A change in temperature (T) or gradient steepness (b) can result in changes in reversed-phase selectivity (α). The magnitude of these changes in α will vary with other separation conditions (column, pH, etc.) and with sample type. In this paper, selectivity changes as a function of T and b are discussed and a simple treatment that allows changes in selectivity to be compared quantitatively for different samples and HPLC conditions is developed. Following papers in this series will apply this theory to arrive at conclusions concerning the use of temperature and gradient steepness in HPLC method development. The present treatment assumes that gradient-steepness selectivity (measured by the parameter S) does not change significantly with temperature. Data for a wide range of compound types and conditions are provided in support of this assumption.

The ability of temperature and gradient steepness to change band spacing has been investigated for several ionizable samples that include 8 substituted benzoic acids, 9 substituted anilines, 22 basic drugs, 9 structurally-related herbicide impurities, 7 chlorophylls and 72 peptides and proteins. Mobile phase pH was also varied to determine the effect of sample ionization on temperature and gradient-steepness selectivity.

The separation of nine un-ionized samples was studied as a function of temperature (T) and gradient steepness (b). Selectivity values Δlog α∗ were obtained for 160 compounds, ranging from nonpolar hydrocarbons to very polar drugs. Selectivity varied markedly with sample type: nonpolar compounds such as aromatic hydrocarbons and fatty acid methyl esters generally showed only modest changes in band spacing as temperature or gradient steepness was varied. More polar samples exhibited larger changes in α (Δlog α∗) when temperature and/or gradient steepness wee changed, but the largest values of Δlog α∗ for these non-ionized samples are less than the average value of Δlog α∗ for the ionized samples of Part III [1]. Poly-functional silane (“polymeric”) columns exhibit slightly increased b- and/or T-selectivity for some samples.

The DrylabG software (LC Resources, Lafayette, CA, USA) was applied for optimization of HPLC resolution of some nucleosides in the reversed-phase systems. Two preliminary runs based on linear gradient range of acetonitrile (ACN) from 0% (pure buffer) to 20% and of methanol (MeOH) from 0% to 50% are shown to be satisfactory for optimization of the resolution. A good agreement between simulated and experimental chromatograms was observed.

A single gradient elution run with water (A) to acetonitrile (B) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on RP-HPLC. For a broad range of sample types, that includes both very hydrophilic and hydrophobic compounds, it was found that isocratic retention is given by log k ≈ log kw −4.2ϕ, where ϕ = 0.01 %B. An initial gradient run allows values of log kw to be estimated for each compound in the sample, which then permits compound retention to be approximated as a function of either isocratic or gradient experimental conditions.

The use of an initial gradient run in this way provides a rational basis for the subsequent development of a final HPLC method. The present approach is based on a wide range of sample types and different reversed-phase columns; for this reason it is expected to be reasonably general and accurate.