Preparative countercurrent chromatography for isolation of charge density-fractionated heparin

A preparative method is described for continuous flow fractionation of heparin according to charge density in two-phase systems of butanol-hexadecylpyridinium chloride/aquaous NaCl. The separation is carried out on a "Slowly-Rotating Coil" device for countercurrent chromatography with 6 coil units and a sample capacity of 80 mg heparin. A turbidometric method is described which can be used to rapidly quantify the distribution of heparin. Characterization of fractions showed that linear charge density, binding constant for acridine orange, and specific activities in a "global" plasma anticoagulant assay and in a specific activities in a specific biochemical thrombin-inhibition assay varied with the NaCl concentration in the eluting phase. Some partial resolution of the two functional activities was observed. The fractions are much more homogeneous in composition than are the usual heparin preparations and may be useful as standards or in structure-function studies.