Thin-layer ion-exchange chromatography of proteins.

Thin-layer chromatography (TLC) is one of the simplest and most convenient techniques to separate small molecules. Of a variety of TLC separation modes, only size-exclusion was successfully used to separate proteins. In this paper, adsorption-TLC was used to separate proteins. The net charges were calculated for four model proteins, albumin, transferrin, lactoferrin and lysozyme, under different pH values. The suitable pH values for separation were determined according to the results from such calculations. Then, the adsorption isotherms of the four proteins were measured to deduce the ionic strength for appropriate elution conditions. Optimal conditions, 0.01 M bicine and pH 8.50, and a three-step elution process (1st step 0.01 M NaCl, 2nd 0.025 M NaCl, and 3rd 0.10 M NaCl), were obtained. Finally, the four model proteins were successfully separated under these elution condition.