Heparin Chromatography

Commercial heparin is not a homogeneous substance. The isolation of the active component still appears to be a problem and usually requires several different procedures. In order to see if it would be possible to simplify the purification, we studied and compared the chromatography of porcine heparin by a systematic approach using various types of adsorbants, gels and programmed elution techniques employing a Beckman Spectrochrom 130 chromatographic analyzer. A total of 20 combinations were attempted with 200 mg of heparin (169 u/mg) being chromatographed in each case. The fractions of each peak were tested by several methods and included the activated partial thromboplastin time, recalcification time, thrombin time, anti-factor Xa, chromogenic, USP assay, agarose gel electrophoresis and the measurement of metachromatic activity. It was found that dextran gel with an average dry diameter of 75-100 um produced the best chromatograms whose peaks were well resolved. The medium molecular weight fraction of the second peak contained half of the applied heparin. This peak contained all the anticoagulant activity besides reacting very strongly for metachromatic activity. The smaller and larger molecular fractions found in the other three peaks gave very weak metachromatic activity and were devoid of anticoagulant activity. These results have important implications in the preparation and clinical use of heparin.