Heterogeneous Adsorption in Chiral LC

Sammanfattning: The adsorption of chiral drugs on proteins immobilized on silica shows an heterogeneous adsorption behavior on multiple sites. The theory of nonlinear chromatography was used for a systematic study on this behavior - linear chromatography can only measure the "global" retention. Frontal analysis in the staircase mode was used for studies on the adsorption of model compounds on two different protein columns; β-blockers to cellobiohydrolase I and analytes of different protolytic classes to α1-acid glycoprotein. It was found that the compounds were retained at different kinds of sites, nonselective and enantioselective, and the adsorption was best fitted by the bi-Langmuir model.With increasing the eluent pH the interaction of both enantiomers of the amines to both types of sites was increased on α1-acid glycoprotein. In the case of cellobiohydrolase I (Cel7A) only the interaction of the second eluted enantiomer increased, resulting in a dramatic increase of the enantioselectivity with increasing pH. When increasing the eluent pH the linear retention of the second eluted enantiomer switches from exothermic to endothermic behavior on Cel7A, this point depends on the relative retention on endothermic enantioselective sites. The hydrophobicity of the compound was found to be an important property regarding retention behavior for both enantiomers and on both types of sites on Cel7A. A very interesting feature was observed: 2-phenylbutyric acid has a maximum of retention at an intermediate eluent pH on α1-acid glycoprotein and from nonlinear studies it was shown that this originates from maximum interaction strength at the enantioselective sites. For both columns, there is a 1:1 stochiometry at the enantioselective site.Protein columns sometimes shows slow and heterogeneous kinetics, and simulation studies were performed to illustrate the consequences of such a behavior. The relations between (i) the coefficient of kinetics (ii) the column length and (iii) the linear flow rate were investigated. A conclusion for the homogeneous case, is that the solution of all skewed peaks is perfectly Gaussian, if the column hold-up time is high enough.