DETERMINATION OF BINDING CONSTANTS BY FLOW-INJECTION GRADIENT TECHNIQUE

A new method is based on the flow injection gradient equipped with an acousto-optic tunable filter (AOTF) detector has been developed for th e determination of binding constants of substrates with cyclodextrins or micelles. The concentration gradient produced by this method elimin ates the need to prepare series of mixtures in which the concentration s of one of the reactants are varied. The fast scanning ability of the AOTF enables the detector to record the whole absorption spectrum of the mixture as it flows through the flow cell. Binding constants can b e rapidly determined. The effectiveness of the method was evaluated by determining binding constants of inclusion complex formation between beta-cyclodextrin (beta-CD) and phenolphthalein and of the incorporati on of organic molecules (e.g., p-toluenesulfonic acid (PTS) and benzop henone (BP)) into micelles (CTAB) under steady state condition (no flo w) and compared with values determined under the flow injection gradie nt. The method is particularly effective for charged substrates, e.g., the binding constants obtained between PTS and CTAB and between pheno lphthalein and beta-CD. Neutral substrate, BP, gives relatively poor a ccuracy and reproducibility, and these may be due to the adsorption of the substrate onto the flow device.