HPLC method development for the quantification of a mixture of reacting species: the case of lactose

Preparative and analytical chromatography are impaired by analytes that undergo a chemical reaction during the chromatographic separation, leading to peak distortion and systematic errors during the subsequent quantification phase. The pitfalls are highlighted through a combination of analytical results and numerical simulations. Two different quantification strategies for partially overlapping and reacting peaks are compared. A novel method development strategy based on the valley-to-peak ratio instead of the more common resolution is proposed. The method has been used to experimentally investigate the chromatographic behavior of a mutarotating sugar, lactose. The separation of the unprotected lactose isomers, α and β, has been optimized using a C18 column and pure water as the mobile phase. Phase dewetting phenomena during method development have also been studied and discussed.