Measurement and use of retention data from high-performance gradient elution : Correction for “non-ideal” processes originating within the column

Abstract

Under “ideal” conditions it is possible to model retention in gradient elution so as to be able to calculate retention times, t g, as a function of isocratic retention in corresponding liquid chromatographic systems. In this paper we consider various “non-ideal” processes that lead to errors in calculated values of t g. The more important of these are solvent demixing due to uptake of one mobile phase component by the column packing, non-linear plots of log k′ vs. gradient time or mobile phase composition and changes in column dead-time, t 0, due to changes in mobile phase composition and flow-rate. Expressions are derived to correct for these various “non-ideal” effects, including equipment limitations discussed in the preceding paper. Calculated values of t g for reversed-phase gradient elution systems then agree with experimental values to within ± 1 % (1 standard deviation) of the total gradient time, t G. These results should prove useful in (a) improving the precision of retention in gradient elution (which should be comparable to that in isocratic elution), (b) using gradient elution for more efficient method development in isocratic procedures and (c) better understanding gradient separations of macromolecules such as proteins.