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Molecular weight determination of unfractionated heparin
Molecular weight determination of unfractionated heparin
Heparin is a polydisperse, heterogeneous polysaccharide derived from animal tissue. Heparin has been used as an anticoagulant for over sixty years and one of the fundamental parameters for characterising unfractionated heparin (UFH) is its molecular weight (MW) and MW distribution. The molecular weight of heparin ranges from 3 to 50 kDa, but, typically falls within the 10 to 20 kDa range for unfractionated heparins used in medical applications. In light of heparin contamination issues in 2008, the United States Pharmacopeia (USP) has proposed to include heparin molecular weight determination for the stage 3 heparin monograph revisions. Many high-performance size-exclusion chromatography (SEC) systems have been developed and represent the most commonly used method in the MW determination of UFH. Scientific Protein Laboratories (Waunakee, WI, USA) have developed an SEC system coupled with multiangle light scattering (MALS) for the determination of absolute MW and MW distribution of UFH and heparin derivatives. MALS eliminates the need for well-characterised molecular weight standards derived from heparin, and the SPL procedure represents an improvement over the column calibration techniques used in other, relative MW determination methods. This method uses an Agilent LC-1200 series HPLC, a 0.1M ammonium acetate mobile phase, a Shodex SB-804HQ analytical column (8.0 x 300 mm) with a Shodex SB-G guard column, and a Wyatt miniDAWN TREOS light scattering detector plus Wyatt Optilab rEX refractive index detector. The specific refractive index increment (dn/dc) and second virial coefficient (A2) measurements can also be determined using the Optilab rEX and miniDAWN TREOS detectors, respectively.
Wyatt Technology Corp.

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