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THE CAUSE OF ACIDITY OF FRESH MILK OF COWS ANDA METHOD FOR THE DETERMINATION OF ACIDITY.BY LUCIUS L. VAN SLYKE AND ALFRED W. BOSWORTH.

(From the Che mica l Laboratory of the New York Agric ultura l Experime ntStatio n, Geneva, N. Y.)

(Received for publica tion, July 11, 1914.)The usual method employed in determining the acidity of milk

is to add a few drops of a solution of phenolphthalein as indicatorto 100 cc. of milk and then titrate with 6 NaOH. By the useof this method it is found that 100 cc. of milk, when strictly fresh,will require the addition of 15 to 20 cc. of the alkali in order toproduce a faint but permanent pink coloration.

The acidity of fresh milk has been commonly attributed to thepresence of acid phosphates and casein, and we will now considerthe relation of these constituents to milk acidity.

That the acidity of milk is due to the presence of acid phosphates(M HzP04) is indicated by the fact that milk is strongly alkalineto methyl orange. Further, it is well known that phosphatescan not be titrated with any degree of accuracy in the presenceof calcium salts, due to the fact that some of the insoluble dical-cium phosphate (CaHP04), which is formed during the titration,hydrolyzes, changing into calcium hydroxide and phosphoricacid, and then the calcium hydroxide unites with more dicalciumphosphate, forming tricalcium phosphate (CaaPzOs). Thesefacts may be represented by the following equations:

(1) CaHPOJ + 2 Hz0 * Ca(OH)z + HaPOd(2) 2 CaHPOd + Ca(OH)z+ CarPzOs + 2 H20.

That tricalcium phosphate is formed during the titration of anysolution containing phosphoric acid and calcium salts is easilydemonstrated by an analysis of the precipitate always appearing;this precipitate is tricalcium phosphate, which is characterizedby its appearance, varying from a flocculent to a gelatinous con-

1 Cameron and Hurst,: Journ. Ame r. Chem. Sot., xxvi, p. 905, 1904.73

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Acidity of Cows Milkdition according to the concentration of the calcium and phos-phates in the solution.

Dibasic phosphates are neutral to phenolphthalein and mono-phosphates are acid to this indicator; phosphoric acid, therefore,acts as a diabasic acid to phenolphthalein. In the reactionrepresented above, we have, in place of the original molecule ofneutral dicalcium phosphate, one molecule of free phosphoric acid,whereby the acidity as measured by titration is increased overwhat it would be if no such reaction occurred. These factsserve to explain some results obtained by us in connectionwith the study of certain problems relating t,o milk.

We have found that when we titrate whole milk with alkali,in the usual way and then similarly titrate the serum obtainedby filtering the milk through a porous procelain filter, the titrationfigure given by the whole milk is about double that obtained withthe serum. For example, 100 cc. of whole milk may show anacidity of 17 cc. of $ alkali, and 100 cc. of serum, 8 cc. Thisdifference has ordinarily been interpreted as being due to the acid-ity of milk-casein, but in a future paper we shall show that caseinis present in fresh milk as a calcium caseinate that is neutral tophenolphthalein. The other constituents removed from themilk by filtering through porous porcelain are fat and dicalciumphosphate, both of which also are neutral to phenolphthalein.From the illustration given above, the titration figure of theresidue on the filter would appear to be 9 (17-8) for 100 cc. of milk,though in reality the reaction is neutral. We believe that thecause of this discrepancy is to be found in the dicalcium phosphatewhich is present in the whole milk but which is not present inthe serum. Its presence in the milk permits the formation ofrelatively large amounts of phosphoric acid and tricalcium phos-phate, requiring the use of increased amounts of $ alkali (17 cc.)to neutralize the milk, as compared with the amount (8 cc.)needed to neutralize the serum. We have been led by such re-sults to believe that the acidity of milk, as usually determined, isabout twice what, it should be.

The disturbing influence of calcium salts in the presence ofphosphates has been studied by Folir? in connection with the

2 Amer. Journ. of Physiol., ix, p. 265, 1903.

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L. L. Van Slyke and A. W. Bosworth 75determination of acidity in urine; he was able largely to overcomethe difficulty by the addition of neutral potassium oxalate, bywhich the calcium is removed in the form of the insoluble oxalate.He showed that by this preliminary treatment, correct titrationfigures could be obtained for monocalcium phosphate, whichwithout such treatment gives figures that are remote from thecalculated acidity.

Making use of Folins procedure, and, before titrating withalkali, adding to milk some saturated solution of neutral potas-sium oxalate, we are able to obtain figures which conform moreclosely to the results indicated as accurate by other considerations.

The method, as modified by us for the determination of acidit.yin milk, whether fresh or otherwise, is as follows:Measure 100 cc. of milk into a 200 cc. Erlenmeyer flask, add 50

cc. of distilled water and 2 cc. of a saturated solution of neutralpotassium oxalate, allow the mixture to stand not less than twominutes and then titrate with & NaOH. Since most solid potas-sium oxalate is acid, care must be taken to prepare a solution thatis really neutral, which may be done in the following way: Asaturated solution of ordinary potassium oxalate is prepared anddecanted from the solid residue. To this solution is added 1cc. of phenolphthalein solution and then, drop by drop, enoughnormal NaOH solution to produce a permanent faintly pinkcoloration.

In the following table is given the acidity of 21 samples of milkfrom individual cows, as determined by the two methods, withand without addition of neutral potassium oxalate.

SUMMARY.

The acidity of fresh milk is due to the presence of acidphosphates. Titration of phosphoric acid with alkali, in thepresence of calcium salts, results in hydrolysis of dicalcium phos-phate formed during the titration, whereby free calcium hydro-oxide and phosphoric acid are first formed and then calciumhydroxide unites with more dicalcium phosphate to form insolubletricalcium phosphate. As a result of these reactions more alkaliis required to make a solution, containing calcium and phos-phoric acid, neutral to phenolphthalein than is required in the

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76 Acidity of Cows Milkabsence of calcium. The calcium must be removed previous totitration by treatment of 100 cc. of milk with 2 cc. of saturatedsolution of neutral potassium oxalate.

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Before addition of neutral Afte r addition of neutralpotassium oxalate potassium oxalatecc. cc.

15 6.415.2 7.015.6 6.816.0 6.817.0 8.017:o 8.017.2 8.017.6 9.017.8 8.818.0 9.018.2 9.618.4 9.618.4 9.418.6 9.418.6 9.419.0 9.419.2 10.019.4 10.420.0 9.822.0 12.823.8 14.0

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