Spectrophotometric determination of pKa values based on a pH gradient flow-injection system

This paper describes a pH gradient flow-injection method for fast spectrophotometric determination of acidity constants. The flow-injection system consists of a three-channel manifold in which the sample bolus is injected between acidic and basic zones. Therefore, the front of the flow-injection peak is made acidic while the tail of the peak is alkalinized, and consequently, a pH gradient from acidic to basic medium is generated along the flow-injection peak in an easy and reproducible way. The whole procedure is composed of two steps; first, the in situ determination of the pH gradient profile by using a standard compound with a known pK(a) value, and second, this pH gradient profile is used to calculate the pK(a) of an unknown compound. An alternating least squares multivariate curve resolution method is used in both steps to resolve the concentration profiles of the acidic and basic species in the standard and in the unknown samples which are the basis of the 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 obtained using the proposed procedure are consistent with those listed in the literature. (C) 2000 Elsevier Science B.V. All rights reserved.