hydrolysable tannins, no aldehydic cross-linkage between collagen and tannin could ... Key words: vegetable tannin; hide collagen; vegetable tannin-aldehyde ...
THE REACTION OF VEGETABLE TANNIN-ALDEHYDECOLLAGEN: A FURTHER UNDERSTANDING OF VEGETABLE TANNIN-ALDEHYDE COMBINATION TANNAGE ZHONGBING LU, XUEPIN LIAO, BI SHI The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, P. R. China Summary Using mimosa as vegetable tannin agent and oxazolidine as aldehydic tanning agent, the approaches of hydrothermal analysis, breaking of hydrogen bond and hydrophobic bond as well as chemical modifications of collagen were employed to investigate the effects of hydrogen bond, hydrophobic bond and side amino group of collagen on vegetable tanning, aldehyde tanning and vegetable-aldehyde combination tanning. The results indicated that the aldehyde should still predominantly react with the side amino group of collagen even though it was added after vegetable tanning. But the aldehyde could further react with vegetable tannin and thus form cross-linkage between collagen and vegetable tannin if there are high-activity nucleophilic sites in tannin molecules. As a result, the synergistic effect of the combination tanning on increasing hydrothermal stability of collagen fibers was achieved and the combination stability of vegetable tannin and collagen was remarkably strengthened. This mechanism is appropriate for describing the combination tanning with condensed tannins that contain nucleophilic sites. But for hydrolysable tannins, no aldehydic cross-linkage between collagen and tannin could be observed. In general, this research revealed that the actual features of vegetable-aldehyde combination tanning should be different from those previously assumed. According to the mechanism suggested in this paper, all of the phenomena relating to vegetable tannin -aldehyde-collagen interaction can be well understood. Key words: vegetable tannin; hide collagen; vegetable tannin-aldehyde combination tannage; tanning mechanism; hydrothermal stability
Introduction As the preferred tannage in the modern leather industry, chrome tanning has many advantages over other tannages, such as, low cost, convenient to operation, high quality of leather and, particularly, high shrinking temperature. However it is more and more difficult to comply with ever emerging regulations with respect to the chrome content of effluent as well as the disposal of chrome containing solid wastes such as sludge, shaving, leather trimmings and buffing dust [1]. So alternative tannages should be taken in
Author to whom correspondence should be addressed. 1
consideration. Extensive research has been undertaken by many scientists in this field and some alternative options have been proposed, among which the combination tanning of vegetable tannin and aldehyde may be a good choice. The reason is that the requirement of shrinking temperature is well satisfied and that the effluent can be treated biologically with low cost. For example, when the combination of mimosa and glutaraldehyde was used, the shrinking temperature was about 95-96C[2]. Using the combination of mimosa and oxazolidine, which reacts with tannin in its ring-open process, the shrinking temperature obtained was around 115C[3]. In order to optimize the technology of vegetable tannin-aldehyde combination tanning, many researchers have been investigating the mechanism of these tannages. But due to the complexity of the vegetable tannin-aldehyde-collagen system, most studies of them were mainly focused on two components system, such as vegetable tannin-collagen, aldehyde-collagen and vegetable tannin-aldehyde [4]. It has been confirmed that the reaction between vegetable tannin and collagen is mainly on the basis of cooperative effect of hydrogen bond and hydrophobic bond, along with the physical adsorption of colloidal tannin, and the former is the main factor for improving the hydrothermal stability of collagen [5]. It has also been verified that aldehyde (e.g. oxazolidine E, Hodgson Ltd UK) can react with condensed tannins under appropriate conditions and the reaction is taking place on C-6 or C-8 positions of A-ring of the tannins[6]. Based on these results, the reaction mechanism of vegetable tannin-aldehyde combination tannage had been elucidated and can be illustrated as in Figure 1. That is, vegetable tannins penetrate into the collagen matrix and combine with collagen through hydrogen bond and hydrophobic interaction. The subsequent reaction is taking place between aldehydic agent and vegetable tannin, resulting in polymerization [7]. In this way, a multiple-bond tanned matrix can be created within the collagen fibres and therefore high hydrothermal stability can be achieved. C2H5
O
N
C2H5
O
+
P
HOH2C
T
N
P Oxazolidine E
CH2OH
vegetable tannins interact with collagen via hydrogen bonds
T
H2C
CH2
T
P
Fig.1 Assumed mechanism of vegetable tannin – aldehyde combination tannage
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However it is not convincible for us to explain some phenomena by this assumed mechanism. For example, if the way of vegetable tannin-collagen association has not been virtually changed in the combination tannage (as shown in Figure 1), how can the hydrothermal stability and washing resistance of the leather be enhanced that mach? The present investigation is focused on the real reaction mechanism of the vegetable tannin-aldehyde-collagen system, which would be greatly helpful, not only to promote the technology of vegetable tannin-aldehyde combination tannage, but also to make some new functional material based on the reaction of this system.
Experimental Materials Hide powder was obtained from Chinese Institute of Chemistry and Industry of Forest Products (moisture 13%, ash 0.26%, pH5.5). The mimosa was kindly provided by Wumin Tannin Extract Corporation (Guangxi, China) and had not been treated by chemicals. Its tannin content is 71%. Oxazolidine was received from Guanghong Stock Company (Taiwan), industrial grade. Other chemicals were analytical grade.
Methods Deamination[8]. Hide powder (100g) was soaked in distilled water overnight, then 100g NaNO2 was added, followed by adding 100ml glacial acetic acid. A stream of CO2 was passed through the solution in order to minimize oxidation. After 24h, another 100g NaNO2 and 100ml glacial acetic acid were added and the reaction was allowed to proceed for another 24h. Finally, the collagen was rinsed with water, washed by aqueous solution of NaCl (10%, w/v) to remove acid and to minimize swelling, washed to pH 5.0 with distilled water and dehydrated with acetone. Esterification[9]. 10g hide powder was treated with 50ml 10% alcoholic dimethyl sulphate and 150ml 2% sodium borate in a Darmstadt tannin analysis bottle (The presence of the alcoholic ensured that only one liquid phase was present). After shaking at 60 r.p.m for 1h, the solvent was filtered off and replaced by fresh reagent. This procedure was repeated five times to enable the reaction to be completed. The product, which was in a highly swollen condition, was soaked in 10% salt solution overnight, washed with distilled water and dehydrated with acetone. Vegetable tanning of hide powder. All the offers were based on dry weight of hide powder. Hide powder was soaked in distilled water overnight, followed by normal processes of pickling and depickling
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(pH =5.2). Then 1500% distilled water and 50% mimosa were added in the vessel, stirring at 35C for 12h and rinsed with distilled water. Combination tanning of hide powder.
10g (dry weight) hide powder tanned by mimosa was added
into 150ml 20g/L oxazolidine E solution and the pH was adjusted to 6.5. After stirring at 20C for 1h, the temperature was raised to 60C, then stirring for 4h and finally rinsed with distilled water. Aldehyde tanning. 10g (dry weight) depickled hide powder was added into 150ml 20g/L oxazolidine E solution and the pH was adjusted to 6.5. The reactant was stirred at 35C for 12h and then rinsed with distilled water. Hydrogen bond breaking. The hide powder samples were soaked in 1M, 2M, 3M and 8M urea solutions respectively for 24h and then rinsed with distilled water. Hydrophobic bond breaking. The hide powder samples were soaked in 10% and 20% (w/w) n-propanol solutions respectively for 24h and then dried under open air. Differential scanning calorimetry (DSC) studies. The temperature and enthalpy values of the instrument were calibrated by using indium and zinc before measurement. These measured values agree with standard values reported in literature (error﹤1%). The heating rate was maintained at 5C/min. The onset temperature Ts and the peak temperature Tp were recorded automatically. The average value of twice-measured results was used for analysis. All of the experiments were performed on PC200 DSC (differential scanning calorimetry) supplied by NETZSH Company (German). Amino analysis. The hide powder samples were hydrolyzed by using 6M HCl at 110C in vacuum for 24h and then analyzed by the 835-50 amino acid analytical machine of Hitachi Company (Japan).
Results and Discussion 1.The influence of hydrogen bond breaking on hydrothermal stability of collagen The effect of urea solutions on hydrothermal stability of hide powders can be determined by DSC analysis. As an example, the influence of urea solutions on DSC curves of the combination tanned sample is shown in Figure 2. The values of Ts ( the onset temperature) and Tp (the peak temperature) of different hide powders treated by urea solutions are given in Table I. As to raw hide powder, collagen is primarily stabilized by numerous hydrogen bond between collagen fibres. Therefore the urea, acting as hydrogen breaking agent, will make a remarkable impact on the hydrothermal stability of collagen, as seen in Table I. In fact, when the concentration of urea solution was
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increased to 8M, no obvious apex could be detected, which may imply that the triple helical structure of collagen has collapsed.
DSC /mW/mg 1.00
exo
0.95
3M
0.90
2M
0.85
8M
0.80
1M
0.75
0M
0.70 0.65 0.60 0.55 0.50 0.45 60
70
80
90
100
110
120
Temperature /℃
Fig. 2 The DSC curves of combination-tanned hide powder treated by urea solution
Table I The influence of urea solution on hydrothermal stability of collagen （℃） concentration hide powder vegetable tanned aldehyde tanned combination tanned of urea hide powder hide powder hide powder solution Ts
Tp
Ts
Tp
Ts
0
61.7
65.8
84.5
87.6
80.8
83.2
107.8
110.0
1M
58.0
62.8
78.4
84.2
77.2
80.2
107.0
109.5
2M
53.6
60.5
75.8
80.4
76.0
79.2
106.8
109.2
3M
50.1
58.0
72.2
76.4
74.9
78.7
106.2
108.5
8M
—
—
65.0
70.7
73.8
77.5
103.7
106.4
Tp
Ts
Tp
“—“ no obvious apex
As for vegetable tanned sample, the breaking of hydrogen bond also has notable impact on the hydrothermal stability of collagen. It is because that not only the hydrogen bond formed by collagen structure itself, but also the multiple hydrogen bond between vegetable tannin and collagen could be destroyed after treating with urea solutions, resulting in the decrease of the tanning effect. Especially, when 5
8M urea solution was used, the hydrothermal stability of vegetable tanned sample was close to that of raw hide powder, that is to say, the tanning effect nearly disappeared. But in case of aldehyde tanned sample, there is no significant difference between the values of hydrothermal stability of collagens before and after the treatment. It is because the tanning effect of aldehyde is based on covalent cross-linkage between collagen fibres, in other words, the hydrothermal stability of the aldehyde tanned sample depends mainly on the status of the covalent cross-linkage, and so the destruction of hydrogen bond has less effect on the hydrothermal stability of aldehyde tanned sample. Even after treated by 8M urea solution, the sample’s Ts and Tp were still 73.8C and 77.5C, only decreased by 7C and 5.7C. These experimental results agree with the results of previous studies[10-12]. However, based on these data and the observation on the change in the hydrothermal stability of combination tanned sample, it can be found that the previous assumed mechanism of vegetable tannin-aldehyde combination tannage may not be entirely correct. According to the presently assumed mechanism, as shown in Figure 1, the multiple hydrogen bond reaction between collagen and vegetable tannin is still the basis of the tanning effect of combination tannage. Therefore, the hydrogen bond breaking agents should have the similar detanning effect on vegetable tanned sample and on combination tanned sample, especially when the concentration of urea is high. The only difference should be that it is the vegetable tannins washed from the former and the aldehyde cross-linked tannins from the latter. But this is clearly not consistent with the above experimental results. Table I and Figure 2 show that the hydrothermal stability of the combination tanned sample is not only the highest, but also inert to the effect of urea solutions. Even treated by 8M urea solution, the Ts of the sample only decreases by 4C. So it can be concluded that the hydrogen bond contributes little to the high hydrothermal stability of vegetable tannin-aldehyde combination tannage.
2 The influence of hydrophobic bond breaking on hydrothermal stability of collagen The intermolecular hydrophobic bond in collagen makes contribution to the structural stability. At the same time, hydrophobic interaction is also a way in which vegetable tannins react with collagen[5,12]. It is believed that hydrophobic forces are weakened in the presence of nonpolar groups from the solvent environment [13]. So the values of Ts and Tp were shifted after the samples were treated by n-propanol solutions, as shown in Table II. It can be seen from Table II that when treated by 10% n-propanol solution, Ts of raw hide powder decreased by 7C. When the concentration of n-propanol was raised to 20%, Ts of raw hide powder 6
increased somewhat, which may be due to the increase of desolvation. As to the tanned samples, 10% and 20% n-propanol solutions have nearly the same effect on the variation of hydrothermal stability, which shows that the contribution of hydrophobic interaction on hydrothermal stability of the tanned hide powders is limited and most of the hydrophobic forces had be destroyed by using 10% n-propanol solution.
Table II The influence of n-propanol solution on hydrothermal stability of collagen（℃） concentration of n-propano
hide powder
Ts
vegetable tanned hide powder
Tp
Ts
Tp
aldehyde tanned hide powder Ts
Tp
combination tanned hide powder Ts
Tp
0
61.7
65.8
84.5
87.6
80.8
83.2
107.8
110.0
10%
54.7
65.0
79.2
84.0
77.9
81.5
106.2
108.9
20%
58.0
66.0
78.6
83.8
78.3
80.8
106.5
108.9
It can also be found that the decreases of Ts of the three tanned samples were different after treated by n-propanol solution. The sequence of decreasing degree is vegetable tanned sample>aldehyde tanned sample>combination tanned sample, which is the same order as that of samples treated by urea solutions. In fact, the hydrothermal stability of combination tanned sample varied only 1.6C with the treatment of 10% n-propanol solution. These results are consistent with those of hydrogen bond breaking experiments and both of them show that the mechanism of combination tannage is greatly different from that of vegetable tanning. The high hydrothermal stability of the combination tanning is not mainly based on the multiple hydrogen bonds and hydrophobic forces between vegetable tannin and collagen, but probably is due to covalent cross-linkage in the vegetable tannin-aldehyde-collagen system.
3 The influence of chemical modification on hydrothermal stability of collagen The content of typical amino acids in the hide powder before and after chemical modification were given in Table III. The values of Ts and Tp of raw hide powder and the modified hide powders treated by various tannages were given in Table IV.
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TableIII The content of typical amino acids in the hide powders before and after chemical modifications (number of amino acid /1000 amino acids ) amino acid
lysine
hydroxylysine
Arginine
Hydroxy-proline
Proline
hide powder
26
8
46
62
129
deaminated hide powder
1.5
0.8
42
61
130
esterified hide powder
16
8
45
60
128
Table IV Ts and Tp of hide powders tanned by different tannages（℃） raw hide powder deaminated hide powder esterified hide powder tannage Ts Tp Ts Tp Ts Tp control
61.7
65.8
60.8
65.7
—
—
vegetable tanning
84.5
87.6
81.0
85.4
81.6
84.5
aldehyde tanning
80.8
83.2
62.0
66.5
78.0
81.8
tannin-aldehyde tanning
107.8
110.0
84.0
87.2
104.0
106.5
‘—’no obvious apex
It can be seen from Table IV that chemical modification of the side groups of the collagen have little influence on the hydrothermal stability of vegetable tanned samples. According to the mechanism of vegetable tanning, vegetable tannins interact with collagen predominantly via multiple hydrogen bonds. Collagen has plentiful groups which can form hydrogen bond with vegetable tannin, including the peptide bonds of collagen and the side chains containing hydroxyl, amino, carboxyl groups. In general, the quantity of peptide bonds is relatively much more than that of others, so the vegetable tanning effect mainly depends on the interaction of hydrogen bonds between vegetable tannins and the main chains of collagen[14]. Consequently the chemical modifications on some side groups would have no obvious influence on vegetable tanning effect. When the hide powder samples were tanned by aldehyde, Ts of raw hide powder and esterified hide powder were 80.8C and 78C respectively, which are very close. But the Ts of deaminated hide powder was only 62C. This can be explained by the mechanism of aldehyde tanning, as that aldehydic tanning agents can react with the side amino of collagen through Mannich reaction and form covalent cross-linkage between collagen fibres. As shown in Table III, the content of lysine and hydroxylysine residues in deaminated hide powder descended from 26/1000 and 8/1000 to 1.5/1000 and 0.8/1000 respectively. As a result there was almost no aldehyde tanning effect in the deaminated hide powder.
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Based on the results above and further analysis of the situation of combination tanned samples, some interesting and meaningful findings could be obtained. As mentioned in the experimental part, the procedure of the combination tannage was as follows: hide powder was first tanned by vegetable tannin and then retanned by aldehydic tanning agent (oxazolidine E). It can be seen from Table IV that there was little difference in hydrothermal stability among the three vegetable tanned samples. However, after retanning by oxazolidine E, the Ts of raw hide powder and esterified hide powder could be enhanced to 107.8C and 104C respectively whilst the Ts of deaminated hide powder was only 84C, only increased by 3C. These data indicated that even collagen has been sufficiently tanned by vegetable tannins, the side amino groups of collagen are still indispensable to achieve high hydrothermal stability in the aldehydic retanning, implying that the reaction between aldehyde and the amino groups is strongly involved in the function of combination tanning. This conclusion is different from the previous elucidated mechanism which assumed that the synergistic effect of the combination tannage mainly depends on the reaction between aldehyde and vegetable tannins combined with collagen, as illustrated in Figure 1. However, it doesn’t mean that aldehyde would not react with vegetable tannins in any case in the retanning. As given in Table IV, the Ts of raw hide powder increased by 22.8C after vegetable tanning and 19.1C after aldehyde tanning. If there is no reaction taking place between aldehyde and vegetable tannins, the value of ∆Ts of the combination tanned sample should be not more than the sum of the Ts variations of the single tannages, that is, ∆Ts≦41.9C (22.8+19.1C). In fact, ∆Ts is 46.1C, which indicated that the reaction between aldehyde and vegetable tannins also contributes to the synergistic effect of combination tanning. To sum up, the reaction mechanism of vegetable tannin-aldehyde-collagen can be illustrated in Figure 3. The model in Figure 3 shows good consistence with all of the results in this paper and can be well applied to explain the reason of washing resistance and high hydrothermal stability achieved by vegetable tannin-aldehyde combination tannage.
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C2H5 HOH2C H N
N H 2C
H O
C
HO
OH
OH OH
CH2OH O
HO
H OH OH
CH2
HO
OH H
H
HO HO
H
OH
OH
H OH HO
NH O
2HC N
H HO HO
Hide Collagen
OH
H
H O
OH
O
OHOH HO
Hydrogen bond
HOH2C OH
C2 H5
CH2
C CH2 OH
Fig. 3 The crosslinking model of vegetable tannin - aldehyde combination tannage
Figure 3 reveals that the vegetable tannin-aldehyde combination tannage is based on the multiple hydrogen bonds between vegetable tannins and collagen and on the covalent bonds between aldehydic tanning agent and the side amino groups of collagen. Moreover, when there are highly nucleophilic sites in tannin molecules, they can act as another crosslinking ligand of the aldehydic tanning agent. As a result, the synergistic effect of the combination tanning increases the hydrothermal stability of collagen fibers. Nevertheless, not all kinds of vegetable tannins can act as crosslinking ligand in the reaction model shown in Figure 3. As to condensed tannins, in which C-6 and C-8 of A ring show highly nucleophilic reactivity[12], they can easily react in the way as shown in Figure 3. But as to hydrolysable tannins, which do not have highly nucleophilic reactive sites on the molecules, no aldehydic cross-linkage between collagen and tannin could be formed. This has been proved by our previous work [3] and the data were given in Table V. It can be seen from Table V that when condensed tannins were used in the combination tannage, the hydrothermal stability of collagen was much higher than those of the combination tannage using hydrolysable tannins. Furthermore, when compared with the control experiment, it can be found that the variance of Ts (∆Ts) in the condensed tannin-aldehyde combination tannage is larger than the sum of ∆Ts of single tannages, which indicated that the mechanism was consistent with the model shown in Figure 3. When hydrolysable tannins were used in the combination tannage, ∆Ts of the combination tannage is obviously less than the sum of ∆Ts of single tannages, indicating that the hydrolysable tannins didn’t take part in the cross-linkage. In
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other words, in the combination tannage using hydrolysable tannins, the vegetable tannin and aldehyde react with collagen individually, without interaction between themselves and thus no synergistic effect can be observed. Moreover, because of their competition in reaction with collagen, the ∆Ts of the combination tannage is less than the sum of ∆Ts of single tannages.
Table 5 The shrinkage temperature (Ts) of the sheepskin leathers tanned by combination [3] tannage using 20% tannin extract and 4% oxazolidine(℃) tannin extraxt
hydrolysable tannin valonia myrobalan chestnut
Ts
88
88
condensed tannin mimosa quebracho gambier
85
114
101
103
Table 6 The influence of sequence in combination tannage on shrinkage temperature(℃) [3] Mimosa (20%)
Quebracho (20%)
Chestnut (20%)
Myrobalan (20%)
Oxazolidine offer
Ts1
Ts2
Ts1
Ts2
Ts1
Ts2
Ts1
Ts2
4%
114
97
101
92
85
81
88
78
Ts1 --- tanned by tannin extract then retaned by oxazolidine Ts2 --- tanned by oxazolidine then retaned by tannin extract
It has also been found in our previous work [3] that when the sequence of combination tannage was reversed, the shrinking temperatures were lower than those of the original sequence, as given in Table 6. This influence is consistent with the combination tanning mechanism revealed above and can be explained as follows. (1) Tanned by oxazolidine first would tighten the collagen fibres, which may prevent tannin extract from penetrating and therefore the interaction between collagen fibres was weakened. (2) Most of the aldehydic tanning agents have been consumed though covalent bonding with amino groups of collagen during aldehyde tanning process and therefore, when retanned with vegetable tannins, the probability of forming collagen-aldyhyde-tannin cross-linkage is remarkably reduced, which results in the decrease of the synergistic effect.
Conclusions 1. In the vegetable tannin-aldehyde-collagen system, vegetable tannins mainly interact with collagen via multiple hydrogen bonds and meanwhile aldehydic tanning agents interact with collagen via covalent bonds. However if there are highly nucleophilic sites on tannin molecules are present, the covalent crosslinkage can be formed between collagen and vegetable tannins via bridge bond of aldehyde. As a result, the
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synergistic effect of the combination tanning in increasing hydrothermal stability can be achieved. 2. Whether the adehydic tanning agents are able to form covalent cross-linkage between collagen and vegetable tannins depends on the molecular structures of vegetable tannins. There are some highly nucleophilic sites in condensed tannin molecules and thus it is easy for them to react in this way. But for the combination tannage using hydrolysable tannins, no aldehydic cross-linkage between collagen and vegetable tannin could be observed. So it is better to use condensed tannins in the vegetable tannin-aldehyde combination tannage. This rule is probably meaningful for us to prepare other new materials by means of the reaction of vegetable tannin-aldehyde-other material.
Acknowledgment This project was financially supported by High Tech Research and Development (863) Programme (2001AA647020) and The key Research Program of Science and Technology in Sichuan Province (01ZQ052-52).
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10. Russell, A. E., Shttleworth, S. G., Williams-wynn, D. A., Future studies in vegetable tannage[J], J. Soc. Leather Technol. Chem., 1967, 51(10): 349-361. 11. Usha, R., Ramasami, T., Effect of crosslinking agent (basic chromium sulfate and formaldehyde) on the hydrothermal and thermomechanical stability of rat tail tendon collagen fibre[J], Thermochimica Acta, 2000, 356: 59-66. 12. Shi Bi, Di Ying, Plant polyphenol [M]. Science Press, Beijing, 2000. 13. Usha, R., Ramasami, T., Influence of hydrogen bond, hydrophobic and electrovalent salt linkages on the transition temperature, enthalpy and activation energy in rat tail tendon(RTT) collagen fibre[J], Thermochimica Acta, 1999, 338: 17-25. 14. Santhanam, P. S., Navudamma, Y., Studies on the phenolic constituents of babul: Ⅱ Fixation on chemically modified protein substrates[J], Leather Science, 1968, 15:1-5.
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