MARKED DIFFERENCES BETWEEN ACETONITRILE/WATER AND METHANOL/WATER MOBILE-PHASE SYSTEMS ON THE THERMODYNAMIC BEHAVIOR OF BENZODIAZEPINES IN REVERSED-PHASE LIQUID-CHROMATOGRAPHY

Two different methods were used to determine the separation factor alpha at different temperatures and the Gibbs-Helmholtz parameters (Delta(Delta H),Delta(Delta S)) Of two adjacent benzodiazepines on a chromatogram were obtained from plots of ln alpha versus 1/T. We first studied each factor (fraction of water phi in the ACN/water mixture and column temperature T), which controls the retention mechanism, and then we examined the simultaneous variation of all these factors. The changes in Delta(Delta H) and Delta(Delta S) in relation to a volume fraction of water phi in an ACN/water mixture were examined. In the ACN/water system, Delta(Delta H) was fairly constant in the acetonitrile region of phi less than or equal to 0.52 and appears to be a roughly linear function of phi for phi greater than or equal to 0.52. In this system Delta(Delta S) is approximately a parabolic function of phi with an optimum at phi = 0.52. The retention mechanism of ten benzodiazepines was found to be significantly different in the methanol/water and ACN/water mixtures. The separation optimization of these ten benzodiazepines was then considered. A fraction of water of 0.43 in the ACN/water mixture and a column temperature of 44 degrees C gave the most efficient separation conditions in the ACN/water mixture.