Batchwise and flow-injection methods for thermo-spectrophotometric determination of acetylcholine and choline with tetrabromophenolphthalein ethyl ester

Abstract A selective spectrophotometric method was established for the determination of acetylcholine (Ach) and choline (Ch) by liquid—liquid and thermochromism of ion association complexes. This method is based on the formation of ion association compounds with tetrabromophenolphthalein ethyl ester (TBPE · H) and thermocromism of ion associates in the organic phase, where selectivity can be improved. The absorbance of blue Ach and/or Ch ion association complexes does not vary with temperature changes, but that of the red amine complexes disappears when the temperature of the absorbance measurements is kept at 45°C. A two-line manifold was constructed for the flow-injection method. The sample is injected into the buffered stream adjusted to pH 11 and mixed with the extracting TBPE · H dichloroethane solution. The organic phase separated with a polytetrafluoroethylene porous membrane passes through a temperature-controlled micro flow cell (45°C) and the absorbance of the organic phase is measured. The sample throughputs were 36 h −1 for both Ach and Ch. The calibration graphs were linear in the range 6.25 × 10 −5 –7.5 × 10 −6 mol dm −3 for Ach and 1.25 × 10 −6 –1.5 × 10 −5 mol dm −3 for Ch. The relative standard deviation was 1.2% for 5 × 10 −6 mol dm −3 Ach ( n = 5_ and 1.2% for 1 × 10 −5 mol dm −3 Ch ( n = 8).