ANION-EXCHANGE HPLC OF FIBRINOGEN AND FIBRINOGEN DEGRADATION PRODUCTS USING pH GRADIENTS

Abstract

Factors were studied which affect the resolution of fibrinogen and fibrinogen degradation products (FDPs) in weak anion-exchange HPLC employing pH gradients. One factor studied was the effect of the buffer capacity of the mobile phase on resolution. A linear pH gradient generated by a gradient chromatofocusing technique, which has an approximately equal buffer capacity throughout the gradient pH range, was found to only marginally separate fragment D and fibrinogen. In contrast, a gradient of an elution buffer of diethanolamine/phosphate, which is strongly buffered in the pI range of fragment D but less so in the pI range of fibrinogen, yielded much better resolution. Another factor studied was the effect of pre-generating a pH gradient within the column prior to chromatography. A pre-generated column pH gradient was found to significantly improve resolution, as a single peak for native fibrinogen was resolved into two peaks. This work also showed different chromatographic profiles for fibrinogen from different commercial sources, indicating different proportions of native and denatured/degraded fibrinogen in the samples.

ACKNOWLEDGMENTS

This work was supported by the Established Full-Time Faculty Research Development Program and a Research and Creative Activities Grant from the Graduate College at Cleveland State University.