Anion-exchange HPLC of fibrinogen and fibrinogen degradation products using pH gradients

Factors were studied which affect the resolution of fibrinogen and fibrinog en degradation products (FDPs) in weak anion-exchange HPLC employing pH gra dients. One factor studied was the effect of the buffer capacity of the mob ile phase on resolution. A linear pH gradient generated by a gradient chrom atofocusing technique, which has an approximately equal buffer capacity thr oughout the gradient pH range, was found to only marginally separate fragme nt D and fibrinogen. In contrast, a gradient of an elution buffer of dietha nolamine/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 wit hin 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 chr omatographic profiles for fibrinogen from different commercial sources, ind icating different proportions of native and denatured/degraded fibrinogen i n the samples.