Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.