Photometric measurement of plasma pH
Journal of Biological Chemistry
The advantages of the spectrophotometric technique in applications of indicator methods were pointed out in 1924 by Brode (1) and by Holmes (2, 3) who emphasized the accuracy of readings, the freedom from error from phenomena of dichromatism, the ease with which corrections can be made for color or turbidity in the material examined, and the small amounts of fluid that can be used. Over comparator technique the photometric has also the advantage that the latter does not require the continuous
... re the continuous use of numerous standards. Nevertheless the spectrophotometer has not found general application in measurement of plasma pH. Perhaps one reason is that the careful studies of Robinson and Hogden (4) on the optical density curves of phenol red in serum, buffered to known pH levels by mixing with phosphate and Verona1 buffers, showed peculiar effects of the plasma proteins on the density curves of the dye. The wave-length at which the dye showed maximal optical density was shifted from its normal value of 560 ml.c, and dye densities measured at 560 rnp were significantly decreaed by the presence of the proteins. The effect was greater when phosphate was used than when Verona1 buffers were employed. Results in the present paper show that when human plasma is diluted with 20 volumes of neutral 0.9 per cent NaCl solution containing phenol red, the only buffers present being those of the plasma, effects of the proteins on the optical density of the dye, such as those observed by Robinson and Hogden in serum diluted wit.h phosphate or Verona1 buffer solution, either do not occur, or occur only to such an extent as to balance a slight increase in pH caused by diluting the plasma with the NaCl solution. Consequently it is possible to estimate human plasma pH from the optical density of phenol red in the diluted plasma with a standard deviation, from the pH determined in undiluted plasma by the hydrogen electrode, of only f0.02 pH unit. The indicators, temperature control, and conditions for diluting the plasma are the same as those used by Hastings and Sendroy (5) in colorimetric plasma pH measurements by means of test-tube comparators, but changes in so many details have been found expedient in adapting the procedure to the spectrophotometer that their description appears desirable.