Determination of ion-exchange equilibrium parameters of amino acid and protein systems by an impulse response technique

Abstract

The application of an impulse technique to determine the Langmuir isotherm parameters of amino acids and proteins in ion-exchange chromatography is described. The ion-exchange column is approximated as many stages in series. Within each stage. the mass transfer rate of a given solute between mobile and stationary phases is finite. Responses to pulse and step changes are calculated using a fast and stable algorithm. The equilibrium parameters are estimated by comparing the responses with the experimental data. The sensitivity of the response with respect to equilibrium and mass transfer parameters was studied. The accuracy of estimation of the equilibrium parameters depends strongly on the pulse volume and pulse concentration. A methodology for selecting the proper pulse parameters was developed and successfully used to determine the equilibrium parameters of phenylalanine and bovine serum albumin. The stage model represents pulse data as closely as a more detailed rate equation model while requiring two orders of magnitude less computation time. This technique offers a fast alternative to the conventional batch equilibrium and frontal analysis methods for determining equilibrium isotherms. The impulse technique requires fewer laboratory manipulations than the batch method and, as a result, the data are less scattered. More important, it requires only 1% as much substrate as batch methods and is of value in estimating the equilibrium parameters of costly biochemicals.