Spectrophotometric determination of pK(a) values based on a pH gradient flow-injection system

This paper describes a pH gradient flow-injection method for fast spectroph otometric determination of acidity constants. The flow-injection system con sists of a three-channel manifold in which the sample bolus is injected bet ween acidic and basic zones. Therefore, the front of the flow-injection pea k is made acidic while the tail of the peak is alkalinized, and consequentl y, a pH gradient from acidic to basic medium is generated along the flow-in jection peak in an easy and reproducible way. The whole procedure is compos ed of two steps; first, the in situ determination of the pH gradient profil e by using a standard compound with a known pK(a) value, and second, this p H gradient profile is used to calculate the pK(a) of an unknown compound. A n alternating least squares multivariate curve resolution method is used in both steps to resolve the concentration profiles of the acidic and basic s pecies in the standard and in the unknown samples which are the basis of th e calculations. The method is tested using several nucleic acid components. An additional advantage of the proposed method is that no pH experimental measurement is needed for the fast determination of pK(a) values. Results o btained using the proposed procedure are consistent with those listed in th e literature. (C) 2000 Elsevier Science B.V. All rights reserved.