Application of dendrimer-functionalized ion-exchange stationary phases for the retention and separation of low-molecular-weight heparins

Abstract

The disaccharides of low-molecular-weight heparins (LMWH) obtained by enzymatic depolymerization most frequently require a determination method that uses separation techniques. Therefore, a new method that applies sorbents as stationary phases in liquid chromatography was developed. The dendrimer phases were synthesized based on silica gel using methylamine and 4-butanediol diglycidyl ether as monomers, which facilitated specific and selective interactions between the solid phase, analyzed compounds and mobile phase owing to the quaternary ammonium groups present in their structure. We obtained sorbents with different numbers of dendrimer layers (from 1 to 4). For all the obtained sorbents, the LMWH retention depended on the number of functional groups on the surface of the stationary phase. An increased number of dendrimer layers on the column surface resulted in increased retention of LMWH disaccharides. The influence of mobile phase pH (2.5, 3.0 and 3.5) and temperature (25, 35 and 45 °C) on the efficiency of LMWH separation was also tested. The optimal analysis conditions obtained for eight disaccharides using the sorbent with three dendrimer layers (3L) and a mobile phase pH of 3.5 at 25 °C were successfully used to determine these substances in commercially available enoxaparin samples.