Optimization of gradient reversed phase chromatographic peak capacity for low molecular weight solutes

A general protocol for optimizing peak capacity for the separation of low molecular weight molecules under gradient elution conditions has not yet been developed. By studying the effects of gradient time, flow rate, temperature, final eluent composition, and column length on peak capacity, a protocol has been developed for the optimization of a separation of small molecules such as those seen in metabolomic studies. The strategy developed employs the Linear-Solvent-Strength Theory (LSS Theory) [1] to predict retention, building on an approach for the optimization of the peak capacity of large molecules (peptides) in fixed column format separations [2].

► The effect of practical parameters on peak capacity for small molecules is studied. ► A protocol is put forth to maximize peak capacity under RPLC gradient conditions. ► The success of the protocol strongly depends on the solute set's k′wk′w values. ► The selection of an appropriate column length is crucial to the protocol. ► Success of the protocol is confirmed by the results seen with a complex sample.