OPTIMIZATION OF CNTs PRODUCTION USING FULL FACTORIAL ...

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International Journal of Nanoscience Vol. 9, No. 3 (2010) 181–192 c World Scientific Publishing Company DOI: 10.1142/S0219581X10006648

OPTIMIZATION OF CNTs PRODUCTION USING FULL FACTORIAL DESIGN AND ITS ADVANCED APPLICATION IN PROTEIN PURIFICATION N. M. MUBARAK∗,§ , FARIDAH YUSOF∗,¶ , M. F. ALKHATIB∗ , EMAD AMEEN∗ , M. KHALID∗ , AL SAADI. MOHAMMED∗ , A. MUATAZ† , I. Y. QUDSIEH‡ and W. RASHMI∗ ∗Nanoscience and Nanotechnology Research Group (NANORG) Department of Biotechnology Engineering, Faculty of Engineering International Islamic University Malaysia, P. O. Box 10 50728 Kuala Lumpur, Malaysia †Department of Chemical Engineering Head of Nanocarbon Research Unit Centre of Research Excellence in Nanotechnology King Fahd University of Petroleum and Minerals P. O. Box 5050, Dhahran-31261, Saudi Arabia ‡Department of Chemical Engineering, Jazan University P. O. Box 114, Jazan-45142, Saudi Arabia §[email protected] ¶[email protected] Revised 12 January 2010 Carbon nanotubes (CNTs) have been successfully synthesized by using in-house fabricated Double Stage Chemical Vapor Deposition (DS-CVD) technique, using acetylene (C2 H2 ) and hydrogen (H2 ) as the precursor gases. The purity, morphology and the structure of CNTs were then characterized using Field Emission Scanning Microscope (FESEM), Transmission Electron Microscope (TEM) and Thermogravimetric Analysis (TGA). The effects of the process parameters were examined whereby the experimental design of the investigation was conducted using Design Expert Version 6.0.8. The statistical analysis reveals that the optimized conditions for the best CNTs yield production at 850◦ C reaction temperature, 60 min reaction time, with gas flow rates at 40 and 150 ml/min for C2 H2 and H2 , respectively. The CNTs produced were successfully used as column chromatographic media. Due to its nanosized structured dimension, CNTs’ have tremendously large surface area and that lead to highly efficient protein purification. Skim latex protein has been used as the model protein and we aim to recover useful proteins and enzymes from this known wasteful material. During the purification, the process parameters such as pH and ionic strength of the running buffer were optimized to enhance

§

Corresponding author.

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protein purification. Results reveal that CNTs behave efficiently as a hydrophobic interaction chromatographic media. Keywords: CNTs; DS-CVD; functionalization; skim latex serum; protein purification.

1. Introduction Ever since CNTs discovered by Iijima1 in 1991, they have been treated as the most promising nanostructured materials. The prospect of developing novel carbon-based nanomaterial has excited worldwide interest among researchers. CNTs have been of great interest; both from fundamental point of view and for potential applications because of their amazing mechanical,2,3 thermal,4 electric and magnetic properties.5,6 They have large number of potential applications, which includes flat panel field emission displays,7 nanoelectronic devices,8 chemical sensors,9 hydrogen storage10,11 and scanning probe tip.12 Various methods to grow CNTs have been developed, including laser ablation,13 arc discharge14 and Chemical Vapor Deposition (CVD).15 In this study, CNTs were produced using double stage CVD (DS-CVD)16 with acetylene (C2 H2 ) and hydrogen (H2 ) as the precursor gases. In order to produce the best CNTs yield in terms of amount and purity, the process parameters were statistically optimized with respect to reaction temperature, reaction time, and gas flow rates for C2 H2 and H2 . Produced CNTs were submitted to purification to remove unwanted impurity, such as left over catalyst, via oxidation with nitric acid and sulfuric acid after which they were functionalized. It has been reported that the purification of CNTs

Fig. 1.

by acid washing creates open end termini in the structure that are stabilized by the carboxyl and hydroxyl groups left bonded to the nanotube at the end termini and/or the sidewall defect sites.17 Carboxylic group can also be introduced at the tube surface which can covalently bind proteins. This can be carried out via a two-step process of diimideactivated amidation between the carboxylic acid groups on the surface CNTs and amine groups on proteins. These functionalized CNTs as well as the unfunctionalized batches were used as a media for column chromatography. We envisaged that due to its nanosized dimension, CNTs provide large surface area, making it a suitable media to purify proteins. In this study, we attempted to use CNTs to purify proteins sourced from skim latex serum. Skim latex serum is recovered from skim latex, a by-product of natural latex concentrate industries, which are usually considered as a waste, thus lavishly thrown away. Skim latex, obtained upon centrifugation as illustrated in Fig. 1, contains a dry rubber content between 3% and 7% with very low dirt content. Skim latex serum is the nonrubber aqueous portion of latex as a result of acid coagulation or membrane filtration. The serum contains a rich source of nitrogen, carbohydrates, proteins, lipids and trace metals. Some of these proteins are important enzymes which has great

Production of skim latex.

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Optimization of CNTs Production Using Full Factorial Design

demand in pharmaceutical, food and detergent industries. Hence, there is need in the art to improve the efficiency and yield of protein separation.18

2. Materials and Methods 2.1. Material There were three types of gases used in the production of CNTs namely, H2 (99.99% purity), C2 H2 (99.9%) and Ar (99%). Ferrocene was used as the catalyst. Skim latex was supplied by the Rubber Research Institute Malaysia, Malaysian Rubber Board, Sungai Buloh, Selangor.

2.2. Methods 2.2.1. Production of CNTs The production of CNTs was carried out on the hot-wall of DS-CVD reaction chamber. The catalyst, ferrocene, was placed at the centre of the first furnace and a ceramic boat where the growth of CNTs took place 40 cm ahead of the second furnace. The system was initially flushed with Ar in order to ensure oxygen free environment. In the mean time, the second furnace was heated to the desired reaction temperature. Heating was continued until a steady state condition was achieved. The flow of Ar was then stopped and heating of the first furnace till 150◦ C was initiated. The flow for C2 H2 along with H2 was immediately opened. The reaction was carried on for the desired time period and on completion, the total amount of CNTs produced in the ceramic boats as well as in the walls of the second furnace were weighed separately. They were then acid purified, functionalized and characterized using FESEM (JEOL JSM 6700F), TEM (JEOL JEM 2010), and TGA (Pyris diamond TG/DTA).

2.2.2. Statistical approach of CNTs production To optimize the process parameters of CNTs production with respect to achieving high purity and yield, a statistical approach was adopted. We have chosen to optimize four process parameters namely reaction time, reaction temperature and flow rates of the precursor gases, C2 H2 and H2 . In order to minimize the experimental runs, the design of experiment was conducted using statistical software Design Expert Version 6.08. After selecting half factorial, four parameters at four levels were duplicated and the computed design revealed a total of

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27 experimental runs. Results of runs were further statistically analyzed by the same computer software. Produced CNTs were analyzed for purity and morphology. Before they were used as the chromatographic media, CNTs were submitted for acid purification and functionalization.

2.2.3. Preparation of skim latex serum Skim latex serum was prepared by first submitting skim latex to acidification by acetic acid to promote the coagulation of small rubber particles. Centrifugation at 10 000 rpm was then carried out which leads to separation of cell debris, clear serum and coagulated latex. The clear serum was submitted to dialysis (with 10 kD MWCO) against a buffer solution to remove low molecular weight solutes.

2.2.4. Purification of skim latex by CNTs For column preparation, the CNTs were first mixed with 20% alcohol. The CNTs suspension was poured into a Bio-Rad (USA) column measuring (1×10 cm) to a height of 2 cm. It was then washed with 5 column volumes of distilled water and further equilibrated with 3 column volumes of running buffer, 50 mM Tris–HCl, pH 7.0. The column was then assembled on AKTAprime (GE, Healthcare, USA) liquid chromatography system for the purification processes. We performed the purification of skim latex serum by functionalized CNTs column and compared the performance to the nonfunctionalized CNTs column. Guided by the functional group that existed on the CNTs in both cases we performed purification processes on functionalized CNTs as in ion exchange chromatography (IEC) procedure. Whereas purification by nonfunctionalized CNTs were performed as in hydrophobic interaction chromatography (HIC). For column preparation, the functionalized carbon nanotubes was gently stirred and allowed to settle or homogenized with the binding buffer (Buffer A) inside the column. When the column was packed, it was washed with distilled water and two times binding buffer. pH and concentration of the binding buffer must be similar to the sample. For functionalized CNTs, the skim latex sample will be dialyzed against binding buffer that does not contain ammonium sulphate and eluted by buffer that contains 2 M ammonium sulphate. To perform this ion exchange chromatography, the method templates used was the ion exchange/Gradient elution. For the AKTAprime

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Volumes of buffer required.

Steps

Nonfunctionalization of CNTs (ml)

Functionalization of CNTs (ml)

Equilibrium Sample size Wash 1 Elution Wash 2 Per fraction

10 2 20 40 10 2

10 2 20 40 10 2

system, the volumes of buffer required for each step of the purification were set accordingly as tabulated in Table 1. Automatically, the column was equilibrated with predetermined pH and ionic strength of each buffer. The column was finally eluted with linear gradient of 2 M ammonium sulphate. The column preparation for the filtration of skim latex serum using hydrophobic interaction chromatography is similar to the ion exchange chromatography except in this type of chromatography; the nonfunctionalized CNTs will be used as the columns resin. When the column was packed, it was washed with distilled water two times binding buffer; pH and concentration of the binding buffer must be similar to the sample. For nonfunctionalized CNTs, the skim latex sample will be dialyzed against binding buffer that does contain 2 M ammonium sulphate and eluted by buffer that does not contain any salt. To perform this hydrophobic interaction chromatography, the method templates used was the hydrophobic interaction chromatography/gradient elution. As we were comparing the capacity between functionalized and nonfunctionalized CNTs as chromatographic media, the binding buffer for each case is different. Binding buffer for nonfunctionalized CNT included 2 M ammonium sulfate as the neutral salt. Serum samples for both chromatographies were prepared accordingly. Fractions of 2 ml of each were collected during the elution period.

3. Results and Discussion 3.1. Characterization of the produced CNTs Results reveal that we have successfully produced high purity and high yield CNTs. The CNTs were then characterized by using FESEM, TEM, TGA and FTIR. FESEM images in Figs. 2(a) and 2(b),

(a)

(b) Fig. 2. FESEM image of CNTs produced at a reaction temperature of 850◦ C (a) at different magnifications (b).

show the raw CNTs before functionalization at different magnifications, i.e., 1000 and 1000 000 nm scale. Images show that the produced CNTs could be observed in vertical alignment with the diameter of CNTs ranging from 31–36 nm. At low magnification some of impurities, such as amorphous carbon and excess catalyst particles, can be seen on the surface of CNTs. These high yield and high purity CNTs can be further used in protein purification. FESEM images (Figs. 3(a) and 3(b)) show that after functionalization of CNTs at the reaction temperature of 850◦ C with different magnification scales (1 µm and 100 nm), it could be clearly observed that oxidized CNTs appeared shorter than raw CNTs. Moreover, the surface of the raw CNTs is smoother than the oxidized CNTs due to the agglomeration of CNTs tube caused by the acid treatment. The figure shows that the surface of CNTs became agglomerated, which created an open

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(a)

(a)

(b)

(b)

Fig. 3. (a) FESEM image of CNTs produced at a reaction temperature of 850◦ C after functionalization, (b) FESEM image at a reaction temperature of 850◦ C at different magnifications.

Fig. 4. (a) TEM image of CNTs at a reaction temperature of 850◦ C, (b) TEM image of CNTs produced at a reaction temperature of 850◦ C at different magnifications.

end that could be attached to the functional group which is possibly formed, especially at the walls and any available defective sites. These functional groups could be carboxylic or amine groups. Figure 4(a) shows the High Resolution Transmission Electron Microscope (HRTEM) images of the production of CNTs at a reaction temperature of 850◦ C at different magnification scales. It was carried out to characterize the structure of nanotubes. From the images, it was clearly observed that all the nanotubes were hollow and tubular in shape. In some of the images, catalyst particles, (arrow) could be seen trapped inside the carbon nanotubes. Figure 4(b) shows the HRTEM images of CNTs at reaction temperature of 850◦ C at different magnification scale. From Fig. 4(b), a highly ordered crystalline structure of CNTs was observed. The clear fringes of graphitic sheets were well separated by

10 nm and aligned with a tilted angle of about 2◦ toward the tube axis. The HRTEM image shows that there were about 19 graphitic walls of the multilayer CNTs grown at 850◦ C. Hence, the CNTs were observed to be multiwalled. Thermogravimetric analysis (TGA) of the raw CNTs was conducted to examine the purity of the CNTs by observing the changes in the mass of CNTs as a function of temperature and a function of time. As revealed by Fig. 5, TGA analysis showed a single peak at one distinct zone, implying high purity of the CNTs. Moreover, the peak corresponds to the decomposition of one element only, since no other peaks were observed. There was a very slight loss of weight between 50◦ C and 200◦ C, which correspond to the loss of water from the catalyst support. The weight loss from 500◦ C to 650◦ C was due to the oxidation of CNTs. The observed flat

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TGA analysis of CNTs produced at a reaction temperature of 850◦ C.

Fig. 5.

profiles between 650◦ C and 850◦ C showed that the metal catalyst and support were not volatile below 800◦ C and thus remain as residue of TGA. The weight loss indicates the purity and yield of CNTs and the higher the weight loss, the higher is the purity of CNTs. From the TGA analysis, we concluded that the optimized run yield a CNTs batch which is almost 95% pure.

Figure 6 shows the FTIR spectra (Perkin– Elemer ATR method) of CNTs before and after functionalization. Results indicated that the functionalization has been successfully occurred on the surface of CNTs. Although quite broad, the absorption peak by carboxylic group can be observed at a range at 1700–1900 cm−1 . This is in accordance to the values given in the literature.19

227 3 20 7718 49 24 21

0.96

17 00

15 08 94 0

0.94

84 1

Funct-CNTs

0.92 0.90 0.88

513 61 0 54 4 53 1

37 74

0.86

a

0.84

Absorbance

0.82 0.80

b

0.78 0.76 0.74 0.72

26 69 37 44

20 42

50 7

18 69

63 2

0.70 0.68

Raw CNTs

0.66 0.64 0.62 0.60 0.58 0.56 0.54 4000.0

3600

3200

2800

2400

2000

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1400

1200

1000

800

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400.0

Wavenumber (cm-1)

Fig. 6.

FTIR absorption spectra, (a) functionalized CNTs, (b) nonfunctionalized CNTs.

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3.2. Statistical analysis of CNTs production

the best equation that represents the optimized model.

Four levels Full Factorial Design was employed to attain all possible combinations of all the input factors with minimal number of experimental runs that could possibly optimized the response within the region of three-dimensional observation spaces. The results from the experiments were analyzed using analysis of variance (ANOVA) using Design Expert Version 6.0.8. The variables such as reaction temperature, reaction time, and gases flow rates were employed for the analysis in the design. The response is the amount of CNTs yield. The results from the analysis for this design are given in Table 2. The Fisher F -test value signifies how the mean square of the regressed model compares to mean square of the residuals (errors). The greater the F value, the more efficient the model will be. A very low probability (P value) demonstrates a very high significance for the regression model. The F -test which is 56.88 implies that model is significant. Analysis of variance (ANOVA) shows an F test value of less than 0.05, implying that the model is also significant. Furthermore, the value of the correlation coefficient (R2 = 0.98) and the value of the adjusted determination coefficient 2 = 0.97) are both considered high, indicating (RAdj a high significance of the model. Equation (1) shows

Table 2. Source

Yield = + 149.06 + 42.81A + 25.31B − 10.94C − 13.44D + 7.81AB − 4.69AC − 8.44AD − 5.94BC + 4.0CD

(1)

where, the response yield was the yield of CNTs produced, A was the coded value of reaction temperature, B was the coded value of reaction time, C and D were the coded value of H2 and C2 H2 , respectively. The coefficients with one factor represent the effect on the particular factor, while the coefficients with two factors represent the interaction between the two factors. The positive sign infront of the terms indicates synergistic effect whereas negative sign indicates antagonistic effect. The coefficient values from the equation of regression model show that reaction temperature is giving higher positive effect compared to reaction time towards CNTs yield. On the other hand, both flow rates of C2 H2 and H2 are giving negative effects. It can aslo be noted that there are positive interactions between reaction time and reaction temperature and between flow rates of C2 H2 and H2 , whereas negative interactions are shown between reaction temperature and flow rate of C2 H2 , between reaction temperature and flow rate of H2 and between

ANOVA results for selected full factorial model for yield.

Sum of squares

Model A B C D AB AC AD BC CD

50 201 30 189 10 764 2139 3164 1139 451 1314 689 351

Curvature Residual Lack of fit Pure error Cor total R-Squared Adj R-Squared Pred R-Squared Adeq precision Std. Dev.

1076 784 771 12.67 52 062 0.986 0.967 0.89 26.54 9.90

187

DF 9 1 1 1 1 1

1 1 1 8 6 2 12

Mean square 5577 30 189 10 764 2139 3164 1139 451 1314 689 351 1076 98 128 6.3

F value

Prob > F

Status

56.88 307 109 21.8 32.2 11.6 4.6 13.4 7.03 3.58

0.002 0.003 0.002 0.0016 0.0005 0.009 0.064 0.029 0.09 0.012

Significant

10.98 20.31

Significant 0.0234 0.047

Significant

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Predicted value of CNTs yield

350 300

y =0.9825x +2.9183 2 R = 0.996

250 200 150 100 50 0 0

50

100

150

200

250

300

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Experimental value of CNTs yield

Fig. 7. Relationship between predicted and experimental values of yield of CNTs.

reaction time and flow rate of C2 H2 . Thus, in short, the coefficient values obtained show that all the process parameters chosen to be investigated in the optimization experiments influenced the yield of CNTs in the production process. The effects of each interaction process parameter were further analysed by statistical software. All terms are included in the model to give the optimum fit of the experimental data. Equation (1) was used to predict the output of CNTs yield and this is compared with the observed values as shown in Fig. 7. In the figure, relationship between the theoretical values and the experimental values for CNTs yield is showed. It was clearly shown that theoretical values obtained were quite close to the experimental values, indicating that the model developed was successful in bridging the correlation between process parameters to the yield of CNTs.

The three-dimensional plot shown in Fig. 8 shows the interaction between the reaction time, the reaction temperature and yield of CNTs produced. The figure indicates that within the ranges tested, the production of CNTs increases as reaction temperature and reaction time increases. Figure 9 shows the interaction between CNTs yield and the gases flow rates. The figure indicates, within the ranges tested, the yield of CNTs increases as the flow rate of C2 H2 increases, provided the flow rate of H2 decreases.

3.3. Purification of skim latex serum protein Chromatography of both small molecules and macromolecules is widespread in biochemistry. Two of the most common techniques used are ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC). IEC uses chromatographic media that is able to separate molecules based on charges whereas HIC uses chromatographic media that is able to separate molecules based on the degree of hydrophobicity. In this work, we used two different types of CNTs batches as the chromatographic media to separate skim latex serum. Guided by the functional groups available on the surface, we used CNTs differently; functionalized CNTs as IEC media and raw CNTs as HIC media. After producing enough, followed by acid purifying and functionlization, CNTs were used as the chromatographic column media in two

DESIGN-EXPERT Plot yiled X = A: temp Y = B: time

225 190.938

Actual Factors C: h2 = 165.00 D: c2h2 = 70.00

156.875

yiled

122.813 88.75

60.00

850.00

47.50

787.50

35.00

B: time

725.00

22.50

662.50 10.00

Fig. 8.

A: temp

600.00

A 3-D interaction plot of yield of CNTs, reaction time and reaction temperature.

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DESIGN-EXPERT Plot yiled X = C: h2 Y = D: c2h2

181.563 168.556

Actual Factors A: temp = 728.38 B: time = 35.00

155.549

yiled

142.542 129.535

180.00

100.00 172.50

85.00 165.00

70.00

D: c2h2

157.50

55.00 40.00

Fig. 9.

C: h2

150.00

A 3-D interaction plot of yield of CNTs, flow rates of C2 H2 and H2 .

ways, namely nonfunctionalized and functionalized. Guided by the available functional group on the CNTs surfaces, purification of proteins were either conducted as in HIC or IEC. The data produced from the AKTA prime is called chromatogram. It is a graph that monitors the signal in the UV detector over time. The detector is a device that measures the absorbance at 280 nm of the flow and through that it flows out of the column. The signal increase and cause the chromatogram to display a “peak”. Each peak in the chromatogram indicates the presence of proteins in the sample. Generally, the chromatogram that results from running the experiment consists of two peaks: the first one is designated to the unbounded protein and the second one to the eluted proteins. In these experiments, the response to be monitored is the area under the curve of the second peak. This is because, the second peak is the measure of positive interaction or binding that occurs between proteins and the column media. A large second peak indicates a good interaction of protein with CNTs, which when carefully eluted will resolve in enhanced purification. The area under the curve of the second peak is automatically generated from the analysed chromatogram data.

3.3.1. Protein purification using nonfunctionalized CNTs As the main functional group that exists on the surface in nonfunctionalized CNTs is carbon, which is hydrophobic, protein purification was

carried out as according to the Hydrophobic Interaction Chromatography (HIC). Protein separation by hydrophobic interaction chromatography was dependent upon interactions between the protein itself, the CNTs matrix and the surrounding solvent which is usually aqueous. Increasing the concentration of buffer salt of a solution, by addition of a neutral salt (e.g., ammonium sulphate or sodium chloride) increased the hydrophobicity of protein molecules that explained the hydration of salt ions in solution results, in an ordered shell of water molecules forming around each ion. It attracted water molecules, which in turn help to unmask hydrophobic domains on the surface of the protein. The increase in concentration of buffer salt enhances the surface hydrophobicity of protein molecules. From the studies, the neutral salt sodium chloride results showed that the efficiency of protein binding to the column was less, due to less ionic strength. Whereas 2 M ammonium sulphate (AS) was the best salt concentration for purification of protein from skim latex due to its high concentration of buffer salt. It enhanced the surface hydrophobicity of protein molecules. Protein samples were therefore best applied to hydrophobic interaction columns under conditions of high concentration of buffer salt. As they percolated through the column, proteins may be retained via hydrophobic interactions. In HIC, the attraction was between the hydrophobic side chain of amino acid that made up protein and the hydrophobic molecules of the chromatographic media (stationary phase). When

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B U

Fig. 10. Chromatographic protein profile of skim latex serum on nonfunctionalized CNTs as media; U, unbound protein: B, bound protein.

the salt concentration decreased during the elution period, the surface of water molecules became a mask. Hydrophobic domains on the surface of protein made the binding tighter on the surface of CNTs. Figure 10 shows a protein separation profile on nonfunctionalized CNTs, performing as an HIC media. Bound proteins were eluted by decreasing the concentration of the neutral salt. Elution of two peaks, as appeared, are labelled as unbound (U) proteins and bound proteins (B). Unbound protein may be hydrophilic and thus have no interaction with the hydrophobic CNT matrix, thus washed out immediately in the early stage of the elution. Bound proteins are hydrophobic proteins which get bounded and are only eluted by decreasing the concentration of neutral salt. As the concentration of neutral salt was decreased, the protein hydrophobic surfaces were masked, pumping them to be released from CNTs matrix. The less hydrophobic protein will be eluted first and the most hydrophobic protein will be eluted last and thus the phenomena will separate the hydrophobic protein based on the charge of its hydrophobicity. Area under the peak two or unbounded protein peak is the response to be considered when evaluating the efficiency of purification values. Results show that besides the concentration of added neutral salt, pH and concentration of the running buffer do influenced the

level of binding protein thus, the difference in the area under the curve of unbound protein peak, pH and concentration of running buffer. Proteins are amphoteric molecules that contain large number of acid and basic groups on the surface. The changes vary with pH and salt concentration of the environment, which will affect the total net charge of the protein.20 Although the changes will be hidden, once neutral salts are added, the full net effect of hydrophobicity will be different with different pH and the salt concentration of running buffer.

3.3.2. Protein purification using functionalized CNTs In this work, the capacity of protein purification using CNTs as media in IEC was evaluated. IEC used functionalized CNTs as the chromatographic media to separate molecules based on charges. The functional groups that exist on the surface of CNTs was amine groups which are attached between amine and CNTs surface. The principle of IEC which is negatively charged sample components were absorbed on the stationary phase and thus separated from positively charged and uncharged sample components. The absorbed components were eluted by increasing the concentration of buffer salt

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U

B

Fig. 11. Chromatographic protein profile skim latex serum on functionalized CNTs as media: U, unbound protein, B, bound protein.

of the mobile phase matrix that contained covalently attached positive group. During the protein purification, the functional group exist on the surface of CNTs, which is positively charge and negatively charge protein molecules would be interaction between functional groups protein side chains was temporary ionic interaction between charges and functional group. Figure 11, shows a protein separation profile on functionalized CNTs, performing as IEC media. Bound proteins were eluted by increasing the concentration of the neutral salt. The area under the curves of the protein profiles during elution when compared, suggest that nonfunctionalized CNTs performed more efficiently as HIC media than functionalized CNTs as IEC media. Unbound protein has less positively charge bound to the column in the early stage of graph. Bound protein has high negative charge, which was eluted by increasing the concentration of the neutral salt. The efficiency of purification using functionalized CNTs was less as observed from area under the elution peak where very slight protein was bound to the column. However, the capacity was dependent on pH and concentration of buffer salt. Purification using covalent functionalization of CNTs was less as compared to that using nonfunctionalization CNTs.

4. Conclusion CNTs have been successfully produced by DSCVD and the statistical analysis reveals that the optimized conditions for the best yield CNTs production is 850◦ C reaction temperature, 60 min reaction time with gas flow rates of 40 and 150 ml/min for C2 H2 and H2 , respectively. The TGA analysis shows that the purity of CNTs produced as about 95% purity. FESEM and TEM analyses reveal that the uniformly dispersed CNTs have diameters ranging from 35 to 45 nm. This work has also demonstrated that CNTs can perform as a column chromatographic media in IEC and HIC. Chromatographic separation of our model protein, skim latex serum, shows that CNTs can be more efficiently used as HIC media as compared to IEC. Commercialized HIC media are known to be expensive thus is not cost effective to be used in up-scale protein separation work. This work shows that CNTs produced cheaply can replace the high cost commercialized HIC media. The nano-sized structured CNTs lead to large surface area, thus it may be functioning as a better chromatographic media than the commercialized product. Results show that as usual the efficiency of the protein purification is dependent upon pH and the ionic strength of the running buffer. CNTs have

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many uses and this work adds to another dimension in the numerous applications of CNTs.

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