Coupling high-performance size exclusion and ion chromatography for the analysis of low-molecular-mass heparin

Abstract

A column-switching method, coupling high-performance size exclusion chromatography (HPSEC) and ion chromatography (IC), was developed for the analysis of glycosaminoglycans. Since the requirements of the recommended mobile phases for each system are considerably different, mobile phase composition and transfer conditions were studied in order to enable characterization of both the molecular mass averages and the free anion contents in the sample by coupling HPSEC and IC systems. Electrolyte solutions of ionic strength higher than 0.1–0.2 M are usually used with derivatized silica-based TSK-GSW (Supelco) columns in order to avoid extra-SEC effects. In this work, an HPSEC eluent of low ion strength provided mass averages comparable to those obtained in more concentrated eluents, and enabled the transfer of elution fractions to the IC system. Switching-intervals were studied to select the optimum transfer fractions for the determination of sulphate and phosphate. Low detection limits, 50 ng and 100 ng, good repeatability values (relative standard deviation, %R.S.D.), 1.56% and 3.37%, and long-term precision values (%R.S.D.), 2.42% and 3.50% of the coupled method, were obtained for sulphate and phosphate respectively. The method was applied to the analysis of a low-molecular-mass heparin.