of organic co-solvent generally decreases the probability The kinetic behavior of human serum albumin ( HSA ) adfor adsorption during the elution time of a protein peak sorbed on a reversed-phase support was studied. With a phos- (8). Even a partial desorption of the proteins already adphate buffer eluent ( pH 7.4 ) , the sharp elution front charactersorbed may be observed (9). When a protein is adsorbed, izes a fast kinetic adsorption process with a high apparent admost of the external polar side chains of the macromolecule sorption rate. In presence of 20% acetonitrile added to the eluent are supposed to remain immersed in the eluent and only the apparent adsorption rate is about 60 times as low as that some hydrophobic side chains can diffuse among the alkyl found for the first adsorption step in pure buffer. The largest chains of the support ( 10 ) .
column capacity is found with 20% acetonitrile in buffer ; for Numerous investigations of protein structural conformalarger organic solvent contents, a decrease of both the apparent adsorption rate and the column capacity are observed with in-tions in reversed phase liquid chromatography have already creasing amounts of acetonitrile in the buffer. In order to better been reported. Various spectroscopic techniques, such as understand the chromatographic behavior of HSA on this type circular dichroism (11) and fluorescence (12, 13), were of support, we studied the stuctural infrared characteristics of used. To further define the nature of the adsorption mechathe protein in solution. Fourier transform infrared spectra show nism of human serum albumin (HSA) on a C6 reversedthat acetonitrile induces some structural changes of the protein phase support, an extensive Fourier transform infrared in solution and competes with alkyl chains for the interaction (FTIR) spectroscopic study has been carried out. It revealed with HSA explaining the slow adsorption kinetic process obthe extent of HSA conformational changes in the eluent served in presence of the organic solvent in the eluent. The when acetonitrile is added but also when the protein is irremore compact protein structure found with 20% acetonitrile is versibly adsorbed on the support (14). These results all correlated with the larger amount of protein adsorbed at this emphasize the ability of acetonitrile to solvate various doaqueous buffer -organic solvent composition.