DETERMINATION OF POLYANIONIC MACROMOLECULES BY SIZE-EXCLUSION CHROMATOGRAPHY

This report presents an extension of a method developed for determinat ion of dextran sulfate in rat serum. The drug is a negatively charged polysaccharide with a molecular mass of 8000. It is fractionated by mo lecular size and separated from serum components by high-performance s ize-exclusion chromatography. Sensitive detection is achieved by the p ost-column complexation of the analyte with 1,9-dimethylmethylene blue (DMMB). A metachromatic complex is formed; the absorbance maximum of the complex is shifted from that of the free dye. Various glycosaminog lycans and other macromolecular polyanions interact with DMMB. Several can be determined using the chromatographic conditions developed for dextran sulfate. The method provides a simple procedure for quantitati on of these compounds. Compared to spectrophotometric assays, less sam ple preparation is required, selectivity is enhanced, and molecular ma ss information is provided. With modification of eluent composition, d ye concentration, and detection wavelength, the method can be validate d for determination of additional compounds.