Molecular mass determination of low-molecular-mass heparins. Application of wide collection angle measurements of light scattering using a high-performance gel permeation chromatographic system equipped with a low-angle laser light-scattering photometer.

A high-performance gel permeation chromatographic system with on-line low-angle laser light-scattering detection (HPGPC-LALLS) was used to determine molecular masses of low-molecular-mass heparins (LMMHs). Measurements at wide and narrow collection angles were compared and the application of the HPGPC-LALLS method to small molecules, with molecular masses in the range 1000-10,000, was assessed. The molecular mass averages of fractionated heparins and commercially available LMMHs were also determined by ordinary HPGPC analysis using an LMMH molecular-mass calibrant, supplied by the National Institute for Biological Standards and Control for the calibration of columns. The LALLS intensity at the routinely used narrow collection angle (theta col = 1 degree) about doubled at the wide collection angle (theta col = 2 degrees) and the signal-to-noise ratio was improved. The present study thus indicates that wide collection angle measurement of light scattering allows the application of the HPGPC-LALLS method to very small biopolymers of molecular mass < 10,000.