the determination of calcium by alealimetric titration · double precipitation method (p. 224). g...

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)

The investigation of McCruddenl in 1909 is the starting point of practically all methods which have in the meantime been devised for the determination of calcium in urine, or in other biological 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 concentration 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).

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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 solution, 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 magnesium oxalate will then come down as welL3v4 That this unde- 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 temperature 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).


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C. H. Fiske and M. A. Logan 213

oxalate small enough to permit complete precipitation of magnesium 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 conditions 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 (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 cases ashing 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).


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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. 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 prepared 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 contents of the bottle are thoroughly mixed by shaking. The standard 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 hydroxide 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).


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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 suction. 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 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 commercial article proves unsatisfactory. As far as our own experience goes, it is useless to spend time testing commercial ammonium hydroxide, which evidently takes up calcium from the soft glass bottles in which it is marketed, and to some extent 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 precipitation of 0.25 to 0.75 mg. of calcium (at a final volume of 25 cc.)


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TABL

E I

Met

hods

of

Tes

ting

and

Purif

ying

Re

agen

ts

Reag

ent

Teat

for

Ca

Alco

hol..

. .

. . .

. . .

. . .

. . .

. Ev

apor

ate

100

cc.

to

dryn

ess;

te

st

resid

ue*

Amm

oniu

m

chlo

ride.

. .

.

Igni

te

5 gm

. so

lid

salt

in p

latin

um;

test

re

sidue

* ‘I

hydr

oxid

e..

. . .

“ ox

alat

e.

. .

.

Evap

orat

e 50

cc

. to

dr

ynes

s in

pl

atin

um;

test

res

idue

* No

te

st

nece

ssar

y

Nitr

ic

acid

.. . .

. . .

. .

. . .

. .

Oxa

lic

acid

....

......

......

Sulfu

ric

acid

....

......

....

Supe

roxo

l. ...

......

......

.

Evap

orat

e 50

cc

. co

ncen

trate

d ac

id

to d

ryne

ss;

test

re

sidue

*

Igni

te

5 gm

. cr

ysta

ls

in p

latin

um;

test

re

sidue

* Ev

apor

ate

10 c

c. 1

0 N

solu

tion

and

igni

te;

test

re

sidue

* Ev

apor

ate

10 c

c. to

dr

ynes

s;

test

re

sidue

*

Teat

for

ba

se

Metho

d of

purifi

catio

n

Igni

te

1 gm

. so

lid

salt

in p

latin

um.

Dis

solv

e re

sidue

wi

th

1 cc

. 0.0

2 N

HC

l, an

d tit

rate

wi

th

0.02

N

NaO

H to

tu

rnin

g po

int

of

met

hyl

red.

At

le

ast

0.9

cc.

0.02

N

NaO

H sh

ould

be

requ

ired

Evap

orat

e 10

cc

. co

ncen

trate

d ac

id

to

dryn

ess

in

plat

inum

. Ad

d 0.

5 cc

. 2.

5 pe

r ce

nt

oxal

ic ac

id,

evap

orat

e to

dr

ynes

s,

igni

te,

and

proc

eed

atr

in

case

of

am

mon

ium

ox

alat

e Sa

me

a8 f

or a

mm

oniu

m

oxal

ate

Dist

illatio

n

Recr

ysta

llizat

ion

from

wa

ter

See

p.

214

Recr

ysta

llizat

ion

from

wa

ter

Dist

illatio

n

Recr

ysta

llizat

ion

from

wa

ter

Dist

illatio

n

Vacu

um

dist

illatio

n

* Th

e te

st f

or c

alciu

m

is a

s fo

llow

s:

To

the

resid

ue

left

afte

r ev

apor

atio

n or

ig

nitio

n ad

d 1

drop

of

con

cent

rate

d ni

tric

acid

an

d 3

cc. o

f wa

ter.

Pour

in

to

a sm

all

coni

cal

cent

rifug

e tu

be

(15

cc.

capa

city

), ne

utra

lize

(to

met

hyl

red)

wi

th

calci

um-fr

ee

amm

oniu

m

hydr

oxid

e,

add

2 cc

. of

3 pe

r ce

nt

amm

oniu

m

oxal

ate

solu

tion,

le

t st

and

over

nigh

t, an

d ce

ntri-

fu

ge.

No s

edim

ent

shou

ld

be d

etec

tabl

e on

car

eful

ex

amin

atio

n of

the

tip

of th

e ce

ntrif

uge

tube

.

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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.

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 phosphate 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).


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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 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 glass is dissolved with 2 cc. of N nitric acid, added first to the test- tube, and Ohen poured 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


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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

Calcium*

mg. per cc.

0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02, 0.01 0.01 0.03 0.03 0.02 0.02 0.02 0.02 0.02

-

-

_-

-

Magnesiumt Phosphorust

mg. per cc. mg. per cc.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.36 9 0.36 9 0.24 6 0.24 6

0.24 6

0.24 6 0.24 0

-7

-

- I VOlUIIlC

cc.

10 10 w 50 50 50 10 10 20 20 10 10 50s 50 10 10 500

-

Ca present

ml.

0.300 0.300 1.000 1.000 1.000 1.000 0.200 0.200 0.200 0.200 0.300 0.300 1.000 1.000 0.200 0.200 1.000

Ca found

ml.

0.300 0.298 1.002 1.002 1.004 0.994 0.200 0.198 0,. 200 0.200 0.29411 0.29211 1.018 1.014 0.202 0.200 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.

11 These 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 standard 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.


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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

Calcium*

nag. per cc.

0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.01

-

I . _

-

Composition of solution

kfagnesium’ Phos- phorus’ Sulfurt

nag. per cc. “8. pe7 cc. nag. per cc.

0 0 8 0 0 8 0 0 16 0 0 16 0.36 9 12 0.36 9 12 0.24 3 8 0.24 3 8 0.24 6 16 0.24 6 16 0.24 6 8 0.24 6 8

-

-

-i

-

Volume Ca preeent Ca found

cc.

10 10 10 10 10 10 10 10 10 10 20 20

WJ.

0.200 0.200 0.200 0.200 0.300 0.300 0.200 0.200 0.200 0.200 0.200 0.200

-

-

WT.

0.200 0 202

* 0.200$ 0.202$ 0.300$ 0.298$ 0.202 0.200 0.200$ 0.202$ 0.202 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


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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 satisfactory 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 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 destroyed by digestion with a little sulfuric acid (with the aid of nitric acid), but the analysis is likely to be spoiled by the formation 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


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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 boiling 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 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

“%:c” ““l$p”

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 calcium 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).


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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 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 sides of 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 residue is entirely colorless, and the flame should be removed before the evolution of oxygen-from the residual hydrogen peroxide-

I4 By 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 transferring the material at any time.


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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 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 magnesium, 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 preliminary 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-


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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 subsequent 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

ma. m7.

0.52 0.54 0.87 0.86 1.12 1.11

TABLE VI

Analysis of Known [email protected] 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

m9.

0.200 0.200 0.200* 0.400 0.400

Ca found

ma.

0.193 0.200 0.200 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 phosphorus, 145 mg. of sulfur, and 110 mg. or more of sodium chloride


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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. Methods of effecting this separation under the most extreme conditions 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 alkalimetric titration of the oxide.


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Cyrus H. Fiske and Milan A. LoganURINE

APPLICATIONS TO THE ANALYSIS OF PHOSPHATE, AND SULFATE, WITHTHE PRESENCE OF MAGNESIUM,

THE PRECIPITATION OF CALCIUM INBY ALKALIMETRIC TITRATION: II.

THE DETERMINATION OF CALCIUM

1931, 93:211-226.J. Biol. Chem.

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