Benefits of solvent concentration pulses in retention time modelling of liquid chromatography

The advantages and disadvantages of the use of isocratic experimental designs including transient increments of organic solvent (i.e., pulses) in the mobile phase(s) of lowest elution strength are explored with modelling purposes. For retained solutes, this type of mixed design offers similar or better predictive capability than gradient designs, shorter measurement time than pure isocratic designs, and retention model parameters that agree with those derived from pure isocratic experiments, with similar uncertainties. The predicted retention times are comparable to those offered by models adjusted from pure isocratic designs, and the solvent waste is appreciably lower. Under a practical standpoint, mixed designs including pulse(s) can be easily constructed by replacing the slowest isocratic runs with runs containing a pulse of short duration at an intermediate time. This allows the elution of the fastest solutes with appreciable retention in the initial sector of the elution program, previous to the pulse, and the elution of the slow solutes after the pulse, also in acceptable times. The fitting of the retention data obtained with pulses is simpler compared to gradient elution, and involves solving the integral equation of gradient elution, simplified by the presence of isocratic sectors. Experiments involving pulses reveal the existence of discrepancies in the predictions for solutes eluting in the nearby of the pulse, offered by the fundamental equation of gradient elution when this is solved using numerical integration. The correction of such discrepancies implies the inclusion of intra-column delays, in the arrival of changes in the concentration of organic modifier in the gradient to the instantaneous position of the solute, along the whole migration.