Automated SEC of Low Molecular Mass Heparins According to Pharmaeuropa

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2008, Volume 0, Issue 0

Low molecular mass heparins are salts of sulphated glucosaminoglycans having a mass average molecular mass less than 8000. At least 60% of the total mass has a molar mass less than 8000.

PSS Polymer Standards Service GmbH

Low molecular mass heparins are salts of sulphated glucosaminoglycans having a mass average molecular mass less than 8000. At least 60% of the total mass has a molar mass less than 8000.

Figure 1

Low molecular mass heparins are obtained by fractionation or depolymerization of natural origin heparin. Batch-to-batch consistency is ensured by demonstrating that the mass average molecular mass and the mass percentage within defined molar mass ranges lower than 8000 are not less than 75% and not higher than 125% of the mean value stated as type specification.

Figure 2

Size exclusion chromatography (SEC, also knows as gel permeation chromatography GPC) is a relative method that enables the measurement of the mass average molecular weight and the percentages within defined molar mass ranges with just one measurement. For calibrating the system a low-molar-mass Heparin for calibration (CRS) is available. This substance can be used to establish a heparin calibration curve that enables the measurement of the absolute molecular masses precisely and with high accuracy.

Experimental:

SEC/GPC analysis was performed on a PSS GPC 1100 System consisting of

  • an isocratic pump

  • an autosampler with variable injection volume

  • an ultraviolet spectrophotomer (UV) working at 234 nm

  • a differential refractometer (RI).

This WinGPC Unity heparin module permits the creation of calibration curves. Because the UV signal is proportional to the number of eluting molecules while the RI is proportional to the mass eluting, the following equations hold true:

This relationship can be used for the determination of the response factor f. If the number average molecular weight of the calibration substance is given (e.g. as 3700), the factor f can be calculated for the whole sample. Consequently, the molar masses in each slice can be calculated from the UV and RI chromatograms according to:

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