CHROMATOGRAPHIC CHARACTERIZATION OF ION-EXCHANGERS FOR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF PROTEINS .1. CHROMATOGRAPHIC DETERMINATION OF LOADING CAPACITY FOR LOW-MOLECULAR AND HIGH-MOLECULAR MASS ANIONS

The capacity of silica-based anion exchangers was determined by three independent methods (potentiometric titration, calculation from elemental analysis data and frontal analysis with sodium nitrate). All three methods gave almost identical results. Breakthrough experiments with sodium nitrate were carried out at different concentrations and flow-rates. The capacity of a large-pore ion exchanger for macromolecular anions such as chondroitin sulphate and bovine serum albumin (BSA) was also determined by frontal analysis. The capacity is much lower than that for small anions and is influenced by the irreversible adsorption of sample ions onto the stationary phase. The capacity for the linear polyanion of chondroitin sulphate was independent of its concentration in the carrier solution (within the range 1-10 g/l). For both biological samples the binding capacity depends on the eluent velocity; the capacity for chondroitin sulphate clearly increases with decreasing flow-rate, whereas globular BSA shows little dependence of capacity on flow-rate. The influence of band spreading on the determination of binding capacity by frontal analysis is discussed.