A procedure for the rapid determination of mixtures of nucleic acid components from the analysis of spectrophotometric multivariate data obtained with continuous-flow and flow injection pH-gradient systems is proposed. Three flow systems have been developed and assayed in which an on-line pH gradient is generated from the mixing and controlled dispersion of acidic and basic titrant solutions. Quantitative determinations of any particular analyte in the unknown samples in the presence of interferences is performed with a single pure standard for this analyte. They are carried out using an alternating least squares multivariate curve resolution procedure. The methods proposed have been validated using synthetic and real sample mixtures. The results obtained are concordant with the labeled values, and the relative prediction errors are around 5%.