Enhancement of retention predictions in reversed-phase liquid chromatography using reference compounds

The use of reference compounds to correct errors associated with the preparation of mobile phase is studied, in order to enhance modelling of retention for optimisation purposes in reversed-phase liquid chromatography. The method fits individual retention models for a set of reference compounds, which are present in all injections. In second step, mobile-phase composition is corrected for each chromatogram, according to the fitted models of the reference compounds. The enhanced precision provided by the large number of replicates for the reference compounds is partially propagated to the retention models of the analytes. The approach is assayed in the separation of amino acids, after pre-column derivatisation with o-phthalaldehyde (OPA) and N-acetylcysteine (NAC) to form isoindoles, using acetonitrile–water mobile phases and a C18 column. In this example, the reference compounds were three by-products present in the reagent mixture, which produce peaks usually eluting before the analytes. Detection of isoindoles and reference compounds was performed at 335 and 254 nm, respectively. In other samples, similar procedures can be developed by adding one or more external inert compounds to the sample solution. The approach is especially useful for situations where the experimental work is carried out along a rather long period, and random variations in the instrument, reagents and environmental conditions are expected.