Analysis of chitosan molecular weight profile by electrophoresis in a porosity step gradient polyacrylamide gel

Abstract

Chitosan is biocompatible and biodegradable natural biopolymer widely applied in various fields of biology, medicine, and pharmacy, however, its effects significantly depend on the degree of polymerization (DP) and the degree of deacetylation (DDA) of polymer chains. Evaluation of the chitosan chain diversity by DP requires the use of a highly expensive method of high-performance size exclusion chromatography. The aim of our study was to determine the molecular weight profile of chitosan specimens by the use of electrophoresis in a porosity step gradient polyacrylamide gel and to evaluate the efficacy of this method in monitoring the purification of chitosan fragments and its derivatives. Two types of step gradient porosity gels were used: 1) gels of layers with acrylamide concentration 2.5, 3.5, 5.0, 10.0, 15.0, 20.0 % w/v for native chitosan or its high molecular fragments; 2) gels of layers with acrylamide concentration 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 % w/v for low molecular chitosan fragments. The main amount of molecules from the chitosan pool was localized in the type 1 gel in the region of 550-40 kDa and distributed among three bands, which in different samples differed significantly in percentage. Electrophoresis of chitosan fragments fractionated by gel permeation chromatography provided a clear separation of medium molecular weight fragments (50–400 kDa) in type 1 gel and of low molecular weight fragments (3–40 kDa) in type 2 gel. Thus the method of chitosan electrophoresis in a step-gradient porosity of polyacrylamide gel was developed which permits to characterize the molecular weight profile of chitosan specimens polymer chains and is effective in monitoring the isolation of chitosan fragments by gel penetration chromatography of molecular weights from 3 to 400 kDa.