methods are undoubtedly the most accurate known, they have the disadvantage in that they require for their execution more time and larger quantities of material than the volumetric methods do.

Of the considerable number of acidimetric methods described, many of which are based on the titration of the ammonium phospho-molybdate precipitate, the one presented by Neumann seems to be quite generally in vogue. Not only is Neumann's method preferably employed in biological investigations for the determination of phosphoric acid in nucleic acids, lecithins, phosphatides, animal and plant tissues, etc., but in ores, steel, iron, phosphate rocks, and fertilizers just as well.

The Incorrectness of the Factor Employed in Neumann's Acidimetric Method.

9

Despite the fact that Neumann's method has been in use already for over ten years, it was only very recently that Heubner 8 and Wardlaw have shown the factor 0.554-as proposed by Neumann and applied in his method for the calculation of phosphoric acid-to be incorrect. The experimental evidence presented by Heubner demonstrated that the above-mentioned factor, leading as it does to the not negligible error of about three per cent., is to be replaced by the factor 0.57, which is more nearly correct. From this it would seem that previous records in the literature of phosphoric acid estimations according to Neumann's method must have been too low. The latter method being of great practical value as well as of general interest, a solution of the question as to which of the two factors-Neumann's or Heubner's is correct seemed to be desirable, if not imperative. Moreover, additional data, it was hoped, might help to clear up the discrepancy between the factor theoretically deduced by Neumann and the factor empirically secured by Heubner. Furthermore, this question had a specific interest for us, inasmuch as we had applied Neumann's acidimetric method to our work on the chestnut bark disease prior to the publication of Heubner on the factor to be applied in Neumann's method.

'A. Neumann, Z. physiol. Chem., 37, 129 (1902-03); 43, 35 (1904-05); Gregersen, Z. physiol. Chem., 53, 453 (1907); Bang, Bioch. Z., 32, 443 (1911). Biochem. Z., 64, 393 (1914).

The Theory of Neumann's Method.

Neumann based his method on the fact that the yellow precipitate of ammonium phosphomolybdate obtained from a nitric acid solution under certain conditions has-as was demonstrated by the work of Hundeshagen 10-the composition: 12 MoO3, (NH4) 3PO4, 2 HNO3. Taking into consideration that a sharp and exact titration of the yellow precipitate against standard alkali, with phenolphthalein as indicator, can be accomplished only after the ammonia has been removed, we obtain, on doubling the above formula, the following equation: 24 MoO3, 2 (NH4)3PO4, 4 HNO3 + 56 NaOH = 24 Na2M0O4+2 Na2HPO4+4 NaNO3 +32 H2O+(6 NH). This means that 56 molecules of NaOH required to neutralize the yellow precipitate and to remove the ammonia correspond to 2 Na,HP, (= 142 grs. P2O5). By using NaOH for titration we can express the above data as follows: 112 litres of NaOH = 142 grs. P2O, hence I c.c. NaOH is

n

2

n

2

n

2

equivalent to 0.554 milligramme P. It is this factor (0.554) that proved to be about three per cent. lower than the average factor (0.57) to which the work of Heubner has led.

Heubner's factor having been fully corroborated by the work of one 11 of us, as giving very nearly correct results under the conditions outlined in the paper, the question seemed of considerable interest as to what causes the deviation from the theoretical factor 0.554. Several reasons may be responsible for this difference. In the first place it has been stated in the literature by various investigators that the composition of the yellow precipitate may vary under different conditions of precipitation. Further, it is altogether not out of the question that in Neumann's method the ammonium phosphomolybdate is not absolutely insoluble in the liquid from which it is thrown down, in which case the filtrate from the yellow precipitate must still contain some phosphorus. Moreover, the yellow precipitate formed may, though to a small extent, be lost in the course of the various manipulations incident to that method. This may especially be due to the effect of the wash water on the yellow precipitate. It is with these questions that we shall chiefly be concerned in this paper.

10 Z. analyt. Chem., 28, 141 (1889).

"Jodidi, S. L., Journ. Amer. Chem. Soc., 37, 1708 (1915).

Data Concerning the Solubility of the Ammonium Phosphomolybdate Precipitate in Water.

A review of the literature in question shows that there is considerable divergency of opinion as to whether or not the yellow precipitate is soluble in water. Hundeshagen,12 who was one of the first to investigate into the nature of the ammonium phosphomolybdate precipitate with the object in view of elaborating a volumetric method for the estimation of phosphoric acid, is of the opinion that only ice-cold water should be used for the washing of the yellow precipitate, since in case water of room temperature is used it is hard to prevent traces of the precipitate from going through the filter or into the solution.

Pemberton 13 draws from his experiments the conclusion that there is no danger of loss in washing the yellow precipitate with

water.

Isbert and Stutzer 14 hold that the ammonium phosphomolybdate is insoluble in cold water.

Eggertz 15 states that the solubility of the ammonium phosphomolybdate in pure water is 1: 10,000, to which Hundeshagen remarks that in his experience the solubility is considerably greater.

Neumann 16 maintains that the yellow precipitate is altogether insoluble in the liquid in which it was thrown down, and is so difficultly soluble in very cold water that only a minute amount of it is dissolved after it has been in contact with the water for a long while.

Wardlaw 17 states that by evaporating down the washings extremely slight amounts of the precipitate were found to have dissolved.

Our observations made during the work reported in the first paper 18 of this series pointed to slight solubility of the ammonium phosphomolybdate in water. It seemed desirable quantitatively to ascertain just how much of the yellow precipitate is dissolved or otherwise lost in the course of the various manipulations.

12 Z. analyt. Chem., 28, 164 (1889).

13

Journ. Amer. Chem. Soc., 15, 386 (1893).

14 Z. analyt. Chem., 36, 583 (1897).

15 Z. analyt. Chem., 28, 161 (1889).

18 Z. physiol. Chem., 37, 132 (1902-03).

"Jour. Proc. Roy. Soc. New South Wales, 48, 83 (1914).

18 Jour. Amer. Chem. Soc., 37, 1708 (1915).

In the washing of the yellow precipitate several factors are evidently involved; namely, the quantity of wash water applied, its temperature, the duration of its contact with the precipitate, which again in some measure depends upon the physical properties of the yellow precipitate as well as of the filter. While the operator has full control of the first two factors (quantity of wash water and its temperature), he is confronted with the fact that the lastnamed factors are more or less incidental.

EXPERIMENTAL.

For our experiments we have used the phosphates of sodium, potassium, and ammonium as well as the free phosphoric acid. The data obtained according to Neumann's acidimetric method were calculated to the results found by the usual gravimetric 19 method in which the phosphoric acid is precipitated as magnesium ammonium phosphate and weighed as pyrophosphate. Inasmuch as more recent work of Gooch,20 Neubauer, 21 Järvinen,22 Schmitz,23 Jörgensen, 24 and others has demonstrated that when the phosphoric acid is precipitated in the cold it is quite difficult to obtain a pure precipitate of MgNHPO4, which is, however, the case when the precipitation takes place in a hot solution, it was deemed necessary to check up all of the analyses made according to the gravimetric method as given in Fresenius by the newer method of Schmitz.25 Both methods were further checked up by the conversion of the alkali orthophosphates into the corresponding pyrophosphates and metaphosphates respectively. In order to avoid errors due to imperfectly-graduated vessels, we have used throughout these experiments the same burette of 50 c.c. and the same flask of 250 c.c. which were found to be accurately graduated and to closely agree with each other. Further, we have used for each series of experiments the same stock solution, which was usually sufficiently dilute and kept in a well-stoppered flask. Whenever possible the portions of the solution to be analyzed were simultaneously measured out. Again, in order to exclude

19

"Fresenius, “Quantitative Anal.," 6th edition, 1, 402 (1903).

20 Z. anorg. Chem., 20, 135 (1899).

"Z. angew. Chem., 1896, 439.

22 Z. anal. Chem., 43, 279 (1904); 44, 333 (1905).

the influence of individual errors as much as possible it was deemed advisable to base the conclusions upon a considerable number of, in the most cases, concordant analyses, the average of which could be considered as reliable. The precipitation of the ammonium phosphomolybdate was accomplished exactly in the manner described in a previous paper. 26 When modifications were applied they will be mentioned.

SERIES A.

Nine grammes of primary potassium phosphate (KH,PO1) were dissolved in water, the solution made up to about five litres and thoroughly shaken. Eight portions of this solution of 250 c.c. each were subjected to gravimetric analysis according to Fresenius's method, eight like portions according to Schmitz's method, while five portions of 50 c.c. each were converted into metaphosphate by ignition. The data obtained are summarized in the table below.

Hence, I c.c. of the solution contains 0.41171 mg. P (a), or 0.41025 mg. P (b), or 0.41106 mg. P (c).

Titration According to Neumann's Method.

Thirty portions of the above solution of 25 c.c. each were subjected to titration by Neumann's method. It is a matter of course that all numbers of this as well as of the other series were treated exactly in the same manner, with the only difference that the quantities of water applied for washing the yellow precipitate varied with the different numbers. The data are presented in

"Jodidi, S. L., Journ. Amer. Chem. Soc., 37, 1709 (1915).