The Molar Extinction Coefficient of 2,6-dichlorophenol Indophenol.

Widely varying values for the molar extinction coefficient of 2.6-dichlorophenoi indophenol have been reported by various workers 1-9. A possible source of this variation may be the different samples of DCIP used, due either ~o the presence of impurities or to the presence of mixtures of the anhydrous and hydrated forms of DCIP sodium salt. However. failure to realise that DCIP is a weak acid with pK 5.9 may also be a cause of the variability. This communication deals with (a) the pH dependence of the molar extinction coefficient of DCIP at 6oo m/z, (b) a method for calculating this coefficient at any pH, (c) the apparent and thermodynamic dissociation constants, and (d) the instability of the dye at acid pH values and in alkaiine g!ycine buffers. The majority of these measurements was made with a solution of DCIP prepared in the following way. A sample of DCIP sodium salt 1British Drug Houses) was dried to constant weight over P20a at 8o ° and 2o mm pressure for 24 h to obtain the anhydrous form. A stock solution of DCIP in water was prepared, and diluted with 9 volumes of the appropriate buffer, to give an ionic strength of o.I after mixing. The absolute concentration of DCIP in these solutions was determined bv comparison of the absorbancies at several pH values with those obtained for a solution of DCtP which had been standardised by titration with ascorbic acid of known purity. The sample was 95.1% pure calculated on the basis of the anhydrous salt. Sodium acetate-acetic acid buffers were used over the range pH 3.9-5.2, sodium phosphate buffers for pH 3.0 and pH 5.7-7.9, Na4P2OT-HC1 was used for pK 8. 4, and sodium carbonate-bicarbonate buffers for pH Io.4 and pH Ix.o. The pH of the dye solutions was measured at 26 ° with a carefully standardised null-point pH meter: pK values were correct to within ± 0.o2 pH units. Spectrophotometric measurements were made at 26 ° with an Optica CF4 grating spectrophotometer which had been calibrated for