Crosslinking - what crosslinking?15 October 2003
Abstract Hydrothermal isometric tension (HIT) is a technique in which the forces generated during and after the shrinking transition for wet collagen are measured. The rate of increasing tension is related to the crosslink density and the rate of relaxation following shrinking is related to the stability of the crosslink. From the measured rates of increasing force during heating in paraffin, there is a correlation with perceived crosslinking by polyphenol, before and after crosslinking by metal ions or aldehydic agents. What is clear is that the results for raw collagen and aluminium (III), oxazolidine and chromium (III) tanned collagen are the same. This is direct supporting evidence for the proposal that chromium (III) does not crosslink collagen. The mechanism depends on the creation of a supramolecular matrix around the triple helix. Rates of relaxation of tension correlate with the stability of the interaction between the applied tanning agent and the collagen. Weak hydrogen bonding and electrostatic interaction exhibits fast relaxation, but the covalent reaction from oxazolidine exhibits no relaxation. The development of a comprehensive and inclusive mechanism of the tanning effect has resulted in a theory based on the creation of a structured matrix around the triple helix. Conventionally imagined crosslinking is not part of the mechanism. Comparison between inorganic tanning and organic tanning (crosslinked polymer or polyphenol) demonstrates that the two mechanisms are essentially identical. Introduction The origin or basis of the tanning effect has received surprisingly little attention in the technical literature. It is surprising because the cause of different tanning mechanism effects is at the heart of the global leather industry. For example, why do vegetable tanning reactions only raise the shrinkage temperature of collagen to 75-85°C, depending on the tannin, but chromium (III) sulfate produces 110-130°C, depending on conditions? About 100 years ago, it was assumed that vegetable tannins simply coated the fibre structure and, thereby, changed its properties. Later it was proposed that the primary interaction is via multiple hydrogen bonding, with some influence of hydrophobic bonding. It has always been assumed that chromium (III) reacts covalently, to form complexes, based on the studies of Bjerrum. Later it was demonstrated that reaction occurs primarily at the carboxyl groups and this reaction is the source of increased hydrothermal stability1. It was taken for granted that the mechanism involved crosslinking the collagen structure, although there was no indication which elements of the hierarchical structure are linked. About 50 years ago, Gustavson2 published data which he interpreted as showing that the linking species in chrome tanning is a 33% basic dichromium complex, in which the two chromium ions are linked via two hydroxy bridges and a sulfate bridge. Furthermore, only about 10% of the bound chromium was in the form of multipoint or crosslinking complexes. This remained the accepted wisdom for the remainder of the 20th century. Recently, some changes to that view were proposed, based on research conducted at University College Northampton. By formulating some empirical rules regarding the nature of the new structure imposed on collagen by tanning chemistries3, it is possible to explain their relative effects on the hydrothermal stability and even predict the effects of new tannages. That approach is consistent with the theory of the 'polymer in a box'4, used to understand the stability of collagen itself. Moreover, considering the structural studies of Berman5, in which it was shown that the supramolecular water is nucleated at the hydroxyproline moieties and creates a structural matrix, the imposed structure from tanning must create its own matrix6. That model does not necessarily invoke the inclusion of crosslinking, in the conventionally imagined way. The latest thinking To make progress in understanding the nature of the tanning mechanism, it is necessary to have a probe into the structure of leather at the molecular level. However, it is only recently that appropriate techniques have been applied. Using extended X-ray analysis fine structure (EXAFS), radical new insights into the chrome tanning mechanism have been gained7. It has been shown that the average bound chromium species is a linear chain of four chromium ions, linked by oxy bridges, but there is no evidence to indicate that sulfate ion is bound to chromium. This is in direct contradiction to the Gustavson model8, which therefore calls into question his conclusions about the degree of crosslinking in chrome tanned leather. Unfortunately, EXAFS cannot provide information in this regard. Hydrothermal isometric tension is not a new technique: it relies on measuring the forces generated up to, during and after the shrinking transition. A sample strip is held between fixed jaws, attached to a transducer, to quantify the changing forces within the material. The experiments were conducted on specimens that were saturated with water and then immersed in liquid paraffin, as the heating medium. Applying the technique to raw collagen and a variety of leathers has yielded some intriguing results, shown in Figures 1-39. The temperature of the onset of shrinking relates to the conventionally measured shrinkage temperature and, in these cases, the values obtained were as expected. However, there were marked differences in the way the forces increased during the thermal transition and the way the forces decreased after the transition: the former has been attributed to the crosslink density and the latter has been attributed to the crosslink stability. The most noteworthy observation is that the crosslink density for chrome tanned collagen is practically the same as the crosslink density of raw collagen, not significantly different from the effects of aluminium (III) or oxazolidine, but clearly different to those agents that can crosslink due to their multiplicity of sites capable of interacting simultaneously with collagen. The natural conclusion is that chromium (III) does not crosslink collagen. The latest studies on organic tanning, based on in-situ polymerisation by oxazolidine of flavonoid polyphenols9, have demonstrated that the reaction depends not only on crosslinking the polyphenol molecules, but also on binding the polyphenolic matrix to the collagen structure covalently. Furthermore, from other recent studies using model polyphenolic molecules, it appears as though the role of hydrophobic bonding between polyphenols and collagen may be more important than we have recognised hitherto10. These observations lead to the conclusion that the matrix theory can be simplified. The matrix is composed of the primary tanning agent, covalently bound to collagen, interacting with the supramolecular water and any other secondary tanning agent or auxiliary. Whilst the matrix may be regarded as crosslinking the fibre structure, it is not necessary to consider crosslinking by individual components of the matrix as a fundamental requirement of the stabilising mechanism. That is not to say that crosslinking does not occur: indeed it is probable that it does occur to some degree, depending on the tanning reagent and the availability of reaction sites. However, this work indicates that conventional crosslinking is not a prerequisite of hydrothermal stability, particularly high hydrothermal stability. Consequences In inorganic tanning, particularly chromium (III) tanning, it is clear that the three component system must be considered when we seek to improve the process. The role and effect of the counterion have not been taken into account in the past, simply because sulfate ion is so effective in the mechanism. However, it is now possible to contemplate alternative approaches that do not rely on sulfate, which would be beneficial in terms of the environmental impact of leathermaking. In addition, choice of counterion or auxiliary tanning agent can determine the hydrothermal stability, producing shrinkage temperatures up to 155°C, as found in mineralised collagen in bone11: this might be useful in the field of high performance materials. In organic tanning, it is clear that there are two requirements: the polymer must fit into a matrix, which may react with collagen via multiple bonding interaction, but which must include some covalent bonds. If those requirements are satisfied, high hydrothermal stability will be achieved, regardless of the specific chemistry involved. It is difficult to see how, in the future, those criteria can be used for prediction of tanning effect without additional information: it is likely that the only truly successful source of steric and thermodynamic data will be through creating a computer model of a substantial portion of the collagen molecule. Conclusions In the search for a viable theory of tanning, we have moved from the point of view that hydrothermal stability is a specific property of individual tanning chemistries, to the notion that it derives from a matrix interaction at the triple helix. This has been presented as two similar matrix forming types of chemistry, inorganic and organic. However, these most recent studies have allowed the thinking to go beyond that perception. The requirements for both types of reaction are identical, regardless of chemical type. Therefore, there is a single tanning mechanism, applying to all chemistries: the only difference between reactions that give different shrinkage temperatures is the degree to which they conform to the requirements for high hydro-thermal stability. The implications are clear. This new understanding of the mechanism of mineral tanning allows the development of novel approaches to chrome tanning, for example the optimum manipulation of or even the elimination of sulfate to reduce the environmental impact. The search for new, high stability tanning methods can be directed more precisely, targeting the parameters that are known to lead to high stability, ie the interaction with collagen within the supramolecular matrix around the triple helix.