Micro-volume flow titration and screening the dissociation constants (pKa) of weak acids

A micro-volume, monosegmented flow analysis system has been developed for titration of weak acids and for determining dissociation constants (pKa) in the range 3-6, using pH indicators and spectroscopic detection. The system consists of a piston pump, a peristaltic pump, a detector and a 250µm i.d. reaction silica capillary. The analytical procedure for determining the acid concentration involves the sequential introduction of air, solutions of phenolphthalein indicator, acid sample, NaOH, and finally air again, using the piston pump. The various plugs disperse axially into each other in the reaction capillary when pumping with the peristaltic pump is started. The acid sample will then be partially neutralised and the appearance of the dramatic pH change across the combined liquid segment, bracketed by the air segments, is registered by measuring the indicator absorbance at 552nm. In the trials reported here, the acid concentration range was 35-100mM, the sample volume less than 1µl, the time needed less than 2min, and the R.S.D. values obtained were in the range 0-2%. Two screening methods for the determination of pKa values were also developed. The first of these consumes less than 200nl of the sample, but requires prior knowledge of the molar sample concentration. In this case, an indicator mixture, exhibiting a linear absorbance change as a function of pH at 598nm is used instead of phenolphthalein. The second approach, used when no information about the acid concentration is available, involves both indicator solutions and is based on determining the pH at the half-neutralisation point. The sample volume needed, in this case, is less than 1µl.