Determination of Modification Degree of Succinylated Gelatin by Size Exclusion Chromatography Coupled with Multi Angle Laser Light Scattering

The clinical effectiveness of succinylated gelatin as a plasma substitute depends on its molar mass and modification degree of its free amino group. In this study, a novel method was developed to determine the modification degree of succinylated gelatin. Succinylated gelatin was derivatized with trinitrobenzenesulfonic acid (TNBS). The molar masses of gelatin and succinylated gelatin were determined before and after derivatization with TNBS using size-exclusion chromatography coupled with multi angle laser light scattering (SEC-MALLS). The modification degree was obtained by estimating the free amino groups of succinylated gelatin before and after succinylation. Human serum albumin was used as a model protein to validate the method. The determined value was consistent with the predicted value. Actual samples were analyzed with the developed method; the mean modification degree of succinylated gelatins was 38%. This research indicates that size-exclusion chromatography multi angle laser light scattering coupled with the trinitrobenzenesulfonic acid derivatization is a feasible method for the determination of the modification degree of succinylated gelatin.