THE DETERMINATION OF CALCIUM BY ALEALIMETRIC

TITRATION
II. THE PRECIPITATION OF CALCIUM IN THE PRESENCE OF
MAGNESIUM, PHOSPHATE, AND SULFATE, WITH APPLI-
CATIONS TO THE ANALYSIS OF URINE

BY CYRUS H. FISKE AND MILAN A. LOGAN*

(From the Biochemical Laboratory, Harvard Medical School, Boston)

(Received for publication, February 4, 1930)

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The investigation of McCruddenl in 1909 is the starting point
of practically all methods which have in the meantime been de-
vised for the determination of calcium in urine, or in other bio-
logical products where the chief difficulty lies in the separation of
this element from both phosphate and magnesium. Such products
-and particularly urine-may however contain, in proportion to
the calcium, much larger quantities of these interfering substances
than were present in any of the known mixtures tested by McCrud-
den. In addition to the sources of error which have so far been
generally recognized, there is in the case of urine of low calcium
content a further complication in the fact that an excess of sulfate
or phosphate tends to increase the solubility of calcium oxalate,
or at least to delay precipitation.
As far as the analysis of urine is concerned, it is a matter of some
consequence to note that the technique recommended by McCrud-
den for this special purpose2 differs from his original procedure’
in some respects, especially in the concentration of oxalate present
during the precipitation. In the urine method the oxalate concen-
tration used is less than 0.02 N, an amount which has been found
too small to bring about complete precipitation of the calcium
from mixtures of high magnesium content, owing to the effect of

* Fellow of the Forsyth Dental Infirmary.

1 McCrudden, F. H., J. Biol. Chem., 7,83,201 (1909-10).
2 McCrudden, F. H., J. Biol. Chem., 10,187 (1911-12).
211
212 Determination of Calcium in Urine

magnesium salts on the solubility of calcium oxalate.3 Since the

prevention of contamination by magnesium ammonium phosphate
is another function of the precipitating agent, it is clear that either
high or low results, depending upon the composition of the solu-
tion, may be expected if the calcium is precipitated with only a
slight excess of oxalate.
The difficulties associated with t.he separation of a small amount
of calcium from a much larger amount of magnesium cannot be
remedied by merely increasing the concentration of oxalate to
the point where the precipitation of calcium is complete, for mag-
nesium oxalate will then come down as welL3v4 That this unde-

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sirable result may be forestalled by the use of a large excess of the
precipitant, leading to the formation of soluble complex magnesium
ammonium oxalates, has long been known in principle,s but the
device has not been used to good effect until quite recently, and so
far has been applied only to the relatively simple problem of
separating calcium from magnesium alone. In the method of
Bobtelsky and Malkowa-Janowskaja,G apparently the best one yet
suggested for the technical analysis of magnesite and related
products, a tremendous excess of ammonium oxalate is prescribed
(15 to 25 gm. of the solid salt per 100 cc. of solution). For micro-
analytical work this arrangement is not suitable, in the first place
because the precipitation and filtration must be conducted at or
near the boiling point, and also owing to the fact that magnesium
cannot be determined in the filtrate in the presence of so large a
quantity of oxalate, which consequently must first be decom-
posed.’ We have accordingly endeavored to determine whether a
pure calcium oxalate precipitate can be obtained at room tempera-
ture in quantitative yield, in the presence of large amounts of
phosphate and magnesium (and also sulfate), using an amount of

* Blssdale, W. C., J. Am. Chem. Sot., 31, 917 (1909). Fischer, W. M.,
2. anorg. u. allg. Chem., 163,63 (1926).
4 Rodt, V., and Kindscher, E., Chem. Ztg., 46, 953 (1924). Luff, G., 2.
anal. Chem., 66,439 (1924-25).
6 Souchay, A., and Lenssen, E., Ann. Chem., 100, 308 (1856). Wittstein,
G. C., Z. anal. Chem., 2,318 (1863).
6 Bobtelsky, M., and Malkowa-Janowskaja, Z. anger. Chem., 40, 1434
(1927).
7 See Hall, W. T., J. Am. Chem. Sot., NJ,2704 (1928).
 C. H. Fiske and M. A. Logan 213

oxalate small enough to permit complete precipitation of magne-

sium ammonium phosphate from the filtrate if desired.
The urine of fasting animals may contain, per mg. of calcium, as
much as 12 mg. of magnesium, 150 mg. of phosphorus, and 150 mg.
of sulfur.s As a result of numerous experiments on pure salt
mixtures of corresponding composition9 it has been found that
0.16 N oxalate, the equivalent of 1 per cent oxalic acid, will suffice
(at pH 5) to precipitate the calcium completely and to prevent
contamination by magnesium, provided that the following condi-
tions are fulfilled: (1) that the concentration of calcium in the
solution is not less than 0.01 or more than 0.03 mg. per cc., and

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(2) that the solubility of magnesium oxalate is further increased
by the addition of ammonium chloride (or nitrate), which has
practically its maximum effect at a concentration of 0.5 N.
The procedure which we have adopted for the precipitation of
calcium oxalate and for the subsequent steps of the analysis, con-
cluding with the titration of the oxide,“’ will now be described.
In many casesashing is required before the calcium is precipitated,
but as the preliminary treatment varies with the nature of the
material to be analyzed this aspect of the subject will be discussed
later under separate headings.

Reagents
Ammonium Chloride, 6 ~-268 gm. are dissolved in water and
made up to 1 liter.
Oxalic Acid, 6.5 Per Cent-25 gm. of crystalline oxalic acid are
dissolved in water and made up to 1 liter.
Ammonium Oxalate, S Per Cent-30 gm. are dissolved in water
and made up to 1 liter. The solution should be filtered the day
after it has been prepared.

* In special cases (e.g., in the urine of animals fed large quantities of

meat) the conditions may be even more extreme. The determination of
calcium in such material can be carried out more rapidly by the use of a
double precipitation method (p. 224).
g Hecht has reported good results with mixtures containing calcium,
magnesium, and phosphate in similar proportions, but his work unfor-
tunately cannot be duplicated inasmuch as explicit directions are not given
(Hecht, G., Biochem. Z., 143,342 (1923)).
lo Fiske, C. H., and Adams, E. T., J. Am. Chem. Sot., 63, 2498 (1931).
214 Determination of Calcium in Urine
Sulfuric Acid, Approxim&ly 10 ~-285 cc. of concentrated
sulfuric acid (sp. gr. 1.84) are added to 800 cc. of water.
Concentrated Nitric Acid (Sp. Gr. l.@).
Nitric Acid, Approximately l ~-63 cc. of concentrated nitric
acid are added to 960 cc. of water.
Ammo?ium Hydroxide, 6 ~-This is prepared by adding con-
centrated ammonium hydroxide from a dropping funnel to stick
sodium hydroxide, and collecting the gas in water (in a cooled
receiver). The solution thus prepared is diluted to the desired
concentration and kept in a paraffined bottle.
Hydrogen Peroxide, $0 Per Cent-Merck’s superoxol.

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Alcohol, 96 Per Cent.
Methyl Red-Saturated alcoholic solution.
0.02 N Hydrochloric Acid.
0.02 N Sodium Hydro&‘de--A parafhned bottle” of 1 liter capa-
city is provided with a Zhole rubber stopper bearing (a) a soda-
lime tube, and (b) a parafhned glass tube long enough to reach
nearly to the bottom of the bottle. The upper end of the glass
tube, which projects several cm. beyond the stopper, is drawn out
to a bore of about 1 mm., and is closed with a piece of rubber
tubing and a short glass rod. The bottle is nearly filled with
distilled water, to which is added a sufficient amount of freshly pre-
pared saturated sodium hydroxide solution to make a liter of
approximately 0.02 N alkali. The rubber stopper with its attach-
ments is at once inserted and sealed in with paraffin, and the con-
tents of the bottle are thoroughly mixed by shaking. The stand-
ard alkali is withdrawn as needed by connecting the tip of a
micro burette with rubber tubing, and applying suction12 or pres-
sure as preferred.
The precautions given above for the exclusion of carbon dioxide
from the standard alkali may fail of their purpose unless the same
degree of care is exercised in keeping the solution free of silicate,
which like carbonate detracts from the sharpness of the end-
point. For this reason the saturated solution of sodium hydro-
xide from which the standard is to be prepared should be made by
dissolving alkali (of the purest quality obtainable) in water in a
metal dish (preferably platinum), since an appreciable amount of
u Paraffin of fairly high melting point (about 55”) should be used.
u Folin, O., and Peck, E. C., J. Biol. Cha., 58,287 (1919).
 C. H. Fiske and M. A. Logan 215

silicate may be introduced by even brief contact with glass while

the solution is still hot. The thoroughly cooled solution may be
transferred to a cylinder and let stand until the sediment has
settled out, but this takes time and is not free from risk. A
quicker, and safer, method is to filter off the sediment with suc-
tion. For this purpose a Jena glass filter is more convenient than
a Buchner funnel, but the first portion of the filtrate may contain
some silicate derived from the glass filter, and should be discarded.
The remainder of the filtrate is collected in a test-tube, and is used
immediately for the preparation of the standard.
By this procedure we have had no difficulty in preparing even

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0.01 N alkali which will give substantially the same titration value
(within 0.2 per cent) whether the indicator used is methyl red or
phenolphthalein. Standards made from saturated alkali which
has been prepared in glass receptacles in our experience will not
meet this test.

Testing and Purijkation of Reagents

All the above reagents, with the exception of the standard alkali
and acid, must be calcium-free. It is important that their purity
in this respect be established in each case by a definitely specified
procedure known to be delicate enough to reveal a significant
amount of calcium. In addition, those reagents which are used
for washing the calcium oxalate precipitate and for converting it
to calcium oxide must be substantially free from impurities of any
sort that will leave an alkaline residue on ignition.
The details of the various tests required are given in Table I,
together with the method of purification to be used when the com-
mercial article proves unsatisfactory. As far as our own ex-
perience goes, it is uselessto spend time testing commercial ammo-
nium hydroxide, which evidently takes up calcium from the soft
glass bottles in which it is marketed, and to someextent even from
hard glass. Dilute (5 N) ammonium hydroxide soon becomes
quite worthless as a reagent for the determination of calcium if
kept in an unparaffined receptacle.

Method
Precipitation-The directions following apply to the precipita-
tion of 0.25 to 0.75 mg. of calcium (at a final volume of 25 cc.)
 TABLE I
Methods of Testing and Purifying Reagents
Reagent Teat for Ca Teat for base Method of purification

Alcohol.. .. .... ... ... ... Evaporate 100 cc. to dryness; Distillation
test residue*
Ammonium chloride. . . . Ignite 5 gm. solid salt in platinum; Recrystallization
test residue* from water
‘I hydroxide.. . . . Evaporate 50 cc. to dryness in See p. 214
platinum; test residue*
“ oxalate. . . . No test necessary Ignite 1 gm. solid salt in platinum. Recrystallization
Dissolve residue with 1 cc. 0.02 from water
N HCl, and titrate with 0.02 N
NaOH to turning point of

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methyl red. At least 0.9 cc.
0.02 N NaOH should be required
Nitric acid.. . . . . . . . . . . . . Evaporate 50 cc. concentrated Evaporate 10 cc. concentrated Distillation
acid to dryness; test residue* acid to dryness in platinum.
Add 0.5 cc. 2.5 per cent oxalic
acid, evaporate to dryness,
ignite, and proceed atr in case
of ammonium oxalate
Oxalic acid ................ Ignite 5 gm. crystals in platinum; Same a8 for ammonium oxalate Recrystallization
test residue* from water
Sulfuric acid .............. Evaporate 10 cc. 10 N solution and Distillation
ignite; test residue*
Superoxol. ................ Evaporate 10 cc. to dryness; test Vacuum distillation
residue*
* The test for calcium is as follows: To the residue left after evaporation or ignition add 1 drop of concentrated nitric
acid and 3 cc. of water. Pour into a small conical centrifuge tube (15 cc. capacity), neutralize (to methyl red) with
calcium-free ammonium hydroxide, add 2 cc. of 3 per cent ammonium oxalate solution, let stand overnight, and centri-
fuge. No sediment should be detectable on careful examination of the tip of the centrifuge tube.
 C. H. Fiske and M. A. Logan 217
from unashed material, or from the slightly acid solution of the
ash when dry ignition has been used. The most suitable recep
tacle for the precipitation is a large lipped Pyrex test-tube (200 X
25 mm.).
To the sample, diluted with water to a volume of about 10 cc.,
are added 2.5 cc. of 5 N ammonium chloride and 10 cc. of 2.5 per
cent oxalic acid. The mixture is then gradually neutralized with
approximately 5 N ammonium hydroxide to pH 5, the final adjust-
ment being made if necessary with a few drops of the oxalic acid
solution. The test-tube is shaken gently for 3 minutes after the
calcium oxalate has begun to separate, and let stand overnight.

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If the material available contains less than 0.25 mg. of calcium
the precipitation must be conducted on a smaller scale. In this
case water is added in sufficient quantity to bring the volume to
4 cc. (instead of lo), and the calcium is precipitated by adding 1 cc.
of ammonium chloride, 4 cc. of oxalic acid, and the requisite
amount of ammonium hydroxide. The procedure otherwise is
that described above.
Filtration-On the following day the calcium oxalate is filtered
off with suction through the small filtration tube described some
years ago by one of usI3 for handling magnesium ammonium phos-
phate precipitates. This consists of a glass tube, about 120 mm.
long and of 8 mm. bore, flanged at one end and constricted at the
other so as to leave a hole 2 mm. in diameter. As calcium oxalate
precipitates may be finely divided, a tight filter is required. The
filtration tube, which is supported by a rubber stopper in the neck
of the suction flask, is provided at the lower (constricted) end
with a mat of ashless paper pulp, made of paper of loose texture
(e.g. Schleicher and Schiill No. 589, black ribbon) and 3 or 4 mm.
thick, which must be well packed, especially at the outer edge.
A piece of glass tubing, not less than 6 mm. in external diameter
and cut off squarely at the lower end, may be used (as a pipette)
for transferring the requisite amount of pulp to the filtration tube,
as well as for packing down the mat, which should be so tight that
the rate of filtration does not exceed 3 cc. per minute with the
suction pump on full.
An equally effective filter, which may be removed more readily
from the filtration tube and is likewise easier to burn, may be
I3 Fiske, C. H., J. Biol. Chem., 46,285 (1921).
218 Determination of Calcium in Urine

made in the following way. A thin pulp mat (1.5 mm.) is first
prepared in the manner just described. On top of this is placed a
disc of ash-free filter paper (of the sort designed to retain fine
precipitates), 8 mm. in diameter, cut out with a cork bore of the
proper size. The disc is pressed down on the mat with the same
glass tube that has been used to manipulate the paper pulp, or
better with a glass rod of the same diameter which also is cut off
squarely and not fire polished. The solid rod is likewise a more
convenient instrument for transferring the paper disc to the
bottom of the filtration tube.
The precipitate is washed with four 2 cc. portions of 3 per cent

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ammonium oxalate solution. The filtration tube, with its stopper,
is then removed from the suction flask, and the tip rinsed off with
water to remove any material that may have crept up the out-
side. The precipitate and the mat are transferred, through the
hole in the lower end of the filtration tube, to a small platinum
dish or crucible, with the aid of a sharpened nichrome wire and
about 1 cc. of water. Any calcium oxalate still adhering to the
glassis dissolved with 2 cc. of N nitric acid, added first to the test-
tube, and Ohenpoured through the filtration tube into the platinum
dish. The apparatus is finally rinsed, in the same order, with two
2 cc. portions of water.
Conversion to Calcium Oxide-The contents of the platinum
dish are evaporated to dryness on the steam bath, leaving the
calcium largely in the form of nitrate. The residue of calcium
nitrate must now, before it is ignited, be reconverted to the
oxalate.1° 0.5 cc. of 2.5 per cent oxalic acid solution (base-free)
is consequently added, and the water is once more removed by
evaporation on the steam bath. The dish is next supported on a
triangle and heated with a micro burner flame, the temperature
being gradually raised until the paper chars and finally begins to
glow. At this stage the flame is removed until the glowing stops,
and then applied at intervals until the paper is completely carbon-
ized. The ignition is finished by heating the dish for a few seconds
with the full flame of the micro burner.
Titration-As soon as the dish is cool enough to handle a drop
of methyl red (saturated alcoholic solution) is added. If the dish
has not become too cold the alcohol evaporates immediately,
leaving the residue dyed yellow. 1 cc. of 0.02 N hydrochloric acid
 C. H. Fiske and M. A. Logan 219

is now introduced, and the dish rotated gently. Particles of

calcium oxide remaining undissolved can readily be distinguished,
by their yellow color, from immaterial traces of filter paper ash,
TABLE II
Determination of Calcium
-
Alone and in Presence of Magnesium
-
and Phosphate

- -7
I VOlUIIlC Ca present Ca found
Calcium* Magnesiumt Phosphorust
_-
mg. per cc. mg. per cc. mg. per cc. cc. ml. ml.
0.03 0 0 10 0.300 0.300
0.03 0 0 10 0.300 0.298

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0.02 0 0 w 1.000 1.002
0.02 0 0 50 1.000 1.002
0.02 0 0 50 1.000 1.004
0.02 0 0 50 1.000 0.994
0.02 0 0 10 0.200 0.200
0.02, 0 0 10 0.200 0.198
0.01 0 0 20 0.200 0,.200
0.01 0 0 20 0.200 0.200
0.03 0.36 9 10 0.300 0.29411
0.03 0.36 9 10 0.300 0.29211
0.02 0.24 6 50s 1.000 1.018
0.02 0.24 6 50 1.000 1.014
0.02 0.24 6 10 0.200 0.202
0.02 0.24 6 10 0.200 0.200
0.02 0.24 0 500 1.000 1.012
- - -
* The standard calcium solutions were prepared by dissolving Kahl-
baum’s calcium carbonate (zur Analyse) in nitric acid.
t Added ae magnesium lactate which was prepared by neutralizing lactic
acid with Merck’s Blue Label magnesium oxide and recrystallizing from
water. The salt contains 3 molecules of water of crystallization.
$ Added ae monopotassium phosphate (purified by recrystallization
from water).
0 When the volume exceeds 20 or 25 cc. the precipitation is conductedin
a lipped 100 cc. Erlenmeyer flask or conical beaker.
11These analyses are included to show that precipitation may be incom-
plete in the presence of a large amount of phosphate if the mixture is shaken
for only 3 minutes after precipitation has begun.

which cannot be brought into solution. In case 1 cc. of the stand-

ard acid does not suffice to dissolve the oxide readily, a second cc.
is added and the dish manipulated until solution is complete. A
small stirring rod may be used, but is unnecessary.
220 Determination of Calcium in Urine

The excess of acid is titrated with 0.02 N sodium hydroxide from

a micro burette to the full yellow color of the indicator, only
0.005 cc. being added at a time as the end-point is approached.
1 cc. of 0.02 N acid is equivalent to 0.4007 mg. of calcium.
ResultsWith the concentration of calcium within the limits of
0.01 to 0.03 mg. per cc., the method can be used, with an error not
exceeding 1 per cent, for the analysis of mixtures containing, per
TABLE III
Determination of Calcium in Presence of Magnesium, Phosphate, and fidJ*ate
-
Composition of solution -i
- -

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Volume Ca preeent Ca found
Calcium* I kfagnesium’ Phos- Sulfurt
phorus’
. _
nag. per cc. nag. per cc. “8. pe7 cc. nag. per cc. cc. WJ. WT.
0.02 0 0 8 10 0.200 0.200
0.02 0 0 8 10 0.200 0 202
0.02 0 0 16 10 0.200 * 0.200$
0.02 0 0 16 10 0.200 0.202$
0.03 0.36 9 12 10 0.300 0.300$
0.03 0.36 9 12 10 0.300 0.298$
0.02 0.24 3 8 10 0.200 0.202
0.02 0.24 3 8 10 0.200 0.200
0.02 0.24 6 16 10 0.200 0.200$
0.02 0.24 6 16 10 0.200 0.202$
0.01 0.24 6 8 20 0.200 0.202
0.01 0.24 6 8 20 0.200 0.202
- - - -
* See Table II.
t Added as sulfuric acid. In these experiments the concentration of
ammonium sulfate (after the acid had been neutralized with ammonium
hydroxide) was 0.5 N or higher. The addition of ammonium chloride was
therefore omitted.
$ Shaken for 15 minutes after the calcium oxalate began to precipitate,
and let stand overnight.

mg. of calcium, 12 mg. of magnesium, 200 mg. of phosphorus, and

an even larger quantity of sulfur (Tables II and III). When the
concentration of phosphorus exceeds 6 mg. per cc., or that of sulfur
8 mg. per cc., the calcium may not be completely precipitated if
the directions given are adhered to. Under such conditions,
however, the tendency to delayed precipitation is made evident
by the fact that the calcium oxalate does not begin to separate at
once. By shaking for 15 minutes (instead of 3 minutes) after the
 C. H. Fiske and M. A. Logan 221

first appearance of a precipitate, correct results can be obtained

with even larger amounts of phosphorus and sulfur present (Table
III).

Determination of Cal&m in Human Urine

Digestion with sulfuric and nitric acids is a thoroughly satis-
factory method for ashing urine provided that the amount of
organic matter is not too large in proportion to the calcium, and
may be used except when the calcium intake is very low or when
the diet contains an excess of any substance (e.g. phosphate) that
interferes with calcium absorption. Because ammonium sulfate

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tends to retard the precipitation of calcium oxalate, the amount of
sulfuric acid that may be used with safety for this purpose is
relatively small. Its effect on the precipitation will naturally be
added to that of the sulfate already present in the urine. A very
considerable amount of organic matter can eventually be des-
troyed by digestion with a little sulfuric acid (with the aid of
nitric acid), but the analysis is likely to be spoiled by the forma-
tion of a precipitate-evidently metaphosphate-which will not
dissolve in water. This difficulty will not infrequently arise in
the case of urine obtained from experimental animals when the
kidneys are excreting very little calcium, in which event a special
method which permits destruction of the organic matter at a
lower temperature must be used (aide injra). Ashing with sulfuric
and nitric acids is on the other hand permissible with human urine
of almost any kind, and is carried out as follows: A sample of urine
containing between 0.25 and 0.75 mg. of calcium is boiled down in a
large lipped Pyrex test-tube (200 X 25 mm.) with 1 cc. of 10 N
sulfuric acid until white fumes appear. While the mixture is
kept gently boiling, concentrated nitric acid, a drop or two at a
time, is allowed to run down the wall of the test-tube, and the
boiling is continued until most of the excess nitric acid has been
driven off, as indicated by the reappearance of white fumes. This
treatment is continued until the residue is colorless. When the
tube has cooled, 10 cc. of water and 2.5 cc. of 5 N ammonium
hydroxide are added, and the excess ammonia is neutralized with
dilute nitric acid until the phosphate precipitate has just disap-
peared. 10 cc. of 2.5 per cent oxalic acid are now introduced,
and the mixture is slowly neutralized to pH 5 with ammonium
222 Determination of Calcium in Urine

hydroxide, as described above. The analysis is continued in the

usual way.
Direct Precipitation-Calcium may also be precipitated from
urine without ashing, following the directions given on p. 217.
This method appears to give quite satisfactory results with ordi-
nary human urine, but the character of the precipitate is such that
the filter is soon clogged, and the analysis consumes more time
than it would if the urine had been ashed. The filtration difficulty
can be eliminated by heating the mixture for 15 minutes in a boil-
ing water bath after the reagents have been added and the volume
has been made up to 25 cc. In this way a considerable amount of

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time may be saved if a large number of samples must be analyzed
TABLE IV
Analysis of Human Urine
Each figure given is the average of duplicate analyses agreeing within
0.8 per cent or less.
Treatment Sample 1 Sample 2

““l$p”
“%:c”
Dry ashing..................................... 13.55 38.2
Ashed with 1 cc. 10 N HzSO+ with aid of HNO, 13.53 37.8
Direct precipitation at room temperature.. . . . 13.58 37.7
“ “ 15 min. in boiling water
bath......................................... 13.52 37.9

at once, but the direct precipitation method (with heat) cannot

be used unless the urine is quite free of material (other than cal-
cium oxalate) which might be precipitated under the conditions
used and carry down inorganic matter. Even traces of albumin,
for example, interfere. The isoelectric point of the denatured
urinary protein is not far from pH 5, and the protein coagulum is
by no means free from ash. The urine must also be quite fresh,
for bacteria contaminating the calcium oxalate precipitate will
cause erroneous results.
All three of the procedures mentioned have been carried out on
two samples of human urine, the calcium content of which had
been determined by the dry ashing method. The results are
identical throughout within less than 1 per cent (Table IV).
 C. H. Fiske and M. A. Logan 223
Special Method for Ashing Urine Low in Calcium
Whenever a sample of urine containing the requisite amount of
calcium cannot be ashed easily with sulfuric and nitric acids, and
particularly when the residue is not completely soluble in water, a
more effective oxidizing agent must be used. Of the reagents
tested for this purpose, including nitric, hydrochloric, and sulfuric
acids, potassium chlorate, and hydrogen peroxide in various com-
binations, we have had the best success with a mixture of nitric
acid and hydrogen peroxide (Merck’s superoxol).
Nitric Acid-Superoxol Oxidation-The urine (containing between
0.1 and 0.3 mg. of calcium) is first evaporated to dryness in a large

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lipped Pyrex test-tube (200 X 25 mm.) by means of a current of
heated air.14 The test-tube is then warmed by supporting it in a
clamp about 4 cm. above the tip of a low micro burner flame, and
the oxidation started by adding a few drops of concentrated
nitric acid and 0.5 cc. of superoxol. The contents of the test-tube
are in this way completely converted into foam. From this
point on, until the last stage of the oxidation, the flame is applied
only to the sidesof the test-tube (which is partly filled with foam),
and is kept in motion so that no spot is continuously heated for
more than a fraction of a second. Meanwhile more superoxol is
introduced, a few drops at a time, and in addition at intervals a
drop or two of nitric acid. The contents of the tube should
consist entirely of foam throughout this stage. In from 5 to 15
minutes the liquid should have become nearly colorless, indicating
that most of the organic matter has been destroyed. At this
point the heating is interrupted until the foam subsides,whereupon
a few more drops of superoxol are added and the burner is returned
to its former position, with the tip of the flame 4 cm. from the
bottom of the test-tube. The heating is continued until the resi-
due is entirely colorless, and the flame should be removed before
the evolution of oxygen-from the residual hydrogen peroxide-

I4By means of apparatus which has been devised by Logan (Logan,

M. A., J. Biol. Chem., 88, 761 (1930)) 40 cc. or more of urine, for example,
can be evaporated in a test-tube of this size with almost no attention and
without the slightest risk of loss by spattering or bumping. Evagoration,
ashing, and precipitation may consequently all be carried out without trans-
ferring the material at any time.
224 Determination of Calcium in Urine

has altogether ceased. This last phase of the oxidation takes

about 1 minute.
The clear mass left at the bottom of the test-tube, when it has
cooled, is dissolved with 3 cc. of water. The solution is made
alkaline with 1 cc. of 5 N ammonium hydroxide, and the excess
alkali just neutralized with N nitric acid, which is added drop by
drop until the precipitate of phosphate redissolves. The calcium
is precipitated in the customary manner by adding 4 cc. of 2.5 per
cent oxalic acid and adjusting the reaction to pH 5 with ammonium
hydroxide, enough water being added in the meantime so that the
final volume will be approximately 10 cc. The rest of the process

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has already been described.

Double Precipitation Method

The urine of cats on a diet consisting solely of large amounts of
meat may contain, per mg. of calcium, as much as 70 mg. of mag-
nesium, 500 mg. of phosphorus, and 500 mg. of sulfur. The
precipitation method outlined above is in this case useless, and
presumably would not be reliable for the analysis of urine from any
species under similar conditions. The following procedure, in
which the calcium is first separated from most of the interfering
substances, including the bulk of the organic matter, by a prelim-
inary precipitation with oxalate and alcohol, gives satisfactory
results.
An amount of urine containing from 0.2 to 0.4 mg. of calcium is
transferred to a 100 cc. conical centrifuge tube and pure powdered
oxalic acid (10 mg. for each cc. of urine) added. The oxalic acid
is dissolved by rotating the tube and heating in a water bath, and
the contents are then neutralized (to pH 5) with 5 N ammonium
hydroxide. To precipitate the calcium, 0.5 volume of 95 per cent
alcohol is now run in rapidly from a pipette, and the tube is rotated
until the alcohol and urine are completely mixed. A very fine
precipitate begins to appear almost at once.
After about 5 minutes the centrifuge tube is placed in a boiling
water bath until the temperature of the contents has reached
about 70”, when it is removed from the bath and rotated for 3
minutes.
On the following day the calcium oxalate is thrown down by
centrifugation, and the supernatant fluid poured off. The sedi-
 C. H. Fiske and M. A. Logan 225

ment is dissolved and transferred to a large lipped Pyrex test-

tube (200 X 25 mm.) with five 1 cc. portions of N nitric acid. 4 or
5 drops of 10 N sulfuric acid are added, and the mixture is boiled
down and ashed. Since the amount of organic matter now present
is very small the oxidation takes only a short time. The subse-
quent treatment of the residue is identical with that followed
after nitric acid-superoxol oxidation (vi& supra).

TABLE V
Analysis of 24 Hour Urines of Meat-Fed Cats by Double Precipitation Method
Ca, ashed Ca, not ashed

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ma. m7.
0.52 0.54
0.87 0.86
1.12 1.11

TABLE VI
Analysis of Known Mix@v.s by Double Precipitation Method
The calcium salt was added to a mixture containing 22 mg. of magnesium
(as lactate), 180 mg. of phosphorus (KHIPOI), 145 mg. of sulfur (added as
H&IO, and neutralized with NH,OH), and 110 mg. of sodium chloride.
Ca present Ca found

m9. ma.
0.200 0.193
0.200 0.200
0.200* 0.200
0.400 0.400
0.400 0.402
l 2.6 gm. of sodium chloride were present.

The analyses in Table V show that this double precipitation

method gives substantially the same results whether the urine has
been ashed or not. Destruction of the organic matter may hence
be omitted, except for the mild oxidation required to remove the
organic impurities carried down with the first calcium oxalate
precipitate. As a further check on the procedure a series of
analyses has been run on known solutions containing from 0.2
to 0.4 mg. of calcium, 22 mg. of magnesium, 180 mg. of phospho-
rus, 145 mg. of sulfur, and 110 mg. or more of sodium chloride
 Determination of Calcium in Urine

(Table VI). Needless to say, the materials from which the salt
mixtures were prepared were carefully purified to insure freedom
from calcium. The magnesium lactate and the monopotassium
phosphate were recrystallized from water, the sodium chloride was
precipitated three times with calcium-free alcohol (see Table I),
and the sulfuric acid was redistilled.

SUMMARY

The separation of calcium (as oxalate) in the presence of large

amounts of phosphate, sulfate, and magnesium is discussed with
special reference to the analysis of urine of low calcium content.

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Methods of effecting this separation under the most extreme con-
ditions likely to be encountered in urine and other biological
material are described, in conjunction with a procedure for the
final analysis of the calcium oxalate precipitate, based on alkali-
metric titration of the oxide.
THE DETERMINATION OF CALCIUM
BY ALKALIMETRIC TITRATION: II.
THE PRECIPITATION OF CALCIUM IN
THE PRESENCE OF MAGNESIUM,
PHOSPHATE, AND SULFATE, WITH
APPLICATIONS TO THE ANALYSIS OF
URINE
Cyrus H. Fiske and Milan A. Logan
J. Biol. Chem. 1931, 93:211-226.

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