Use of reversed polarity and a pressure gradient in the analysis of disaccharide composition of heparin by capillary electrophoresis

A capillary electrophoresis method with reversed polarity, combining both t he application of a voltage and a pressure gradient between the buffer vial s, was developed for the analysis of eight heparin-derived Delta-disacchari des obtained by enzymatic depolymerization. A 60 mM formic acid buffer at p H 3.40 was selected as running electrolyte, with an applied voltage of -15 kV and an overimposed pressure gradient (3.45.10(-3) MPa) for 6 min from in let to outlet starting at 20 min. Figures of merit such as run-to-run and d ay-to-day precision, and limits of detection were established. The electrop horetic method was applied to the analysis of depolymerization products of different kinds of heparins. The composition of the depolymerization buffer was selected in order to reduce baseline distortions in the electrophoreti c separation, thus a buffer solution containing 30 mM Tris, 50 mM sodium ch loride, and 3 mM calcium chloride at pH 7.10 was used. Percentages of molar disaccharide compositions for unfractionated heparins from porcine, bovine and ovine intestinal mucosa, and bovine lung were determined. In addition, low-molecular-mass heparins from bovine and porcine intestinal mucosa were analysed as well. (C) 1998 Elsevier Science B.V. All rights reserved.