Studied in Transgenic Plants. Plant Cell Environ., 17, 507 (1994). 10. T. Brennan, C. Frenkel: Involvement of Hydrogen Peroxide in Regulation of Senescence in Pear. Plant Physiol., 59, 411 (1977). 11. S. Gilmour, R. Hajela, M. Thomashow: Cold Acclimation in Arabidopsis thaliana. Plant Physiol., 87, 745 (1988). 12. J. Jarillo, A. Leyva, J. Salinas, J. M. Martinez-Zapater: Low Temperature Induces the Accumulation of Alcohol Dehydrogenase mRNA in Arabidopsis thaliana, a Chilling-tollerant Plant. Plant Physiol., 101, 833 (1993). 13. M. Uemura, R. A. Joseph, P. L. Steponkus: Cold Acclimation of Arabidopsis thaliana. Effect on Plasma Membrane Lipid Composition and Freeze-induced Lesions. Plant Physiol., 109, 15 (1995). 14. R. McKown, G. Kuroki, G. Warren: Cold Responses of Arabidopsis Mutants Impaired in Freezing Tolerance. J. Exp. Bot., 47, 1919 (1996). 15. N. Harpham, A. Berry, E. Knee, G. Roveda-Hoyos, I. Raskin, I. Sanders, A. Smith, C. Wood, M. Hall: The Effect of Ethylene on the Growth and Development of Wild-type and Mutant Arabidopsis thaliana (L.) H e y n h. Ann. Bot., 68, 55 (1991). 16. I. Sergiev, D. Todorova, V. Alexieva, E. Karanov, A. Smith, M. Hall: Rosette Leaf Senescence in Wild Type and an Ethylene Insensitive Mutant of Arabidopsis thaliana during

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38. J. Leipner, P. Stamp, Y. Frachebound: Artificially Increased Ascorbate Content Affects Zeaxanthin Formation but not Thermal Energy Dissipation or Degradation of Antioxidants during Cold-induced Photooxidative Stress in Maize Leaves. Planta, 210, 964 (2000). 39. T. Okuda, Y. Masuda, A. Yamanaka, S. Sagisaka: Abrupt Increase in the Level of Hydrogen Peroxide in Leaves of Winter Wheat Is Caused by Cold Treatment. Plant Physiol., 97, 1265 (1991). 40. T. Prasad, M. Anderson, B. Martin, C. Steward: Evidence for Chilling-induced Oxidative Stress in Maize Seedlings and a Regulatory Role for Hydrogen Peroxide. Plant Cell, 6, 65 (1994). 41. D. O’Kane, V. Grill, P, Boyd, R. Burdon: Chilling, Oxidative Stress and Antioxidant Responses in Arabidopsis thaliana Callus. Planta, 198, 371 (1996). 42. S. Ivanov, T. Konstantinova, D. Parvanova, D. Todorova, D. Djilianov, V. Alexieva: Effect of High Temperatures on the Growth, Free Proline Content and Some Antioxidants in Tobacco Plants. Compt. Rend. Acad. Bulg. Sci., 54 (7), 71 (2001). 43. D. Todorov, V. Alexieva, E. Karanov: Effect of Putrescine, 4PU-30 and Abscisic Acid on Maize Plants Grown under Normal, Drought and Rewatering Conditions. J. Plant Growth Regul., 17, 197 (1998). 44. I. Moskova D. Todorova, V. Alexieva, S. Ivanov, I. Sergiev: Effect of Exogenous Hydrogen Peroxide on Enzymatic and Nonenzymatic Antioxidants in Leaves of Young Pea Plants Treated with Paraquat. Plant Growth Regul., 57, 193 (2009). 45. S. M. A. Zobayed, F. Afreen, T. Kozai: Temperature Stress Can Alter the Photosynthetic Efficiency and Secondary Metabolite Concentrations in St. John’s Wort. Plant Physiol. Biochem., 43, 977 (2005). 46. M. Seppanen, K. Fagerstedt: The Role of Superoxide Dismutase Activity in Response to Cold Acclimation in Potato. Physiol. Plant., 108, 279 (2000). 47. L. Jahnke, M. Hull, S. Long: Chilling Stress and Oxygen Metabolizing Enzymes in Zea mays and Zea diploperennis. Plant Cell Environ., 14, 97 (1991). 48. D. Todorov, E. Karanov, A. Smith, M. Hall: Chlorophyllase Activity and Chlorophyll Content in Wild and eti5 Mutant of Arabidopsis thaliana Subjected to Low and High Temperature. Biol. Plant., 46, 633 (2003). Received 12 January 2010 Revised 17 February 2011

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Oxidation Communications 35, No 3, 662–673 (2012) Effect of nanoparticles in biological systems

Noble Metal Nanoparticles K. A. Stancheva Department of Inorganic and Analytical Chemistry, ‘Prof. Dr. Assen Zlatarov’ University, 8010 Bourgas, Bulgaria E-mail: [email protected] ABSTRACT The rapid development of synthesis of noble metal nanoparticles over the last 10–15 years has given a broad arsenal of opportunities to the scientists, beginning from the well-known nanoparticles, ‘nanoclouds’, ‘nanospheres’, ‘nanostars’, ‘nanonecklaces’, ‘nanorods’, ‘nanowires’, ‘nanosensors’, ‘nanochips’, ‘nanocells’. The unique nanoparticle properties have expanded their applications in a wide range of fields such as chemistry, physics, medicine, material science, biology, etc. Nanoparticles of gold, platinum and silver have found applications in electronics, catalysis and ceramics, in pharmacy, in the production of polymers with better properties, in optics and others. The resonance optical properties of nanoparticles, along with the high specificity of biomolecular recognition, have opened a new avenue for applications in biomedical diagnostics, targeted drug delivery, optical imaging and photothermal therapy of cancer cells in vitro. The analysis of literature survey has shown that the major contemporary research directions are on the development of new methodologies for synthesis of nanocrystal structures having definite sizes and shapes. Multitude experimental works on synthesis of noble metal nanopatricles have been proposed. The properties of nanoparticles depend on many controllable, variable parameters. The use of a control of a synthesis of nanoparticles of noble metals having engineered properties is determined by the strong influence of their size, shape, structure and aggregates on their physical chemical properties as well as on their resonance and spectral characteristics. Keywords: noble metal nanoparticles, synthesis, properties, characterisation. AIMS AND BACKGROUND Gold, silver, platinum – one of the first metals discovered by the people, that story of the studying began several centuries ago. The research investigation of colloidal noble metal particles has been traditionally associated with the name of Faraday, though solutions of colloidal gold had been well known to the alchemists in search 662

for Panacea – a connection between the elixir of life and the philosopher stone. Even in the 16th century Paracelsus used colloidal gold solution in alcohol tincture in herbs as a curative medicament in his medical practice. Colloidal gold was used to colour glasses and ceramics in antiquity. A famous example of this is the Lycurgus cup, assumed to be made in Rome around the 4th century BC, which is currently on display at the British Museum (Fig. 1).

Fig. 1. The Lycurgus cup, appears red in transmitted light (right) and green in reflected light (left) due to the presence of gold/silver colloids

Current Trends The noble metal nanostructures have been intensively studied within the past decade. Nanosized materials have been an important subject in theoretical and applied sciences; the unique properties of nanoparticles enlarged their application in a broad ranges of different fields, including chemistry, physics, biology, materials science, medicine, catalysis and so on1–3. Nano-engineered products with noble nanoparticles have applications as semiconductor nano-crystallites for use in microelectronics, in ceramics, catalysis and pharmacy, in production of polymers with enhanced functional properties, for transparent coatings with UV/IR absorption properties, as abrasionresistant materials and optical lenses, etc. Noble nanoparticles have well characterised physical properties, such as coatings on glasses and ceramic to produce a desired hue. Besides the melting temperature of the glasses and ceramics is lowered and energy consumption is dramatically lowered4. Noble metal layers supported on metal oxide surfaces have diverse applications ranging from catalysis to microelectronics. Pt nanoparticles supported on yttriastabilised zirconia are used in solid oxide fuel cells5. Ag nanoparticles deposited on glass are used as optical sensors6. During the last years the noble metal nanoparticles and their composites are widely used as effective transformers of biospecific interactions. The resonance optical 663

properties of nanoparticles are successfully applied for development of biochips and biosensors. Such devices are from great interest in biology (determination of DNA, proteins, and metabolic processes), medicine (screening drugs, revealing antibodies and antigens, infection diagnostic) and technique (Fig. 2).

Fig. 2. Antimicrobial activity of silver nanoparticles: 1 – colonies of E. coli grown plates supplemented with silver nanoparticles: 0 (a), 10 (b), 20 (c), and 50 (d) mg/ml (Ref. 7); 2 – transmission electron micrograph of E. coli cell treated with 50 µg/ml of silver nanoparticles in liquid Luria–Bertani (LB) medium for 1 h (Ref. 7)

Nanoparticles of noble metals have variety of applications in nanobiotechnology and nanomedicine, owing to the potency of spectral position and amplitude of their plasmon resonance (PR). The plasmon resonance is a strong absorbance at a specific frequency, which induces the collective oscillation of electrons at the surface of the metal particles. For nanoparticles, localised surface plasmon oscillations can give rise to the intense colours of suspensions or sols containing the nanoparticles. The ruby colour, for example, is caused by excitation of a collective oscillation of valence electrons in gold nanoparticles. The wavelength of the PR depends on the size, shape, topology and the dielectric environment of the metal clusters (Fig. 3).

Fig. 3. Gold nanoparticles solutions with different sizes of the particles

Nanoparticles of noble metals exhibit strong absorption bands in the ultravioletvisible light regime that are not present in the bulk metal. Shifts in this resonance due to changes in the local index of refraction upon adsorption to the nanoparticles can also be used to detect biopolymers such as DNA or proteins and it has application for detection of biospecific conjugated macromolecules in express clinical diagnostic (Fig. 4). 664

Fig. 4. Spectral characteristics: a – 3, 10, 20, 50 nm gold nanoparticles, and b – 20 nm silver nanoparticles obtained under different conditions and solvents

Recently in the medicine colloid particles having spherical forms (nanospheres) are used. The use of nonspherical or/and inhomogeneous particles (for instance, the gold nanoshells7,8, nanorice9,10 and nanostars11) as well as one-dimension chains or planar arrays formed by solid or polymer-coated spheres12,13, bispheres14, nanoholes15, nanorods16, or island films17 opens new possibilities for controlled spectral tuning the light plasmon resonance (LPR) and for enhanced detection sensitivity to the biospecific molecular binding on nanometal surface. Applications of PR nanostructures to biomedical science and practice are based on 2 principles: (1) the biospecific targeting of tissues or cells, and (2) the optical PR. Nobel nanowires have ~20 nm thickness and 200 to 2000 nm length with and absorptions from the near to mid-IR. They improve solar cell efficiencies, optics, and nanoelectronics. These ultra-long nanodevices exhibit tremendous photothermal properties. Nanowires are an extension of the technology for nanorods. Gold nanorods have recently found huge successes in cancer therapy. Gold nanorods are also used for blood testing in diagnostics; as optical contrast agents in imaging, in material science, optics, and for improving the density of optical data storage in compact disks. Silica (core)/gold, platinum or silver (shell) nanospheres represent multilayered sphere with a silica core, gold, silver or platinum nanoshell. Silica(core)/gold(shell) nanoclouds have applications to photothermal therapy of tumors. Effect of such nanoparticles on the laser photothermolysis of tumor of dogs and cats is studied in vivo by the local injection of nanoparticles around the tumor18–20. The laser phototermolisis in vivo of cat and dogs shows that the temperature in the volume region of nanoparticles localisation can substantially exceed the surface temperature recorded by the thermal imaging system20. The authors demonstrates effective optical destruction of cancer cells by local injection of PR gold nanoshells followed by continuous wave semiconductor laser irradiation at wavelength 808 nm. About 0.1 ml of the nanoshell suspension was injected having particle concentration 5×10–9 µg/ml and 1×10–9 µg/ml. In this work a positive therapeutic effect was observed as a result of undertaken complex therapy20 (Fig. 5).

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Fig. 5. Photograph (a) and thermogram (b) of cutaneous squamous cell carcinoma of external ear canal20

The initial experiments on animals and living cells showed that the cancer cells that are injected with nanoparticles die as at the same time the healthy cells are not affected. The great challenge before the scientists is discovery of proteins that are able to be connected with cancer cells, but are not able to do the same with the living cells. Recently the scientists created biosensors with nanorods21–23. Such sensors allow monitoring in vivo biological process within single living cells and the cell biological processes21. Gold nanoparticles are developed as contrasting agents for photo-acoustical medical imaging. The scientists study cancer cells by treating them trough conjugated gold nanorods with antibodies (glycoproteins). Their absorption peak can be adjusted toward near IR (700–900 nm) area for an augmentation of their penetrated capacity in depth in the living cell22,23. METHODS OF SYNTHESIS OF NOBLE METAL NANOSTRUCTURES Nobel nanoparticles are attractive due to their comparatively easy methods of synthesis and modification as well as their size, shape, distribution are properties dependent. Several classes of synthesis methods exist thus displaying different characteristics of the nanoparticles. Basically nanoparticles can be spectroscopically characterised on the basis of their sizes and the concentration of the synthesised nanoparticles can be revealed. The synthesis metods of noble nanoparticles are divided in 2 major groups: disperce methods based on the dispersion of the metal and condensation methods (chemical, photo-, radio-chemical, in the presence of nucleuses, surfactants, etc.), where the corresponding salt is formatted the presence of the nanoparticles from the reduction of the metals ions. Different chemicals are used for reducing agents in the process of nanoparticles production, as they are either inorganic such as sodium/potassium borohydrate24, hydrazine25 and salts of tartarate26, or organic ones like sodium citrate27, ascorbic acid28,29 and amino acids capable of being oxidised30,31. Then the nanoparticles formed need to 666

be stabilised for further use. Various reagents have been reported to serve as stabilising agents. These include polymers such as different kinds of polyethyleneglycol32,33, polyvinyl alcohol34, polyvinyl pyrilidon35,36 and the surfactant, viz. sodium dodecelsulphate37–39, triton40 and carbohydrates-like chitosane41, etc. METHODS OF SYNTHESIS OF NANOPARTICLES

Gold nanoparticles. The usual synthetic route to prepare gold nanoparticles involves the reduction of a suitable metal salt in solution in the presence of a stabiliser. The size and morphology of the gold nanoparticles can be tuned by varying the concentration ratio of capping agent to metal salts and by choosing a suitable reducing agent. A weak reducing agent, such as citrate or tartarate, will favour a slow reaction allowing particle growth over a long period to yield facetted and small nanoparticles, whereas with relatively strong reducing agents, such as formamide (methanamide) or o-anisidine, bigger and generally spherical nanoparticles will form. Active reducing agents are ascorbic acid and formaldehyde, can reduce the gold (III) as metal even in normal temperature and even in acid medium. Compexon III reduces the gold in heating or irradiation of the solution by UV light, β- or γ-rays; in alkali solution Au (III) can be reduced without heating. Gold nanoparticles can be synthesised and stabilised by peptides, proteins, DNA and chemical/biological polymers. The most popular example for nanoparticles production is the reduction of chloroauric acid (HAuCl4) by sodium citrate (Na3C6H5O7), as the medics use the method of Frens42 and the biologists use the method of Turkevich43. For Au nanoparticles can be written the equation: [HAuCl4] + 3e– = Au° + H+ + 4Cl–.

(1)

Silver nanoparticles. Nearly monodisperse silver nanoparticles can also be synthesised via reduction of a suitable Ag salt, usually AgNO3 or silver acetate, in aqua solution in the presence of a stabiliser. NaBH4, sodium citrate, potassium bitartarate, dimethyl formamide, ascorbic acid, and alcohols are some of the reducing agents that have been successfully used. Precipitation can be done by reducing silver nitrate solution with ascorbic acid in the presence of stabilising agent: 2Ag+ + C6H8O6

2Ag0 + C6H6O6 + 2H+.

(2)

Long-chain n-alkanethiols are the most common protective agents employed to stabilise silver colloids, even though aromatic amines such as aniline, carboxylic acids, and polymers have been also employed. Gold nanoparticles also can be prepared by these agents and many variations of this method have been developed in the past decade. Platinum and palladium nanoparticles. The usual technique for the synthesis of platinum and palladium nanoparticles is the chemical reduction of the metal salt, usually a chloride, in the presence of a stabiliser. A variety of reducing agents have 667

been successfully employed, including NaBH4, potassium bitartarate, superhydrides as lithium triethylborohydride, amines, and alcohols. The most popular procedure for platinum nanoparticles synthesis is the reduction of PtCl62– by citrate. The most common capping agents are n-alkanethiols and polymers, but a few examples of Pt and Pd nanoparticles capped with amines, alkyl isocyanides, and cyclodextrins have also been reported. Platinum particles of varying shapes are synthesised by reduction in the presence of CTAB as a stabiliser. Using this method, cubes, octahedral, and porous particles have been realised. NANORODS SYNTHESIS

Formation of spherical and cylindrical nanostructures in isotropic medium is related to the spontaneous aggregation of the nearly spherical nucleuses stabilised. To obtain no spherical nucleuses syntactical anisotropic conditions are created. Such a method is a synthesis of noble metallic soles in porous materials such as oxides of Si and Al, and the reduction of the metals is electrochemical44,45. Another variant of synthesis of nanorods is a reduction of micel by surfactants. The solution of surfactant cetrimonium bromide (CTAB) ((C16H33)N(CH3)3Br) is widely applied. CTAB capable to form different kind of micelles – spherical, cylindrical, plastic. The nucleuses employed are ultra disperse (3 nm) which are synthesised by a reduction of the corresponding noble metal salt with NaBH4 (Refs 46–48). Electrochemically obtained Au nanorods are used as nucleuses around which a thin gold, silver or platinum nanoparticles film is formed by meance of a reduction with ascorbic acid in appropriate environment49.

Fig. 6. (1) TEM images of gold nanorods after Au overgrowth with AgNO3 (a –50 µl; b – 75 µl) addition49 and (2) TEM images of thin gold nanowires produced with a photochemical reduction process using Au nanorods49. The arrows in (2) indicate the possible nanorod seeds

NANOSPHERES SYNTHESIS

Spherical nanoclouds represent thin layer of metal on dielectric nucleus having diameter ~100 nm. They absorb light in red or near IR spectral area and have a high light diffraction as these their properties make them perspective in diagnostic and therapeutic instrumentation. 668

Spherical nanostructures can be produced in 4 stages: (1) obtaining of spherical Si nucleuses; (2) functionalisation of their surface with amino groups; (3) absorption of metal particles on the amino groups; (4) condensation of metal centers50,51. METHODS FOR IDENTIFICATION AND CHARACTERISATION

Basically nanoparticles can be spectroscopically characterised on the basis of their sizes and the concentration of the synthesised nanoparticles can be revealed. IDENTIFICATION

Research, development, production, and applications of nanotechnology require the following facilities: UV-vis. spectroscopy. The formation of nanoparticles is followed by scanning the solution containing noble nanoparticles at the wave length ranged from 400–800 nm using UV-vis. spectrophotometer. The absorption is a function of the size and concentration of the particles (Figs 7 and 8). 2.5

2.0

c 1.5

a

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0.5

0.0 200

d

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absorbance

absorbance

2.0

300

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500

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wavelength (nm)

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900

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0.0 200 300 400 500 600 700 800 900

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Fig. 7. Absorption spectra of silver hydrosols recorded at different reaction times: 15 s (a), 30 s (b), 1 min (c), 3 min (d), and 7 min (e) (end of reaction)52

Fig. 8. Absorption spectrum of platinum particles of aged samples (1 × 10–4 M K2PtCl4 and 0.2 ml of 0.1 M polymer)52

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The optical properties of noble nanoparticles are decidedly dependent on: particle size, interparticle distance, type of surfactant, shape of the nanoparticle, dielectric constant of the dispersion medium. Transmission electron microscopy (TEM). The analysis of the synthesised noble nanoparticles is carried out on the film coated drop of nanoparticles employing transmission electron microscopy (Fig. 9).

Fig. 9. TEM image of gold nanoparticles synthesised by the modified Turkevich method as employing L-tryptophane as reducing agent53

Scaning electron microscopy (SEM). The SEM is a type of electron microscope that images the sample surface by scanning it with a high-energy beam of electrons. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample surface topography, composition and other properties such as electrical conductivity (Fig. 10).

Fig. 10. SEM image of gold nanoclouds having diameter 220 nm (PR 900 nm) and TEM image of gold nanorods15×50 nm (PR 780 nm)54,55

CONCLUSIONS A major current research direction is to develop methodology for size- and shapeselective noble nanocrystal growth. There have been extensive efforts focusing on the development of new synthetic methods for making nanoparticles with uniform sizes and aspect ratios. Fundamental studies on the synthesis of noble metal nanoparticles are directed to their use in areas such as chemistry, physics, material science, biology and so on.

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ACKNOWLEDGEMENT The author wish to extend her sincere appreciation to the Ministry of Education and Science – Bulgaria for the grant allocation! References   1. K. Watanabe, D. Menzel, N. Nilius, H. J. Freund: Photochemistry on Metal Nanoparticles. Chem. Rev., 106, 4301 (2006).   2. O. Salata: Nanoparticles – Known and Unknown Health Risks. J. Nanobiotechnol., 2, 1 (2004).   3. C. A. Mirkin, T. A. Taton: Materials Chemistry: Semiconductors Meet Biology. Nature, 405, 626 (2000).   4. G. De, S. K. MEDDA, S. DE, S. PAL: Metal Nanoparticle Doped Coloured Coatings on Glasses and Plastics through Tuning of Surface Plasmon Band Position. Bull. Mater. Sci., 31, 479 (2008).   5. Y. I. Park, P. C. Su, S. W. Cha, Y. Saito, F. B. Prinz: Thin-film SOFCs Using Gastight YSZ Thin Films on Nanoporous Substrates. J. of Electrochem. Soc., 153, A431 (2006).   6. A. J. Haes, S. L. Zou, G. C. Schatz, R. P. van Duyne: A Nanoscale Optical Biosensor: the Long Range Distance Dependence of the Localized Surface Plasmon Resonance of Noble Metal Nanoparticles. J. of Phys. Chem. B, 108, 109 (2004).   7. I. SONDY, B. SALOPEK-SONDY: Silver Nanoparticles as Antimicrobial Agent: A Case on E. coli as a Model for Gram-negative Bacteria. J. Colloid and Interface Sci., 275, 177 (2004).   8. Y. Sun, Y. Xia: Gold and Silver Nanoparticles: A Class of Chromophores with Colors Tunable in the Range from 400 to 750 nm. Analyst, 128, 686 (2003).   9. L. A. Trachuk, S. A. Vrublevsky, B. N. Khlebtsov, A. G. Melnikov, N. G. Khlebtsov: Optical Properties of Gold Spheroidal Particles and Nanoshells: Effect of the External Dielectric Medium. In: Optical Technologies in Biophysics and Medicine (Ed. V. V. Tuchin). VI Proc. SPIE, 5771, 1 (2005). 10. H. Wang, D. W. Brandl, F. LE, P. Nordlander, N. J. Halas: Nanorice: A Hybrid Plasmonic Nanostructure. Nano Lett., 6, 827 (2006). 11. C. L. Nehl, H. Liao, J. H. Hafner: Optical Properties of Star-shaped Gold Nanoparticles. Nano Lett., 6, 683 (2006). 12. P. K. Jain, S. Eustis, M. A. El-Sayed: Plasmon Coupling in Nanorod Assemblies: Optical Absorption, Discrete Dipole Approximation Simulation, and Exciton-coupling Model. J. of Phys. Chem. B, 110, 18243 (2006). 13. C. J. Murphy, A. M. Gole, S. E. Hunyadi, C. J. Orendorff: One-dimensional Colloidal Gold and Silver Nanostructures. Inorg. Chem., 45, 7544 (2006). 14. S. Enoch, R. Quidant, G. Badenes: Optical Sensing Based on Plasmon Coupling in Nanoparticle Arrays. Opt. Express, 12, 3422 (2004). 15. S. H. Chang, S. K. Gray, G. Schatz: Surface Plasmon Generation and Light Transmission by Isolated Nanoholes and Arrays of Nanoholes in Thin Metal Films. Opt. Express, 13, 3150 (2005). 16. M. B. Cortie, X. Xu, M. J. Ford: Effect of Composition and Packing Configuration on the Dichroic Optical Properties of Coinage Metal Nanorods. Phys. Chem. Chem. Phys., 8, 3520 (2006). 17. G. Kalyuzhny, M. A. Schneeweiss, A. Shanzer, A. Vaskevich, I. Rubinstein: Differential Plasmon Spectroscopy as a Tool for Monitoring Molecular Binding to Ultrathin Gold Films. J. of Am. Chem. Soc., 123, 3177 (2001). 18. D. P. O’Neal, L. R. Hirsch, N. J. Halas, J. D. Payne, J. L. West: Photo-thermal Tumor Ablation in Mice Using Near Infrared-absorbing Nanoparticles. Cancer Lett., 209, 171 (2004). 19. W. R. Glomm: Functionalized Gold Nanoparticles for Application in Biotechnology. J. of Dispers. Sci. Technol., 26, 389 (2007).

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Oxidation Communications 35, No 3, 674–683 (2012) Biological section

Phenolic Compounds and Antioxidant Capacities of Dried Raspberry from Serbia, Extracted with Different Solvents M. N. Mitic*, M. V. Obradovic, D. A. Kostic, A. N. Pavlovic, J. M. Brcanovic Department of Chemistry, Faculty of Sciences and Mathematics, University of Nis, 33 Visegradska Street, P.O.Box 224, 18 000 Nis, Serbia E-mail: [email protected] ABSTRACT The antioxidant capacity and phenol content of 3 dried raspberry samples from different geographical regions were studied. Three solvent systems were used (methanol, ethanol and acetone) at same concentrations (70%) and with 100% deionised water in the presence 0.1% HCl. The antioxidant capacity of the dried fruit extracts was evaluated using 2,2-diphenyl-1-picrylhydrazyl radical-scavenging assays. The efficiency of the solvents used to extract phenols from the 3 dried raspberry samples varied considerably. The polyphenol content of dried raspberry samples was 9.82 to 16.73 mg gallic acid equivalent/g of dried fruit. The high phenol content was significantly correlated with high antioxidant capacity. The following compounds were identified and quantified using HPLC-DAD: 2-anthocyanins, 2-flavonols, 2-flavan-3-ols and 3-hydroxycinnamic acids in all dried raspberry samples. Generally, raspberry fruits are a rich source of phenolics, which shows an evident antioxidant capacity. Keywords: dried raspberry, phenolic profile, antioxidant capacity, HPLC-DAD. AIMS AND BACKGROUND Berry fruits are very rich sources of bioactive compounds such as polyphenolics, anthocyanins, ascorbic acid and minerals. The chemical composition of berry fruits can be highly variable depending on the cultivar, growing location, ripeness stage, harvest, and storage conditions1. Higher intakes of phenolics and other antioxidant compounds from foods are associated with reduced risk of cancer, heart disease, and stoke2,3. Raspberry (Rubus idaeus L.), member of the Rosaceae family, provide delicious fruits that can be consumed fresh or as ingredient in processed products such *

For correspondence.

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as ice cream, jam, jelly, marmalade, purees, fruit juices, liquors, wines, dried fruits, etc. In Serbia, raspberry is one of the most important berries, grown extensively on a commercial basis primary for export. Dried fruits retain most of the nutrition value of fresh fruits. Dried fruit are fruits where a large portion of their original water content has been removed either naturally, through sun drying, or through the use of specialised dryers or dehydrators. Dried fruits has a long tradition of use dating back to the 4th millennium BC in Mesopotamia, and are appreciated because of their sweet taste, nutritive value and long shelf-life. Today, dried fruit consumption is widespread. The specific nutrient content of the different dried fruits reflects their fresh counterpart and the processing method (e.g. traditional dried fruits versus sugar infused dried fruit). Dried fruit are an excellent source of polyphenols and phenolic acids. Different dried fruits have unique phenolic profiles. There are several methods established for the extraction of polyphenols from fruit materials. These methods vary in solvents and conditions used. The extraction method is essential for the accurate quantification of phenolic content and antioxidant capacity. This fact makes it hard to compare data from literature reports due to the reason mentioned earlier4. Naczk and Shahidi5 identified a group of factors that influence the quantification of phenolics in fruit materials. The chemical nature of the phenolic compounds, the extraction method employed and the assay method were some of those factors. Several methods for the analysis of polyphenols have been proposed in literature, most of which are based on high performance liquid chromatography (HPLC) coupled with either a photodiode array detector (DAD) or a mass spectrometer. Since UV-vis. detection depends on the chemical structure of a molecule, several wavelengths could be selected monitoring. Red-coloured anthocyanins show an absorbance maximum at around 520 nm; yellow-coloured flavonols display an absorbance maximum at around 360 nm; hydroxycinnamic acids can be specifically detected by their high absorbance around 320 nm. Flavan-3-ols show no specific absorbance and have a maximum around 280 nm, as do all the above-mentioned phenolics. In contrast, flavan-3-ols have fluorescence properties that the other polyphenols do not. This is the reason, why the use of a fluorescence detector has been proposed for analysing flavan-3-ols6. The objective of this present study was to evaluate dried raspberries cultured from different location in Serbia in terms of their biochemical characteristics, and to investigate the effects of different solvent extraction systems on the content of phytochemicals such as phenolics, flavonoids, and anthocyanins. EXPERIMENTAL Chemicals. Standards of catechin, epicatechin, quercetin, kaemferol and the phenolic acid standards, such as gallic, ferulic, p-coumaric and caffeic acids, were purchased from Sigma Chemicals Co. (St. Louis, Mo). Cyanidin-3-glucoside and 675

cyanidin-3-rutinoside were purchased from Extrasynthèse (Ganay, France). 2,2diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Steinheim, Germany). 6-hydroxy-2,5,7,8-tetramethylchromancarboxylic acid (Trolox) and the Folin–Ciocalteu phenol reagent were obtained from Merck (Darmstadt, Germany). Other chemicals and solvent were of analytical grade. Fruit samples. One raspberry cultivar from 3 different geographical regions: Velika Plana (sample 1), Niska Banja (sample 2) and Vlasina (sample 3) were analysed. Berries were harvested of the 2nd decade of June 2010. Plantations were built on sandy loam soil type. Samples were taken from 5 bushes and dried at room temperature until constant weight. Firstly, raspberry samples (10.00 g) were homogenised in a blender, and samples (each weighed 3.00 g) were extracted with the methanol–water system, ethanol–water system and acetone–water system (80% methanol, ethanol or acetone) containing 0.1% HCl volumes (20, 10 and 10 ml, respectively), 3 times in the further course. The samples were mixed in an ultrasound bath during the extraction procedure. Such obtained extracts were filtered using the Buchner funnel and Watman No 1 filter paper. The solid residues were rinsed for several times in order to gain transparent extracts. Finally, the obtained dried raspberry extracts were collected in a graduated flask of the same volume of 50 ml. Determination of total phenolic compounds. The Folin–Ciocalteu reagent was used to determine the total phenolic compounds7. A volume of 1 ml of dried raspberry extract, diluted 5–6 times with methanol (to obtain absorbance within the range of the prepared calibration curve), was mixed with 0.5 ml of the Folin–Ciocalteu reagent previously diluted with distilled water (1:2). A volume of 2 ml of 20% sodium carbonate solution was added to the mixture, shaken thoroughly and diluted to 10 ml by adding distilled water. The mixture was left to stand for 120 min and the blue colour formed was measured at 760 nm with a spectrophotometer (UV-vis. spectrometer Agilent 8454). Gallic acid was used as a standard for the calibration curve. The concentrations of gallic acid in the solution, from which the curve was prepared, were 0, 50, 100, 150, 250 and 500 mg/l (R2=0.998). The content of TP was expressed as mg of gallic acid equivalent (GAE) per 1 g of dried fruit (d.f.). All measurements were carried out in 3 repetitions. Determination of total flavonoids. The total flavonoids (TF) assay was done as previously described by Yang et al.8 with minor modifications. A volume of 1 ml of diluted extracts or standard solution of catechin (50–500 mg/l) was placed in a 10-ml volumetric flask, then 4 ml of dd H2O, and after 5 min 0.3 ml of NaNO2 (5 %) and 1.5 ml of AlCl3 (2 %) were added. The mixture was shaken and 5 min later 2 ml of 1M solution of NaOH were added, again well shaken. The absorbance was measured at 510 nm against the blank. The results were calculated according to the calibration curve for catechin (R2=0.999). The content of TF was expressed as mg of catechin equivalent (CE) per 1g of dried fruit. All samples were analysed in triplicate. 676

Measurement of the DPPH. scavenging activity. The free radical scavenging capacity of dried raspberry extracts was determined according to the previously reported procedure using the stable DPPH radicals9. The method was based on the reduction of stable DPPH nitrogen radicals in the presence of antioxidants. An aliquot (2.5 ml) of dried fruit extracts or methanol solution of Trolox (10–30 mM) was mixed with 2.5 ml of 0.1 mM DPPH methanol solution. The mixture was thoroughly vortexes, kept in the dark for 30 min, and after that the absorbance was measured at 515 nm against a blank of methanol without DPPH. The results were calculated according to the calibration curve for Trolox (R2=0.996). DPPH values, derived from triplicate analyses, were expressed as mmol of TE per 1g of dried fruit. HPLC-DAD determination of phenolics composition. The individual phenolics were analysed by the direct injection of the extracts (previously filtered through a 0.45-µm pore size membrane filter) into an Agilent 1200 chromatographic system equipped with a quaternary pump, and Agilent 1200 photodiode array detector with radiofrequency identification tracking technology for flow cells, an UV-lamp, an Agilent 1200 fluorescence detector for multi-wavelength detection, online acquisition of excitation (ex) and emission (em) spectra, an 8 µl flow cell, and automatic injector and Chemstation software. The column was thermostated at 30oC. After injecting 5 µl of sample extract, the separation was performed in the Agilent-Eclipse XDBC-18 4.6×150 mm column. Two solvents were used for the gradient elution: A-(H2O+5% HCOOH) and B-(80% ACN (acetonitrile) +5 % HCOOH+H2O). The elution program used was as follows: from 0 to 28 min, 0.0% B, from 28 to 35 min, 25% B, from 35 to 40 min, 50% B, from 40 to 45 min, 80% B, and finally for the last 10 min again 0% B. The detection wavelengths were 280, 320, 275/322 (λex/λem), 360 and 520 nm. Identification and quantification of the various phenolic compounds were made by means of calibration curves obtained with standard solutions of cyanidin-3-glucoside, quercetin, kaempferol, catechin, epicatechin, caffeic acid, p-coumaric acid and ferulic acid. Results are expressed as mg per 100 g of dried fruit. Statistical analysis. The data were reported as mean ±standard deviation (SD) for triplicate determinations. The significance of inter-group differences was determined by the analysis of variance (ANOVA). The p value of 18 > 17 > 16> 15 > 9 > 10 > 8 > 12 > 13 > 11 > 14 > 5 > 7 > 6 > 2>3>4>1. S. sorbrinius 21 > 19 > 20 > 16 > 17 > 18 > 15 > 10 > 12 > 14 > 9 > 8 > 13 > 11 > 7 > 6 > 3>2>4>1>5. S. artimidis 19 > 21 > 20 > 16 > 18 > 17 > 15 > 7 > 8 > 10 > 12 > 13 > 14 > 9 > 7 > 5 > 6 > 11 > 4 > 2 > 3 > 7 . Perusal of Table 1 shows that 2-hexylphenol (21) exhibits highest activity against P. gingivalis and S.sorbrinius, whereas 4 pentylphonol (19) displays the highest active against S. artemidis. The lowest activity (–log MIC) is shown by phenol (1) against P. gingivalis and S. artemidis and by 2-ethylphenol (5) for S.sorbrinius. Otherwise, activities show nearly the same orders, which means that the order of activity is independent of the bacteria, i.e. they do not show any structure–activity relationships. Therefore, we have followed regression analysis for establishing structure–activity relationships and proposing most appropriate models, using method of maximum R2. Regression analysis is performed on distance-based and extended-connectivity type topological indices presented in Table 2. Before multiple regression analysis is undertaken it is necessary to normalise the data in certain ways in order to make the detection of statistically significant correlation easier. The best way is to determine effective number of correlating parameters; such data resulted into statistically significant models. Detailed regression analysis yielded models which are summarised in Table 3. In order to discuss these models we have followed the rule of thumb11,12, and hence, we have used 4 descriptors as the maximum. Therefore, on this basis we discuss our results as follows.

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Table 3. Regression parameters and quality of correlations for modelling bacterial growth inhibition (–logMIC) using distance-based and extended-connectivity-type indices

Model

Parameters

 1  2  3  4  5

Har Har, Jhetm ZM1, H, Jhetm Jhetz, Jhetm, Jhetv, Jhetp W, Jhetz, Jhetm, Jhetv, Jhetp

 6  7  8  9 10

Har Pol, Har Pol, Har, Jhetz Pol, Har, Jhetz, Jhete W, Jhetz, Jhetv, Jhete, Jhetp

11 12 13 14 15

Har ZM2, Jhetp ZM2, Har, Jhetp ZM2, Har, J, Jhetv ZM2, Jhetp, Har, J, Jhetv

S.e. (standard error) P. gingivalis 0.2009 0.2019 0.2064 0.1873 0.1916 S. sobrinius 0.2907 0.2637 0.2525 0.2487 0.2721 S. artemidis 0.2054 0.1827 0.1752 0.1658 0.1680

R2

R2A

F

0.9114 0.9153 0.9163 0.9352 0.9364

0.9068 0.9059 0.9016 0.9189 0.9152

195.5103 97.2518 62.0734 57.6808 44.1867

0.8016 0.8453 0.8661 0.8777 0.8628

0.7912 0.8281 0.8425 0.8471 0.8171

76.7663 49.1825 36.6544 28.7080 18.8652

0.8712 0.9035 0.9162 0.9294 0.9320

0.8645 0.8927 0.9014 0.9117 0.9094

128.5529 84.2364 61.9780 52.6364 41.1330

MODELS FOR P. gingivalis

The perusal of Table 3 shows that one-parametric model based on Harary index was found as below: Model 1 –log MIC = –3.9818 ± 0.2478 (0.0177) Har, N = 21, SE = 0.2009, R2 = 0.9114 R2A = 0.9068, F = 195.5103.

(1)

Here and thereafter, N is the number of compounds used, SE – standard error, R – multiple correlation coefficient; R2A – adjustable R2, and F – the Fisher statistics. From Table 3 we also observed that as we go for higher order regression, i.e for a bi-parametric model we obtain the following model as follows: Model 2 –log MIC = –3.0527 ± 0.2313 (±0.0177) Har –0.23573 (±0.2599) Jhetm, N = 21, SE = 0.2019, R2 = 0.9153, R2A = 0.9059, F = 97.2518.

(2)

This model is statistically not significant as the coefficient of correlation is smaller than the standard error for Jhetm topological index.

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Therefore, we can conclude that for P. gingivalis the activity –log MIC can be estimated by one-parametric model using Har, the Harary index. Equation (1) also shows that by increasing the value of Har, –log MIC activity also increases. MODELS FOR S. sorbrinius

To obtain statistically significant models, persual of Table 3 shows that as we pass from 1- to 5-parametric models (6 to 10), R2 as well as R2A increase gradually up to model 9, i.e. for 4-parametric model. This model includes correlating parameters as the polarity number (Pol), Har, Jhetz and Jhete. As we have proceeded for 5-parametric model R2 and R2A both decrease, it means that model 10 is not significant to model the activity. The activity –log MIC can be best predicted by model 9, which equation is as follows: Model 9 –log MIC = – 10.5513 – 0.4185 (±0.1725) Pol + 0.7155 (±0.2179) Har +171.7819 (±138.3204) Jhetz – 170.5441 (±38.4200) Jhete,

(3)

N = 21, SE = 0.2487, R2 = 0.8777, R2A = 0.8471, F = 28.7080.

To look the contribution of correlating parameters for estimating the –log MIC activity, we can see that positive coefficient of topological indices H and Jhetz indicates that increase in the value of these 2 indices increase the activity of –log MIC. On the other hand, negative coefficient of Pol and Jhete indices shows that decrease in their value results in increase of –log MIC. MODELS FOR S. artemidis

As we compare antibacterial activity for P. gingivalis and S. sorbrinius the behaviour of set of compounds is quite different for exhibiting antibacterial activity against S. artemidis. Detailed analysis (Table 3) has shown that in this particular case the quality of model goes on increasing as we pass from 1-parametric model to 4-parametric model. There is gradual increase in R2 from 0.8721 to 0.9293 and increase in R2A is from 0.8644 to 0.9117. However, when we go for 5-parametric model, it is found statistically insignificant as R2A declined to 0.9093 value. Therefore, using 4-parametric model 14 the equation which exhibits the estimation of –log MIC is the following: Model 14 –log MIC = – 6.6495 + 0.2790 (±0.1003) ZM2 – 0.8236 (±0.3544) Har + 3.2216 (±2.0078) J – 5.7809 (±0.0763) Jhetv

(4)

N = 21, SE = 0.1658, R2 = 0.9294, R2A = 0.9117, F = 52.6364.

Here, the significance of positive and negative signs of correlation coefficient of correlating parameter remains the same as discussed earlier.

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Table 4. Comparison of observed and estimated values of –log MIC (mM) against P. gingivalis using model 1 (equation (1))

Compound No  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21

observed –1.453 –1.178 –1.267 –1.267 –0.612 –0.691 –0.637 –0.342 –0.292 –0.297 –0.438 –0.342 –0.435 –0.468 –0.124 –0.045 0.310 0.328 0.854 0.523 0.886

–log MIC (mM) estimated –1.598 –1.128 –1.140 –1.145 –0.707 –0.724 –0.733 –0.302 –0.322 –0.333 –0.302 –0.224 –0.247 –0.258 0.074 0.062 –0.247 0.209 0.456 0.733 0.886

residual 0.145 –0.050 –0.127 –0.122 0.095 0.033 0.096 –0.040 0.030 0.036 –0.136 –0.118 –0.188 –0.210 –0.198 –0.107 0.557 0.119 0.398 –0.210 0.000

Table 5. Comparison of observed and estimated values of –logMIC (mM) against S. sorbrinius using model 9 (equation (3))

Compound No 1  1  2  3  4  5  6  7  8  9 10 11 12

observed 2 –1.459 –1.333 –1.170 –1.333 –1.867 –0.867 –0.852 –0.729 –0.711 –0.565 –0.810 –0.590

–log MIC (mM) estimated 3 –1.703 –1.314 –1.179 –1.085 –1.295 –1.022 –1.120 –0.812 –0.711 –0.638 –0.687 –0.723

residual 4 0.244 –0.019 0.009 –0.248 –0.572 0.155 0.268 0.083 0.000 0.073 –0.123 0.133 to be continued

690

Continuation of Table 5

1 13 14 15 16 17 18 19 20 21

2 –0.785 –0.692 –0.493 –0.124 –0.124 –0.19 0.523 0.260 0.620

3 –0.472 –0.586 –0.225 –0.148 –0.472 –0.448 0.376 0.254 0.718

4 –0.313 –0.106 –0.268 0.024 0.348 0.258 0.147 0.006 –0.098

Table 6. Comparison of observed and estimated values of –logMIC (mM) against S. artemidis using model 14 (equation (4))

Compound No  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21

observed –1.327 –0.885 –0.885 –0.869 –0.515 –0.547 –0.481 –0.167 –0.342 –0.167 –0.856 –0.297 –0.297 –0.297 –0.033 0.328 0.060 0.208 0.796 0.456 0.745

–log MIC (mM) estimated –1.107 –0.887 –1.032 –0.937 –0.522 –0.639 –0.524 –0.142 –0.258 –0.140 –0.639 –0.219 –0.308 –0.173 0.135 0.244 –0.308 0.077 0.556 0.608 0.841

residual –0.220 0.002 0.147 0.068 0.007 0.092 0.043 –0.025 –0.084 –0.027 –0.217 –0.078 0.011 –0.124 –0.168 0.084 0.368 0.131 0.240 –0.152 –0.096

691

Fig. 1. Correlation of observed and estimated –log MIC (mM) of phenols using model 1 (equation (1)) for P. gingivalis bacteria

Fig. 2. Correlation of observed and estimated –log MIC (mM) of phenols using model 9 (equation (3)) for S. sorbrinius bacteria

Fig. 3. Correlation of observed and estimated –log MIC (mM) of phenols using model 14 (equation (4)) for S. artemidis bacteria

692

CONCLUSIONS Using these models, we have calculated –log MIC against each of the bacteria and compared them with the observed (experimental) antibacterial activity. The comparison is shown in Table 4. The predictive ability of the proposed statistically significant models (1, 9 and 14) for the activity, –log MIC is shown in Figs 1–3 for each of the bacteria. We can, therefore, conclude that distance-based topological indices, Har, Pol and ZM2 together with extended connectivity type J, Jhetz, Jhete, Jhetv topological indices are the better parameters for modelling –log MIC of some phenols used in the present study. Also, that the better results are obtained in multi-parametric models. REFERENCES   1. S. SHAPIRO, B. GUGGENHEIM: Inhibition of Oral Bacteria by Phenolic Compounds. Part I. QSAR Analysis Using Molecular Connectivity. Quant. Struct.-act. Relat., 17, 327 (1998).   2. S. SHAPIRO, B. GUGGENHEIM: Inhibition of Oral Bacteria by Phenolic Compounds. Part II. QSAR Analysis Using Molecular Connectivity. Quant. Struct.-act. Relat., S., 17, 338 (1998).   3. M. CHRISTINA, E. WILLIAM, J. ACREE, M. H. ABRAHAM: Correlation of Minimum Inhibitory Concentration towards Oral Bacterial Growth Based on the Abraham Model. QSAR Comb. Sci., 25 (10), 912 (2006).   4. S. CHATTERJEE, A. S. HADI, B. PRIZE: Regression Analysis by Examples. 3rd ed. Wiley, New York, 2000.   5. H. J. WEINER: Structural Determination of Paraffin Boiling Points. Am. Chem. Soc., 69, 17 (1947).   6. A. T. BALABAN: Topological Indices Based on Topological Distance in Molecular Graph. Pure Appl. Chem., 55, 199 (1983).   7. A. T. BALABAN, P. V. KHADIKAR, C. T. SUPURAN, A. THAKUR, M. THAKUR: QSAR Study on pKa of Sulphonamides Using Balaban Index (J ): A Physicochemical Approach. Bioorg. Med. Chem. Lett., 17 (15), 3966 (2005).   8. A. J. SHUSTERMAN, A. S. JOHNSON, C. HANSCH: Correlation of Mutagenicity of 1,1-dimethyl3-(X-phenyl)triazenes with Molecular Orbital Energies and Hydrophobicity. International J. of Quantum Chemistry, 36, 19 (1989).   9. http://www.talete.mi.it/help/dragon_help/index.html?IntroducingDRAGON. 10. http://www.acdlabs.com/download/. 11. M. S. TUTE: History and Objectives of Quantitative Drug Design in Advances in Drug Research (Eds N. J. Harter, A. B. Simmord). Vol. 6, Academic Press, London, 1971, p. 1. 12. C. HANSCH (Ed.): Comprehensive Drug Design. Pergamon Press, New York, 1990, p. 19. Received 3 October 2009 Revised 4 November 2009

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Oxidation Communications 35, No 3, 694–707 (2012) Biological section

QSAR Approach Insight the Structural Requirement of Substituted Quinazolinones Derivatives as Angiotensin II Receptor Antagonists M. C. Sharmaa*, S. Sharmab, D. V. Kohlia Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, 470 003 Sagar (M.P.), India b Department of Chemistry, Chodhary Dilip Singh Kanya Mahavidyalya, 477 001 Bhind (M.P.), India E-mail: [email protected] a

ABSTRACT A quantitative structure activity relationship (QSAR) study on a series of 2, 3, 6-substituted quinazolinones as angiotensin II receptor antagonists was made using combination of various physicochemical descriptors (thermodynamic, electronic and spatial). Several statistical expressions for 2D QSAR models were developed using stepwise multiple regression analysis. The output of the present research work is interesting; the 2D QSAR studies indicated contribution of different physicochemical descriptors. The statistically significant best 2D-QSAR model having correlation coefficient r2 = 0.8029 and cross-validated squared correlation coefficient q2 =0.7214 with external predictive ability of pred_r2 = 0.8942 was developed MLR with the descriptors like PSA(EX) P&S, slogp, kappa1 and T_N_Cl_5. The study suggested that substitution of group at R1, R2 position on quinazolinones ring with hydrophobic nature and low bulkiness are favourable for the antihypertensive activity. The quantitative structure activity relationship study provides important structural insights in designing of potent antihypertensive agents. Keywords: QSAR, quinazolinones, angiotensin II, AT1, MLR, antihypertensive agents. AIMS AND BACKGROUND The renin–angiotensin–aldosterone system plays an integral role in the pathophysiology of hypertension because it affects the regulation of fluid volume, electrolyte balance and blood volume. Renin, an enzyme produced primarily by the juxtaglomerular cells *

For correspondence.

694

of the kidney, catalyses the conversion of angiotensinogen into an inactive substance, angiotensin I (Ang-I). Angiotensin-converting enzyme (ACE) then converts Ang-I to the physiologically active angiotensin II (Ang-II), which causes potent vasoconstriction, aldosterone secretion and sympathetic activation. All of these actions contribute to the development of hypertension1–3. Angiotensin II receptor antagonists act by binding to specific membrane-bound receptors that displace Ang-II from its type 1­receptor subtype (AT1). These drugs, therefore, function as selective blockers4. Ang-II effects are mediated by AT1 receptors. These receptors are widespread in organs and tissues but are found predominately in vascular and myocardial tissue, the liver, the adrenal cortex (i.e. the zona glomerulosa tissue, which secretes aldosterone) and some areas of the brain. Angiotensin II receptor antagonists are primarily used for the treatment of hypertension where the patient is intolerant of ACE inhibitor therapy. More recently, they have been used for the treatment of heart failure in patients intolerant of ACE inhibitor therapy, particularly candesartan. Irbesartan and losartan have trial data showing benefit in hypertensive patients with type II diabetes, and may delay the progression of diabetic nephropathy. Candesartan is used experimentally in preventive treatment of migraine5. Computational chemistry has developed into an important contributor to rational drug design. Quantitative structure activity relationship (QSAR) modelling results in a quantitative correlation between chemical structure and biological activity. Numerous data sets which are reported in the literature were subjected to QSAR analysis in order to design novel angiotensin II receptor antagonists 6–12. One of the authors (M. C. Sharma) has developed a few QSAR studies to predict biological activity of different group of compounds13–24. The observed selection of test set molecules was made by considering the fact that test set molecules represents a range of biological activity similar to the training set. In addition, a wide range of structural diversity of compounds in the test set permits us to evaluate the extrapolative accuracy of the QSAR models. To gain insight into the structural and molecular requirement influencing the Ang-II antagonistic activity, herein we depict QSAR analysis of some 2, 3, 6-substituted quinazolinones derivative for Angiotensin II AT1 receptor antagonist activity. The relevance of the model used for the design of novel derivatives should be assessed not only in terms of predictivity, either internal or external, but also in terms of their ability to provide a chemical and structural explanation of their binding interaction. These results should provide guidelines for design of more potent and selective Angiotensin II AT1 receptor antagonist. EXPERIMENTAL DATA SET

The Angiotensin II receptor antagonist activity data of a series of 2, 3, 6-substituted quinazolinones angiotensin II receptor antagonists were taken from the reported work25. The general structure of these analogues is shown in Table 1 where the structural features and activity of the respective compounds under study are listed. Previously 695

QSAR studies of these series12 are based on different approach. Here QSAR studies are carried out by VLife science MDS. The biological data were converted to negative logarithmic scale (pIC50) in mathematical operation mode of software to reduce skewness of data set and then used for subsequent QSAR analysis as dependent variables. Training set (17 compounds) and the test set (6 compounds) were selected by considering the fact that the test set compounds represents structural diversity and a range of biological activities similar to that of training set and subsequently used as the dependent variable for the QSAR analysis (Table 2). Table 1. Structures and biological activity of substituted quinazolinone derivatives

R1 N N

R2 Com. No R1 MCS1 COOH MCS2* CN4H MCS3 CN4H MCS4 CN4H MCS5 CN4H MCS6* CN4H MCS7 CN4H MCS8 CN4H MCS9* CN4H MCS10 CN4H MCS11 CN4H MCS12* CN4H MCS13 CN4H MCS14 CN4H MCS15* CN4H MCS16 CN4H MCS17 CN4H MCS18 CN4H MCS19* CN4H MCS20 CN4H MCS21 CN4H MCS22 CN4H MCS23 CN4H

R3

O R2

CH3 CH3 CH3 OCH3 NH2 NHCH3 N(CH3)2 NHSO2CF3 NHAc NHCOC4H9 NHCO2CH2Ph NHCON(CH3)iPr CH3CHOH (CH3)2COH CH2NH2 (CH3)2COCH3 CH2OCH3 CH2OPh CH3CH(OCH3) H N(CH3)CO2CH2Ph NHCONHiPr CH2OH

R3 nBu nBu nPr nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu nBu

IC50(nM)a   92    4   10    5    1.3    2    5 120    9    2    4.3    0.10    9    9   15    6   18 130    7   10    0.83    0.75   10

pIC50b 1.963 0.602 1.000 0.698 0.113 0.301 0.700 2.079 0.954 0.300 0.633 –1.000 0.954 0.954 1.176 0.778 1.255 2.113 0.845 1.000 –0.080 –0.124 1.000

a IC50 or inhibition of specific binding of rabbit aorta receptor; b –log IC50 to generate equation; *indicates the compounds considered in the test set in QSAR.

696

Table 2. Selected physicochemical parameters of substituted quinazolinone derivatives

slogp

XlogP

5.219 6.535 6.491 4.936 5.324 4.512 5.96 4.934 6.415 5.588 6.091 5.573 5.963 4.469 5.072 5.853 5.812 5.12 5.183 5.766 4.622 4.546

151.27 157.7 169.58 138.9 143.5 136.26 152.63 138.88 159.76 143.67 126.66 132.88 137.49 131.39 140.89 150.12 147.72 140.88 128.26 166.73 159.18 134.292

SssssCEindex 19.0671 20.1185 20.8337 17.6733 17.8466 17.1733 18.79 17.546 20.1185 18.084 15.4735 16.6353 17.1353 16.6353 17.546 18.622 19.668 17.6733 16.2415 21.4249 19.923 17.173

chi5chain 2 2 4 1 1 1 2 2 2 2 1 1 1 1 1 1 1 1 1 2 3 1

T_C_Cl_4 T_N_Cl_5 4 4 4 4 4 4 4 4 5 4 5 4 4 4 4 4 4 4 3 4 4 4

1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 1 1 0

T_O_O_2 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MOLECULAR DESCRIPTORS AND 2D MODEL

All computational work was performed on Apple workstation (dual-core processor) using Vlife MDS (Ref. 26) QSAR plus software developed by Vlife Sciences Technologies Pvt. Ltd. Pune, India, on windows XP operating system. All the compounds were drawn in Chem DBS using fragment database and then subjected to energy minimisation using batch energy minimisation method. Energy minimisation and geometry optimisation were conducted using Merck molecular force field (MMFF) and atomic charges, maximum number of cycles were 1000, convergence criteria (RMS gradient) was 0.01 and medium dielectric constant of 1 by batch energy minimisation method. Conformational search was carried out by a systemic conformational search method. Complete geometry optimisation was performed taking the most extended conformations as starting geometries. The basis of energy minimisation is that the drug binds to effectors/receptor in the most stable form, i.e. minimum energy state form. Most stable structure for each compound was generated and used for the calculation of the various 2D descriptors like physicochemical, structural, topological, electro-topological, the Baumann alignment-independent topological descriptors, etc. using VLife MDS 697

software. Pre-processing of the independent variables (i.e. descriptors) was done by removing the invariables (descriptors that are constant for all the molecules), which resulted in 227 descriptors in the descriptor pool. The relationship between biological activities and various descriptors (physiochemical and alignment-independent) were established by sequential multiple regression analysis (MLR) using VLife MDS 3.5 in order to obtain QSAR models27–28. QSAR MODELS DEVELOPMENT AND VALIDATION

This is done to test the internal stability and predictive ability of the QSAR models. Developed QSAR models were validated by the following procedure. Internal validation. A molecule in the training set was eliminated, and its biological activity was predicted as the weighted average activity of the k most similar molecules. The cross-validated r2 (q2) value was calculated using the following relation: q2 = 1 –

∑(yi – ŷi)2 ∑(yi – ymean)2

,

(1)

where yi and ŷi are the actual and the predicted activities of the i-th molecule in the training set, respectively, and ymean – the average activity of all molecules in the training set. However, a high q2 value does not necessarily give a suitable representation of the real predictive power of the model for antihypertensive molecules. So, an external validation is also carried out in this study. The external predictive power of the model is assessed by predicting pIC50 value of 15 test set molecules, which are not included in the QSAR model development. The predictive ability of the selected model is also confirmed by pred_r2 or rCVext2. External validation. The predicted r2 (pred_r2) value was calculated using the following equation: pred_r2 = 1 –

∑(yi – ŷi)2 ∑(yi – ymean)2

,

(2)

where yi and ŷi are the actual and predicted activities of the i-th molecule in test set, respectively, and ymean – the average activity of all molecules in the training set. Both summations are over all molecules in the test set. The pred_r2 value is indicative of the predictive power of the current model for external test set. Randomisation test. To evaluate the statistical significance of the QSAR model for an actual data set, we have employed a one-tail hypothesis testing. The robustness of the QSAR models for experimental training sets was examined by comparing these models to those derived for random data sets. Random sets were generated by rearranging biological activities of the training set molecules. The significance of the models obtained was derived based on calculated Z score.

698

Zscore =

(h – μ) σ

,

where h is the q2 value calculated for the actual data set; μ – the average q2, and σ – its standard deviation calculated for various iterations using models build by different random data sets. The probability (α) of significance of randomisation test is derived by comparing Zscore value with Zscore critical value, if Zscore value is less than 4.0; otherwise it is calculated by the formula as given in literature. For example, a Zscore value greater than 3.10 indicates that there is a probability (α) of less than 0.001 that the QSAR model constructed for the real dataset is random. The randomisation test suggests that all the developed models have a probability of less than 1% that the model is generated by chance. The robustness of a QSAR model was checked by Y-randomisation test. In this technique, new QSAR models were developed by shuffling the dependent variable vector randomly and keeping the original independent variable as such. MULTIPLE LINEAR REGRESSION (MLR) ANALYSIS

Multiple regressions estimates the values of the regression coefficients by applying least squares curve fitting method. For getting reliable results, dataset having typically 7 times as many data points (molecules) as independent variables (descriptors) is required. The regression equation takes the form Y = b1x1 + b2x2 + b3x3 + c,

where Y is the dependent variable; the ‘b’ – regression coefficients for corresponding ‘x’ (independent variable); ‘c’ – a regression constant or intercept. In the present study QSAR model was developed using multiple regression by forward-backward variable selection method with pIC50 activity field as dependent variable and 227 physicochemical descriptors as independent variable having cross-correlation limit of 5. Selection of test and training set was done by sphere exclusion method. Stepwise multiple regression analysis was used to generate QSAR equations. Random selection method and sphere exclusion method were used for the selection of the training and test set. For variable selection stepwise forward-backward method was used. A suitable statistical method coupled with a variable selection method allows analyses of this data in order to establish a QSAR model with the subset of descriptors that are most statistically significant in determining the biological activity29,30. RESULTS AND DISCUSSION 2D-QSAR study was performed using multiple regressions analysis (MLR) method. 2D QSAR equations were selected by optimising the statistical results generated along with variation of the descriptors in these models. The best regression 5 equa699

tions obtained using MLR method is represented. It is simple to interpret 2D-QSAR MLR equation where each descriptor contribution can be seen by the magnitude and sign of its regression coefficient. A descriptor coefficient magnitude shows its relative contribution with respect to other descriptors and sign indicates whether it is directly (+) or inversely (−) proportional to the activity. Biological activity data, (IC50 nM) were converted to negative log dose in mol (pIC50) for QSAR analysis. In order to derive a reasonable QSAR equation, the obtained equations were evaluated by the predictive results for the dataset. Removal of outliers improved the correlation coefficient of the QSAR equations. Following statistical measure was used to correlate biological activity and molecular descriptors; n number of observations (molecules); k – number of variables; optimum component, number of optimum components in the model; r2 – coefficient of determination; q2 – cross-validated r2 (by leave one-out); pred_r2 – r2 for external test set; Zscore – Zscore calculated by the randomisation test; best_ran_q2 – highest q2 value in the randomisation test; best_ran_r2 – highest r2 value in the randomisation test; F-test (the Fischer value) for statistical significance; SEE – standard error of estimate of the model; SECV – standard error of cross-validation; and SEP – standard error of external test set prediction. Statistical values indicated that equations with outliers. The following statistical parameters were considered for comparison of the generated QSAR models: correlation coefficient (r), squared correlation coefficient (r2), predictive r2 for external test set (pred r2) for external validation. A set of 23 compounds (Table 1) was considered for 2D QSAR analysis for 2, 3, 6-substituted quinazolinone substituents on the basis of structural similarity. Dataset of 23 molecules (Table 1) was divided into training (17) and test (6) set compounds. Selection of the training set and the test set molecules was done manually by considering the fact that test set molecules represent a range of biological activity similar to that of the training set. Thus, the test set was a true representative of the training set. The test set consisted of 6 compounds, namely, MCS2, MCS6, MCS9, MCS12, MCS15 and MCS19. In order to develop 2D QSAR, the data set was subjected to a multiple linear regression analysis. This resulted into several correlation equations between the log IC50 values as a dependent variable and several quantifying parameters as an independent variable. Equation was considered as model for antagonistic activity on angiotensin II AT1 receptor antagonists. Model 1 as the statistically significant model using the multiple regressions analysis method with 0.6437 as the coefficient of determination (r2) was considered. Model 1 can explain 64.37% of the variance in the observed activity values. It shows an internal predictive power (q2= 0.561) of 57% and a predictivity for the external test set (pred_r2 = 0.7083) of about 71%. The F-test value of 32.81 shows the overall statistical significance level for 99.99% of the model, which means the probability of failure for the model, is 1 in 10 000. The descriptor T_2_Cl_6 is the number of double-bonded atoms separated from the chlorine atom by 6 bonds. It is another influential alignment-independent descriptor suggesting that the presence of substituents with chlorine on the phenyl ring at the ortho-position will lead to an 700

increase in activity. The next most important descriptor, which influences the activity variation, is SssssCE-index and is inversely proportional to the activity and indicates the presence of aromatic ring substituents at the R2 position of the quinazolinone ring which is detrimental for the activity. Its positive value suggests that increasing the number of such carbons will lead to better angiotensin II receptor activity. The next important descriptor which influences the activity is positive contributing H-Donor Count that is the number of hydrogen atoms is inversely proportional to activity and thus adding hydrogen atoms are favourable for the activity.The statistically significant model (model 1) as shown in Table 3 with multiple regression analysis method. The above model is validated by predicting the biological activities of the test molecules in Table 4. The plot of observed versus predicted activities for the test compounds is represented in Fig. 1a. From Table 4 it is evident that the predicted activities of all the compounds in the test set are in good agreement with their corresponding experimental activities and optimum fit is obtained. Model 2 using the multiple regression analysis method with 0.8746 as the coefficient of determination (r2) was considered using the same molecules in the test and training sets. The model can explain 87% of the variance in the observed activity values. The model shows an internal predictive power (q2= 0.7379) of 73% and a predictivity for the external test set (pred_r2 = 0.851) of about 86%. The F-test value of 57.876 shows the overall statistical significance level for 99.99% of the model. The statistically significant model (Model 2) is shown in Table 3 with multiple regression analysis method. The above model is validated by predicting the biological activities of the test molecules as indicated in Table 4. The plot of observed versus predicted activities for the test compounds is represented in Fig. 2a. In accordance with model 2 shows a positive correlation with chi3Cluster, XlogP, kappa1, chi0 a negative correlation. It is apparent from the equation that the Chi5 is a molecular connectivity index descriptor. Molecular connectivity, a quantification of topology, is detrimental to biologic activity in the models. The next descriptor XlogP is a surface descriptor indicating the hydrophobic fraction of the total van der Waals (vdW) surface area and is calculated by the Audry method using (XlogP). The total hydrophobic area summarises the contributions of the quinazolinone ring substituents. The hydrophobic surface area is positively correlated with the activity. This indicates that the molecules possessing greater hydrophobic surface area have increased activity. The other descriptor (K1) is the first kappa shape index: (n −1) (n − 3)2/p23 for odd n, and (n − 3)(n − 2)2/p23 for even n which is a physicochemical descriptor belonging to the subclass of the kappa shape indices. The shape descriptor is negatively correlated with biological activity. So an increase in the (K1) value increases the activity. The last descriptor chi3Cluster in model represents to signify simple 3rd order cluster chi index in a compound. Model 3 generated using the multiple regression analysis method with 0.8426 as the coefficient of determination (r2) was considered using the same molecules in the test and training sets. The model can explain 84% of the variance in the observed activity values. The model shows an internal predictive power (q2= 0.7116) of 71% and a predictivity for the external test set 701

(pred_r2 = 0.821) of about 82% (Fig. 3c). The F-test value of 45.90 shows the overall statistical significance level for 99.99% of the model. The descriptor T_N_Cl_5 is another influential alignment-independent descriptor, which suggests that the presence of chlorine substituents on R2 position would also increase the activity. As a positive contributing descriptor, T_C_Cl_4 is an alignment-independent descriptor (Table 3) influencing activity variation and is directly proportional to activity and indicates the presence of chlorine group at the R3 position of the quinazolinone ring would lead to a positive effect on the antihypertensive activity. The next descriptor, T_2_2_6 is an alignment-independent type descriptor, which value signifies the 2-bonded atoms separated from any other 2-bonded atom by 6 bonds in a molecule. The other positively signed parameter T_2_2_6 provided the information that any 2-bonded atom separated from any other 2-bonded atom by 6 bonds distance in a molecule significantly improves the antihypertensive activity. Table 3. Developed 2D-QSAR models for substituted quinazolinone derivatives

n(training/test) k r2 q2 pred_r2 F-test SEE SECV SEP best_ran_r2 best_ran_q2 Zscore_ran_r2 Zscore_ran_q2 α_ran_r2 α _ran_q2 Descriptor 1 Descriptor 2 Descriptor 3 Descriptor 4

702

2D-Model 1 2D-Model 2 2D-Model 3 2D-Model 4 2D-Model 5 17/6 17/6 17/6 17/6 17/6  8  8  8  8  8   0.643   0.874   0.842   0.802   0.726   0.561   0.737   0.711   0.721   0.658   0.708   0.851   0.821   0.894   0.803 32.81 57.876 45.90 39.61 51.37   0.112   0.120   0.601   0.218   0.301   0.310   0.261   0.191   0.290   0.297   0.021   0.298   0.319   0.109   0.477   0.325   0.108   0.217   0.359   0.402   0.116   0.219   0.468   0.420   0.314   1.012   7.650   9.046   5.380   3.539   1.214   4.351   5.106   6.129   1.952   0.014   0.010   0.014   0.07   0.03 0.5. The F-test reflects the ratio of the variance explained by the model and the variance due to the error in the regression. High values of the F-test indicate that the model is statistically significant. Internal and external validation. Internal validation is carried out using ‘leave-oneout’ (LOO) method21. The cross-validated coefficient, q2, is calculated using the following equation: q2 = 1 –

∑(yi – ŷi)2 ∑(yi – ymean)2

,

(1)

where yi and ŷi are the actual and the predicted activities of the i-th molecule in the training set, respectively, and ymean – the average activity of all molecules in the training set. However, a high q2 value does not necessarily give a suitable representation of the real predictive power of the model for antihypertensive molecules. So, an external validation is also carried out in this study. The external predictive power of the model is assessed by predicting pIC50 value of 15 test set molecules, which are not included in the QSAR model development. The predictive ability of the selected model is also confirmed by pred_r2 or rCVext2. pred_r2 = 1 –

∑(yi – ŷi)2 ∑(yi – ymean)2

,

(2)

where yi and ŷi are the actual and predicted activities of the i-th molecule in test set, respectively, and ymean – the average activity of all molecules in the training set. Both summations are over all molecules in the test set. The pred_r2 value is indicative of the predictive power of the current model for external test set. Random sets were generated by rearranging the activities of the molecules in the training set and the significance of the models was derived based on the calculated Zscore (Ref. 22). Zscore =

qorg2 – qa2 qstd2

,

where q2org is the q2 value calculated for the actual data set; q2a – the average q2, and q2std – the standard deviation of q2, calculated for various iterations using different random data sets. The probability (a) of significance of the randomisation test is 728

derived by using calculated Zscore value for various iterations using models build by different random datasets. RESULTS AND DISCUSSION The various 2D-QSAR models were developed using MLR method. 2D-QSAR equations were selected by optimising the statistical results generated along with variation of the descriptors in these models. The fitness/pattern plots were also generated for evaluating the dependence of the biological activity on various different types of the descriptors. The frequency of use of a particular descriptor in the population of equations indicated the relevant contributions of the descriptors. The contribution charts for all the significant models are presented in Fig. 1a–c, which gives the percentage contribution of the descriptors used in deriving the model. The model shows that in a multivariant model, a dependent variable can be predicted from a linear combination of the independent variables. The value is less than 0.01 for each physiochemical parameter involved in model generation. The data showed overall internal statistical significance level better than 99.9%.

Fig. 1. Plots of actual versus predicted activity for: model 1 (a), model 2 (b) and model 3 (c); black dots () represent traning set, gray dots test set molecules ()

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Model 1 pIC50= –1.9436(± 0.0353) SsClcount + 0.3909(± 0.0401) T_2_O_3 + 0.9284(± 5.8037) SAMostHydrophobic –0.9578 n = 19, degree of freedom = 12, r2 = 0.8918, q2 = 0. 7108, F-test = 32.9542, r2 SE = 0.2409, q2 SE = 0.3079, pred_r2 = 0.8031, pred_r2SE = 0.9514.

Following statistically significant models, considering the term selection criteria as r2. The statistically significant model (Model 1) with coefficient of determination (r2) = 0. 8918 (which corresponds to value of r =0.2409) was considered as a model for antihypertensive. The model showed an internal predictive power (q2=0.7108) of 71% and predictivity for external test set (pred_r2 =0.8031) about 80%. The overall statistical significance level was found to be better than 99.9% as it exceeded the tabulated F1, 21α0.001= 32.9542. The developed model reveals that the descriptor T_2_O_3 (i.e. pair of any double bonded atom with any atom separated by 4 bonds) plays most important role (~45%) in determining antihypertensive activity, which mainly indicates the relationship with reference to variation in different substitution patterns (mono-, di-, tri-) on the phenyl ring. The next 2 most important factors governing variation in the activity are SsClcount (20%) and SAMostHydrophobic (~20%) and both are inversely proportional to the activity. The descriptor SAMostHydrophobic area is the vdW surface descriptor showing hydrophobic surface area calculated by the Audry method using SlogP and it describes the most hydrophobic value on the vdW surface of the molecule. The positive sign of this descriptor reveals that the hydrophobicity of the molecules is detrimental for the 2,3-dihydroquinazolinones derivatives as antihypertensive activity. The model revealed that SsClcount as topological parameter signifies the total number of chlorine atoms connected with one single bond and positive coefficient of the descriptor suggests that activity of 2,3-dihydroquinazolinones derivatives may be improved by increasing the number of chlorine atoms present in the quinazolinone nucleus at R1 site. The plots of calculated versus actual values of pIC50 and contribution chart are shown in Fig. 1a. The predicted (LOO) activities of the compounds by the above model are shown in Table 3. Model 2 pIC50= +0.7961 (± 0.0709) hydrogen count + 0.6246 (± 0.4961) Mol: wt: + 0.3909 (± 0.0401) T_N_O_1 + 5.3074 n = 19, degree of freedom = 13, r2 = 0.7987, q2 = 0.7511, F-test = 57.6726, r2 SE = 0.2286, q2 SE = 0.2694, pred_r2 = 0.7269, pred_r2SE = 1.1869.

The statistically significant model (Model 2) with coefficient of determination (r ) = 0.7987 (which corresponds to value of r =0.2286) was considered as a model for antihypertensive activity. The model showed an internal predictive power (q2=0.7511) of 75% and predictivity for external test set (pred_r2 =0. 7269) about 72%. The overall statistical significance level was found to be better than 99.9% as it exceeded the tabulated F= 57.6726. The developed model reveals that the descriptor hydrogen 2

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count is an element count descriptor which calculates the number of hydrogen atoms in a compound and plays most important role (~58%) in determining antihypertensive activity. The next 2 most important factors governing variation in the activity are Mol: wt: (~35 %) and T_N_O_1 (~20%) and both are inversely proportional to the activity. The descriptor which influences the activity (Mol: wt:) is inversely proportional to the activity and indicates the presence of bulky group (H, methyl) at R1 and R2 position which is detrimental for the activity. The next positive contributing descriptor, T_N_O_1 is influencing activity variation and is directly proportional to activity and indicates the presence of methoxy, –NH, NH2 side chain at the R position of the quinazolinone ring would lead to a positive effect on the antihypertensive activity. The plots of calculated versus actual values of pIC50 and contribution chart are shown in Fig. 1b. The predicted (LOO) activities of the compounds by the above model are shown in Table 3. Model 3 pIC50= – 0.1487(± 0.0016) T_T_T_3 + 0.4835(± 0.0809) T_N_O_6 – 1.6841(± 0.0839) Dipole Moment +1.5084(± 11.7543) PolarizabilityAHC + 1.7456 n = 19, degree of freedom = 11, r2 = 0.8730, q2 = 0.7118, F-test = 18.8957, r2 SE = 0.3198, q2 SE = 0.4817, pred_r2 = 0.6909, pred_r2SE = 0.8687.

The model developed explains 87% variance as indicated by the value of r2 = 0. 8730 which corresponds to value of r = 0.85. The model had internal and external predictive power of 71% (q2 = 0.7118) and 69% (pred_r2 = 0.6909), respectively. The model showed statistical significance level better than 99.9% as it exceeded the tabulated F2,20α0.001= 18.8957. The descriptor, T_T_T_3 is an alignment-independent type descriptor, which value signifies the triple bonded atoms separated from any other triple bonded atom by 3 bonds in a molecule. The other positively signed parameter T_T_T_3 provided the information that any triple bonded atom separated from any other triple bonded atom by 3 bonds distance in a molecule significantly improves the antihypertensive activity. The polarisabilityAHC descriptor signifies the molecular polarisability using sum of atomic polarizabilities using the atomic hybrid components (AHC). The descriptor has a positive coefficient in the model, suggesting that the increased polarizable groups in the molecules have significant activity on the antihypertensive activity. Lastly descriptor dipole moment signifies dipole moment calculated from the partial charges of the molecule. Thus molecules with greater charge separation (i.e. greater number of electronegative atoms) have large values of dipole moment and negative coefficient of this descriptor in model indicates that increase in the number of electronegative atoms and thus charge separation in molecules is beneficial for antihypertensive activity. The plots of calculated versus actual values of pIC50 and contribution chart are shown in Fig. 1c. The predicted (LOO) activities of the compounds by the above model are shown in Table 3.

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Model 4 pIC50= + 0.3481(± 0.0006) T_2_O_3 – 0.1080(± 0.0001) hydrogen count + 0.3621(± 0.0103) T_2_2_3 + 17.8466(± 4.2547) SA-average –11.5699 n = 15, degree of freedom = 10, r2 = 0.9648, q2 = 0.9036, F-test = 54.7661, r2 SE = 0.1640, q2 SE = 0.2712, pred_r2 = 0.7044, pred_r2SE = 0.8055.

The model explained 90% of variance (r2 = 0.9648) having an equivalent value of r = 0.9648. The internal and external predictivity of the model was 90% (q2=0.9036) and 70% (pred_r2=0.7044), respectively. This model also explained statistical significance better than 99.9% as the obtained F-value exceeded the tabulated F1,20α0.001= 54.7661. The model includes descriptors T_2_O_3 (60%) contributing negatively to biological activity, number of double bounded atoms (i.e. any double bonded atom, T_2) separated from oxygen atom by 3 bonds in a molecule and 2 other descriptors, viz. hydrogen count number of hydrogen atoms in a compound and T_2_2_3. The descriptor SA-average is one of the estate contributors and it defines the electrotopological state indices for number of average hydophobicity function value. This descriptor has ~30% contribution in biological activity and indicates that electrondonating groups as substituents. The predicted (LOO) activities of the compounds by the above model are shown in Table 3. Model 5 pIC50= + 0.2608(± 0.0013) SsCH3Count–0.8686(± 12.1891) T_O_Cl_5 + 1.3693(± 0.0239) SulfursCount – 0.1907(± 0.0026) H-donor Count + 10.5399 n = 15, degree of freedom = 9, r2 = 0.7848, q2 = 0.6273, F-test = 17.1159, r2 SE = 0.2665, q2 SE = 0.6273, pred_r2 = 0.6845, pred_r2SE = 0.6593.

The statistically significant model using the multiple linear regression analysis method was performed, which resulted in a coefficient of correlation of 7848 and an internal predictive power of 78%, with the good external predictivity of 68%. The internal and external predictivity of the model was 62% (q2=0.6273) and 68% (pred_r2  = 0.6845), respectively. This model also explained statistical significance better than 99.9% as the obtained F-value exceeded the tabulated F1,20α0.001= 17.1159. The predicted (LOO) activities of the compounds by the above model are shown in Table 3. The model includes descriptors SsCH3Count (40%) contributing positively to biological activity and 3 other descriptors, viz T_O_Cl_5, SulfursCount and H-donor Count. The important descriptor T_O_Cl_5 is one of the estate contributors and it defines the number of oxygen atoms (single double or triple bonded) separated from chlorine atom by 5 bond distance in a molecule. The next SsCH3Count defines the total number of –CH3 group connected with single bond. The positive coefficient of this descriptor signifies the importance of methyl group for 2, 3-dihydroquinazolinones. Molecules having greater number of methyl groups have more potent antihypertensive activity. Lastly SulfursCount descriptor has ~30% contribution in biological activity

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and indicates the number of sulphur atoms in a compound as substituent at R1 position in the quinazolinone moiety. CONCLUSIONS This work reveals how the antihypertensive activities of various 2,3-dihydroquinazolinones may be treated statistically to uncover the molecular characteristics which are essential for high activity. The parameters that govern the activity are interdependent and modifying any of them will affect both activities simultaneously, thus appropriate substituent modulation will help achieve designing of promising new 2,3-dihydroquinazolinones-based compounds with antihypertensive activity. From these charts one can decide the extent and direction (positive or negative) of descriptors in contributing to the biological activity of molecules and thus help in designing new compounds with improved biological profile. From the above 2D-QSAR study of 2,3-dihydroquinazolinones derivatives having angiotensin II AT1 receptor antagonists , it was actual that Model 1 developed by MLR method has improved statistical significance and predictive ability with descriptors like SsClcount, T_2_O_3 ,SAMostHydrophobic in compare to the other 4 models developed by the MLR method. Hence, from the result it was revealed that optimum combination of substitution having increase in SsClcount and SAMostHydrophobic increase in T_2_O_3 enhances the activity. These features may be helpful in development of more safer and potent 2,3dihydroquinazolinones derivatives having antihypertensive activity. It is necessary that the proposed models should have both the statistical quality as well as good predictive power therefore all the expressions were tested for internal and external validation. Both the validations put forward decision-making input for selection of QSAR models. Internal validation was carried out using leave-one-out (LOO) cross-validation method, boot-strapping technique and randomised biological activity test while external validation was confirmed with test set data. Furthermore, we hope that the current study provides better insight into the designing of more potent 2,3-dihydroquinazolinones as antihypertensive agent in the future before their synthesis. ACKNOWLEDGEMENT The authors wish to express their gratitude to V-life Science Technologies Pvt. Ltd. for providing the software for the study, and Head, School of Pharmacy, Devi Ahilya Vishwavidyalaya for providing facilities to carry out the work. REFERENCES 1. C. M. FERRARIO: The Renin–Angiotensin System: Importance in Physiology and Pathology. J. Cardiovasc. Pharmacol., 15 (Suppl. 3), 51 (1990). 2. M. B. Vallotton: The Renin–Angiotensin System. Trends. Pharmacol. Sci., 8, 69 (1987). 3. C. NAHMIAS, A. D. STROSBERG: The Angiotensin AT2 Receptor Searching for Signal-transduction Pathways and Physiological Function. Trends Pharmacol. Sci., 16, 223 (1995).

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  4. K. H. BERECEK, S. J. KING, J. N. WU: Angiotensin-converting Enzyme and Converting Enzyme Inhibitors. Cellular and Molecular Biology of the Renin–Angiotensin System. CRC Press, Boca Raton, FL, 1993, 183–220.   5. C. HANSCH: A Quantitative Approach to Biochemical Structure–Activity Relationships. Acc. Chem. Res., 2, 232 (1969).   6. A. M. DOWEYKO: QSAR: Dead or Alive? J. Comput. Aided. Mol., Des., 22, 81 (2008).   7. M. C. SHARMA, D. V. KOHLI, S. SHARMA, S. C. CHATURVEDI: Two Dimensional-quantitative Structure–Activity Relationships – 2,3-diarylthiophenes as Selective COX-1-2 Inhibitors. Digest. J. Nanomat. Biostruct., 4 (3), 459 (2009).   8. M. C. SHARMA, D. V. KOHLI, S. C. CHATURVEDI, S. SHARMA: Molecular Modelling Studies of Some Substituted 2-butylbenzimidazoles Angiotensin II Receptor Antagonists as Antihypertensive Agents. Digest. J. Nanomat. Biostruct., 4 (4), 843 (2009).   9. M. C. SHARMA, D. V. KOHLI, N. K. SAHU, S. SHARMA, S. C. CHATURVEDI: 2D-QSAR Studies of Some 1, 3,4-thidiazole-2-yl-azetidine-2-one as Antimicrobial Activity. Digest. J. Nanomat. Biostruct., 4 (2), 339 (2009). 10. M. C. SHARMA, S. SHARMA, D. V. KOHLI, S. C. CHATURVEDI: Molecular Modelling Studies Atom Based of 3-bromo-4-(1-H-3-indolyl)-2, 5-dihydro-1H-2, 5-pyrroledione Derivatives Antibacterial Activity against Staphylococcus aureus. Der. Pharmacia. Letters, 2 (1), 1 (2010). 11. M. C. SHARMA, S. SHARMA, D. V. KOHLI, S. C. CHATURVEDI: Prediction of Anti-HIV Activity of Non-nucleoside Inhibitors of Human Immuno Deficiency Virus-I Derivatives: Molecular Modelling Approach. Arch. Appl. Sci. Res., 2 (1), 134 (2010). 12. K. SAHU, M. C. SHARMA, V. K. MOURYA, D. V. KOHLI: QSAR Studies of Some Side Chain Modified 7-chloro-4-aminoquinolines as Antimalarial Agents. Arab. J. Chem., 2011 (in press) DOI:10.1016/j.arabjc.2010.12.005 13. M. C. SHARMA, S. SHARMA, N. K. SAHU, D. V. KOHLI: 3D QSAR kNNMFA Studies on 6-substituted Benzimidazoles Derivatives as Non-peptide Angiotensin II Receptor Antagonists: A Rational Approach to Antihypertensive Agents. J. Saud. Chem. Soc., 2011 (in press) DOI:10.1016/j. jscs.2011.03.005 14. M. C. SHARMA, S. SHARMA, N. K. SAHU, D. V. KOHLI: QSAR Studies of Some Substituted Imidazolinones Derivatives Angiotensin II Receptor Antagonists Using Partial Least Squares Regression (PLSR) Based Feature Selection. J. Saud. Chem. Soc., 2011 (in press) DOI:10.1016/j. jscs.2011.03.012 15. J. I. LEVIN, P. S. CHAN, T. BAILEY, A. S. KATOCS, Jr., A. M. VENKATESAN: The Synthesis of 2,3-dihydro-4(1H)-quinazolinone Angiotensin II Receptor Antagonists. Biorg. Med. Chem. Lett., 4 (9), 1141 (1994). 16. MDS: Molecular Design Suite: VLife Sciences Technologies, Pvt. Ltd., Pune, India, 2003. See www. vlifesciences.com. 17. A. T. BALABAN: Highly Discriminating Distance-based Topological Index. Chem. Phys. Letters, 89, 399 (1982). 18. D. PLAVSIC, M. SOSKIC, N. LERS: On the Calculation of the Molecular Descriptor. J. Chem. Inf. Comput. Sci., 38, 889 (1998). 19. D. BONCHEV, N. TRINAJSTIC: Topological Characterization of Molecular Branching. J. Quantum. Chem., 67, 4517 (1977). 20. L. B. KIER, L. H. HALL: Valence Connectivity Indices. Eur. J. Med. Chem., 12, 307 (1997). 21. R. D. CRAMER, D. E. PATTERSON, J. D. BUNCE: Comparative Molecular Field Analysis (CoMFA) 1. Effect of Shape on Binding of Steroids to Carrier Proteins. J. Am. Chem. Soc., 110, 5959 (1988). 22. W. ZHENG, A. TROPSHA: Novel Variable Selection Quantitative Structure–Property Relationship Approach Based on the k-nearest Neighbor Principle. J. Chem. Inf. Comput. Sci., 40, 185 (2000). Received 15 December 2010 Revised 29 January 2011

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Oxidation Communications 35, No 3, 735–741 (2012) Biological section

Screening of Antimicrobial Activity in Few Marine Macroalgae Collected from the Rameswaram– Mandapam Coast of India R. Shrivastavaa*, S. Mitrab, M. Sahab Bhopal Memorial Hospital and Research Center, 462 038 Bhopal, (M.P.) India E-mail: [email protected] b School of Biotechnology, Chemical and Biomedical Engineering, VIT University, 632 014 Vellore, (TN) India a

ABSTRACT The aim of present study was to evaluate chemical and antibacterial activity of commonly found marine macroalgae (Rhodophyceae and Phaeophyceae) from the Rameshwaram–Mandapam coast of India against MTCC Escherichia coli, Staphylococcus aureus and Salmonella typhi by using broth dilution, agar well diffusion, and spectrophotometric analysis. Among the different solvent extracts of marine macroalge, i.e. chloroform-methanol, diethyl ether and water, the chloroform-methanol extracts of Dictyota dichotoma, showed maximum inhibition against S. aureus and S. typhi, at a meager concentration of 0.35 mg/ml. The Dictyota dichotoma extract reduced the viable E. coli count in a log phase culture by 103 times and against S. typhi in 47%. In conclusion, water-methanol extract of Dictyota dichotoma could be used as potential antimicrobial agents to treat or prevent different bacterial infections. Keywords: chloroform-methanol extract, macroalgae, antibacterial activity. AIMS AND BACKGROUND Investigatory studies on marine macroalgae have shown that they contain bio-active compounds that exhibit anti-microbial activities against potent pathogens. Extensive chemical investigations of extracts from marine organisms have led to discovery of variety of secondary metabolites with anti-microbial activities against human pathogens. Lipophilic solvent extracts from marine macroalgae have been investigated as a source of substances with pharmacological implications. Although the ocean represents the centre of biological diversity with 34 of 37 phyla of life represented (compared to only 17 on land), prospecting marine resources for biotechnological use, particularly in *

For correspondence.

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drug discovery, is a relatively recent activity. Seaweeds, especially brown and the red algae are expected to contain bioactive compounds1. Lipophilic solvent extracts from marine algae have been investigated as a source of substances with pharmacological implications2,3 have shown antibacterial activity in organic solvent extract of 6 species of marine algae against multi-drug resistant bacteria. Sastry et al.4 also reported antibacterial activity in the organic solvent extracts of seaweeds against gram-positive and gram-negative pathogenic bacteria. Extensive chemical investigations of extracts from marine organisms have led to the discovery of a variety of secondary metabolites with antimicrobial activities against human pathogens5,6. While chemically-mediated disease resistance is well documented among terrestrial plants7–9, little is known about the antimicrobial functions of secondary metabolites produced by marine plants. However, a high number of novel bio-products with useful and sometimes unique pharmacological properties have been described and some of them are in late stage of clinical trials1. The aim of the present study was to evaluate antimicrobial activity of extract of different algal species, namely Sargassum tenerrimum, Dictyota dichotoma, Gracilaria crassa and Gelidiella acerosa, against different bacterial species of Escherichia coli MTCC 521, Salmonella typhi MTCC 733, and Staphylococcus aureus MTCC 737. EXPERIMENTAL Macroalgal material. Four species were collected from the Rameswaram and Mandapam coastal region in India (Table 1). The identification was done by CMFRI, Mandapam, India. Table 1. Macroalgal species and collection points

No Species 1 Sargassum tenerrimum 2 Dictyota dichotoma 3 Gracilaria crassa 4 Gelidiella acerosa

Class Place Location Phaeophyceae Thonithurai, east of the gulf of Mannar Mandapam Phaeophyceae north of Thonithurai, Palk bay Mandapam Rhodophyceae near Pisasu Munai, Palk bay Vadakadu, Rameswaram Rhodophyceae Vedalai, Mandapam the gulf of Mannar

The collected macoalgae from the Rameshwaram–Mandapam coast in India were thoroughly washed with fresh seawater to remove all extraneous matters and immediately deep frozen in the field with salt-ice mix. The deep frozen material was kept in the laboratory in a deep freeze until further use. The frozen material was thoroughly washed in many changes of glass-distilled water to remove salts and other matter. This cleaned material was then dried in folds of blotting paper to remove the excess moistures, before preparing extracts.

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Microorganisms. Microorganisms were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. The anti-bacterial activity was tested against Escherichia coli MTCC 521, Salmonella typhi MTCC 733, and Staphylococcus aureus MTCC 737. Extraction. Macroalgae were minced and subjected to extraction using diethyl ether, water and chloroform–methanol (1:1). The chloroform–methanol solvent system showed the maximum activity so all the algal samples were then minced and subjected to extraction using 1:1 chloroform–methanol mixture in a 1:4 dry weight:solvent mixture ratio. The filtrates were evaporated up to dryness in a rotary evaporator at 37oC to yield the stock and this was subsequently dissolved in 1% DMSO. From this stock, the concentration of the extract in mg/ml was calculated. Broth dilution assay. A modified version of a bacterial growth inhibition assay10,11 was used to assess the antibacterial effects of the extracts against the pathogenic bacteria Escherichia coli and Salmonella typhi. Instead of a stationary culture, a growing log phase culture that is permitted to result from an inoculation was chosen for testing antibacterial property. The algal extract was diluted using serial dilution technique in nutrient agar such that the concentration changes as 10–1, 10–2 and 10–3 and so on. This method is particularly useful while doing preliminary screening and selecting the most efficient anti-bacterial agent among all tested. 4 µl log phase culture of the bacteria was added to each test tube. The control was made with serially diluting 1 ml of DMSO. After 24 h, test against E. coli was plated for viable cell count and the minimum concentration showing appreciable inhibition (near to 50% inhibition or IC50) was determined. For calculation of percent inhibition in S. typhi by using spectrophotometer the initial (t=0 h) and at t =14, 16, 18, and 20 h OD values were determined in controls and with extract of macroalage at 595 nm and compared with each other and calculated percentage inhibition by change of turbidity. Agar well diffusion assay. This method as described12 was used to test against the pathogenic bacterium Staphylococcus aureus. A well was punctured into the agar plated Petri plate and was filled with 20 µl of algal extract dissolved in DMSO. The control was kept with well filled with DMSO only and after the incubation, inhibition diameters (in mm) against algal extract were determined by using calliper square. RESULTS AND DISCUSSION Recently, much attention has been directed towards extracts and biologically active compounds isolated from seaweeds. Marine macroalgae have received comparatively less bioassay attention. On the contrary, there are a number of seaweeds with economic potential13. The review of literature in some studies shows that methanol extraction appears more effective, particularly in terms of antimicrobial activity than n-hexane 737

and ethyl acetate2, whereas in others, chloroform has been shown to be better than methanol and benzene4. In the present study, a crude extract of marine macroalgae with mixture of chloroform–methanol was prepared and quantified. The concentration of crude stock is higher in S. tenerrimum and Gelidiella acerosa as compared to other macro marine algae (Table 2). It was evident from the experience of the previous study and the fact that the use of organic solvents always provides a higher efficiency in extracting antimicrobial activities, as compared to water extraction. Preliminary experiments also show that this chloroform–methanol mixture is more potent in extracting than diethyl ether or water. Table 2. Sample quantification

No 1 2 3 4

Sample S. tenerrimum Gracilaria crassa Gelidiella acerosa Dictyota dichotoma

Concentration (crude stock) (mg/ml) 1.00 0.40 1.00 0.35

Broth dilution test was carried against a growing log phase culture of the pathogenic E. coli and minimum concentration showing appreciable inhibition could be found in the 10–2 dilution of the extracts. The concentration of the extracts at this dilution and the extracts showing the best inhibitory effect are tabulated in Table 3. The inhibitory effect was analysed by plating for viable cell (after a day incubation) from the tests against the control. As can be clearly seen from the table, Sargassum tenerrimum, Dictyota dichotoma, Gracilaria crassa, Gelidiella acerosa were best able to show inhibitory effect. All of them were able to reduce the number of colonies by 103 times. Gracilaria crassa, Sargassum tenerrimum extracts were more potent than others. Against a control with 6×(1012) CFU (at 24th hour), the former reduced the viable cell to 6.6×(109) and the later to 7.2×(109) CFU, respectively. Table 3. Broth dilution test (against E. coli)

S. No 1 2 3 4 5

Sample Control S. tenerrimum Gelidiella acerosa Dictyota dichotoma Gracilaria crassa

Conc. of extract (mg/ml) – 1.00 1.00 0.3l 0.40

Cell count (at 10–2 dil.) >300 (TNTC)   66 290 312   72

CFU/ml 6 × 1012 6.6 × 109 29 × 109 31.2 × 109 7.2 × 109

Similarly, the well diffusion test was carried out for all the algae against the pathogenic Staphylococcus aureus and the results are tabulated in Table 4. Dictyota dichotoma was the only species showing appreciable activity with a diameter of the zone of inhibition as 10 mm. This activity was shown at a concentration of only 0.35 mg/ml. 738

Table 4. Well diffusion test (against Staphylococcus aureus)

No

Sample

Zone of inhibition (mm)

1 2 3 4

S. tenerrimum Gracilaria crassa Gelidiella acerosa Dictyota dichotoma

– – – 10

Note: implies less than 3 mm.

Spectrophotometricaly, the effects of extracts of marine macroalgal species were studied on growth kinetics of S. typhi (Tables 5 and 6). The respective concentration of the algal extract can be derived from the table of crude concentration shown before 10–2 dilution given more promising result than10–3 dilution. Dictyota dichotoma, Gelidiella acerosa, Sargassum tenerrimum were the most effective at minimum concentration of 0.35, 1, and 1 mg/ml, respectively, and gave a percentage inhibition (change in OD value) of 47, 42 and 46%, respectively. These extracts drove the growing culture into an early stationary phase and resulted in a stunted log phase. At 10–3 dilutions none of them gave proper inhibition which could be attributed to the very small concentration. The inhibition was demonstrated at the log and stationary phase of the bacterium and not at the inoculation or early phase indicating that the antibacterial compound may be antimetabolite and may be of phenolic origin. Table 5. Effect on growth kinetics of S. typhi for 10–2 dilution (values included after negating from the initial value of each)

Series Sample 1 2 3 4 5

Control G. crassa S. tenerrimum G. acerosa Dictyota dichotoma

Initial 0 (0.008) 0 (0.101) 0 (0.186) 0 (0.101) 0 (0.845)

14th hour 16th hour 18th hour 20th hour % Inhibition 0.374 0.514 0.548 0.628 – 0.308 0.374 0.369 0.388 38 0.215 0.281 0.327 0.340 46 0.211 0.303 0.345 0.365 42 0.252 0.384 0.367 0.335 47

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Table 6. Effect on growth kinetics of S. typhi for 10–3 dilution (values included after negating from the initial value of each)

Series Sample 1 2 3 4 5

Control G. crassa S. tenerrimum G. acerosa Dictyota dichotoma

Initial 0(0.009) 0(0.028) 0(0.033) 0(0.028) 0(0.072)

14th hour 16th hour 18th hour 0.371 0.343 0.328 0.325 0.309

0.514 0.410 0.450 0.445 0.426

0.565 0.480 0.514 0.529 0.524

20th hour 0.612 0.437 0.515 0.546 0.557

% Inhibition – 28.5 15.8 10.8 08.9

The survey done shows the presence of highly potent antimicrobial agent in Gracilaria crassa, Dictyota dichotoma, Sargassum tenerrimum viewing their abundance in nature these crops can be harvested to yield economically, antibacterial agents potent against clinically important bacteria. The relative less concentration of extracts of Gracilaria and Dictyota showing appreciable inhibition may support their economic use. The survey indicates the potential use of the algal bioactive compounds for disinfection. The activity is potent enough to decrease the number of viable cells by more than 103 at a sparse concentration of 0.35 mg/ml, too. It is clear from the above survey that Dictyota has emerged as the favourite showing a broad spectrum of action particularly in case of the studied pathogenic organisms. In case of S. typhi, the extract of this algae drives the culture to an early death phase. The resulting compound may be stated as an antimetabolite owing to the nature which showed shunted log phase and early stationary phase. Dictyota dichotoma have long being acknowledged to show a broad spectrum activity. Nair et al.12 also stated that methanol extract of Dictyota to be effective against Bacillus subtilis 14 also stated that Dictyota showed anti-microbial activity against marine pathogens and saprophytes. CONCLUSIONS Higher concentration of chloroform–methanol extract of Dictyota dichotoma was observed which showing antimicrobial activity against different bacterial species,

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i.e. Escherichia coli MTCC 521, Salmonella typhi MTCC 733, and Staphylococcus aureus MTCC. ACKNOWLEDGEMENTS Authors thank Dr. Sumit Kumar Mitra, Geological Survey of India for the help to locate the samples and to CMFRI Mandapam, India, to provide the algal samples. We are also grateful to Dr. Lazar Mathew, Dean, VIT University, Vellore, for the full support and encouragements. REFERENCES   1. IUCN GLOBAL MARINE PROGRAMME: IUCN Bioprospecting Marine Resources Conservation Concerns and Management Implications. 2004.   2. C. FEBLES, A. ARIAS, A. HARDISSON, M. C. GIL-RODRIGUEZ, A. SIERRA LOPEZ: In vitro Study of Antimicrobial Study in Algae (Chlorophyta, Phaeophyta and Rhodophyta) Collected from the Coast of Tenerife. Anuario del Estudios Canarios, 34, 181 (1995) (in Spanish).   3. I. MARASNEH, M. JAMAL, M. KASHASNEH, M. ZIBDEH: Antibiotic Activity of Marine Algae against Multi-antibiotic Resistant Bacteria. Microbiol., 83, 23 (1995).   4. V. M. V. S. SASTRY, G. R. K. RAO: Antibacterial Substances from Marine Algae-successive Extraction Using Benzene, Chloroform and Methanol. Bot. Marina, 37, 357 (1994).   5. K. L. RINEHART, Jr., P. D. SHAW, L. S. SHIELD, J. B. GLOER, G. C. HARBOUR, M. E. S. KOKER, D. SAMAIN, R. E. SCHWARTZ, A. A. TYMIAK, D. L. WELLER, G. T. CARTER, M. H. G. MUNRO, J. R. G. HUGHES, H. E. RENIS, E. B. SWYNENBERG, D. A. STRINGFELLOW, J. J. VARVA, J. H. COATS, G. E. ZURENKO, S. L. KUENTZEL, L. H. LI, G. J. BAKUS, R. C. BRUSCA, L. L. CRAFT, D. N. YOUNG, J. L. CONNOR: Marine Natural Products as Sources of Antiviral, Antimicrobial, and Antineoplastic Agents. Pure Appl. Chem., 53, 795 (1981).   6. J. L. REICHELT, M. A. BOROWITZKA: Antimicrobial Activity from Marine Algae: Results of a Large-scale Screening Programme. Hydrobiologia, 116/117, 158 (1984).   7. J. L. INGHAM: Phytoalexins and Other Natural Products as Factors in Plant Disease Resistance. Bot. Rev., 38, 343 (1972).   8. J. L. INGHAM: Disease Resistance in Higher Plants. Phytopathol. Z., 78, 314 (1973).   9. R. HAMMERSCHMIDT: Phytoalexins: What Have We Learned after 60 Years. Ann. Rev. Phytopathol., 37, 285 (1999). 10. J. KUBANEK, P. R. JENSEN, P. A. KEIFER, C. SULLARDS, W. FENICAL: Seaweed Resistance to Microbial Attack: a Targeted Chemical Defense against Marine Fungi. Proc. Natl. Acad. Sci. USA, 100, 6919 (2003). 11. Ch. S. VAIRAPPAN: Potent Antibacterial Activity of Halogenated Metabolites from Malayasian Red Algae, Laurencia majuscule (Rhodomelaceae, Ceramiales). Biomol. Eng., 20, 255 (2003). 12. R. NAIR, T. KALARIYA, S. CHANDA: Antibacterial Activity of Some Selected Indian Medicinal Flora. Turk. J. Biol., 29, 41 (2005). 13. A. T. CRITCHLEY, R. D. GILLESPIE, K. W. G. ROTMANN: Seaweed Resources of South Africa. In: Seaweed Resources of the World (Eds A. T. Critchley, M. Ohno). Japan International Cooperation Agency, Yokosuka, 1998, 413–431. 14. S. ENGEL, M. P. PUGLISI, P. R. JENSEN, W. FENICAL: Antimicrobial Activities of Extracts from Tropical Atlantic Marine Plants against Marine Pathogens and Saprophytes. Marine Biology, 149, 991 (2006). Received 30 April 2010 Revised 10 July 2010

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Oxidation Communications 35, No 3, 742–750 (2012) Environmentally friendly processes

Phenolic Content of the Effluents from the Petroleum Industry in Albania B. Seitia*, D. Topia, A. Korpaa, A. Lamea, K. Xhaxhiua, I. Beqirajb Department of Chemistry, Faculty of Natural Sciences, University of Tirana, Tirana, Albania E-mail: [email protected] b Department of Industrial Chemistry, Faculty of Natural Sciences, University of Tirana, Albania a

ABSTRACT Albanian oil industry has fashioned a remarkable economic landscape for the country, however on the negative side, petroleum exploration and production also have adverse effects on the environment of the host communities and the subsistence flora and fauna life and farm, which is the traditional means of livelihood of the people. On the other scale, when considered in respect of its negative impact on the socioeconomic life and the environment of the immediate oil bearing local communities and its inhabitants, it has left a balance sheet of ecological and socio-physical disaster. Effluent generated by the industries is one of the sources of pollution. Present in discharged effluents by oil industry, phenols are responsible for different toxic effects on the human health. Phenols index is a means to address evaluation of the phenols content in the effluents. The notion phenols index includes reaction in the presence of 4-aminoantypirine and phenols, which form an organic complex capable to absorb the light in the UV-vis. intervals. In addition, analytical method such as standard method ISO 6439, 1990 was applied for measuring the phenols content. Sampling sites were selected for monitoring. The reported results of phenol content were compared with International standards. Keywords: environmental pollution, phenols index, effluents. AIMS AND BACKGROUND Albania is considered a rich country in natural resources. Electricity system is based on diversified sources of primary energy and supply. Oil industry has fashioned a remarkable economic landscape for the country, however on the negative side, pe*

For correspondence.

742

troleum exploration and production also has adverse effects on the environment of the host communities and the subsistence flora and fauna life and farm, which is the traditional mean of livelihood of the people. Petroleum is used mostly, by volume, for producing fuel oil and petrol, both important ‘primary energy’ sources. Petroleum deposits input is 62% of the total energy. Impacting unavoidably the environment, oil deposits have been intensively exploited for 70 years1. Oil industry is quite developed in regions such as Fieri, Ballshi and Kucova. The annual oil production is 520 000 t. This paper aims at evaluating phenols content using phenol index of effluents from the oil industry applying ISO 6439 (1990) standards. The quantity of the effluents discharged from the industry plays a significant role not only for the content of phenols in water but also for their degree of distribution. The average annual discharges in water for some resources (decanting plants) are as follows: Marinza 171 550 m3 year–1; Sheqishta 139 633 m3 year–1; Gorishti 135 378 m3 year–1; Visoka 106 729 m3 year–1; Kashi 400 504 m3 year–1; Usoja 400 749 m3 year–1, and Kucova 6618 m3 year–1 (Ref. 2) (Fig. 1). TOXICOLOGY OF PHENOLIC COMPOUNDS

Studies have approved that phenol is a carcinogenic compound3. The International Agency for Research on Cancer (IARC) and the EPA have determined that phenol is not classifiable as human carcinogen. Phenol, C6H5OH, is an organic compound that contains a hydroxyl group that is attached directly to a benzene ring. The phenol can be met in abundance by effluents of oil industry origin. In addition, phenol and its derivatives, being basic structural unit for a variety of synthetic compounds, belong to a group of common environmental contaminants. Their presence even in very low concentrations (ppb) can be an obstacle to the use of water. Phenols cause unpleasant taste and odour of drinking water. It is classified as toxic compound and can exert negative effects on different biological species. When a substance is released either from a large area, such as an industrial plant, or from a container, such as a drum or bottle, it enters the environment. Such a release does not always lead to exposure. Only when someone comes in contact with the substance, it is exposed to it. Someone might be exposed by breathing, eating, or drinking the substance, or by skin contact. If someone is exposed to phenol, many factors will determine whether he/she will be harmed or not. Phenol can be found in air and water after release from the manufacture, use, and disposal of products containing phenol. Phenol in soil is likely to move to groundwater4. Long-term exposure to phenol at work places has been associated with cardiovascular diseases. Ingestion of liquid products containing concentrated phenol can cause serious gastrointestinal damage and even death. Application of concentrated phenol to the skin can cause severe skin damage3. Short-term exposure to high levels of phenol has caused irritation of the respiratory tract and muscle twitching in animals. Longer-term exposure to high levels of phenol caused damaged to the heart, kidneys, liver, and lungs in animals. Drinking water with extremely high concentrations of phenol has caused muscle tremors, difficulty in walking, and death 743

in animals. Short-term application of phenol to the skin has produced blisters and burns in animals. There is no evidence that phenol causes cancer in humans. The monohydric phenols include cresols, xylenols and dihydric phenols, to the last ones also belong the cathecoles and resorcinoles. The residues, which contain phenols ‘phenolic residues’ often contain other compounds too, such as, ammonium, cyanide, organic acids, aldehides, etc.5 The toxic effects of phenol exposure in humans and biodiversity of aquatic species are unavoidable. Phenol and its vapours are corrosive to skin, eyes and respiratory tract6.

Fig. 1. Geography of oil exploitation in Albania

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EXPERIMENTAL ISO 6439/1990 provides information about drinking waters, surface waters, brines (saline waters), domestic waters and industrial waters. After a preliminary distillation the test samples were analysed according to specific application by direct colorimetric method and by chloroform extraction method. The term ‘phenol index’ as used in this International standard includes only phenols which react with 4-aminoantipyrine under the conditions specified to give coloured compounds which absorb light in the UV-vis. range. It is difficult to define various phenols. Phenolic compounds containing 1,4-substituent such as carboxyl, halogen, hydroxyl, methoxyl or sulphonic acid, do coloured product complex with 4-aminoantipyrine. Hence the phenol index includes only those phenolic compounds which can be determined under specified conditions. During the reaction of the 4-aminoantipirine the phenol is oxidised to 1,4benzoquinone and the last one mentioned creates an imino group, after the reaction depicted in Fig. 2. NH2

H3 C

H3 C

N

N O

+

OH

K3 [F e (CN)6 ]

N phenol

O

H3 C

H3 C

N

O N

4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylideneamino)2-phenyl-1,2-dihydro-pyrazol-3-one

Fig. 2. Chemical reaction between phenolic compounds and 4-aminoantypirine

The percentage composition of the various phenolic compounds present in a given test sample is unpredictable. It is obvious, therefore, that a standard containing a mixture of phenolic compounds can not be made applicable to all tests. It represents the minimum concentration of phenolic compounds after preliminary distillation7. Chloroform extraction is a common laboratory technique used for the determination of phenol index without the need for dilution from 0.002 to 0.10 mg/l phenol when the coloured product is a concentrated extract in the chloroform phase by applying the phenol as a standard. The samples obtained were extracted by different effluents from petroleum industry. The samples were extracted from: (i) water discharges from oil industries; (ii) plant of oil decantation and, and (iii) the main water supplies crossing the regions where the oil industries operate.

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RESULTS AND DISCUSSION The information provided in this paper is a tool to address evaluation of phenol content. Results obtained report various phenols content in effluents from oil industry. In addition, they provide the basis for data interpretation8,9. Monitoring sites are: the Gjanica river and the Roskoveci-Hoxhara collector. The international standard ISO (ISO 5667, 1980) is a tool to address selection of samples. Phenol evaluation was based on: (i) national, international standards7,10, and many noted papers4,11. The toxicity of phenols, cresols and xylenes is similar and for this reason the phenols are employed as model substances. In this work the results are expressed by means of the ‘phenol index’ parameter, which expresses the content of phenol and other components in 1 l of sample, referring to the degree of coloured that they give with the 4-aminoantipirine7. Decision of Council of Ministers (DoCM) 177&WB/IFC imposes 6–9 pH values and 0.5 mg/l phenols content in effluents. The analyses of wastewaters from oil industry are given for a 1-year period, divided into 4 months sub-periods. Tables 1–6 report the outcomes of investigation carried out in the sampling sites. Table 1 reports the wastewater discharge values obtained by the center of oil processing (COP) in Patosi. The discharge wastewaters of this center obtained from the Visoka decantation plant have a pH 7–7.5 and a phenols content of 0.13–1.92 mg/l. In the meantime, referring to the waste water discharge of year 2005 the pH is 7–7.5 and the phenols content is 0.4–1.82 mg/l. An increase of the phenols content can be seen. These water discharges are a pollution source for the Gjanica river12. Tables 2 and 3 report the outcomes of the investigations carried out in COP Marinza and Sheqishta. The source Marinza discharges its wastewaters in the decantation plant and is characterised by pH value of 7–8.5 and phenols content of 0.30–1.58 mg/l. For the year 2005 the parameters are: pH 6.5–7.5 and phenols content 0.3–0.75 mg/l. The results show that the phenols content has been increased, whereas the pH values have decreased. Investigations carried out in Sheqishta report: pH 7–8 and phenol content 0.9–1.6 mg/l. Compared with the parameters of the year 2005 (pH 7–7.5 and phenols content 0.22–0.70 mg/l), it can be said that the level of phenols varies around the same values13. These water discharges are a potential source for the pollution for the main collector of RoskoveciHoxhara. Table 4 reports the results obtained from the investigations carried out in COP Ballshi. The oil sources of Ballshi and Drenova discharge their waters in the decantation plant of Usoja. Effluents discharged here have these parameters: pH 7.2–8.5 and phenols 0.18–2.94 mg/l. Compared with the parameters of the year 2005 (pH 7–8 and phenols1 0.20–1.79 mg/l), a worsening of the environmental parameters can be concluded, which makes a high pollution potential for the Gjanica river. Effluents from decantation plant in Kashi provided the following data: pH 7.5–8.5 and phenol content 1.44–1.47 mg/l. Comparing the afore-mentioned data with those for the year 2005 (pH 7.5–8 and phenols 1.2–1.60 mg/l), it can be stated that this plant is still a potential risk for contamination of the Marusha artificial reservoir. The Gorishti source (Table 5) discharges its waters in the respective decantation plant and 746

the parameters are pH 7–8.5 and phenols content 1.70–1.85 mg/l. Compared with the results of the year 2005 (pH 7–8 and phenols 0.30–1.58 mg/l), an increase of the phenols content can be seen. This makes a potential polluting risk for the Vjosa river. The source of Kucova discharges its wastewaters in the respective decanting plant and the parameters are pH 7 and phenols content of 0.18–0.25 mg/l. Compared with the year 2005 (pH 7 and phenols 0.17–0.2125 mg/l), no major difference can be seen. This indicates that the source of Kucova is in a good condition and does not pose any risk for the hydric environment of the area13. In Table 6 are given the results for the source Tranagat – Patosi, the parameters of which are pH 7–8 and phenols content of 1.72–1.85 mg/l. Compared with the parameters of year 2005 (pH 7–8 and phenols content of 0.17–1.58 mg/l), a slight increase of phenols content is observed, hence this source has also an environmental impact on the area. Table 1. Results of discharge waters of the CCOP Patosi*

Sample 1         Sample 2         Sample 3        

Sampling site Visoka dec. plant (reservoir discharge) Visoka dec. plant (outlet) Zharreza station Visoka groups Gjanica river, Visoka decanting plant Visoka dec. plant (reservoir discharge) Visoka dec. plant (outlet) Zharreza station Visoka groups Gjanica river, Visoka dec. plant. Visoka dec. plant (reservoir discharge) Visoka dec. plant (outlet) Zharreza station Visoka groups Gjanica river, Visoka dec. plant

pH 7.5 7.5 7 7.5 7 7.5 7.5 7 7.5 7 7.5 7.5 7 7.5 7

Phenol (mg/l) 1.92 0.28 0.13 0 2.70 1.93 0.30 0.12 0 2.80 1.92 0.29 0.11 0 2.65

*CCOP – Center of Crude Oil Processing; dec. – decanting. Table 2. Results of discharge waters of the CCOP Marinza*

Sample 1       Sample 2      

Sampling site 1 Marinza dec. plant (reservoir discharge) Marinza dec. plant (outlet) Belina bridge Connection point with Roskoveci-Hoxhara collector Marinza dec. plant (reservoir discharge) Marinza dec. plant (outlet) Belina bridge Connection point with Roskoveci-Hoxhara collector

pH 2 8.5 7.5 7 7 8.5 7.5 7 7

Phenol (mg/l) 3 1.56 0.45 0.50 0.30 1.58 0.49 0.54 0.35

to be continued

747

Continuation of Table 2

Sample 3      

1 Marinza dec. plant (reservoir discharge) Marineza dec. plant (outlet) Belina bridge Connection point with Roskoveci-Hoxhara collector

2 8.5 7.5 7 7

3 1.56 0.46 0.50 0.32

*CCOP – Center of Crude Oil Processing; dec. – decanting. Table 3. Results of discharge waters of the CCOP Sheqishta*

Sample 1       Sample 2       Sample 3      

Sampling site Sheqishta dec. plant (reservoir) Sheqishta dec. plant (outlet) Canal near pump station Kallma Sheqishta dec. plant (reservoir) Sheqishta dec. plant (outlet) Canal near pump station Kallma Sheqishta dec. plant (reservoir) Sheqishta dec. plant (outlet) Canal near pump station Kallma

pH 7.5 7.5 7 7.5 8 7.5 7 8 7.6 7.5 7 7.5

Phenol (mg/l) 0.9 1.5 0.5 0.4 0.9 1.6 0.5 0.4 0.85 1.5 0.5 0.4

*CCOP – Center of Crude Oil Processing; dec. – decanting. Table 4. Results of discharge waters of the CCOP Ballshi*

Sample 1         Sample 2         Sample 3        

Sampling site Usoja dec. plant (reservoir) Usoja dec. plant (outlet) Kashi dec. plant (reservoir) Gjanica river, discharge of the Usoja dec.plant Gjanica river, Ballshi Usoja dec. plant (reservoir) Usoja dec. plant (outlet) Kashi dec. plant (reservoir) Gjanica river, discharge of the Usoja dec.plant Gjanica river, Ballshi Usoja dec. plant (reservoir) Usoja dec. plant (outlet) Kashi dec. plant (reservoir) Gjanica river, discharge of the Usoja dec.plant Gjanica river, Ballshi

*CCOP – Center of Crude Oil Processing; dec. – decanting.

748

pH 8.5 7.5 8 7.2 7.5 8.5 7.5 8 7.5 7.5 8.5 7.5 8 7.2 7.5

Phenol (mg/l) 1.40 0.20 1.47 2.90 2.60 1.45 0.18 1.45 2.94 2.65 1.42 0.20 1.44 2.91 2.65

Table 5. Results of discharge waters of the CCOP Gorishti*

Sample 1       Sample 2       Sample 3      

Sampling site Zharreza station (reservoir) Gorishti station (reservoir) Kucova station (reservoir) Usoja station (outlet) Zharreza station (reservoir) Gorishti station (reservoir) Kucova station (reservoir) Usoja station (outlet) Zharreza station (reservoir) Gorishti station (reservoir) Kucova station (reservoir) Usoja station (outlet)

pH 7 8 7 7.5 7 8.5 7 8 7 8.5 7 7.5

Phenol (mg/l) 1.21 1.80 0.25 0.05 1.08 1.76 0.20 0.08 1.14 1.84 0.18 0.04

*CCOP – Center of Crude Oil Processing; dec. – decanting. Table 6. Results of discharge waters of the CCOP Tranagat-Patosi*

Sample 1       Sample 2       Sample 3    

Sampling site Gorishti dec. plant (reservoir) Gorishti dec. plant (outlet) Discharge in the Vjosa river The discharge point of dec. plant in Vjosa river Gorishti dec. plant (reservoir) Gorishti dec. plant (outlet) Discharge in the Vjosa river The discharge point of dec. plant in Vjosa river Gorishti dec. plant (reservoir) Gorishti dec. plant (outlet) Discharge in the Vjosa river The discharge point of dec. plant in Vjosa river

pH 8 7 7 7 8.5 7 7 7 8.5 7 7 7

Phenol(mg/l) 1.85 1.70 1.75 – 1.86 1.74 1.78 – 1.85 1.72 1.75 –

*CCOP – Center of Crude Oil Processing; dec. – decanting.

CONCLUSIONS Defined in national and international standards, in all sampling sites, except Kucova, phenol index more than 0.5 mg/l has been measured. Effluents discharged from oil industry and surface water system resulted in high phenol content. The main collector Roskoveci-Hoxhara and the Gjanica river resulted as the most contaminated sites. Actions for the minimisation of the polluting level such as monitoring of waters discharge is needed along with the examination of the possibilities for their injection in the exploited wells. In compliance with the standards, a continuous monitoring system as well as the building of new separators to prevent 749

the polluting chemicals of the discharge waters to get their way into the natural hydric ecosystem, is recommended. REFERENCES   1. M.I.E.: Oil and Gas Industry in Albania. Statistical and Scientific, Informational Bulletin, Tirana. Tirana, 2004. 260 p. (in Albanian).   2. S.N.C.H.: Technical and Economical Efficacy on the Existing Decanting Plants and Their Influence on Environment. Fieri, Albania, 2005.   3. IARC: Toxicology of Phenolic Compounds. 1989.   4. S.N.C.H.: Environmental Monitoring in Oil Industry and Annual Environmental Report Compiling. Fieri, 2005. 270 p. (in Albanian).   5. Directive 2000/60/EC: On Individual Values for Dangerous Substances.   6. E. HODGSON: A Textbook of Modern Toxicology. 3rd ed. Wiley Inerscience, New York, 2004.   7. ISO 6439-1990: Water Quality – Determination of Phenol Index by 4-aminoantipyrine Spectrometric Methods after Distillation.   8. MEFEA, UNDP: Albania’s Technology Needs Assessment. 2004.   9. (unfccc.int/files/meetings/seminar/application/pdf/sem_albania_sup2.pdf) 10. S.N.C.H.: Map Compiling of Environmental Contamination of Oil Industry Area and Measures for Amilioration of Actual Situation. Fieri, Albania, 2005 (in Albanian). 11. PHARE: Oil and Gas Production (Onshore). World Bank/IFC ed., 1995. 12. DoCM, No 177: The Liquid Discharging Limits and the Zonal Criteria of the Receiving Hydric Environments; Production of the Crude Oil By-products, 2005 (in Albanian). 13. ASTM, D 1293-99: Standard Test Methods for pH of Water. 14. Oakwood Environmental Ltd.: Full Environmental Benchmark Survey for the Rehabilitation of the Patos-Marinza Oilfield, Albania. Process Review Annex 1, London, 1997. Received 7 March 2012 Revised 31 March 2012

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Oxidation Communications 35, No 3, 751–758 (2012) Environmentally friendly processes

Anodic Oxidation as Green Alternative for Removing Textile Dyes from Synthetic and Real Wastewaters A. M. Sales Solanoa, J. H. Bezerra Rochaa, D. Ribeiro Da Silvaa, C. A. Martínez-Huitlea*, M. Zhoub Universidade Federal do Rio Grande do Norte, CCET – Instituto de Química, Lagoa Nova – CEP 59.072-970, Brazil b College of Environmental Science and Engineering, Nankai University, 94 Weijin Road, 300 071 Tainjin, China E-mail: [email protected]; [email protected] a

ABSTRACT In this work, the treatment of synthetic wastewaters containing Novacron Blue C–D (NB) by anodic oxidation (AO) using boron-doped diamond (BDD) and platinum (Pt) anodes was investigated. Galvanostatic electrolyses of NB synthetic wastewaters have led to the complete decolourisation removal at different operating conditions (current density and temperature). The influence of these parameters was investigated in order to find the best conditions for dyestuff colour removal. According to the experimental results obtained, the AO process is suitable for decolourising wastewaters containing these textile dyes, due to the electrocatalytic properties of BDD and Pt anodes. Energy requirements for removing colour during galvanostatic electrolyses of NB synthetic solution depends mainly on the operating conditions: for example, it passes from 14.53 Wh m–3 at 20 mA cm–2 to 60.75 Wh m–3 at 60 mA cm–2 (pH 8) (data estimated per volume of treated effluent) using BDD anodes. In order to verify the Brazilian law regulations of NB synthetic solutions after electrochemical decolourisation treatment, the Hazen units (HU) values were determined and the total colour removal was achieved; remaining into the regulations. Also, electrical energy cost for removing colour was estimated. Finally, AO process was tested to treat an actual textile effluent. Keywords: the Hazen units, textile dyes, anodic oxidation, green alternative, electrocatalytic activity.

*

For correspondence.

751

AIMS AND BACKGROUND Synthetic dyes are extensively used in many fields of up to-date technology, such as in various branches of the textile industry, leather tanning industry, paper production, food technology, agricultural research, light-harvesting arrays, photo-electrochemical cells and in hair colourings. Due to large-scale production and extensive application, synthetic dyes can cause considerable environmental pollution and are serious healthrisk factors1. Reactive dyes are widely used due to their relatively easy application in the dyeing process and stability during wear. The textile industry produces large quantities of wastewater during the washing and dyeing process that contain large quantities of dye that are not fixed on the fibre surface, and as a result are disposed together with the textile effluent (colouration in water courses, affecting their transparency, gas solubility and can present carcinogenic and mutagenic properties)1, 2. For the removal of dyes from wastewater a wide range of techniques, such as chemical precipitation, chemical oxidation or adsorption with subsequent biological treatment has been developed1. As an innovative green alternative, the electrochemical processes for treating wastewater containing dyes have been recently proposed2. The application of electrochemical technologies for wastewater treatment is benefiting from advantages such as versatility, environmental compatibility (green chemistry) and potential cost effectiveness3,4. This technique has been already used for decolourisation and degrading dyes from aqueous solutions by several green scientific groups2. In the present study, we propose the use of electrochemical technology as a green alternative to remove synthetic dyes from water in order to eliminate their strong colour and their ecotoxicological consequences on aquatic environment, investigating the influence of the applied current density and temperature, on the colour removal of a synthetic solution containing NB (dyes widely used in the North-eastern Brazilian Textile industries, affecting aquatic ecosystems due to their colouration persistence, and also due to their carcinogenic and mutagenic properties1) in order to identify optimum experimental conditions which give high current efficiency and need low energy requirements. EXPERIMENTAL Ultrapure water was obtained by simplicity water purification system. Chemicals were of the highest quality commercially available, and were used without further purification. Na2SO4 and NaOH were purchased from Fluka. Dye was purchased by Brazilian textile industry and no information about structure or chemical formula was available because these are dyes protected by patent restriction. The dyestuff solution was prepared dissolving textile dyes in distilled wastewater containing 0.5 M Na2SO4+0.25 M NaOH, adjusting pH value to 8 using 0.1 M NaOH. The textile dyeing industrial effluent generally contains with 100–150 mg l–1 of dissolved salts and pH 8, for this reason, AO process was tested adjusting pH value and using this dye concentration (190 mg l–1). Synthetic dye solutions contained 190 mg l–1 of NB 752

dye was used, as initial concentration. Bulk oxidations were performed in undivided electrochemical cell, the reaction compartment having a capacity of 400 ml. AO experiments of NB were performed under galvanostatic conditions using a VERSTAT3 potentiostat-galvanostat (Princeton Applied Research). BDD anode was supplied by Adamant Technologies (Neuchatel, Switzerland). BDD was used as the anode, and titanium as the cathode. Ti-supported Pt anode was supplied by Industrie De Nora S.p.A. (Milan, Italy). Both anodes were square, each with 10 cm2 geometrical area. The temperature of the electrolyte was controlled using a water thermostat. Experiments were performed at 25°C for studying the role of applied current density (jappl = 20, 40 and 60 mA cm–2) and, while the temperature effect (25 to 60°C) was studied under a current density of 40 mA cm–2. Experimentally, decolourisation efficiency or percentage of colour removal is determined by the expression: % colour removal = [(ABS0–ABSt)/ABS0]×100,

where ABS0 and ABSt are the average absorbance before electrolysis and after an electrolysis time t, respectively, at the maximum visible wavelength (λmax=600 nm for NB) of the wastewater. Colour removal was monitored by measuring absorbance decrease using a UV 1800 Shimadzu spectrophotometer. HU were determined using a Hach Model DR/2500 spectrophotometer calibrated with a method 8025 (Pt–Co units)5. The energy consumption (EC) per volume of treated effluent was estimated and expressed in kWh m–3. The average cell voltage, during the electrolysis, is taken for calculating the EC, as follows: EC=[(V×A×t)/Vs)],

where t is the time of electrolysis (h); V and A – the average cell voltage and the electrolysis current, respectively; and Vs – the volume (m3)2. RESULTS AND DISCUSSION The visible spectrum of NB showed a maximum absorption peak in the range of visible light which is in accordance with the colour of NB solutions. Thus, the measurement of the colour removal was obtained using an UV-vis. spectrophotometer at 600 nm. In both cases, absorbance was satisfactorily reduced during the AO of NB using BDD and Pt anodes. The intensity of the visible band decreases continuously until its disappearance after about 200 min of electrolysis leading to complete solution decolourisation for BDD and Pt anodes. Furthermore, the changes in absorbance were rapid, indicating that during the first stages of the treatment, there are mechanisms that involve the oxidation of the dye to other more simple organics. AO of these complex molecules can lead to the formation of many intermediates by elimination of chromophore groups prior to the formation of aliphatic carboxylic acids and carbon dioxide, as observed by other authors2. Figure 1 presents the influence of the applied current density (jappl) on the decay of colour, during AO of synthetic waste of NB using Pt and BDD anodes. As can be 753

observed, the complete removal of colour in all cases was achieved; independently on the value of jappl and pH. However, it seems that the decolourisation time depends mainly on the jappl. In fact, colour removal times for BDD and Pt decrease when an increase in the jappl was attained, being more evident for BDD anode. It indicates the complete dye elimination by means of its reaction with electrogenerated •OH radicals on electrode surface2–4,6. This behaviour suggests that the oxidation of NB could be carried out both by direct AO and mediated AO (hydroxyl radicals and adsorption organic compound on anode surface).

Fig. 1. Influence of jappl on the colour removal as a function of the time, during BDD (a) and Pt (b) anodic oxidation of textile NB dye. Operating conditions: [NB] – 190 mg l–1, T–25°C, pH 8.0 and agitation rate – 400 rpm. Inset: Colour removal photographs during electrochemical processes at different jappl values: 20 mA cm–2 (squares), 40 mA cm–2 (circles) and 60 mA cm–2 (triangles)

It is important to remark that Comninellis explained the different behaviour of electrodes in AO process, considering 2 limiting cases: the so-called ‘active’ and ‘nonactive’ anodes6. Typical examples are Pt, IrO2 and RuO2 for the former and PbO2, SnO2 and BDD for the latter. The proposed model assumes that the initial reaction in both kind of anodes corresponds to the oxidation of water molecules leading to the formation of hydroxyl radicals (•OH). But the electrochemical and chemical reactivity of heterogeneous (•OH) is dependent on the nature of the electrode material. Then, the surface of active anodes interacts strongly with •OH (case of Pt) and then, a so-called higher oxide or superoxide, this may occur when higher oxidation states are available for a metal oxide anode, above the standard potential for oxygen evolution2–5. It acts as a mediator in the oxidation of organics, which competes with the side reaction of oxygen evolution via chemical decomposition of the higher oxide species. In contrast, the surface of a non-active anode interacts so weakly with •OH that allows the direct reaction of organics (case of BDD). Therefore, the AO by BDD was faster than Pt anode (Fig. 1). It can be due to absorption steps by dye and oxidation by-products formed during NB elimination at Pt, affecting strongly the decolourisation efficiency and time process. This performance is directly correlated to the influence of the chemical structure of the dye and pH in the EO process, as already confirmed by other authors2. Then, a specific behaviour during AO of NB using BDD and Pt electrodes is expected. In fact, from our results, the influence of the chemical structure of the dye is evident, as can be observed in Fig. 1. 754

Whereas the electrochemical decolourisation of NB seems independent on jappl using Pt; for removing this dye using BDD anode, the process was strongly dependent on jappl values. Thus, our results confirm that not only the chemical structure influences the process, but also the pH values used strongly affect the decolourisation efficiency due to the protonated/unprotonated form adopted by dyes dissolved in solution. For example, Ammar et al.7 investigated the effect of current, dye concentration and pH on the electrochemical oxidation of 100 cm3 of indigo carmine solutions with 0.05 M Na2SO4 at 35°C in a stirred undivided cell with a 3 cm2 Si/BDD anode and a 3 cm2 stainless steel cathode. A faster colour decay of the dye at pH 3.0 was observed with increasing current from 100 and 300 mA. This causes the decrease in time needed for total mineralisation from 600 to 300 min due to the higher production of ●OH that accelerates the oxidation of organics (as explained above). However, the 220 mg dm–3 indigo carmine solution at 100 mA becomes colourless more quickly at pH 10.0 (120 min) than at pH 3.0 (270 min), because the electroactive species in alkaline medium (the unprotonated form) was more easily oxidised than that of acid medium (the diprotonated form) at this dye. According to these results, the behaviour showed by NB dye during AO process can be confirmed. On the other hand, textile industries employ specific methods for removing colour from wastewater (biological or physical chemical treatments) and after that; decolourisation is measured by the Hazen units (HU) (Ref. 5). According to the Brazilian laws, the colour in the residual effluents from textile industry must be lower than 300 HU. Then, total colour removal was achieved in all cases for each dyestuff solutions; remaining into the regulations. Inclusive, the 300 HU limit is attained after short times or 50–70% of AO treatment. These results allow to establish that the AO treatment could be employed as an alternative for removing colour from dye wastewaters. In addition, as can be seen from Fig. 1, photographs of colour removal were taken, during AO process in order to show the elimination of colour; confirming the application of this technology. Figure 2 shows the influence of temperature on the colour decay as function of time by applying 40 mA cm−2 of current density for removing NB under pH 8.0. As can be seen, total colour removal was achieved in both cases. It seems that the temperature has a significant impact on the kinetics of the AO of NB because the rate of colour removal was considerably increased by increasing the temperature (from 25 to 40 or 60°C) using BDD and Pt anodes. The increase of temperature from 25 to 60°C decreases the electrolysis time required for the total colour removal from 20 to 50% depending on anode. A change in the temperature has slight influence on the AO with hydroxyl radicals. However, at Pt anode, a negligible performance in the oxidation rate was observed in the AO of NB by increasing the temperature, maybe due to the competition between oxygen evolution reaction and organic oxidation on anode surface. Table 1 presents the EC (kWh l–1 of colour removed) required for removing 95% of the dye colour at different conditions (jappl and temperature). As can be observed, during the electrolyses of synthetic wastewaters; the EC seems to 755

be proportional to the jappl. Finally, taking into consideration an electrical energy cost of about R$ 0.3 (R$ = the Brazilian currency; taxes excluded) per kWh (Agência Nacional de Energia Elétrica, Brazil), the process expenditure was estimated and reported in Tables 1 and 2 (inset in Fig. 2a, b) in order to show the viability of this process as a green alternative: Cost (R$ m–3)=[EC (kWh l–1)×0.3 (R$/kWh)]

In this frame, as can be observed from the Tables (inset in Fig. 2a and b), the cost of the electrochemical process is diminishing with an increase in the temperature. These data are a significant result, because the textile industrial effluents are generally discharged with temperatures values between 60 and 80°C, and these conditions could be suitable to remove completely the organic pollutants from these wastewaters.

Fig. 2. Influence of temperature on the evolution with time of colour removal during anodic oxidation of NB under alkaline conditions using BDD (a) and Pt (b) anodes. Operating conditions: [NB]0–190 mg l–1, jappl–40 mA cm−2, temperatures 25°C (squares), 40°C (circles) and 60°C (triangles), agitation rate: 400 rpm. Inset a and b (Tables): HU values into the Brazilian laws for treated effluent, energy consumption per volume of treated effluent, decolourisation time and cost process (Brazilian currency), during anodic oxidation of NB for different applied current densities and temperatures, using BDD and Pt anodes, respectively

REAL TEXTILE EFFLUENT In order to verify the efficiency of the electrochemical treatment using Pt and BDD electrodes as an alternative for removing colour, an effluent of an actual Brazilian textile industry was employed. It is mainly composed of NB and amylum (starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. Textile chemicals from starch are used to reduce breaking of yarns during weaving; the warp yarns are sized, especially for cotton. Starch is also used as textile printing thickener) with different additives (specific textile process involving NB). This effluent contains a high concentration of chemical oxygen demand (COD=7068 mg dm−3) and the Hazen units (7504 HU). Its conductivity is 5.90 mS cm−1 and the pH is around 8.2. It is worth noting that these conditions were determined from the effluent without any physicochemical treatment (effluent directly obtained after textile treatment). AO was performed under optimum conditions of current density and temperature established by previous experiments with synthetic 756

dye solutions containing NB (60 mA cm–2 and 60°C). Additionally, temperature condition was chosen, mimicking the actual temperature of the effluent discharged in the industry. As shown in Fig. 3, the decrease with time of the absorbance during galvanostatic electrolysis of real textile effluent (0.5 l) was monitored for the period of 6 h, by applying 60 mA cm−2 of current density under untreated effluent conditions (T=60°) and an colour decay was observed in both cases (Pt and BDD). Nevertheless, a complete decolourisation was only achieved at BDD anode, obtaining 25 HU (see photographs).

Fig. 3. Electrochemical decolourisation process of a real effluent as a function of time using Pt and BDD anodes. Inset: photographs showing colour removal

On the basis of these results, the diamond electrochemical technology can be suitable as an alternative for pre-treatment of textile real effluents. Finally, it is important to mention that COD was reduced to 750 mg/l at 60ºC; confirming the potential efficiency of this dyestuff treatment. CONCLUSIONS These results point out the high performance of AO for treating synthetic dyeing wastewaters compared to other advanced oxidation processes. The EC makes useless BDD-AO for complete elimination of wastewaters polluted with dyes but it can be a feasible process for decolourising wastewaters containing dyes as a pre-treatment process. Finally, taking into consideration electrical energy cost per kWh, the viability of this process as a green alternative was confirmed. Further experiments are in progress in order to improve the current efficiency and reduce the charge required for complete oxidation, performing experiments at a lower current density below to limit value. Also, other parameters such as pH variation and intermediates produced during electrochemical process will be studied and reported in details in a separate paper in a near future.

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ACKNOWLEDGEMENTS The authors thank the financial support provided by PETROBRAS and they also thank Industrie De Nora S.p.A. (Milan, Italy) for providing the Ti/Pt electrodes. The authors thank FAPERN (Programa de infra-estrutura para jovens pesquisadores – PPP) for the financial support provided. REFERENCES 1. E. FORGACS, T. CSERHATI, G. OROS: Removal of Synthetic Dyes from Wastewaters: A Review. Environ. Inter., 30, 953 (2004). 2. C. A. MARTINEZ-HUITLE, E. BRILLAS: Decontamination of Wastewaters Containing Synthetic Organic Dyes by Electrochemical Methods. A General Review. Appl. Catal. B: Environ., 87, 105 (2009). 3. C. A. MARTÍNEZ-HUITLE, S. FERRO: Electrochemical Oxidation of Organic Pollutants for the Wastewater Treatment: Direct and Indirect Processes. Chem. Soc. Rev., 35, 1324 (2006). 4. M. Panizza, G. Cerisola: Direct and Mediated Anodic Oxidation of Organic Pollutants. Chem. Rev., 109, 6541 (2009). 5. Ch. COMNINELLIS: Electrocatalysis in the Electrochemical Conversion/Combustion of Organic Pollutants for Waste Water Treatment. Electrochim. Acta, 39, 1857 (1994). 6. International Organization for Standardization, ISO 2211:1973: Measurement of Colour in Hazen Units (Platinum-Cobalt Scale) of Liquid Chemical Products. 7. S. Ammar, R. Abdelhedi, C. Flox, C. Arias, E. Brillas: Electrochemical Degradation of the Dye Indigo Carmine at Boron-doped Diamond Anode for Wastewaters Remediation. Environ. Chem. Lett., 4, 229 (2006). Received 18 September 2010 Revised 12 October 2010

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Oxidation Communications 35, No 3, 759–766 (2012) Environmentally friendly processes

Biochemical Profiling and Pigment Contents of Different Class of Marine Macroalgae Collected from the Rameswaram–Mandapam Coast in India R. Shrivastavaa*, Sh. Mitrab, M. Sahab Bhopal Memorial Hospital & Research Center , 462 038 Bhopal (MP), India E-mail: [email protected] b School of Biotechnology, Chemical and Biomedical Engineering, VIT University, 632 014 Vellore, (TN), India a

ABSTRACT In this study, 3 taxa from the Chlorophyta, Phaeophyta and Rhodophyta were collected from different depths of the Rameswaram–Mandapam coast, India. A total of 3 specimens from these divisions were used to determine percentage of total protein (TP), total soluble carbohydrate (TSCH) and total lipid (TL) and different pigments, i.e. chlorophylls and phycobilins, etc. Percentage of TP, TSCH and TL and pigment contents varied significantly with respect to the algal taxa, stations and depth distribution. In addition, individual differences were important in all of the measured parameters. The maximum TP were determined in Caulerpa sp. (20.47%). In contrast to TP value, TSCH values were very high; maximum TSCH were determined in Caulerpa sp. (57.81%), but TL is maximum in Sargassum tenerrimum (7.34%) but very low as compared to TP and TSCH. In case of pigments the total chlorophyll is high in Caulerpa sp. and lowest in Sargassum tenerrimum and phycobilins content (phycocyanin and phycoerythrin) is high in Gelidiella acerosa. In conclusion, the variations in TP, TSCH and pigment in 3 taxa of macroalgae were analysed according to station, and environment. Keywords: total protein, total soluble carbohydrate, total lipids, pigments. AIMS AND BACKGROUND In marine ecosystems, macroalgae are ecologically and biologically important. Macroalgal communities provide nutrition, reproduction, and an accommodating environment for other living organisms1–6. Because of these properties, macroalgae are some of the most important organisms maintaining the ecosystem stability. Macroalgal *

For correspondence.

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polysaccharides are used in the food, cosmetics, paint, crop, textile, paper, rubber and building industries. In addition, they are used in medicine and in pharmacology for their antimicrobial, antiviral, antitumor, anticoagulant, antioxident and fibrinolytic properties7–15. According to FAO (Food and Agriculture Organisation), the annual global aquaculture production of marine algae is 6.5 × 106 t (Ref. 15). Macroalgae have been harvested for a long time in the Far East, where they are used in the food industry. Because of their high protein content, protein concentrates (PCs) of seaweeds have become more important for the food industry, especially in developed countries16. Their recent utilisation as an animal feed is on the increase. The use of macroalgae as food for fish larvae has been initiated as an alternative to microalgal cultures. Even though the Rameswaram–Mandapam coast in India is surrounded by seas and the macroalgal industry is as yet undeveloped. Therefore, it has not been possible to profit from the many marine algae that are ready to be harvested. First, macroalgal productivity and contents have to be analysed in order to determine how to profit from them. The quantity of macroalgal pigment is mostly used to define algal biomass which has a very good antioxidant properties. It is also affected by environmental factors. Many studies indicate that extreme environmental factors, e.g. salinity, temperature, nutrients, and intense irradiance, cause a high rate of pigment production17–19. The aim of this study is to determine the total soluble carbohydrates (TSCH), total protein (TP), total lipid (TL) and pigment contents of some macroalgae, which were collected from various depths in the Rameswaram–Mandapam coast in India. EXPERIMENTAL Three species were collected from the Rameswaram and Mandapam coastal region. These species belong to the Phaeophycae, Rhodophycae and Chlorophycae families (Table 1) . The identification is done by CMFRI, Mandapam, India. Table 1. Name and location of algae collected

S. No Name of the species 1 Sargassum tenerrimum 2 3

Gelidiella acerosa Caulerpa sp.

Class Place Phaeophyceae Thonithurai, east of Mandapam Rhodophyceae Vedalai, Mandapam Chlorophycae Near Thonithurai, Mandapam

Location the gulf of Mannar the gulf of Mannar the gulf of Mannar

Determination of biomass. Plants were weighed after blotting (wet weight), dried and reweighed (dry weight).The plants were cleaned of epiphytes and debris and rinsed in the seawater available with the sample. This is to provide ambient salinity. Rinsing in freshwater is not recommended because it can result in different dry weights due to variations in loss of surface salt. The drying was done in the sun or it can also be

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done by suspending over incandescent light bulbs. In case of the latter procedure care was taken that the sample did not get charged. Extraction of pigments. Comparison of photosynthetic pigment levels are useful in studies of shade and sun plants, effects of nutrients in eutrophication studies, a comparisons of photosynthetic rate. Freshly collected samples were weighed and ground using mortar and pestle under low light and temperature, because the pigments are easily degraded by light and heat. Chlorophylls a and b were extracted by grinding the samples in 80% acetone20 and phycoerythrin is extracted in a 0.1 M phosphate buffer (pH 6.5). The extract was centrifuged at 45 000 rpm for 2 to 5 min at 4oC. Estimation of phycobilins. Calculations are given for determination of phycobilins21, phycoerythrin (PE) and phycocyanin (PC) in 0.1 M PO4 buffer (pH 6.8). The absorption peaks measured are 455, 564, 592, 618 and 654 nm. PE (mg/ml) = [(A564 – A592) – (A455 – A592) 0.20] × 0.12 PC (mg/ml) = [(A618 – A645) – (A592 – A645) 0.51] × 0.15

Estimation of chlorophylls. The absorption peaks of chlorophyll a and b are measured and calculated by using equations postulated22. Absorption coefficients of chlorophylls can be found in 90% acetone with a measured absorption peaks of 647 and 664 nm. Chlorophyll a and b (Ref. 23) are measured using the following calculations. chlorophyll a (mg/l) = 11.93 (A664) –1.93 (A647) chlorophyll b (mg/l) = 20.36 (A645) – 5.50 (A664)

Thin layer chromatography of pigments. Thin layer chromatography is performed in order to separate the pigments as described earlier. Thin layer chromatography was carried on a silica plate with 3 samples, namely the extracts of Gelidiella acerosa, Sargassum tenerrimum and Caulerpa sp. The mobile phase was chosen to be petroleum ether: acetone 7: 3 ratio and Rf values were calculated. After TLC the spots were scraped off and dissolved in appropriate solvents, acetone, chloroform and ethanol for carotenoids and continuous absorption spectra were taken and compared with the standards to identify the pigments. Estimation of protein by the Lowry method24. The percent protein concentration was calculated using the equation below. The mg protein for 0.5 ml of solution was determined by comparing the absorption of the sample with a standard curve using 0.00 to 0.25 mg concentration of bovine serum albumin in 1 N NaOH. percent protein = mg protein × (10/mg tissue) × 100.

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Estimation of soluble carbohydrates by the Dubois method25. The percent carbohydrate was calculated according to the equation based on absorptions taken from a standard curve of 0.00 to 0.25 mg glycogen (reagent grade) dissolved in 5% TCA: percent carbohydrate = mg carbohydrate × (50/mg tissue) × 100,

where the dilution of the original sample is mg carbohydrate in 0.2 ml × 50. Estimation of lipids. The gravimetric procedure for total lipid in dried plant material is modified based on that of Freeman et al. (1957) and Sperry and Brand (1955)26,27. The plant material was homogenised in a 2:1 chloroform–methanol (C: M) mixture, washed with water, and the lower lipid phase was separated, dried, and weighed. The initial dry weight of the shell-glass vial was subtracted from the final weight to obtain lipid weight. The percent lipid was calculated using the following equation: percent lipid = mg lipid × (3/2/ mg tissue) × 100

where the numerator is two-thirds of the original sample. RESULTS AND DISCUSSION The results show that the comparative study of percent dry weight is a simple yet useful method for comparing biomass, dominance of a species, or population, as well as allocation of resources to different plants parts. The data in Table 2 show that the biomass of all the species of macroalgae was found to be more or less similar in weight (fresh wt. and dry weight wt.) basis. Thus, the results indicated that at the end of the development, various biochemical process lead to the formation of proteins, lipids, sugars and pigments that seems to be similar in all the varieties. Table 2. Determination of total biomass

Species Caulerpa sp. Gelidiella acerosa Sargassum tenerrimum

Fresh weight (FW) (g) (branches included) 5.50 5.67 5.23

Dry weight (DW) (g) (branches included) 1.98 2.10 2.09

Ratio FW/DW 2.777 2.700 2.502

The results of the comparative biochemical profiling and pigment analysis of the collected marine macroalgae from the phycobilliprotein analysis (Table 3) show that Gelidiella acerosa has the maximum phycoerithrin concentration. This is an expected result as the algae belongs to Rhodophycae family and its red colour is rendered because of the presence of this pigment. However very unlikely, Caulerpa has shown the presence of some amount of the pigment phycoerithrin as well as phycocyanin. This may be not actually phycoerithrin or phycocyanin, but some other compounds having an absorbance peak near that of phycoerithrin. Phycocyanin concentration is found to be very low in Caulerpa and maximum in Gelidiella sp. 762

Table 3. Estimation of phycobilliprotein content

Species Caulerpa sp. Gelidiella acerosa Sargassum tenerrimum

Phycoerithrin (mg) 0.055 0.211 0.096

Phycocyanin (mg) 0.001 0.019 0.010

When it comes to comparing the amount and type of chlorophyll molecule which is present in abundance, chlorophyll a stands out. Highest levels of chlorophyll a has been found in Caulerpa as predicted since it is a Chlorophycae. The total chlorophyll content (Chl. a + Chl. b) of Caulerpa is also the highest among all (Table 4). Table 4. Estimation of chlorophylls content

Species Caulerpa sp. Gelidiella acerosa Sargassum tenerrimum

Chlorophyll a (mg) 0.476 0.438 0.330

Chlorophyll b (mg) Total (Chl.a+b)( mg) 0.327 0.803 0.009 0.447 0.000 0.330

However, Gelidiella has also shown high amount of chlorophyll which is antagonistic to its property of being a Rhodophyte, but in case of Sargassum tenerrimum chlorophyll a is in a high content but chlorophyll b is absent. However, it has been surveyed previously that red algae growing in shallow waters near the estuary or sea coast shows an increase in chlorophyll content and the pigmentation decreases in red and increases in green; this may be a probable solution to the anomalic reading of the data. Table 5. Estimation of carbohydrate, proteins and lipid

Species Caulerpa sp. Gelidiella acerosa Sargassum tenerrimum

% of carbohydrate 57.81 30.65 26.39

% of protein 20.47 14.35 12.51

% of lipids 3.22 6.25 7.34

The TLC results show a wide range of pigment peaks that are present in the samples (Table 5). Even from here proper identification is impossible because various carotenoids even have peaks near the same wavelength and even elutes out simultaneously and can not be differentiated. Moreover, there can be various pigments that do not elute out properly or even if does, can not be viewed by naked eye because their bands can be extremely light. So there is a chance to miss out some pigments. However, from the Rf values and the absorption peak some data can be derived. There is a prominent amount of carotenoids along with fucoxanthin present in Sargassum tenerrimum as is evident from the peak around 469 nm in the 5th fraction and also from the orange colour that is clearly visible. The amount of pigments, particularly carotenoids also sometimes imply to the antioxidant potential of the algae. The 1st 763

fraction of Sargassum and Gelidiella shows a peak around 383 nm that planly implies to carotenes. In the base fraction of Sargassum, that is fraction 6th, other than some carotenoids, it shows the presence of chlorophyll a (corresponding to peaks 308 and 664), and possibly chlorophyll c (624 nm), and chlorophyll d (592 nm) (Chemistry of Natural Products). The 2nd fraction of the acetone extract of Gelidiella shows presence of carotenes, most probably e-carotene (400 nm) and phycoerithrin at 562 nm. The 3rd fraction of the same species shows a predominant peak at 666 nm confirming the presence of chlorophyll a. The 2nd fraction of Sargassum shows the presence of chlorohyll a (664 and 407 nm) whereas its 3rd fraction shows mostly different types of carotenoids with 2 peaks that can not be resolved due to lack of data. The peak at 471 nm can yield a number of carotenoids whose Rf values are near to each other like violaxanthin, neoxanthin, lycopene, etc. The 4th fraction yields absorbance peak of few carotenoids and chlorophyll a in trace. It can be seen that chlorophyll a is getting eluted out with carotenoids sometimes. The 1st fraction of Caulerpa shows carotenoids and chlorophyll a. The 2nd fraction contains predominantly chlorophyll a. The base line fraction or the 3rd fraction shows presence of an appreciable peak at around 453 nm that corresponds to chlorophyll b. The very small peaks that can be found at and around 580 nm may quantify the earlier found trace amounts of phycoerithryn in Caulerpa. The very small peak at 630 nm that is observed may also testify for trace amount of chlorophyll c that may be present. Usually carotenoids give peaks in the range of 380–500 nm, phycoerythrin – around 580 nm, and chlorophylls – around 600–670 nm (the data are compared with standards from Chemistry of Natural Product28). Table 6. Absorption peaks of TLC fractions dissolved in acetone

Species 1st fraction Gelidiella acerosa Sargassum tenerrimum 381, 385, 396, Caulerpa sp. 408, 666, 635

2nd fraction 384, 392, 400, 562 407, 664

3rd fraction 666, 404

4th fraction

405, 483, 471

403, 666

453, 384, 524, 576, 580, 631

464, 524, 530, 664

The results for total carbohydrate, proteins and lipid contents as shown in (Table 6) reveal that Caulerpa has the maximum percentage of protein to its dry weight. The high protein content in Caulerpa has made it a food in the Japaneese and Phillipinian islands. The lowest protein content has been found in Sargassum tenerrimum. The values are in accordance with earlier investigation showing that total protein content in algae ranges within 5–15% . The soluble carbohydrate content for Caulerpa is the highest among the other species. It is low for all the other specimens as expected as they produce substances like alginate (Sargassum), agar (Gelidiella) which are mostly insoluble. However, a relative high quantity of soluble carbohydrates in Gelidiella show that the overall insoluble carbohydrate content may be low and this may be 764

directly proportional to the gel yield and gel strength. However, the gelation property varies depending on seasons and general statement can not be made. The lipid content found has been unusually high over the usual 1–4% lipid content. Sargassum tenerrimum and Gelidiella acerosa showed high lipid content. Considering that most marine plants contain epi- and endophytic microorganisms, it is not possible to rule out these microbes which may contribute to the overall results of the extracts tested. In addition, it has been demonstrated that extraction procedures and the method of storage can significantly alter the composition of a crude extract and may result in false negative or positive assay results29 . CONCLUSIONS The results and discussion made in this paper led to the conclusion that the marine sources are a good source of foods because of their high protein content. Protein concentrates of seaweeds have become more important for the food industry. In addition, they are used in medicine and pharmacology due to their antioxidant properties because the pigments also a good source of antioxidants. ACKNOWLEDGEMENTS Authors thank Dr. Sumit Kumar Mitra, Geological Survey of India, for his help to locate the samples and to CMFRI Mandapam, India, to provide the algal samples. We are also grateful to Dr. Lazar Mathew, Dean, VIT University, Vellore, for the full support and encouragements. REFERENCES 1. M. I. WAHBEH : Amino Acid and Fatty Acid Profiles of Four Species of Macroalgae from Aqaba and Their Suitability for Use in Fish Diets. Aquaculture, 159, 101 (1997). 2. G. G. FOSTER, A. N. HODGSON: Consumption and Apparent Dry Matter Digestibility of Six Intertidal Macroalgae by Turbo sarmaticus (Mollusca: Vetigastropoda: Turbinidae). Aquaculture, 167, 211 (1998). 3. J. FLEURENCE: Seaweed Proteins: Biochemical, Nutritional Aspects and Potential Uses. Trends in Food Sci. & Technol., 10, 25 (1999). 4. W. L. ZEMKE-WHITE, K. D. CLEMENTS: Chlorophyte and Rhodophyte Starches as Factors in Diet Choice by Marine Herbivorous Fish. J. Exp. Mar. Biol. Ecol., 240, 137 (1999). 5. T. R. McCLANAHAN, B. A. COKOS, E. SALA: Algal Growth and Species Composition under Experimental Control of Herbivory, Phosphorus and Coral Abundance in Glovers Reef. Belize. Mar. Poll. Bull., 44, 441 (2002). 6. S. WILSON: Nutritional Value of Detritus and Algae in Blenny Territories on the Great Barrier Reef. J. Exp. Mar. Biol. Ecol., 271, 155 (2002). 7. F. E. ROUND: The Biology of the Algae. 2nd ed. Edward Arnold Ltd., London, 1973. 278 p. 8. Z. CHENGKUI (C. K. TSENG), Z. JUNFU (C. F. CHANG): Chinese Seaweeds in Herbal Medicine. Hydrobiologia, 116/117, 152 (1984). 9. W. FENICAL, V. J. PAUL: Antimicrobial and Cytotoxic Terpenoids from Tropical Green Algae of the Family Udoteaceae. Hydrobiologia, 116/117, 135 (1984).

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10. V. VREELAND, E. ZABLACKIS, B. DOBOSZEWSKI, W. LAETSCH: Molecular Markers for Marine Algal Polysaccharides. Hydrobiologia, 151/152, 155 (1987). 11. R. J. P. CANNELL: Algal Biotechnology. Appl. Biochem. Biotechnol., 26 (1), 85 (1990). 12. K. G. UVEN, K. Y. OZSOY, O. N. ULUTIN: Anticoagulant, Fibrinolitic and Antiaggregant Activity of Carrageenans and Alginic Acid. Bot. Mar., 34, 429 (1991). 13. A. PARKER: Using Elasticity/Temperature Relationships to Characterize Gelling Carrageenans. Hydrobiologia, 260/261, 583 (1993). 14. M. HONYA, T. KINOSHITA, K. TASHIMA, K. NISIZAWA, H. NODA: Modification of the M/G Ratio of Alginic Acid from Laminaria japonica A r e s c h o n g Cultured in Deep Seawater. Bot. Mar., 37, 463 (1994). 15. J. FLEURENCE, E. CHENARD, M. LUCON: Determination of the Nutritional Value of Proteins Obtained from Ulva Armoricana. J. Appl. Phycol., 11, 231 (1999). 16. K. WONG, H. C. K. CHEUNG: Nutritional Evaluation of Some Subtropical Red and Green Seaweeds, Part II. In vitro Protein Digestibility and Amino Acid Profiles of Protein Concentrates. Food Chem., 72, 11 (2001). 17. N. MARIN, F. MORALES, C. LODEIROS, E. TAMIGNEAUX: Effect of Nitrate Concentration on Growth and Pigment Synthesis of Dunaliella Salina Cultivated under Low Illumination and Preadapted to Different Salinities. J. Appl. Phycol., 10, 405 ( 1998). 18. S. BOUSSIBA, W. BING, J. P. YUAN, A. ZARKA, F. CHEN: Changes in Pigments Profile in the Green Alga Haeamtococcus Pluvialis Exposed to Environmental Stresses. Biotechnol. Lett., 21, 601 (1999). 19. M. R. ZUCCHI, O. NECCHI : Effects of Temperature, Irradiance and Photoperiod on Growth and Pigment Content in Some Freshwater Red Algae in Culture. Phycol. Res., 49, 103 (2001). 20. D. I. ARNON: Copper Enzyme in Isolated Chloroplast Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24, 1 (1949). 21. S. BEER, A. ESHEL : Determining Phycoerythrin and Phycocyanin Concentrations in Aqueous Crude Extracts of Red Algae. Australian J. of Marine and Freshwater Research, 36 (6), 785 (1985). 22. J. H. C. SMITH, A. BENITEZ: Chlorophylls: Analysis in Plant Material. In: Modern Methods of Plant Analysis (Eds K. Paech, M. V. Tracey). Springer, Berlin, 1955, 142–196. 23. J. C. MEEKS: Algal Physiology and Biochemistry. Botanical Monographs. Vol. 10. Blackwell Scientific Publications, Oxford, 1974, 161–175. 24. O. H. LOWRY, N. J. ROSEBOROUGH, A. L. FARR, R. J. RANDALL: Protein Measurement with the Folin Phenol Reagent. J. of Biological Chemistry, 193, 265 (1951). 25. M. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, F. SMITH: Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem., 28, 350 (1956). 26. N. K. FREEMAN, F. T. LINDGREN, Y. C. NG, A. V. NICHOLS: Infrared Spectra of Some Lipoproteins and Related Lipids. J. Biol. Chem., (1957). 27. W. M. SPERRY, F. C. BRAND: The Determination of Total Lipids in Blood Serum. J. Biol. Chem., 213, 69 (1955). 28. H. K. LICHTENTHALER, C. BUSCHMANN: Current Protocols in Food Analytical Chemistry. Ch. F4.3.1-F4.3.8, 2001. 29. G. CRONIN, N. LINDQUIST, HAY ME, W. FENICAL: Effects of Storage and Extraction Procedures on Yield of Lipophilic Metabolites from the Brown Seaweeds Dictyota ciliolate and D. menstrualis. Mar. Ecol. Prog. Ser., 119, 265 (1995). Received 23 November 2009 Revised 5 January 2010

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Oxidation Communications 35, No 3, 767–775 (2012) Oxidation of lignine in ionic liquid medium

A Preliminary Study of Oxidation of Lignin From Rubber Wood to Vanillin in Ionic Liquid Medium A. A. Shamsuria*, D. K. Abdullahb Laboratory of Molecular Biomedicine, Institute of Bioscience, University Putra Malaysia, 43 400 UPM Serdang, Selangor, Malaysia E-mail: [email protected] b Department of Chemistry, Faculty of Science, University Putra Malaysia, 43 400 UPM Serdang, Selangor, Malaysia a

ABSTRACT In this study, lignin was oxidised to vanillin by means of oxygen in ionic liquid (1,3dimethylimidazolium methylsulphate) medium. The parameters of the oxidation reaction that have been investigated were the following: concentration of oxygen (5, 10, 15 and 20 ft3 h–1), reaction time (2, 4, 6, 8 and 10 h) and reaction temperature (25, 40, 60, 80 and 100°C). The Fourier transform infrared spectroscopy, high performance liquid chromatography and ultraviolet-visible analyses were used to characterise the product. The results revealed vanillin as the product obtained via the oxidation reaction. The optimum parameters of vanillin production were 20 ft3 h–1 of oxygen for 10 h at 100ºC. In conclusion, 1,3-dimethylimidazolium methylsulphate could be used as an oxidation reaction medium for the production of vanillin from rubber wood lignin. Keywords: oxidation, lignin, vanillin, ionic liquid, rubber wood. AIMS AND BACKGROUND Lignin is second in abundance component of wood after cellulose that can be potential substrates for the production of vanillin. Vanillin has been used as flavouring materials and aroma in food industry and also in the fragrance industry for instance perfumes, hygienic products and to cover up disagreeable scent or taste in drugs as well. It can also be used in chemical intermediary for the production of pharmaceuticals and other fine chemicals1,2. The presence of small quantity of vanillin in vanilla beans or seed pods of the tropical Vanilla orchid makes its extraction limited and not economically feasible3. Thus, the oxidation of lignin to produce vanillin has been investigated

*

For correspondence.

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extensively4,5. In addition, lignin-based vanillin is suitable as flavouring because it has a richer flavour profile than oil-based flavouring1. Lignin can be isolated from lignocellulosic materials by using the Kraft pulping or sulphate process. The process involved the use of concentrated and strong bases, specifically a mixture of sodium hydroxide and sodium sulphide, known as white liquor6. Black liquor that contains lignin fragments after processing of lignocellulosic materials by this isolation process can be used to produce vanillin via oxidation reaction7. However, additional steps are needed after the oxidation reaction to neutralise the high pH value of black liquor in relation to the vanillin recovery. Besides, this process to produce vanillin is no longer accepted because of corrosivity, hazardous and can cause harm to the environment8. Another method has been developed to isolate lignin is organosolvent process9. Nevertheless, this process also has its own drawbacks due to the use of organic solvents that are highly volatile, highly flammable and toxic. Moreover, the process also usually requires high pressures conditions10 and the stateof-the-art equipments are often necessitated for oxidation of lignin since low boiling point of organic solvents that were used as a medium have high vapour pressure. Ionic liquids are considered as green solvents because they indicate some interesting properties such as negligible vapour pressure (being non-volatile), environmentally-benign, non-toxic, recyclable, non-flammable and chemically inert11–14. Ionic liquids are capable to dissolve and blend biopolymers with high efficiency, no severe side-reaction occurred and simplicity in recovery of the products15,16. Since ionic liquids offer a potentially clean medium for carrying out chemical reactions or processes, more attention has been paid on lignocellulosic materials, for example cellulose and lignin17,18. Up to now, the oxidation studies of lignin in ionic liquids medium are less reported, especially by using hydrophilic ionic liquid and in the absence of any catalyst19. In this preliminary study, rubber wood was used as a raw material. The lignin was isolated by means of ionic liquid from rubber wood, the isolated lignin was oxidised to produce vanillin. The process was carried out in hydrophilic ionic liquid medium and no catalyst required for avoiding possible metal-contamination of the product. The product was characterised by the Fourier transform infrared spectroscopy, high performance liquid chromatography and ultraviolet-visible analyses. The effect of experimental reaction conditions on the optimisation of vanillin production was also studied. EXPERIMENTAL MATERIALS

1,3-Dimethylimidazolium methylsulphate and methanol were purchased from Merck. Rubber wood (Hevea brasiliensis) used in the experiments was supplied by local sawmill and wood processing factory in Malaysia. Oxygen gas was obtained from Mox-Linde Gases. Chloroform was procured from Fisher Scientific. All chemicals were used as received. 768

ISOLATION OF LIGNIN FROM RUBBER WOOD

The isolation of lignin from rubber wood by using ionic liquid was done according to a procedure described elsewhere12. Rubber wood was grinded, sieved and kept in the oven to remove moisture. Then, 52.50 g of rubber wood (oven-dry weight) were placed in a glass flask, 5.0 mol of 1,3-dimethylimidazolium methylsulphate were poured into a glass flask. The flask was stirred and heated by placing in oil bath at 100°C for 2 h. After the flask was cooled to the room temperature, the insoluble component in the solution was then separated by filtration under reduced pressure. Then, the soluble lignin was separated from ionic liquid through precipitation with methanol. The isolated lignin was filtered off and washed thoroughly with distilled water for several times. They were collected and dried into a vacuum oven at 85°C for 24 h. The isolated lignin was kept over a desiccant before further oxidation. OXIDATION OF LIGNIN TO VANILLIN

2.0 g of the isolated lignin were dissolved into 18.0 g of 1,3-dimethylimidazolium methylsulphate in a beaker with stirring to prepare lignin solution with concentration of 10 wt.%. The solution was transferred into a round-bottom flask and then the flask was mounted on a rotary evaporator apparatus equipped with oxygen tank for the oxidation reaction. After specific reaction condition, the oxidative mixture was mixed with 50 g of chloroform with stirring in a beaker for 30 min followed by filtration. The filtrate was then transferred to a separation funnel in order to extract the product and separate any lignin solution. The solution at the bottom of the funnel was collected. The product soluble in chloroform was used in the experiments for characterisation. The parameters investigated include the concentration of oxygen (5, 10, 15 and 20 ft3 h–1), reaction time (2, 4, 6, 8 and 10 h) and reaction temperature (25, 40, 60, 80 and 100°C). The experiments were performed in triplicate. CHARACTERISATION

Fourier transform infrared (FTIR). FTIR analyses were carried out by using a Perkin Elmer Spectrum 100 Series spectrometer to determine the presence of vanillin functional groups in the product after the oxidation. The FTIR spectra were obtained by using a universal attenuated total reflectance (UATR) equipped with a ZnSe-diamond composite crystal accessory. The spectra resolution was 4 cm–1, the scanning wave­ number ranged from 4000 to 800 cm–1 and each spectrum was 16 scans. All spectra were rationed against the reference spectrum of background. High performance liquid chromatography (HPLC). The identification of the product was carried out based on qualitative analysis in an Agilent 1200 Series HPLC and a column (5-pm octadecasilane silica, Nucleosil-C18, 150 × 4.6 mm). The separation was obtained using a linear gradient of 2 solvent systems: solvent A (water + acetic acid, 94:6) and solvent B (methanol + acetonitrile + acetic acid, 95:4:1). A linear gradient was run for over 30 min from 0 to 40% B as eluent at a flow rate of 1 ml min–1. 769

The product was detected with a DAD detector at 280 nm by computer comparison of the retention times and peak areas with the standard vanillin. Ultraviolet-visible (UV-vis.) spectroscopy. UV-vis. spectra were measured on a Perkin Elmer Lambda 25 UV-vis. spectrometer in the wavelength range of between 240 and 330 nm with scan speed of 960 nm min–1. Lamp change wavelength was 355 nm and slit width 1.00 nm. A 2.5 ml aliquot of the product was placed directly into the quartz cuvette where the beam could directly pass through it to measure the absorbance (A). RESULTS AND DISCUSSION FTIR CHARACTERISATION

Figure 1 displays the FTIR spectra of the standard vanillin and the product, the bands of spectra are summarised in Table 1 and the chemical structure of vanillin is shown in Fig. 2. The functional groups and corresponding molecular motions are determined based on the specific band. As can be seen in Fig 1, the FTIR spectra of the standard vanillin (a) and the product (b) exhibited strong intensity and broad bands at 3475.26 and 3476.48 cm–1, respectively. These bands could be assigned to the O–H bond stretching of the phenol group, while the bands with medium intensity at 3021.48 and 3022.39 cm–1 are corresponded with the C–H bond stretching of the aromatic ring group. The characteristic C=O bond stretching of the aldehyde group results from medium intensity of the bands at 1644.32 and 1645.27 cm–1. The bands with strong intensity that are caused by C–O bond stretching of the ether group belong to 1214.25 and 1215.42 cm–1. The strong intensity of the bands at 1001.27 and 1002.15 cm–1 are associated with C–O bond stretching of the phenol group. From Table 1 the spectrum bands of the standard vanillin and the product illustrated the equality between them and the presence of the functional groups of vanillin (Fig. 2) are explicitly observed in the product. Hence, the product from the oxidation reaction could be recognised as a vanillin. However, HPLC analyses were additionally conducted to confirm vanillin as the product of oxidation. Table 1. FTIR bands of the standard vanillin and the oxidation product

Standard vanillin (cm–1) 3475.26 3021.48 1644.32 1214.25 1001.27

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Product (cm–1) 3476.48 3022.39 1645.27 1215.42 1002.15

Band origin

Functional group

O–H stretching C–H stretching C=O stretching C–O stretching C–O stretching

phenol aromatic ring aldehyde ether phenol

T (%)

a

b

4000

3500

3000

2500

2000

1500

1000

wavenumber (cm–1)

Fig. 1. FTIR spectra of the standard vanillin (a) and the oxidation product (b)

HO O O Fig. 2. Chemical structure of vanillin

HPLC CHARACTERISATION

Figures 3 and 4 present the HPLC chromatograms of the standard vanillin and the product, respectively. The chromatograms showed a major peak for the retention time of the product (11.312 min) which is nearly the same as the retention time of the standard vanillin (11.318 min). This observation corresponds to the result that was obtained from the FTIR spectra. This qualitative analysis also indicated there was no significant difference in the chromatogram of the product from the oxidation reaction with the standard vanillin. However, the unanticipated high signal/noise ratio of the product believed to be due to the existence of small amount of impurities. Based on the analysis, the peaks of impurities did not show any useful information regarding other compounds because their concentrations are relatively low, thus, they are not considered further. Nevertheless, UV-vis. measurements were conducted to prove the presence of insignificant amounts of impurities in the product.

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Fig. 3. HPLC chromatogram of the standard vanillin

Fig. 4. HPLC chromatogram of the oxidation product

UV-vis. CHARACTERISATION

Figure 5 shows the UV-vis. spectra of the standard vanillin and the product. The absorption peaks for both spectra are almost similar especially for the peaks in the region from 242 to 254 nm. However, the spectrum of the product contained some small absorption peaks arising between 284 and 302 nm in comparison with the standard vanillin. These results suggested that the insignificant absorption peaks could be

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attributed to the amount of impurities that are present in the product in insignificant amounts. These peaks could be assumed negligible as long as the main broad peaks (242 to 254 nm) are not changed to sharp and narrow peaks, this is most likely the main broad peaks are far more significant compared to the other peaks. The UV-vis. results are entirely consistent with the results obtained from the HPLC chromatograms.

absorbance (A)

a

240

b

250

260

270

280

290

300

310

320

330

wavelength (nm)

Fig. 5. UV-vis. spectra of the standard vanillin (a) and the oxidation product (b)

OPTIMUM CONDITIONS FOR OXIDATION OF LIGNIN TO VANILLIN

The product that has been characterised above is obtained under optimum reaction conditions. Lignin undergoes depolymerisation during the oxidation reaction; as a result, the rate of depolymerisation depends on the oxidation conditions. The primary chemical structure of lignin does not changed during the dissolution in ionic liquid. However, it changes along with the formation of vanillin when the concentration of oxygen is greater. The reaction time of the oxidative mixture has to be maximised to extend the depolymerisation. The temperature has also indicated similar effect on oxidation reaction, whereby high temperature increases the solubility of oxygen in the ionic liquid and enhances the lignin depolymerisation rate. Therefore, the optimum parameters of vanillin production were 20 ft3 h–1 of oxygen for 10 h at 100ºC that led to lignin depolymerisation with high efficiency. In contrast, increase in ionic liquid concentration does not result in improvement of the vanillin production for all investigated parameters, the resultant product is almost the same at the end of the oxidation reaction. On the other hand, this preliminary study also showed that the product acquired from these optimum reaction conditions released a scent identical to the standard vanillin. CONCLUSIONS The FTIR spectroscopy results indicated the presence of vanillin functional groups in the product. The HPLC results revealed that the retention time of the product is nearly 773

the same as that of the standard vanillin. UV-vis. spectra demonstrated absorption peak of the product very similar to those of the standard vanillin. The optimum parameters of vanillin production were 20 ft3 h–1 of oxygen for 10 h at 100ºC. In conclusion, the vanillin has been successfully obtained by means of rubber wood-based lignin via oxidation reaction in an ionic liquid (1,3-dimethylimidazolium methylsulphate) medium. ACKNOWLEDGEMENT The authors gratefully acknowledge the technical support and the facilities provided by the Institute of Bioscience (IBS), University Putra Malaysia (UPM). Research for this paper was supported by UPM under the Research University Grant Scheme (05-01-09-0617RU). This paper is dedicated to Prof. Dr. Dzulkefly Kuang Abdullah, the last Head of Laboratory of Industrial Biotechnology, IBS, UPM. We also thank Prof. Dr. Slavi K. Ivanov, Mrs. M. Boneva and anonymous referees for valuable comments on this paper. REFERENCES   1. L. J. Esposito, K. Formanek, G. Kientz, F. Mauger, V. Maureaux, G. Robert, F. Truchet (Eds): Vanillin. Vol. 24: Kirk-Othmer Encyclopedia of Chemical Technology. 4th ed. John Wiley & Sons, New York, 1997, p. 812.   2. M. J. W. Dignum, J. Kerler, R. Verpoorte: Vanilla Production: Technological, Chemical, and Biosynthetic Aspects. Food Rev. Int., 17, 119 (2001).   3. N. J. Walton, M. J. Mayer, A. Narbad: Vanillin. Phytochemistry, 63, 505 (2003).   4. M. ZabkovA, E. A. des Silva, A. E. Rodrigues: Recovery of Vanillin from Lignin/Vanillin Mixture by Using Tubular Ceramic Ultrafiltration Membranes. J. Membr. Sci., 301, 221 (2007).   5. P. Sridhar, J. D. Araujo, A. E. Rodrigues: Modeling of Vanillin Production in a Structured Bubble Column Reactor. Catal. Today, 105, 574 (2005).   6. O. Wallberg, A-S. Jönsson: Separation of Lignin in Kraft Cooking Liquor from a Continuous Digester by Ultrafiltration at Temperatures above 100°C. Desalination, 195, 187 (2006).   7. C. Fargues, A. Mathias, A. Rodrigues: Kinetics of Vanillin Production from Kraft Lignin Oxidation. Ind. Eng. Chem. Res., 35, 28 (1996).   8. M. B. Hocking: Vanillin: Synthetic Flavoring from Spent Sulfite Liquor. J. Chem. Educ., 74, 1055 (1997).   9. J. I. Botello, M. A. Gilarranz, F. RodrIguez, M. Oliet: Preliminary Study on Products Distribution in Alcohol Pulping of Eucalyptus globulus. J. Chem. Technol. Biotechnol., 74, 141 (1999). 10. L. Barbera, M. A. Pelach, I. Perez, J. Puig, P. Mutje: Upgrading of Hemp Core for Papermaking Purposes by Means of Organosolvent Process. Ind. Crop. Prod., 34, 865 (2011). 11. A. A. Shamsuri, D. K. Abdullah: Protonation and Complexation Approaches for Production of Protic Eutectic Ionic Liquids. J. Phys. Sci., 21, 15 (2010). 12. A. A. Shamsuri, D. K. Abdullah: Isolation and Characterization of Lignin from Rubber Wood in Ionic Liquid Medium. Mod. Appl. Sci., 4, 19 (2010). 13. A. A. Shamsuri, D. K. Abdullah: Synthesizing of Ionic Liquids from Different Chemical Reactions. Singap. J. Sci. Res., 1, 246 (2011).

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14. A. A. Shamsuri, R. Daik: Plasticising Effect of Choline Chloride/Urea Eutectic-based Ionic Liquid on Physicochemical Properties of Agarose Films. BioResources, 7, 4760 (2012). 15. A. A. Shamsuri, D. K. Abdullah, R. Daik: Fabrication of Agar/Biopolymer Blend Aerogels in Ionic Liquid and Co-solvent Mixture. Cell Chem. Technol., 46, 45 (2012). 16. H. Xie, S. Li, S. Zhang: Ionic Liquids as Novel Solvents for the Dissolution and Blending of Wool Keratin Fibers. Green Chem., 7, 606 (2005). 17. L. Feng, Z.-I. Chen: Research Progress on Dissolution and Functional Modification of Cellulose in Ionic Liquids. J. Mol. Liq., 142, 1 (2008). 18. Y. Pu, N. Jiang, A. J. Ragauskas: Ionic Liquid as a Green Solvent for Lignin. J. Wood Chem. Technol., 27, 23 (2007). 19. K. Stärk, N. Taccardi, A. Bösmann, P. Wasserscheid: Oxidative Depolymerization of Lignin in Ionic Liquids. ChemSusChem., 3, 719 (2010). Received 18 September 2011 Revised 3 November 2011

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Oxidation Communications 35, No 3, 776–784 (2012) Technological aspects of oxidation processes

Calculation and Prognosis of the Thermodynamic Properties of Rare Earth Tellurites of the Ln2Te4O11 type G. Baikusheva-Dimitrova Department of Inorganic and Analytical Chemistry, ‘Prof. Assen Zlatarov’ University, 1 Prof. Yakimov Street, 8010 Bourgas, Bulgaria E-mail: [email protected] ABSTRACT The perspectives of the rare earth tellurites is in their application in the contemporary technologies, such as the nanotechnologies, silicate industry, as a dielectric and ferroelectric material; in the medicine as additions to different kinds of drugs for curing of hard healing diseases; and also as an agricultural microfertilisers. The aim of the present work is to determine experimentally, calculate and predict the specific molar heat capacity (Cp) of the rare earth tellurites products of the Ln2Te4O11 type synthesised by us. The compositions studied were of the type Ln2Te4O11 where Ln = Y, Er, Yb. They were synthesised in vacuumed ampoules and characterised by chemical and X-ray analyses. The specific molar capacities (Cp) of the compounds studied were determined in the temperature range 399–587 K using differential scanning calorimeter DSC (Setaram, France). The values measured were computer-processed by the linear regression method to obtain empirical formulae for the corresponding compound and the coefficients a, b and c in the equation Cp = a + bT + cT2 were determined. The dependencies of the experimentally measured and the calculated values of Cp for the compounds studied Y2Te4O11, Er2Te4O11 and Yb2Te4O11 had regression coefficients R2 close to unity which means that the calculation procedure used was precise and correct. This provided a possibility to employ regression analysis to predict Cp in temperature ranges for which there are no experimental data. On the other hand, the specific molar heat capacities calculated allowed determination of the temperature dependences of entropy (∆ST0), change of enthalpy (∆HT0 – ∆H2980) and the Gibbs function (∆GT0), as well as to predict the thermodynamic values in non-studied temperature intervals. The data on the thermodynamic properties were necessary for development of industrial technologies for synthesis of compounds containing rare earth elements and products on their basis with preselected properties. Keywords: rare-earth metals, tellurites, thermodynamic, specific molar capacities, thermodynamic methods. 776

AIMS AND BACKGROUND Tellurites of rare earth elements are comparatively new class of inorganic substances attracting still more attention during the last few years1,2. The aim of the present work is to determine experimentally, calculate and predict the specific molar heat capacity (Cp) of the rare earth tellurites products of the type Ln2Te4O11 synthesised by us. On the other hand, the calculated specific molar capacities allow finding the temperature dependences of entropy (∆ST0), change of enthalpy (∆HT0 – ∆H298) and the Gibbs function (∆GT0). The studies on the specific molar capacity allow calculating the thermodynamic values in wide temperature interval3,4. The temperature dependence of Cp at low temperatures is known to be related to the characteristics of the solid matter crystalline structure. The calculation of the Debye temperature from data on Cp provides possibilities to use data on the movement of crystal lattice vibrations and, hence, the strength of the chemical bonds. Besides, the data on Cp allow studies on the versatile processes of arrangement which determine, for instance, the magnetic, ferroelectric properties or superconductivity, as well as the formation of point defects. The rare earth tellurites synthesised were of the type Ln2Te4O11 and their thermodynamic characteristics are necessary for the development of industrial technologies for synthesis of rare earth compounds and products based on them and will be a contribution to an important chapter of inorganic chemistry, such as chemistry of tellurium. EXPERIMENTAL The metal tellurites of rare earth elements of the type Ln2Te4O11 necessary for the experiments were synthesised from tellurium dioxide TeO2 and high purity (99.99%) oxides of rare earth elements: Y2O3, Er2O3 and Yb2O3 through vacuum synthesis in ampoules. For the experimental determination of the thermodynamic values like specific molar capacity, differential scanning calorimeter DSC (Setaram, France) was used by a method described in Refs 3 and 5. The parameters of the empty crucible, reference sample and substance studied were taken into account. The measurements were carried out at a scanning rate of 20oC/min, recorder speed of 5 mm/min and amplifier range 250 V. The working temperature interval was 300–600 K. The main part of DSC is a block of heat-conducting material with 2 seats for flat aluminium ampoules. The block temperature is raised at controlled rate while room temperature is maintained in the outer chamber. The thermal batteries placed near the working seat are connected by bridged circuit. The disturbances in the heat development in the seat are compensated and the consumption of differential power for the heating is registered on a recorder. For the determination of the specific molar capacities of the tellurites studied, the samples were finely ground and sieved through 0.25 mm2 sieve. Then the substance was homogenised and 0.2 to 0.7 g of it were placed in a capsule. 777

The specific molar capacities were determined as follows: 1. Record the base line under hating the 2 empty capsules of the same weight. 2. Record the curve for the capsule with the reference substance –Al2O3. 3. Record the curve for the capsule with the substance studied. Cp =

I m´ I´m

Cp´,

where І´ is the distance from the curve of empty capsule to the reference (Al2O3), cm; І – distance from the curve for empty capsule to the curve for the substance studied, cm; m´ – reference weight, g; m – substance studied weight, g; Ср´ – specific molar capacity of the reference Al2O3, J mol–1 K–1; Ср – specific molar capacity of the substance studied, J mol–1 K–1. RESULTS AND DISCUSSION The experimental results for Cp of the substances of the type Ln2Te4O11, where Ln=Yb, Er or Y studied in the temperature interval 388–587 K are presented in Table 1. Table 1. Experimental results for Cp of Yb2Te4O11 , Er2Te4O11 and Y2Te4O11

T (K) 388 398 408 418 428 438 448 458 467 477 487 497 507 517 527 537 547 557 567 577 587

778

Ср (J mol–1 K–1)

Yb2Te4O11

Er2Te4O11

Y2Te4O11

388.18 393.09 397.38 400.70 396.51 395.83 394.94 385.55 388.43 391.45 378.15 386.98 393.54 393.90 389.73 396.19 396.81 397.35 372.71 373.77 373.48

390.55 386.52 391.47 394.87 389.88 394.98 392.61 388.82 390.93 396.64 383.95 391.30 393.75 395.47 399.18 397.38 395.54 396.98 385.07 392.65 390.68

339.57 341.32 336.30 348.78 363.95 358.61 353.88 355.62 359.45 362.92 364.72 375.17 382.49 386.81 370.56 384.25 382.23 385.31 388.63 384.44 380.66

The values obtained were computer-processed by the linear regression method to derive empiric formulae for the individual substances and the coefficients a, b and c in the general equation: Cp = a + bT + сT2

(1)

The standard molar entropies of Y2Te4O11, Er2Te4O11 , Yb2Te4O11, were calculated by the method of Kelly6,7, the coefficients a, b and c, as well as the regression coefficients R2 for the individual substances are shown in Table 2. Table 2. Standard molar entropy ∆S°298, coefficients a, b and c and regression coefficients R2

Compound

∆S0298 (J mol–1 K–1)

a

b

c

R2

Yb2Te4O11 Er2Te4O11 Y2Te4O11

437.61 451.85 395.31

–0.0016 –0.0009 –0.0011

1.4392 0.8730 1.3032

  66.683 186.14   –8.6007

0.8658 0.8670 0.8992

The plots of the temperature dependencies of the experimentally determined specific molar capacities in the interval 388–587 K and the empiric equations derived for the individual substances are presented in Figs 1–3. specific molar heat capacity Cp (J mol–1 K–1)

400 375 350 325 y = –0.0016x2 + 1.4392x + 66.683 R 2 = 0.8658

300 275 300

350

400

450

500

550

600

650

700

temperature (K)

Fig. 1. Dependence of the experimental results for Cp on temperature for Yb2Te4O11

VSHFLILFPRODUKHDWFDSDFLW\ CS (-PRO–1 K–1)

425

(U27H4O11

400 375 350 y = –0.0009x 2 + 0.873x + 186.14 2 R = 0.867

325 300 300

350

400

450

500

550

600

650

tɟPSHUDWXUH (K)

Fig. 2. Dependence of the experimental results for Cp on temperature for Er2Te4O11

779

sSHFLILFPRODUKHDWFDSDFLW\ (-PRO–1 K–1)

400 Y27H4O11

375 350 325 300

y = – 0.0011x2 + 1.3032x – 8.60 07 R 2 = 0.8992

275 250 300

350

400

450

500

550

600

650

tɟPSHUDWXUH (K)

Fig. 3. Dependence of the experimental results for Cp on temperature for Y2Te4O11

The dependencies between the experimentally determined and calculated by regression specific molar capacities for Y2Te4O11, Er2Te4O11 and Yb2Te4O11 are shown in Figs 4–6. It can be seen from Figs 4–6 that the dependencies of the experimentally measured and calculated by regression specific molar capacities have R2 close to unity which means that the calculation procedure used was precise and correct. It made possible the use of regression analysis to predict the values of Cp at temperatures for which there are no experimental data.

ɋɪFDOFXODWHGYDOXH (-PRO–1 K–1

400

Yb 2Te 4O11

395 390 385 380 375

y = 1.031x – 17.627 2 R = 0.8082

370 365 360 360

370

380

390

400

410

ɋɪ ɟ[SHULPHQWDOO\GHWHUPLQHG (-PRO–1 K–1

ɋɪ FDOFXODWHGYDOXH (-PRO–1 K–1

Fig. 4. Dependence between the measured and calculated by regression specific molar capacity for Yb2Te4O11 402 400 398 396 394 392 390 388 386 384 382 380 380

Er 2Te 4O11

y = 0.8815x + 48.101 R 2 = 0.8238

382

384

386

388

390

392

394

396

398

400

ɋɪ ɟ[SHULPHQWDOO\GHWHUPLQHG (-PRO–1 K–1

Fig. 5. Dependence between the measured and calculated by regression specific molar capacity for Er2Te4O11

780

ɋɪ ( calculated value) (J mol–1 K–1)

385 380 375 370 365 360 355 350 345 340 335 330

Y2Te 4O11

y = 0.7715x + 78.632 R 2 = 0.8644 330

335

34

345

350

355

360

365

370

375

380

385

390

395

ɋɪ (experimentally determined) (J mol–1 K–1)

Fig. 6. Dependence between the measured and calculated by regression specific molar capacity for Y2Te4O11

The specific molar heat capacities calculated by equation (1) allow finding the temperature dependencies of entropy (∆ST0), change of enthalpy (∆H0T – ∆H0298) and the Gibbs function (∆G0T) using the formulae below. The results are presented in Tables 3–5. ∆S 0T = ∆S 0298 +

T

Cp

∫

T 298

∆H 0T – ∆H 0298 =



T

∫ Cp dT ,

298

∆G0T = ∆S0T –

(2)

dT ,

∆H0T – ∆H0298 T

(3) .

(4)

The good agreement between the experimentally determined and calculated values of Cp provided possibility to predict the thermodynamic vales in a non-studied temperature interval. The values of Cp calculated by linear regression for temperatures outside the temperature interval of the experiments, as well as the values of ∆S0T, (∆HºT – ∆Hº298) and ∆GºT calculated by equations (2)–(4) are presented in Tables 3–5. The data on the thermodynamic values allow developing industrial technologies for synthesis of compounds of rare earth elements and products based on them.

781

Table 3. Standard molar thermodynamic values for Yb2Te4O11

T (K)

298.15 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Cp (J mol–1 K–1) 356.69 357.58 377.54 389.50 393.46 389.42 377.38 357.34 329.30 312.27 299.23 290.18 287.14 281.01 286.08 304.98

∆S0T (J mol–1 K–1) 437.61 439.81 493.54 536.78 570.38 594.82 610.41 613.38 615.86 616.87 617.88 619.64 624.03 625.53 633.90 651.66

∆H0T – ∆H0298 (J mol–1) 0.0 661.5 19575.4 39670.5 59746.8 78604.4 95043.1 107863.0 115864.1 125483.7 135207.1 145626.8 158458.2 169125.9 186481.2 214050.2

∆G0T (J mol–1 K–1) 437.61 438.50 490.94 524.08 550.87 590.54 699.09 610.55 613.94 615.27 616.56 618.64 620.04 623.24 630.41 648.57

∆H0T – ∆H0298 (J mol–1) 0.0 679.0 19777.6 39857.9 60244.9 80263.6 99238.9 116496.0 131359.7 143155.0 155725.6 169896.3 181525.5 194301.3 214576.0 234586.3

∆G0T (J mol–1 K–1) 451.85 452.79 496.33 544.30 579.14 600.81 628.36 640.82 652.20 654.54 657.82 660.08 664.30 665.49 672.72 680.30

Table 4. Standard molar thermodynamic values for Er2Te4O11

T (K)

298.15 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

782

Cp (J mol–1 K–1) 366.42 367.04 381.44 391.34 396.74 397.64 394.04 385.94 373.34 356.24 344.64 338.54 328.94 322.84 329.18 334.24

∆S0T (J mol–1 K–1) 451.85 454.11 508.36 551.49 585.73 612.38 632.28 646.01 653.94 656.36 659.48 664.22 665.41 667.74 677.72 686.43

Table 5. Standard molar thermodynamic values for Y2Te4O11

T (K)

298.15 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Cp (J mol–1 K–1) 282.17 283.36 312.37 336.68 355.09 368.00 375.41 377.32 373.73 364.64 350.05 349.96 351.27 342.28 336.69 330.60

∆S0T (J mol–1 K–1) 395.31 397.06 441.64 481.04 515.13 543.87 567.21 585.13 597.61 604.64 606.20 614.84 623.37 624.20 626.33 627.34

∆H0T – ∆H0298 (J mol–1) 0.0 524.2 16217.1 34290.8 539.20.9 74280.7 94546.8 113893.8 131496.7 146530.3 158169.8 175627.4 193848.3 206001.2 219471.4 232031.6

∆G0T (J mol–1 K–1) 395.31 396.29 398.73 402.80 418.63 429.30 449.84 460.30 470.69 485.02 501.34 513.55 525.77 537.97 550.15 562.30

CONCLUSIONS Tellurites of rare earth elements of the type Ln2Te4O11 were synthesised. Their purities and individualities were determined by chemical and X-ray analyses. The results obtained from the chemical analysis were compared to the theoretically calculated values and the agreement was found to be good. The specific molar heat capacities of the tellurites synthesised were determined and for the first time a prediction was made for non-studied compounds using the interpolation method and regression analysis. Using the relationship between the standard entropy heat capacities, the temperature dependencies of the thermodynamic functions were determined. The thermodynamic characteristics of the compounds studied can be used to develop industrial technologies for synthesis of compounds of rare earth elements and products on their basis with predetermined properties. REFERENCES 1. J. DOBROWOLSKY: Les tellurites – des elements des terrea rares. J. Rocz. chem., 40, 1169 (1966). 2. I. BARIN: Thermochemical Data of Pure Substances. Part I and Part II. VCH Verlag GmbH., Weinheim, 1993, 777–972.

783

3. I. L.McNAUGHTON, C. T. MORTIMER: Differential Scanning Colorimetry. Perkin Elmer Corporation Norvalk Connection, 1975, р. 11. 4. R. DEHOFF: Thermodynamics in Materials Science. Taylor&Francis Group, New York, 2006. 5. G. HOHNE, W. HEMMINGER, H. FLAMMERHEIM: Differential Scanning Calorimetry – An Introduction for Practitioners. Springer Verlag, Berlin, 1996. 6. S. P. GORDIENKO, B. V. FENOTACHKA, G. Ch. VIKSMAN: Thermodynamics of Lanthanide Compounds. Naukova Dumka, Kiev, 1979. 7. E. GORDIENKО, R. CLUSTRA, J. van MILTENBURG: Thermodynamics of Lanthanide. J. Chem. Thermodynamics, 17, 1079 (1985). Received 27 December 2011 Revised 29 February 2012

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Oxidation Communications 35, No 3, 785–794 (2012) Technological aspects of oxidation processes

Calculation Methods and the Newton Interpolation Formulae for Analysis and Prediction of Standard Entropies of Tellurites of Rare Earth Elements of the Ln2(TeO3)3 Type G. Baikusheva-Dimitrovaa*, G.Vissokovb Department of Inorganic and Analytical Chemistry, ‘Prof. Assen Zlatarov’ University, 1 Prof. Yakimov Street, 8010 Bourgas, Bulgaria E-mail: [email protected] b Institute of Catalysis, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 11, 1113 Sofia, Bulgaria a

ABSTRACT Data on the standard entropies of tellurites of rare earth elements are scarce and this determined the interest towards these studies. The present paper reports for studies and suggests 4 methods for calculation of the standard entropy of the rare earth tellurites synthesised. An assessment of their accuracy was performed and the relative error rates in the general practice of the Gauss least square laboratory method were found. The dependence DSC of the molecular masses of the tellurites synthesised was investigated and graphically represented. To calculate the standard values of the entropy of the lantanides tellurites studied, the Newton interpolation formulae from the mathematical calculation methods were used for non-equally spaced argument values, applied for analysis and prediction of the standard entropy of formation. Keywords: tellurites, thermodynamic data, entropy, thermodynamic methods. AIMS AND BACKGROUND Entropy is a measure of disorder in a thermodynamic system and represents the number of possible configurations or arrangements of the systems building particles. It is a criterion used to estimate how close to the thermodynamic equilibrium is certain system and it is usually bigger when the chaos and, respectively, its probability, are bigger1. Entropy depends on the nature of the substances. For comparativeness of results obtained, it is generally accepted that entropy is reduced per 1 mol of the substance un*

For correspondence.

785

der standard conditions (pressure of 1 at = 1.013×105 Pa and temperature T = 298.15 K) and this value is called standard entropy ∆S0298. This is an important thermodynamic property of the compounds and it is successfully used in the general inorganic, physical and analytical chemistry to study a number of technological processes. The tellurites of rare earth elements are comparatively new class of inorganic compounds attracting much attention recently2,3. The data on the standard entropies of rare earth tellurites are quite scarce and this stipulated our interest towards these compounds. The present work reports for calculations of the standard entropies of the rare earth tellurites synthesised calculated by 4 different methods and assessment of their precision. The dependence of ∆S0298 on the molecular weights of the tellurites synthesised was studied and graphically presented. To calculate the standard values of the entropy of the lantanides tellurites studied, the Newton interpolation formulae from the mathematical calculation methods were used for non-equally spaced argument values, applied for analysis and prediction of the standard entropy of formation. EXPERIMENTAL For the present study, tellurites of rare earth elements of the type Ln2(TeO3)3 were synthesised by vacuum ampoule synthesis from tellurium dioxide and oxides of rare earth elements Me2O3, where Me = Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Er, Tm and Yb of high purity 99.9%. Four methods for determination of the standard entropy ∆S0298 of the tellurites studied were suggested. The equations allow calculation of tellurites ∆S0298 on the basis of the equations for carbonates and sulphites. The methods used were: 1. Method of Koumok – the determination of ∆S0298 of crystalline substances is reduced to the sum of the entropies of the corresponding ionic increments: ∆S0298 (AmBn) = m Si298(An+) + n Si298(Am–)

(1)

where ∆S0298 is the entropy increment, J mol–1 K–1. 2. Method of Kelly – the standard entropy of crystalline substances is equal to the sum of the entropies of the component oxides. 3. Method of Vineru – the standard entropy of crystalline substances is in linear dependence with the logarithms of the corresponding molecular weights ∆S0298 = a AlgM + b

(2)

4. Method of Larimer – determination of ∆S0298 of crystalline substances is reduced to the sum of the entropies of the corresponding ionic increments with these for positive ions being positive while for the negative ones, the increments depend on the corresponding anion. The precision of the determination of ∆S0298 of the tellurites synthesised are of special importance for theory and practices. The standard entropy of the compounds studied was determined as the arithmetic average of the values obtained from the 4 786

methods. The mean square error (the average standard deviation) σ was calculated and the relative error was found in percentage according to the generally accepted in laboratory practice least square method of Gauss4. ı=

∑ (∆S ) i

i

n(n – 1)

2

; ε% =

σ 0 ∆S 298

× 100 ,

(3)

where ∆S = ∆S0298 – ∆Si, and ∆S0298 is the average arithmetic value of the standard entropy while Si are the values of entropy obtained by the different methods used. The experimental data were processed by the Newton interpolation formulae for non-equally spaced values of the argument from the mathematical calculation methods of analysis4,5. In the interval [x, x], n + 1 points are given x0, x1, …, xn, which are called interpolation angles. The values of some functions at these points are also given: f(x0) = y0; f(x1) = y1; … ; f(xn) = yn.

(4)

F(x0) = y0; F(x1) = y1; … ; F(xn) = yn.

(5)

Pn(x0) = y0; Pn(x1) = y1; … ; Pn(xn) = yn.

(6)

An interpolation function F(x) is drawn, which has the same values as f(x) at the interpolation angles, i.e. Geometrically, it means that a curve y = F(x) of certain type passing through the given system of points must be found. This problem becomes single-valued if a polynomial Pn(xn) of power not higher than n is sought, obeying the conditions (5), so: The interpolation formula obtained is usually used for approximate calculation of the values of given function f(x) for values of the argument x different from the interpolation angles. This operation is called interpolation of the function f(x). We have the following two cases: – interpolation – when the argument values are between the end points x0 and xn (x ∈ [x0, xn]); – extrapolation – when the argument x values can be outside the interval [x0, xn], i.e. x ∉ [x0, xn] which allows better study of the experimental data. The experimental data are usually with non-equally spaced argument x values, i.e. the differences between adjacent values are not equal. The Newton interpolation formula for non-equally spaced argument values has the following general expression: P(x) = y0 + [x0, x1](x – x0) + [x0, x1, x2](x – x0)(x – x1) + … + [x0, x1, …, xn](x – x0)(x – x1)…(x – xn–1),

(7)

where y0 is the initial value of the function; [x0, x1] – divided differences of first order [x0, x1] =

y1 – y0 x1 – x0

;

787

[x0, x1, x2] – divided differences of second order [x1, x2] – [x0, x1]

[x0, x1, x2] =

x2 – x0

;

[x0, x1, … xn] – divided differences of n-th order [x0, x1, … xn] =

[x1, …, xn] – [x0, …, xn–1] xn – x0

.

Polynomial (7) is transformed into n

P(x) = y0 + ∑ Ai i =1

(8)

where Ai = [x0, x1, … , xi]x(x – x1)(x – x2)…(x – xi–1). The divided differences and Ai are presented in tables. The values of Ai were calculated for the interpolation angles selected x0, x1, … , xi. The functions calculated for the interpolation angles using the Newton polynomial coincided with the experimental data, as it was expected. With the Newton interpolation polynomial constructed (8), the values of the function can be calculated at points different from the interpolation angles. By this method, a prediction can be made for values of the property measured for which there are no data but they are within the closed interval [x0, xn]. For points of longer distance, the calculation error (uncertainty) increases. Nevertheless, this method can successfully be used to calculate thermodynamic values of rare earth tellurites. RESULTS AND DISCUSSION The results obtained by the 4 methods used to calculate the standard entropy values of the rare earth tellurites are presented in Table 1. The data necessary5 for the calculations of DS by the 4 methods are as follows: – ionic increments of entropy for the method of Kumouk; – standard entropies of the oxides of the corresponding tellurites of rare earth elements by the method of Kelly; – constants a, b in the equation of by the method of Vineru for the corresponding oxides; – the ionic entropy increments by the method of Latimer. As a result for the values of the standard entropy ∆S0298 calculated for the compounds studied by the 4 methods, the arithmetic average value was taken. The precision of the determination of the ∆S0298 of the tellurite synthesised is of certain importance for theory and practice. To assess the reliability of the results obtained, the relative percent error ε% was calculated. The values obtained are presented in Table 2.

788

Table 1. Standard entropies ∆S0298 of tellurites of rare earth elements

 

Compound Sc2(TeO3)3 Y2(TeO3)3 La2(TeO3)3 Cе2(TeO3)3 Pr2(TeO3)3 Nd2(TeO3)3 Sm2(TeO3)3 Eu2(TeO3)3 Gd2(TeO3)3 Tb2(TeO3)3 Dy2(TeO3)3 Ho2(TeO3)3 Er2(TeO3)3 Tm2(TeO3)3 Yb2(TeO3)3

Methods of determination of ∆S0298 (J mol–1 K–1)

1 317.6 338.2 357.4 372.6 387.4 373.0 376.6 375.2 384.0 394.6 394.4 396.8 392.8 385.8 382.6

2 299.169 321.252 349.49 372.753 377.816 380.745 373.213 368.709 379.071 379.071 371.971 380.368 377.791 362.429 363.551

3 285.972 315.648 337.707 338.153 338.441 339.644 341.794 342.347 344.126 344.681 345.844 346.623 347.360 347.883 349.151

4 289.48 308.74 323.8 323.8 323.8 324.64 326.3 326.3 327.98 327.98 328.82 329.66 329.66 330.5 331.32

Table 2. Arithmetic average values of ∆S0298 of rare earth tellurites, standared deviation σ and relative error ε%

Compound

М

Sc2(TeO3)3 Y2(TeO3)3 La2(TeO3)3 Cе2(TeO3)3 Pr2(TeO3)3 Nd2(TeO3)3 Sm2(TeO3)3 Eu2(TeO3)3 Gd2(TeO3)3 Tb2(TeO3)3 Dy2(TeO3)3 Ho2(TeO3)3 Er2(TeO3)3 Tm2(TeO3)3 Yb2(TeO3)3

616.96 704.86 804.86 807.28 808.86 815.52 827.76 830.98 841.54 844.90 852.04 856.90 861.56 864.90 873.12

∆S0298 298.0 321.0 342.0 351.8 356.9 354.5 354.5 353.1 358.8 361.6 360.2 363.4 361.9 356.6 356.6

σ  7  6  7 12 15 13 12 11 13 15 14 15 14 12 11

ε (%) 2 2 2 4 4 4 3 3 4 4 4 4 4 3 3

The Newton interpolation formulae for non-equally spaced argument values of the mathematical calculation methods were used to find the standard values of the entropy of the lanthanide tellurites studied using their molecular weights. The Newton interpolation polynomial was constructed for 11 angles of interpolation where the value of the polynomial coincides with the arithmetic average values 789

of the standard entropy. For convenience, the molecular weight of La2(TeO3)3 was assumed to be zero (x0 = 0). The molecular weights of the tellurites, the interpolation angles X used and the corresponding values of the standard entropy Y are presented in Table 3. Table 3. Interpolation angles (X) and the corresponding values of entropy (Y)

Compound La2(TeO3)3 Cе2(TeO3)3 Pr2(TeO3)3 Nd2(TeO3)3 Eu2(TeO3)3 Gd2(TeO3)3 Tb2(TeO3)3 Ho2(TeO3)3 Er2(TeO3)3 Tm2(TeO3)3 Yb2(TeO3)3

М 804.86 807.28 808.86 815.52 830.98 841.54 844.90 856.90 861.56 864.90 873.12

  x0 x1 x2 x3 x4 x5 x6 x7 x8 x9 x10

X   0.00   2.42   4.00 10.66 26.12 36.68 40.04 52.04 56.70 60.04 68.26

Y 342.0 351.8 356.9 354.5 353.1 358.8 361.6 363.4 361.9 356.6 356.6

The values of the divided differences A were calculated for the selected interpolation angles x0, x1 …xn. The standard entropy ∆S0298 obtained by the polynomial constructed for the interpolation angles coincided with the arithmetic average values calculated by the 4 methods, as expected when interpolation methods of analysis are used. The divided differences Ai calculated are presented in Table 4. The polynomial suggested in the present paper allows finding the standard entropy of tellurites of molecular weights outside the interpolation interval – and Sm2(TeO3)3 and Dy2(TeO3)3. For compounds for which there are no data in literature, the present interpolation model can successfully be used to determine the standard entropy knowing only their molecular weight. The entropy of Pm2(TeO3)3 was calculated to be 346.66 J mol–1 K–1. The standard entropies (arithmetic average values obtained from the 4 methods used) and the values calculated using the Newton polynomial are shown in Table 5. For the interpolation angles selected, the standard entropies calculated with the polynomial coincide with the arithmetic average values obtained from the 4 methods, as it should be expected. For compounds outside the interpolation angles (Sm2(TeO3)3 and Dy2(TeO3)3), the entropies were calculated by the Newton method. The good agreement with the arithmetic average values for these substances can easily be seen.

790

791

Y

342 351.8 356.9 354.5 353.1 358.8 361.6 363.4 361.9 356.6 356.6

X

 0   2.42   4.00 10.66 26.12 36.68 40.04 52.04 56.7 60.04 68.26

I order   4.04959   3.22785 –0.3604 –0.0906   0.54   0.83333   0.15 –0.3219 –1.5868  0  

III order –0.0216   0.01889   0.00037 –0.0001 –0.0025   0.00081 –0.0065   0.01821

 

II order –0.2054 –0.4355   0.0122   0.02422   0.02109 –0.0445 –0.0283 –0.1581   0.13727

 

 

IV order   0.00155 –0.00054 –1.30E-05 –5.90E-05   0.00011 –0.00031   0.00088

V order –5.70E-05   1.40E-05 –9.40E-07   3.60E-06 –1.20E-05   3.80E-05

Table 4. Divided differences calculated for the interpolation angles selected

VI order   1.77E-06 –3.02E-07   8.70E-08 –3.25E-07   1.19E-06    

 

 

VII VIII IX order order order –3.99E-08   8.29E-10 –2.00E-11   7.16E-09 –2.52E-10   1.20E-11 –7.36E-09   5.23E-10   2.63E-08  

X order 4.40E-13

Table 5. Arithmetic average values of ∆S0298 and ∆S0298 calculated by the Newton polynomial

Compound La2(TeO3)3 Cе2(TeO3)3 Pr2(TeO3)3 Nd2(TeO3)3 Sm2(TeO3)3 Eu2(TeO3)3 Gd2(TeO3)3 Tb2(TeO3)3 Dy2(TeO3)3 Ho2(TeO3)3 Er2(TeO3)3 Tm2(TeO3)3 Yb2(TeO3)3

М 804.86 807.28 808.86 815.52 827.76 830.98 841.54 844.90 852.04 856.90 861.56 864.90 873.12

∆S0298 342.0 351.8 356.9 354.5 354.5 353.1 358.8 361.6 360.2 361.6 363.4 361.9 356.6

∆S0298 342.0 351.8 356.9 354.5 351.7 353.1 358.8 361.6 364.2 361.6 363.4 361.9 356.6

Figure 1 shows the graphical dependence of the standard entropy values calculated by the 4 methods from the molecular weights of the tellurites of rare earth elements synthesised. Obviously, the dependence is too complex due to effects of substances structures and the nature of the bonds between its particles.

values of standard entropy

370

350

330

310

290 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 molecular weight

Fig. 1. Dependence of the values of standard entropy ∆S0298 on the molecular weight of tellurites of rare earth elements

The dependence of standard deviation σ on the standard entropy values calculated is presented in Fig. 2.

792

standard deviation (J mol K–1)

18 16 14 12 10 8 6 4 2 0 290

300

310

320

330

340

350

360

370

standard entropy (J mol K–1)

Fig. 2. Dependence of the standard deviation σ on the values of the standard entropy ∆S0298 of the tellurites of rare earth elements

values of standard entropy (J mol K–1)

Using the Newton interpolation polynomial, it is possible to find the standard entropies of compounds of the same type for which there are no data in literature. This is possible if their molecular weights are within the interval of the polynomial obtained [x0, xn]. This is the way by which the standard entropy of the radioactive Pm2(TeO3)3 was found. The arithmetic average values obtained from the 4 methods and the standard entropy values calculated by the Newton polynomial were compared (Fig. 3). 370 365 360

series 1

theoretical

355

series 2

calculated

350 345 340 800

820

840

860

880

molecular weight

Fig. 3. Graphical comparison of ∆S0298 and ∆S0298 with the Newton polynomial

The Newton interpolation polynomial allows predicting the values for substances of the same type for which there are no data in literature. CONCLUSIONS 1. Tellurites of rare earth elements of the type Ln2(TeO3)3 were synthesised, where Ln are Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Er, Tm and Yb by vacuum ampoule synthesis. The new method suggested allows preparing comparatively pure phases of tellurites of rare earth elements. 2. The standard entropies of the tellurites synthesised were calculated by 4 different methods. The arithmetric average value of ∆S0298 was determined for all the compounds. 793

3. The standard entropy of substances is a function of their molecular (atomic) weight. This dependence is almost linear for the first 3 compounds studied and more complex for the lanthanide tellurites but close by value (351.8 versus 363.4) which, according to our opinion, is related with the individual stability of the f orbitals. The growth of 4f sub-layer results in insignificant changes, as expected. 4. The standard entropies were determined for the first time by mathematical calculation methods of analysis and prediction of properties of compounds of the same type for which there are no data in literature. The precision was estimated and the relative error was found to be small enough which confirmed the precision of the calculations. This approach is valuable for the determination of other properties of all the lanthanide tellurites studied. The calculation of the standard entropy as well as other thermodynamic values is quite important both for the practice and for filling of information on the tellurites of rare earth elements. REFERENCES 1. Physico-mathematical and Technical Encyclopaedia. Bulg. Acad. Sci. Publ. House, Sofia, Vol.1, 1990, p. 830. 2. A. L.VOLOSTCHINA, V. A.OBOLONTSCHIK: Tellurities of Rare Earth Elements. Ukrainskii Chem. J., 48, 1028 (1982). 3. I. BARIN: Thermochemical Data of Pure Substances. VCH Verlag Gesellschaft, Weinheim, Parts 1 and 2, 1993, p. 777, 972, 1406. 4. D. HRISTOZOV: Laboratory Practices of Physics. Nauka i Izkustvo Publ., Sofia, 1990. 5. B. K. KASENOV, A. S. PASHINKIN: Thermodynamic Methods in Inorganic Chemistry. University of Karaganda, 1989. Received 27 December 2011 Revised 29 February 2012

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Oxidation Communications 35, No 3, 795–800 (2012) Technological aspects of oxidation processes

Investigation of the Composition of Synthesised Rare Earth Tellurites of the Ln2Te4O11 Type G. Baikusheva-Dimitrova Department of Inorganic and Analytical Chemistry, ‘Prof. Assen Zlatarov’ University, 1 Prof. Yakimov Street, 8010 Bourgas, Bulgaria E-mail: [email protected] ABSTRACT Rare earth tellurites are comparatively new group of inorganic compounds which recently take the interest of many researchers. The perspectives of these compounds is in their application in modern technologies, such as the nanotechnology, silicate industry, as a dielectric and ferroelectric material; in the medicine – as additions to different kinds of drugs for treating hardly curable diseases; and also as an agricultural microfertilisers. The rare earths tellurites of the type Ln2Te4O11 were synthesised. Purity and individuality of the tellurites synthesised can be controlled chemically. For reliable assessment of the completeness of the course of chemical reactions and the accuracy of the information obtained, 3 determinations for each tellurite were performed and average values were taken. The results of chemical analyses of the compounds studied were compared with theoretically calculated quantities for the tellurites. The absolute error was determined. The experiment shows that the mechanical mixtures of metal oxide and telluric dioxide are homogenised and meet the stoichiometric composition of the studied compounds. Keywords: tellurites, rare earth, metal oxide, tellurium dioxide, chemical analysis. AIMS AND BACKGROUND The fields of use of rare earth tellurites are versatile and important and this necessitates their profound investigation. The perspective of these compounds is in their implementation in modern technologies like nanotechnology, silicate industry, as dielectric and segnetoelectric materials, in medicine – as additives to various drugs for treating hardly curable diseases, as well as microfertilisers in agriculture1,2. The aim of the present work is to synthesise and study compositions of tellurites of rare earth elements of the type Ln2Te4O11. Their purity and individuality were controlled by chemical methods. For reliable estimation of the completeness of the 795

chemical reactions, 3 measurements were performed for each tellurite and the average values ware taken. The results obtained from the chemical analyses of the compounds studied were compared with the theoretically calculated quantities for each tellurite. The absolute and relative per cent errors were determined. EXPERIMENTAL The metal tellurites of rare earth elements of the type Ln2Te4O11 necessary for the experiments were synthesised from tellurium dioxide and oxides of rare earth elements: Sc2O3, Y2O3, La2O3, Ce2O3, Pr2O3, Nd2O3, Sm2O3, Eu2O3, Gd2O3, Tb2O3, Dy2O3, Ho2O3, Er2O3, Tm2O3, Yb2O3 and Lu2O3, all of them of high purity 99.99%. The initial materials were taken in stoichiometric quantities for the corresponding goal compound: Ln2Te4O11. The weighed oxides in quantities corresponding to the stoichiometry of the goal product were mixed and subjected to thorough homogenisation by prolonged grinding in an agate mortar. Then, they were placed in quartz ampoules which were vacuumed and sealed. To find the temperature of the thermal treatment, 50–100 mg of the mechanical mixture were preliminarily subjected to derivatograph analysis. Based on the experimental data obtained, the metal tellurites were synthesised at temperatures 50°C lower than the melting temperature. The compositions of the tellurites synthesised were determined both by chemical and X-ray analyses. The metal ions of the oxides of the type Me2O3 were determined complexonometrically3,4 while the tellurite ions – iodometrically and gravimetrically5. The results obtained from these studies confirmed the end product stoichiometry (Tables 1 and 2). Table 1. Chemical analysis of the metal oxides contained in the rare earth tellurites of the type Ln2Te4O11

Compound

1 1 – Sc2Te4O11 2 – Y2Te4O11 3 – La2Te4O11 4 – Cе2Te4O11 5 – Pr2Te4O11 6 – Nd2Te4O11

Theoretically calculated (mass. %) metal oxide μТ (%) 2 17.76 26.13 33.79 33.96 34.06 34.52

Determined by chemical analysis (mass. %) metal oxide

1 3 17.80 26.09 33.84 33.98 34.10 34.50

2 4 17.77 26.15 33.77 33.95 34.02 34.55

3 5 17.75 26.14 33.87 33.96 34.05 34.53

average value μexp (%) 6 17.77 26.13 33.83 33.96 34.06 34.53

Δμ 

ε (%)

7 0.03 0.04 0.01 0.02 0.04 0.03

8 0.15 0.14 0.04 0.05 0.13 0.08 to be continued

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Continuation of Table 1

1   7 – Sm2Te4O11   8 – Eu2Te4O11   9 – Gd2Te4O11 10 – Tb2Te4O11 11 – Dy2Te4O11 12 – Ho2Te4O11 13 – Er2Te4O11 14 – Tm2Te4O11 15 – Yb2Te4O11 16 – Lu2Te4O11

2 35.33 35.54 36.22 36.43 36.88 37.18 37.47 37.67 38.17 38.40

3 35.35 35.55 36.22 36.40 36.86 37.21 37.48 37.67 38.20 38.44

4 35.30 35.58 36.24 36.45 36.90 37.17 37.39 37.68 38.21 38.42

5 35.33 35.54 36.22 36.43 36.87 37.18 37.47 37.67 38.17 38.40

6 35.33 35.56 36.23 36.43 36.88 37.19 37.45 37.67 38.19 38.42

7 0.02 0.01 0.01 0.03 0.02 0.02 0.03 0.003 0.01 0.02

8 0.07 0.02 0.02 0.07 0.04 0.06 0.09 0.009 0.02 0.05

Table 2. Chemical analyses of the tellurium oxide contained in rare earth tellurites of the type Ln2Te4O11.

Compound

  1 – Sc2Te4O11   2 – Y2Te4O11   3 – La2Te4O11   4 – Cе2Te4O11   5 – Pr2Te4O11   6 – Nd2Te4O11   7 – Sm2Te4O11   8 – Eu2Te4O11   9 – Gd2Te4O11 10 – Tb2Te4O11 11 – Dy2Te4O11 12 – Ho2Te4O11 13 – Er2Te4O11 14 – Tm2Te4O11 15 – Yb2Te4O11 16 – Lu2Te4O11

Theoretically calculated (mass. %) tellurium dioxide μТ (%) 82.24 73.87 66.21 66.04 65.94 65.48 64.67 64.46 63.78 63.57 63.12 62.82 62.53 62.33 61.83 61.60

Determined by chemical analysis (mass. %) tellurium dioxide 1

2

3

average value μexp (%)

82.18 73.75 66.22 66.10 65.90 65.50 64.65 64.48 63.80 63.60 63.20 62.82 62.55 62.30 61.88 61.58

82.26 73.85 66.19 66.02 65.90 65.45 64.68 64.50 63.82 63.55 63.14 62.84 62.50 62.35 61.85 61.62

82.24 73.88 66.21 66.05 65.93 65.51 64.67 64.47 63.75 63.58 63.12 62.82 62.53 62.33 61.81 61.60

82.23 73.83 66.21 66.06 65.91 65.49 64.66 64.48 63.79 63.58 63.16 62.83 62.53 62.33 61.85 61.60

Δμ

ε (%)

0.01 0.04 0.003 0.02 0.03 0.06 0.003 0.02 0.01 0.006 0.007 0.006 0.003 0.003 0.02 0.007

0.02 0.06 0.005 0.02 0.04 0.01 0.005 0.04 0.02 0.01 0.001 0.01 0.005 0.005 0.03 0.001

The X-ray phase and structural analyses of the tellurites were carried out on an apparatus URD – 6 (Germany). The data obtained were treated by the isostructural method to calculate the singony, space group and the parameters of the unit cell of the tellurites studied (Table 3). The synthesis of metal tellurite was considered to be 797

completed when the results obtained on relative intensity and interplanar distances fully comply with literary data. Table 3. Parameters of the unit cell of tellurites of the type Ln2Te4O11

Compound

Sc2Te4O11 Y2Te4O11 La2Te4O11 Ce2Te4O11 Pr2Te4O11 Nd2Te4O11 Sm2Te4O11 Eu2Te4O11 Gd2Te4O11 Tb2Te4O11 Dy2Te4O11 Ho2Te4O11 Er2Te4O11 Tm2Te4O11 Yb2Te4O11 Lu2Te4O11

Space group

Unit cell parameters (Å)

a

b

c

β

C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c C 2/c

12.7952 12.3654 12.2562 12.7341 12.6851 12.6005 12.5600 12.6014 12.4600 12.4263 12.3742 12.3703 12.4262 12.2900 12.2600 12.2605

5.2792 5.1041 5.1312 5.2473 5.2303 5.2165 5.1740 5.1210 5.1420 5.1216 5.1147 5.1052 5.1093 5.0730 5.0690 5.0581

16.4234 16.0995 15.8344 16.4030 16.3291 16.2701 16.1900 16.1981 16.0900 15.9593 16.0983 16.0034 15.8433 15.9400 15.9200 15.9005

106.085 106.010 107.361 106.000 105.951 106.000 106.000 105.810 106.080 105.491 106.121 106.120 105.759 106.100 106.150 106.185

Volume V Number X-ray (cm3) of parti- density D cles (g/cm3) 1065.94 4 5.69 976.70 4 5.87 950.30 4 6.73 1053.52 4 9.11 1041.60 4 6.17 1027.87 4 6.29 1011.36 4 6.48 1005.79 4 6.53 990.54 4 6.70 978.83 4 6.81 978.80 4 6.86 971.00 4 6.94 968.02 4 7.00 954.84 4 11.11 950.32 4 7.24 946.92 4 7.26

It can be seen from the X-ray analyses of the compounds of the type Ln2Te4O11 synthesised that all of them are of the monoclinic singony. RESULTS AND DISCUSSION The compositions of the rare earth tellurites of the type Ln2Te4O11, synthesised were studied. Purity was controlled by chemical analysis. For reliable estimation of the completeness of the chemical reactions and accuracy of the data obtained, 3 measurements were performed for each tellurite and the average value was taken. The results from the chemical analyses were compared to the theoretically calculated ones for each tellurite. The absolute and relative per cent errors were determined. The measurement precision was assessed. The absolute error is the difference between the experimental value of the mass content μexp and the theoretically calculated one μТ: ∆μ = |μexp – μT|

(1)

The relative error of the value was calculated in percent by the formula: ε% =

798

∆μ μ

%=

|μexp – μT| μT

× 100.

(2)

The experimental results obtained from the chemical analyses of the oxides of rare earth elements and TeO2 are presented in Tables 1 and 2 and compared to the theoretically calculated stoichiometric quantities for the corresponding tellurite of the type Ln2Te4O11 . It can be seen that there was a good agreement between the experimental and theoretical values which was considered enough to assert that the mixtures of metal oxide and tellurium dioxide were well homogenised and correspond to pure phases of rare earth tellurites by their stoichiometric composition. The study carried out confirmed the stoichiometry of the end product. The arithmetic average values obtained from the chemical analyses and the theoretically calculated stoichiometric total contents of oxides in the corresponding tellurites are compared in Fig. 1.

Fig. 1. Comparison between the experimentally determined and theoretically calculated total contents of oxides in the corresponding tellurites (numbers 1–16 on the abscissa correspond to the numbering of compounds in Tables 2 and 3)

As can be seen from Tables 1 and 2 and Fig.1 for Ln2Te4O11, there is good accordance between the experimentally determined and theoretically calculated values. This was considered enough to assert that the stoichiometric mixtures of metal oxide and tellurium dioxide corresponded to pure phases of tellurites of rare earth elements. Obviously, the experimental results obtained had values close to 100% which confirmed the precision of the studies. CONCLUSIONS 1. Tellurites of rare earth elements of the type Ln2Te4O11 were synthesised by vacuumed ampoule synthesis. The purity and individuality of the substances were determined both by chemical and X-ray phase analyses. 2. For higher reliability, 3 determinations of the contents of the corresponding oxides were performed for each tellurite and the average value was taken.

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3. The absolute and relative errors were calculated in percent. The error of the results was found to be small which indicates for the high precision of the measurements. 4. The experimentally determined and theoretically calculated values of the oxides contents in the tellurites studied were compared. The experimental results have values close to 100%. This indicated that the compounds synthesised had stoichiometric compositions corresponding to pure phases of tellurites of rare earth elements of the type Ln2Te4O11. REFERENCES 1. A. L.VOLOSTCHINA, V. A.OBOLONTSCHIK: Tellurities of Rare Earth Elements. Ukrainskii Chem. J., 48, 1028 (1982). 2. I. BARIN: Thermochemical Data of Pure Substances. VCH Verlag gesellschaft, Weinheim, Parts 1 and 2, 1993, p.777, 972, 1406. 3. G. SHARLO: Methods of Analytical Chemistry. Inostrannaya Literatira Publ. House, Moscow, 1969. 4. G. SHVARTSENBACH, G. FLYASHKA: Complexonometric Titration. Khimiya, Moscow, 1970. 5. I. NAZARENKO, I. YARMAKOV: Analytical Chemistry of Selenium and Tellurium. Nauka, Moscow, 1977. Received 27 December 2011 Revised 29 February 2012

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Oxidation Communications 35, No 3, 801–804 (2012) New member of the Editorial Board

Presentation of Prof. Alberto D’Amore (the Second University of Naples-sun, Italy) for Including in the List of the Editorial Board of Oxidation Communications Journal Professor Alberto D’Amore is a specialist in the field of characterisation, degradation, stabilisation and modifications of polymers (polymer material science). His papers are focused mainly on mechanical degradation of polymers, viscoelasticity, fatigue properties, structural relaxation, thermodegradation, oxidation, ozonolysis, photooxidative degradation, hydrolysis, biodegradaton and mechanical degradation of polymers. Alberto D’Amore was born in Naples (Italy) on July 25th 1956. He is married with two sons. His home is at Via Roma 48, Somma Vesuviana, 80049 (NA), Italy. Just now he is an Associate Professor of Materials Science and Technology at The Second University of Naples-SUN, Department of Aerospace and Mechanical Engineering, Via Roma, 19, 81031, Aversa (CE), Italy. Prof. Alberto D’Amore is a member of the following scientific societies: SIR (Italian Society of Rheology), AIMAT (Italian Association of Materials Engineering), AIM (Italian Association on Macromolecules). He served as Scientific Secretary of the Italian Society of Rheology (1989–1995), Scientific Secretary of the Working Party on Industrial Rheology – European Federation of Chemical Engineering, Italian Delegate of the Working Party on Industrial Rheology – European Federation of Chemical Engineering (1997–2002). He was the Director of Research Unit of University of Brescia at INSTM – The National Institute of Materials Science and Technology (1999–2002), Member of the Scientific Committee at INSTM and Director of the Research Unit of the Second University of Naples at INSTM (2002–today). He is the Chairman of the ‘International Conference on Times of Polymers (TOP) and Composite’ and is included in the International Scientific Committee of several International Conferences. Professor D’Amore directed several scientific projects coming either from the Italian Government or from Industries. He was selected as Expert Evaluator within the framework of FP7, Expert Evaluator for the Italian Government ‘Ministero dello Sviluppo Economico’ for Industrial Innovation Projects and Expert Evaluator Projects POR (Piani Operativi Regionali) for Region Emilia Romagna, Region Veneto and Region Piemonte. Professor Alberto D’Amore is in the board of Expert Evaluators of ‘Ministero dell’Università e della Ricerca Scientifica’. He was selected as Expert Evaluator by

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The National Authority for Scientific Research (ANCS), based in Bucharest – Romania. Professor D’Amore published more than 130 papers in International Journals and books and edited several international books. Below we would like to give the list of his some the last publications. We would like to recommend Professor Alberto D’Amore to be a member of the Editorial Board of Oxidation Communications Journal. Prof. Slavi Kunev Ivanov – Editor-in-Chief of the Journal Prof. Gennady Efremovich Zaikov – Member of Editorial Board of the Journal from Russia. List of some publications of Prof. Alberto D’Amore: 1. D’Amore A., Grassia L. and Verde P., ‘Modeling the Flexural Fatigue Behavior of Glass Fiber Reinforced Thermoplastic Matrices’ Mechanics of Time-Dependent Materials, DOI, 10.1007/ s11043-012-9192-y (2012) 2. Grassia L., D’Amore A., ‘Calculation of the Shrinkage Induced Residual Stress in a viscoelastic Dental Restorative Material’  Mechanics of Time Dependent Materials, DOI, 10.1007/s11043-012 -9190-0 3. Grassia L, D’Amore A., ‘Finite element calculation of residual stress in dental restorative material’ AIP Conf. Proc. 1459, 312–315 (2012). 4. D’Amore A, Grassia L., ‘Timescales and properties of PSA (pressure sensitive adhesives)’, AIP Conf. Proc. 1459, 341–343 (2012). 5. Martone, A., Grassia L., Zarrelli M., Giordano M, D’Amore A., ‘Enthalpy relaxation of an epoxy matrix/carbon nanotubes’, AIP Conf. Proc. 1459, 347–349 (2012). 6. D’Amore A., Grassia L, Verde P., ‘Modeling the Fatigue Behavior of Glass Fiber Reinforced Thermoplastic and Thermosetting Matrices’, AIP Conf. Proc. 1459, 372–374 (2012). 7. Grassia L., D’Amore A., Verde P., ‘On The Inter-Conversion Between Viscoelastic Material Functions of Polycarbonate’, AIP Conf. Proc. 1459, 375–377 (2012). 8. D’Amore, A., De Maria, G., Grassia, L., Natale, C., Pirozzi, S., ‘Silicone-rubber-based tactile sensors for the measurement of normal and tangential components of the contact force’, J. of Applied Polymer Science, 122 (6), 3758–3770 (2011). 9. Grassia, L., Carbone, M.G.P., D’Amore, A., ‘Modeling of the isobaric and isothermal glass transitions of polystyrene’, J. of Applied Polymer Science, 122 (6), 3752–3757 (2011). 10. Grassia, L., Pastore Carbone, M.G., Mensitieri, G., D’Amore, A., ‘Modeling of density evolution of PLA under ultra-high pressure/temperature histories’. Polymer, 52 (18), 4011–4020 (2011). 11. Grassia, L., D’Amore, A., ‘Isobaric and isothermal glass transition of PMMA, Pressure-volumetemperature experiments and modelling predictions’. J. of Non-Crystalline Solids, 357 (2), 414–418 (2011). 12. Grassia, L., D’Amore, A., Simon, S.L., ‘On the viscoelastic Poisson’s ratio in amorphous polymers’ Journal of Rheology, 54 (5), 1009–1022 (2010). 13. Grassia, L., D’Amore, A., ‘Thermal residual stresses in amorphous thermoplastic polymers’, AIP Conference Proceedings, 1255, 414–416 (2010). 14. Grassia, L., D’Amore, A., ‘Modeling the residual stresses in reactive resins-based materials, A case study of photo-sensitive composites for dental applications’ AIP Conference Proceedings, 1255, 408–410 (2010). 15. Grassia, L., D’Amore, A., ‘Isobaric PVT behavior of Poly(Carbonate) (PC)’ AIP Conference Proceedings, 1255, 417–419 (2010).

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16. Grassia, L., D’Amore, A., ‘On the interplay between viscoelasticity and structural relaxation in glassy amorphous polymers’, J. of Polymer Science, Part B, Polymer Physics, 47 (7), 724–739. (2009). 17. Grassia, L., D’Amore, A., ‘The relative placement of linear viscoelastic functions in amorphous glassy polymers’, Journal of Rheology, 53 (2), 339–356 (2009). 18. D’Amore, A., Grassia, L., Acierno, D., ‘Modelling the yield stress and the Poisson’s ratio of glassy polymers’, E-Polymers, art. no. 052 (2009). 19. Grassia, L., D’Amore, A., ‘Constitutive law describing the phenomenology of subyield mechanically stimulated glasses’, Physical Review E – Statistical, Nonlinear, and Soft Matter Physics, 74 (2), (2006) art. no. 021504. 20. D’Amore, A., Caputo, F., Grassia, L., Zarrelli, M., ‘Numerical evaluation of structural relaxationinduced stresses in amorphous polymers’, Composites Part A, Applied Science and Manufacturing, 37 (4), 556-564 (2006). 21. Zarrelli, M., Partridge, I. K., D’Amore, A., ‘Warpage induced in bi-material specimens, Coefficient of thermal expansion, chemical shrinkage and viscoelastic modulus evolution during cure’, Composites Part A, Applied Science and Manufacturing, 37 (4), 565–570 (2006). 22. Grassia, L., D’Amore, A., ‘Residual stresses in amorphous polymers’, Macromolecular Symposia, 228, 1-15 (2005). 23. Penco, M., Sartore, L., Bignotti, F.,Rossini, M., D’Amore, A., Fassio, F., ’Binary blends based on poly(vinyl chloride) and multi-block copolymers containing poly(ε-caprolactone) and poly(ethylene glycol) segments’, Macromolecular Symposia 180 , pp. 9–22,( 2002) 24. Bignotti, F., Penco, M., Sartore, L.,D’Antone, S., D’Amore, A., Spagnoli, G. ,’Thermal degradation of two classes of block copolymers based on poly(lactic-glycolic acid) and poly(ε-caprolactone) or poly(ethylene glycol)‘, Macromolecular Symposia 180 , pp. 257–266,( 2002) 25. D’Antone, S., Bignotti, F., 23. Sartore, L., D’Amore, A., Spagnoli, G., Penco, M., ‘Thermogravimetric investigation of two classes of block copolymers based on poly(lactic-glycolic acid) and poly(ε-caprolactone) or poly(ethylene glycol)’. Polymer Degradation and Stability, 74 (1), 119–124 (2001). 26. Penco, M., Bignotti, F., Sartore, L., Peroni, I., Casolaro, M., D’Amore, A., ‘Stimuli-responsive polymers based on N-isopropylacrylamide and N-methacryloyl-L-leucine’, Macromolecular Chemistry and Physics, 202 (7), 11500–1156 (2001). 27. Penco, M., Bignotti, F., Sartore, L., D’Antone, S., D’Amore, A., ‘Multiblock copolymers based on segments of poly(D,L-lactic-glycolic acid) and poly(ethylene glycol) or poly(ε-caprolactone), A comparison of their thermal properties and degradation behavior’, J. of Applied Polymer Science, 78 (10), 1721–1728 (2000). 28. D’Amore, A., Caprino, G., Nicolais, L., Marino, G., ‘Long-term behaviour of PEI and PEI-based composites subjected to physical aging’, Composites Science and Technology, 59 (13), 1993–2003 (1999). 29. Alfani, R., Colombet, P., D’Amore, A.,Rizzo, N., Nicolais, L. , ‘Effect of temperature on thermomechanical properties of Macro-Defect-Free cement-polymer composite’, Journal of Materials Science 34 (23) , pp. 5683–5687 (1999) 30. Caprino, G., D’Amore, A., Facciolo, F., ‘Fatigue sensitivity of random glass fibre reinforced plastics’. J. of Composite Materials’, 32 (12), 1203–1220 (1998). 31. Caprino, G., D’Amore, A. ,’ Flexural fatigue behaviour of randomcontinous-fibre-reinforced thermoplastic composites’, Composites Science and Technology 58 (6) , pp. 957–965 (1998) 32. Giordano, M., Calabro, A., Esposito, C., D’Amore, A., Nicolais, L. ‘An acoustic-emission characterization of the failure modes in polymer-composite materials’, Composites Science and Technology 58 (12) , pp. 1923–1928 (1998) 33. Han, Y., D’Amore, A., Marino, .,Nicolais, L., ‘A phenomenological study of the structural relaxation of an inorganic glass (Li2O2SiO2)’, Materials Chemistry and Physics 55 (2) , pp. 155–159, (1998) 34. Han, Y., D’Amore, A., Nicolais, L. , ‘The effect of structural parameters on the enthalpy relaxation of an inorganic glass (Li2O · 2SiO2)‘, Materials Chemistry and Physics 51 (1) , pp. 64–69 (1998)

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35. Di, Y., Acierno, D., D’Amore,.,Nobile, R., Nicolais, L. ’Capillary extrusion behavior of phenolphthalein poly(ether-ether-sulphone) (PES-C)’, Journal of Applied Polymer Science 65 (5) , pp. 951–958, (1997) 36. Netti, P., D’Amore, A., Ronca, D., Ambrosio, L., Nicolais, L., ‘ Structure-mechanical properties relationship of natural tendons and ligaments’, J. of Materials Science, Materials in Medicine, 7 (9), 525–530 (1996). 37. Yang, Y., D’Amore, A., Di, Y., Nicolais, L., Li, B., ‘Effect of physical aging on phenolphthalein polyethersulfone/poly(phenylene sulfide) blend. I. Mechanical properties’, J. of Applied Polymer Science, 59 (7), 1159–1166 (1996). 38. D’Amore, A., Marino, G., Nicolais, L.,Mijovic, J., ‘Polymer composites: Structural relaxation in amorphous glassy polymers’, Polymer News 21 (7) , pp. 233–236 (1996) 39. D’Amore, A., Caprino, G., Facciolo, F. , ‘Correlation between flexural and tensile fatigue response of short glass fibre reinforced plastics’, Advanced Composites Letters 5 (2) , pp. 53–58, (1996) 40. D’Amore, A., Caprino, G., Stupak, P.,Zhou, J., Nicolais, L. ‘Effect of stress ratio on the flexural fatigue behaviour of continuous strand mat reinforced plastics‘, Science and Engineering of Composite Materials 5 (1) , pp. 1–8 , (1996) 41. Zhou, J., D’Amore, A., Zhuang, G.,He, T., Li, B., Nicolais, L. ‘Tension-tension fatigue failure behaviour of poly(phenylene ether ketone) (PEK-C)‘ Polymer  37 (11) , pp. 2103–2111, (1996) 42. Iannace, S., Ambrosio, L., D’Amore, A., Nicolais, L. ‘Nonlinear viscoelasticity of Hydrothane®’, Journal of Materials Science: Materials in Medicine 7 (5) , pp. 305–307, (1996) 43. Mijovic, Jovan, Nicolais, Luigi,D’Amore, Alberto, Kenny, Jose M. ‘Principal features of structural relaxation in glassy polymers A review ‘ Polymer Engineering and Science 34 (5), pp. 381–389 (1994).

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Oxidation Communications 35, No 3, 805 (2012)

ERRATA The affiliations of the authors of the article ‘AN IMPROVED METHOD FOR OXIDATION OF OXIMES WITH POTASSIUM PERMANGANATE ADSORBED ON GRAPHITE REAGENT UNDER VISCOUS CONDITIONS’, published in book 2, vol. 35 (2012) should be: LI-YUN ZHUa, CHEN HUANGb, CHENXIAO SHIc, FANG LINa, CHANGHE ZHANGd, JI-DONG LOUa,e* College of Life Sciences, China Jiliang University, Hangzhou, 310 018 Zhejiang, China E-mail: [email protected] b China Pharmaceutical University, Nanjing, 210 009 Jiangsu, China c School of Environmental and Chemical Engineering, Shanghai University, 200 444 Shanghai, China d Centre for the Research and Technology of Agro-environmental and Biological Sciences (CITAB)/Department of Biology and Environment, Universidade de Trásos-Montes e Alto Douro (UTAD), Apartado 1013, 5001-801 Vila Real, Portugal e Sirnaomics Inc., 401 Professional Drive, Gaithersburg, MD 20879, USA a

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Instruction for authors Starting from 2011, the authors can publish their manuscripts as rapid publication (6 months after the receipt of positive referees comments and the revised version) after they pay a fee of 100 Euro. This does not concern authors whose Universities and Organisations have a subscription or sponsorship to the Journal. The authors receive a hard copy of the Journal issue containing their published article free of charge. Some figures, according to authors decision, can be published coloured in order to make them more understandable to the reader. The additional payment is 75 Euro per printed page. The language of the Journal is exclusively English. Contributions will be con­sid­ered only if they have not been previously published or been submitted elsewhere. Authors are requested to submit manuscripts using double spacing and ca. 60 characters per line and 30 lines per page. Receipt of a contribution for con­sid­er­ation will be acknowledged immediately by the Editorial Office. The acknowledgement will indicate the paper reference number assigned to the contribution. Authors are particularly asked to quote this number on all sub­se­quent correspondence. Organisation

The title page should include the title, authors and their affiliations, complete address of the author to whom correspondence should be sent and an Abstract. Abstract – should not exceed 200 words and should give the subjects and con­clusions of the article and all results of general interest. References and com­pound numbers should not be mentioned in the Abstract. A maximum of five key­words should follow the Abstract. Aims and Background – should include brief and clear remarks outlining the specific purpose of the work and a short summary of the background material including num­bered ref­erences. Experimental – should be sufficiently detailed (but concise) to guarantee re­pro­ducibil­ity. Results and Discussion – should indicate the logic used for the in­ter­pre­tation of data without lengthy speculations. Authors submitting material on purely theo­retical problems or on a new experimental technique might unite the sections Ex­peri­men­tal, Results and Discussion into one section under the heading Discussion. Conclusions – short summary of the main achievements of the research. References – should be typed on separate sheets and numbered in the order as first cited in the text. They should be indicated by superscript Arabic numerals in the text. Abbreviations of journal titles should follow the style used in Chemical Abstracts Service Source Index, 1970 edition and supplement. Se­quence and punctuation of references should be: 1. E. NIKI, M. KUDO, Y. KAMIYA: Reactions at t-Butoxy Radical with Co­balt Ions. Oxid Commun, 1, 33 (1979). 2. M. B. NEIMAN, D. GAL: The Kinetic Isotope Method. Akademiai Kiado, Budapest, 1971. 3. J. A. HOWARD: In: Advances in Free Radical Chemistry. Vol. 4 (Ed. G. H. Williams). Lagos Press, London, 1972, 49–69. 4. C. H. BAMFORD, C. F. H. TIPPER (Eds): Comprehensive Chemical Ki­netics. Vol. 16: Liquid-Phase Oxidation. Elsevier, Amsterdam–Oxford–New York, 1980, p. 264. However, in the list of references to original papers the article title should be also included in accordance with the following example:

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5. D. GAL: Modelling of Processes Proceeding in the Presence of Oxygen: from the Model to Reality. Oxid Commun, 20, 1 (1997). In preparing the list of References attention must be drawn to the following points: (a) Names of all authors of cited publications should be given. Use of ‘et al.’ in the list of references is not acceptable; (b) Only the initials of first and middle names should be given. Tables – each bearing a brief title and typed on a separate sheet, should be numbered in Arabic numerals. The tables should be placed after the list of Ref­erences. Figures and captions – should be grouped together at the end of the manu­script with figures numbered consecutively and captions typed on a separate sheet. Figures (graphs) should be marked by pensil on the margin or at the back with the name of the first author and the beginning of the title. Particular attention is drawn to the use of SI Units, IUPAC nomenclature for compounds and standard methods of literature citation. Manuscripts (without figures) should not exceed 20 typewritten pages. Electronic Submission of Manuscripts

Manuscripts should be submitted in electronic form. Submission not in elec­tronic form may face a delay in publication. All text (including the title page, abstract, keywords, all sections of the manu­script, figure captions, and references) and tabular material should be in one file, with the complete text first, followed by the tabular material. The manuscript must be prepared using MS Word 6.0 and above. Manuscripts in PDF are not accepted. Chemical equations must be supplied using equation editor. Tables must be created using table format feature. Graphics, i.e. figures, schemes, etc. should be in a separate file. The file name should be descriptive for the graphic. Structures and schemes may be supplied in ChemWindow format and other graphics in Microsoft Excel or Microsoft PowerPoint format. Submission of manuscripts

Manuscripts should be sent to the following address: Prof. Dr. Slavi K. Ivanov SciBulCom Ltd., P. O. Box 249, 7 Nezabravka Str., 1113 Sofia, Bulgaria Phone/Fax: +359 2 872 42 65, +359 2 978 72 12 E-mail: [email protected] All manuscripts are subject to critical review and the names of referees will not be dis­closed to the authors. The manuscript sent back to the au­thor for revision should be returned within 2 months in duplicate. Oth­er­wise it will be considered withdrawn. Revised manuscripts are gen­erally sent back to the original referees for comments, unless (in case of minor revisions) the editors accept them without seeking further opinions. Proofs should be corrected and re­ turned as soon as possible. The authors receive CD-ROM containing copy of the book.

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