Fractionation of low-molecular-mass heparin by centrifugal partition chromatography in the ion-exchange displacement mode

Centrifugal partition chromatography in the ion-exchange displacement mode allowed a preparative and efficient fractionation of low-molecular-mass heparins from enoxaparin sodium. Amberlite LA2 — a lipophilic liquid secondary amine — was chosen as a weak anion exchanger. The biphasic system methyl isobutyl ketone–water was selected. Protonated LA2 (10%, v/v) was added to the organic stationary phase. Hydroxide (Na+, OH−) was chosen as a displacer in the aqueous mobile phase. The observed pH and concentration profiles are typical of displacement chromatography, as supported by numerical simulation. The Dubois test for the analysis of sugar content and an analysis of sulfur content (and consequently sulfatation rate) were carried out to monitor the effectiveness of the procedure. Moreover, the fractions were analyzed by high-performance size-exclusion chromatography and the 1H NMR spectra confirmed the fractionation of the sample of enoxaparin sodium.