Gelatin is the heterogeneous product of the denaturation of the mechanical protein collagen, found in the connective tissue and skin of animals. As gelatin is a macromolecular hydrocolloid protein, its properties are affected by variations in pH, temperature and salt concentration. Given the role of the molecular weight profile of gelatin in determining the physicochemical behaviour of gelatin solutions, the objective of this study was to attempt to establish whether the effects of the above-mentioned factors on the desolvation beahviour of gelatin are due to an alteration in the molecular weight profile of the molecule or not, as monitored using high-performance size exclusion chromatography (HPSEC). This was done in an effort to compliment previous studies on the ethanolic coacervation of gelatin solutions, where the phase behavior of the protein was monitored by turbidimetry.
Bovine and cold water fish skin gelatin solutions (0.2% w/w) were prepared by heating aqueous suspensions of undissolved gelatin to 40 °C with stirring for 20 minutes, and the pH adjusted values between pH 4 and pH 8 using dilute hydrochloric acid or dilute sodium hydroxide. The stock gelatin solution was incubated at 37 °C for 1.5 hours before being combined with 8g of pure water or saline solution containing 0.01g or 0.1g of sodium chloride; thus the final solutions contained 0.2% w/w gelatin in 0%, 0.01% and 0.1% salt solutions. The resultant mixtures were incubated for a further 20 minutes before being analysed by size exclusion chromatography on a Shimadzu HPLC fitted with a Waters Corporation Ultrahydrogel column.
From the results of this study, it was seen that the presence of salt has a significant effect on both bovine and cold water fish skin gelatin, with retention times that were shifted from 30 minutes in the absence of salt to 35 minutes in the presence of salt, indicating that the addition of salt decreases the molecular weight of the gelatins. Thus in solution, the added salt appears to cause dissociation of the gelatin polymers to lower molecular weight species which are more soluble, providing an explanation for earlier observations that gelatin solutions, in the presence of added salt, require greater concentrations of desolvating agent for phase separation, when compared to solutions with no added salt.
The pH of the solution was also found to have an effect that was observed to be more significant with regards the B-type bovine gelatin than cold water fish skin gelatin, which has an isoelectric point closer to that of A-type gelatin. Additionally it has been found that the pH of the solution only has an effect on gelatin molecular weight profiles when the range of pH’s being used is greater than the range of the iso-electric point. The IEP of B-type gelatin is narrower than that of A-type gelatins; it therefore follows that bovine gelatin is more susceptible to changes in molecular weight profile as a result of pH alteration than the cold water fish skin gelatin.