HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF PROTEINS ON SEPARON-HEMA .3. SELECTION OF SUITABLE GRADIENT CONDITIONS FOR THE SEPARATION OF PROTEINS BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY

The aim of this work was to verify the possibility of the application of the simple approach to the prediction of the peak maximum position and peak width. The system studied was based on the sorbent Separon HEMA 1000 H benzoyl and on several proteins which were eluted with a decreasing concentration of ammonium sulphate. It was established that the relationships based on the approach according to Yamamoto et al. required correction, because a "limit" concentration of salt exists. A protein leaves the column at this "limit" concentration always if the initial concentration of salt in the gradient is higher than this "limit" value. A knowledge of the "limit" values of salt concentrations together with a knowledge of mutual ln kappa' vs. ](NH4)2SO4[ dependences of the mixture of proteins (determined from isocratic experiments) is necessary in order to choose the elution conditions, e.g., the gradient time, initial and final salt concentrations and in some instances even the length of the column.