THE QUANTITATIVE PRECIPITATION OF CITRIC ACID

BY ADRIAN C. KUYPER
(From the Biochemical Laboratory, State University of Iowa, Iowa City, and
the Laboratory of Physiological Chemistry, the Ohio State
University, Columbus)
(Receivedfor publication, January 7, 1938)

In an attempt (1) to obtain information on t,he nature of the

unidentified constituents of urine from an analysis of its titration

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curve, it was found advantageous to remove citrate by some pre-
liminary treatment. The precipitation of citric acid as the cal-
cium salt from aqueous solution was found to be incomplete. Its
precipitation from urine, however, was found to be almost com-
plete. Further experimentation showed that this more complete
precipitation from urine than from aqueous solution could be
attributed to the phosphate present in the urine. Citrate, cal-
cium, and phosphate apparently formed an insoluble complex.
This behavior was studied in greater detail because it promised
to furnish a means for the complete removal of citrate from aque-
ous solution and also becauseit may be encountered at unexpected
places in the determination of other organic acids. These studies
have shown that the concentrations of calcium and of phosphate
in the solution determine the amount of citric acid precipitated.
When these salts are present in sufficient amounts and suitable
proportions, citric acid is quantitatively precipitated.

EXPERIMENTAL

Solutions containing varying amounts of calcium chloride,

potassium acid phosphate, and citric acid were prepared in cen-
trifuge tubes. Normal sodium hydroxide was added to each tube,
slowly and with constant stirring, in slight excess of the amount
required to make the solution blue to litmus. After standing for
a short time the tubes were centrifuged and the clear supernatant
liquid was decanted from the white gelatinous precipitate. Each
precipitate was dissolved in 1.66 N sulfuric acid; the resulting
405
406 Precipitstion of Citric Acid

solutions were transferred to volumetric flasks of convenient size

and brought to volume with the acid. The amount of citrate
in these solutions was determined from the amount of COZ liber-
ated when they were oxidized with KMnOl in the Van Slyke
manometric apparatus (2).
A number of these determinations are recorded in Table I.
The first two demonstrate the extreme sensitivity of the reaction.
In the presence of only small amounts of phosphate and calcium,
TABLE I

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Injluence of Phosphate and Calcium on Completeness of Citrate Precipitation

Constituents of precipitating solutions*

Citrate
precipitated
Citric acid C&l2 KHzPOn

m!J. m.-eq. ?n.-eq. n&.-e*. per cenf

1 0.035 0.0054 1.10 0 .21 80
2 0.13 0.0203 8.2 1.38 95
3 11.8 0.184 10.9 0.00 2
4 11.8 0.184 10.9 0.69 78
5 11.8 0.184 10.9 1.38 94
6 11.8 0.184 10.9 2.76 98
7 11.8 0.184 10.9 5.49 99
8 11.8 0.184 10.9 7.22 99
9 11.8 0.184 10.9 10.98 19
10 11.8 0.184 10.9 13.74 8
11 11.8 0.184 21.8 10.98 100
12 27.4 0.428 27.4 8.82 101
13 82.2 1.28 27.4 8.82 99
14 109.5 1.71 27.4 8.82 97
15 137.0 2.14 27.4 8.82 94

* In each experiment the indicated amount of citric acid was contained

in 100 cc. of solution. The addition of calcium chloride, potassium phos-
phate, and sodium hydroxide increased this volume from 2 to 20 per cent.
These changes in volume were insufficient to affect the amount of citrate
precipitated significantly.

80 per cent of the citric acid in a 0.035 mg. per cent solution was
precipitated. The use of larger amounts of calcium and phos-
phorus might have been accompanied by more complete precipita-
tion, but this could not be shown experimentally because of the
limits of the analytical procedure. Practically complete pre-
cipitation was obtained from a solution containing 0.13 mg. of
citric acid per 100 cc. with the amounts of calcium and phosphate
indicated.
 A. C. Kuyper 407
The remaining experiments demonstrate more clearly the part
phosphate plays in this precipitation of citrate. When no phos-
phate was present, practically no citrate was precipitated. In-
creased amounts of phosphate resulted in the precipitation of
increased amounts of citrate, until complete precipitation occurred.
Beyond this, citrate precipitation again became incomplete. This
incomplete precipitation occurred whenever (Experiments 9 and
10) the amount of calcium in the mixture was not sufficient to
unite with all the phosphate and citrate. Use of more calcium
(Experiment 11) again produced complete precipitation. In the

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last four experiments, 2.94 mM of phosphate were sufficient to
precipitate quantitatively about 100 mg. of citric acid.
DISCUSSION

It would probably be difficult to obtain significant information

on the structure of the precipitate formed in these experiments.
It might be expected to contain some calcium phosphate; the
variable composition of this compound (3) would complicate the
interpretation of analyses for the mole ratios of phosphate, cal-
cium, and citrate in the precipitate. The fact that citrate is
precipitated only when calcium ion is present in excess of the
amount necessary to react with phosphate and citrate seems to
indicate that it forms an insoluble calcium complex which is read-
ily dissociated. The precipitation of only small amounts of citrate
under certain conditions in which large amounts of calcium phos-
phate are precipitated (Experiments 9 and 10) indicates that
citrate precipitation is not due to adsorption on calcium phosphate.
SUMMARY

When a solution of citric acid, containing suitable amounts of

calcium and phosphate, is made alkaline, the citric acid is quanti-
tatively precipitated. Calcium must be present in excess of the
amount necessary to react with phosphate and citrate. In-
creasing amounts of phosphate are required to precipitate larger
amounts of citrate.
BIBLIOGRAPHY

1. Kuyper, A. C., J. Sol. Chem., 123, 409 (1938).

2. Kuyper, A. C., J. Am. Chem. Sot., 66, 1722 (1933).
3. Lorah, J. R., Tartar, H. V., and Wood, L., J. Am. Chem. Sot., 61, 1097
(1929).
THE QUANTITATIVE PRECIPITATION
OF CITRIC ACID
Adrian C. Kuyper
J. Biol. Chem. 1938, 123:405-407.

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