A STUDY OF THE RETENTION BEHAVIOR OF PROTEINS IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY(I) - THE EFFECT OF SOLVENT AND TEMPERATURE ON RETENTION BEHAVIOR OF PROTEINS IN REVERSED-PHASE CHROMATOGRAPHY

The retention behavior of proteins was investigated by using reversed-phase chromatography (RPC), comparing to the retention behavior of small molecules in RPC. The evaluation was carried out on a SynChropak RP-P(C18) column with 0.1% aq. TFA-organic solvent modifier such as acetonitrile, isopropanol, and ethanol. The Z value (the number of solvent molecules required to displace the solute from the surface) was a general index for the characterization of protein retention as a function of organic concentration over a range of temperature between 5 and 70-degrees-C. Van't Hoff plots provided the basis for evaluating the enthalpic and entropic changes associated with the interaction between protein and the stationary phase. Z values did not change significantly at the range of temperature showing the consistent DELTAH-degrees and DELTAS-degrees values. From these investigation, it was concluded that the retention behavior of proteins in RPC was able to be predicted by the retention parameters applied to small molecules, Furthermore, myoglobin and hemoglobin in RPC as stated above showed a similar retention behavior regardless of their molecular weights.