A Comparison of Molecular Weight Determination of LMWH SEC/MALS vs. SEC/UV-RI

Low-molecular-weight heparins (LMWHs) are achieved by depolymerization or fractionation of natural heparins. They are said to have a mass-average molecular weight of below 8000 and for which at least 60% of the total weight has a molecular mass below 8000.

Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) has been the most typical method of measuring the molecular weight and molecular weight distributions of LMWHs by using the two most basic detection technologies: ultraviolet (UV) coupled with refractive index (RI) detection. However, these detectors symbolize a relative technique so as to establish molecular weights, requiring calibration standards. A newer, absolute technique involves the use of multi-angle light scattering (MALS), which does not need any standards. The European Pharmacopeia (EP) monograph for LMWH stipulates the use of the UV/RI detection technique and offers a proven calibration standard. A number of laboratories worldwide have chosen this technique.

Figure 1. Examples of UV and RI traces for an LMWH sample.

Wyatt has previously developed a SEC/MALS technique and felt it was highly suitable for the analysis of LMWHs. Wyatt has recently implemented the UV-RI method illustrated in the EP monograph and compared the molecular weight results produced for LMWH using each detection type. The adopted technique uses an Agilent LC-1200 series HPLC, Tosoh TSK-gel G2000 SWxl column with Tosoh TSK-gel Guard SWxl, Waters 2487 dual wavelength UV detector, 0.2M Sodium Sulfate pH 5.0 mobile phase, and Wyatt Optilab^® rEX™ refractive index detector. For MALS analysis, the UV detector was substituted with a Wyatt miniDAWN^® TREOS^® detector; all other aspects of the method remained the same.

Conclusion

The results showed that both detection types are appropriate and acceptable for the examination of LMWHs. The molecular weight and distribution results produced using each detection type are comparable. This shows that a SEC/MALS technique could be selected instead of the SEC/UV-RI technique presently required by the EP monograph, and that it would lead to a reduction in time because it removes the need for calibration standards.

Figure 2. Examples of LS and RI traces for an LMWH sample.

This information has been sourced, reviewed and adapted from materials provided by Wyatt Technology.

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