Abstract
In studies involving the fractionation of blood phosphate compounds by means of hydrolysis with N HCl certain difficulties were encountered in the determination of the resulting orthophosphate by Kuttner's colorimetric procedure. 1 The results were usually too low, and analyses of known quantities of P under similar conditions likewise gave values below those expected. Although inclined to attribute our difficulties to the losses of HCl during hydrolysis, we had the same trouble even when we prevented such alterations in concentration. We had no more trouble when we substituted N H2 SO4 for the N HCl. Our analyses on known quantities of P were also entirely satisfactory when H2 SO4 instead of HCl was used for the hydrolysis. This led us to investigate the probable influence of different salts in high concentration on the orthophosphate values determined by the Kuttner method. We discovered subsequently that Rimington 2 had already pointed out the effect which the concentration of various salts used as anticoagulants may exert upon the quantitative determination of P in blood by Brigg's procedure. We, nevertheless, present these results to call attention once more to this important matter, and thus save other investigators the time and trouble it has cost us to find out this simple fact. The warning is perhaps especially needed now, inasmuch as hydrolysis by N HCl is generally employed in the study of various phosphate fractions in blood or muscle.
On neutralizing this extra acid before carrying out the colorimetric test a rather high salt concentration is produced, which in our experiments, on account of the final dilution used, amounted to 0.2 M. Not all salts interfere with the colorimetric reaction, as will be shown presently, but the chlorides and nitrates do interfere seriously.
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