Abstract
Based on the stoichiometric displacement theory for retention of solute (SDT‐R), the changes of free energy of biopolymers during retention process in reversed‐phase high performance liquid chromatography (RPLC) can be divided into two independent fractions, the adsorption and desorption free energy changes. The totally net free energy change and the two fractions of seven kinds of proteins in RPLC were accurately measured. The standard deviation between experimental values and theoretical expectation was less than ±2%. In addition, from the point of view of thermodynamics, with the comparisons of the adsorption and desorption free energy changes between small solutes and biopolymers in RPLC, the following experimental facts were first explained quantitatively: [Robbat, A., Jr.; Liu, T.Y. J. Chromatagr. 1990, 513, 117] The elution range of organic solvent for small solutes in the mobile phase in RPLC is usually broad, while that for biopolymers is very narrow. [Tan, L.C.; Carr, P.W. J. Chromatogr. 1993, 656, 521] Small solutes in RPLC can be eluted with isocratic elution, but biopolymers only with gradient elution. [Tchapla, A.; Heron, S.; Colin, H.; Guiochon, G. Anal. Chem. 1988, 60, 1443] The resolution of small solutes in RPLC depends on column length, while that of biopolymers is almost independent of it.
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