Initial experiments in high-performance liquid chromatographic method development .1. Use of a starting gradient run

A single gradient elution run with acetonitrile (B)-water (A) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on reversed-phase high-performance liquid chromatography. 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 approximate to log k(w) - 4.2 phi, where phi = 0.01 %B. An initial gradient run allows values of log k(w) 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. Predictions based on this initial run can be used to (a) select between isocratic or gradient elution for further experiments and the final method, (b) choose a value of %B to achieve a desired value of k for the initial or final band in isocratic separation and/or (c) choose values of %B for the initial and final mobile phase in gradient elution. 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. Errors in subsequent predictions based on an initial gradient run can also be estimated as a function of experimental conditions, allowing the selection of conditions for minimum error.