Performance Liquid Size Exclusion Chromatography Research Articles

An assay for anti-factor Xa activity of low molecular weight heparins by high performance liquid size exclusion chromatography

The "gold standard" assay for monitoring low molecular weight heparins (LMWHs) activity is the chromogenic-based anti-factor Xa assay. The methodology of an anti-factor Xa assay is that LMWH is added to a known amount of excess factor Xa and excess antithrombin. It will bind to antithrombin and form a triplet complex with factor Xa, inhibiting the activity of factor Xa. However, the residual factor Xa can still hydrolyze chromogenic peptide substrate, releasing the chromophore for photometric detection. The absorbance is inversely proportional to the amount of heparin/LMWH. The results are given in anticoagulant concentration in units/ mL of anti-factor Xa, such that high values indicate high levels of anticoagulation and low values indicate low levels of anticoagulation. Herein, a novel assay method for anti-FXa activity of LMWHs using high performance liquid size exclusion chromatography (SEC) is reported, in which antithrombin III (AT Ill ) was diluted by the buffer solution contained LMWHs. Subsequently, exogenous FXa and p-nitroaniline coupled peptide substrate were added and incubated for a period, separately. The resulting mixture was separated based on size by SEC, and the free chromophore p-nitroaniline can be detected at an absorption maximum of 385 nm without interference from the absorbance of p-nitroanilide substrates. Moreover, the measurements are not influenced by sample opacity or turbidity, so it is possible to test various complex samples, such as plasma. The assay is robust, sensitive, and cost effective.

The application of HPLC–SEC for the simultaneous characterization of NOM and nitrate in well waters

The usefulness of HPLC–SEC for the characterization of well water quality and the identification of surface-water or wastewater percolation into wells was studied. In total, 267 private wells from rural areas of Finland, two surface waters and two wastewater effluents were analyzed for organic matter (Dissolved Organic Carbon (DOC)) and nitrate with conventional methods. High Performance Liquid Size Exclusion Chromatography (HPLC–SEC) was also used for NOM (Natural Organic Matter) and nitrate analysis. High DOC values were found occasionally in both shallow and deep wells. HPLC–SEC with UV-254 detection separated 6 fractions in the wells studied: three High Molecular Weight (HMW) fractions, two Intermediate Molecular Weight (IMW) fractions and a Low Molecular Weight (LMW) fraction. The LMW fraction is an overlap of LMW organic compounds and nitrate. In wells with a high DOC content the chromatograms were typically “surface water”-like, with HMW fractions clearly present and often dominant. Anthropogenic influence on wells was seen in the increase of LMW fraction VI, which shows an increased nitrate concentration. Nitrate concentration can be determined with precision by HPLC–SEC using UV detection at 224 nm.