Accurate and Reproducible Determination of Molecular Weight Distribution of Sodium Heparin U.S.P. By HPLC

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Abstract
Accurate molecular weight averages and molecular weight distributions have been determined for bulk Sodium Heparin, USP. The molecular weight averages were approximately 10,000 daltons (one dalton = one molecular weight unit) and ranged from 3,000 to 17,000 daltons in a given sample. The method uses high pressure liquid chromatography (HPLC) with 5–10 micron particle size controlled porous glass of different pore diameters (40 Å, 100 Å, and 250 Å) and simultaneous detection with a differential refractometer and ultraviolet detector (254 nm). The columns were calibrated with different molecular weight fractions of Sodium Heparin and baseline resolution obtained between 6.5K daltons differences in molecular weight. Analysis time was 25 minutes per sample and the method gave excellent reproducibility for calculated molecular weight averages in a repeated series of analysis. It was determined that the column doing most of the separation in the set was the one packed with 250 Å pore diameter size material.

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