THE DETERMINATION OF CALCIUM IN THE PRESENCE OF MAGNESIUM AND PHOSPHATES: THE DETER- MINATION OF CALCIUM IN URINE.

BY FRANCIS H. McCRUDDEN.

(From the Hospital of the Roclcefeller Institute for Medical Research.)

(Received for publication, August 3, 1911.)

A year ago I published the details of a very accurate method for the quantitative estimation of calcium and magnesium in the presence of phosphates and small amounts of iron.1 In the devel- opment of the method, accuracy was the first consideration, sim- plicity and rapidity having been deemed of secondary importance. During the past winter, in the course of investigations in which many calcium determinations were necessary, a more rapid method appeared desirable. The new method, therefore, has been studied with a view to ascertain to what extent its details could be modi- fied in the direction of ease and rapidity of execution without essential loss of accuracy. The present paper gives a report of these investigations. Part I deals with pure solutions; Part II with modifications advisable in the case of urine.

PART I. THE DETERMINATION OF CALCIUM IN THE PRESENCE OF

MAGNESIUM AND PHOSPHATES.

The method as previously published is carried out as follows:

The solution is brought to a volume of about 100 cc. and two drops of alizarin red are added. Ammonia is then added drop by drop until the red color of the alizarin just disappears (or, in the case of urine, until a pre- cipitate of phosphate just appears). Dilute hydrochloric acid is then added drop by drop until the red color of the alizarin just reappears (or, in the case of urine, until the precipitate of phosphate just disappears). If the solution has become warmed by the neutralization, a few drops of acid

‘This Journal, viii, p. 33, 1910.

187

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188 Determination of Calcium

in excess should be added to insure complete solution of the calcium phos- phate, the solution cooled and the neutralization repeated. After the solution is just acid to alizarin, 10 cc. of G hydrochloric acid and 10 cc. of 2.5 per cent oxalic acid are added and the solution is brought to the boiling point and kept gently boiling until the calcium oxalate is coarsely granu- lar. The flask should be kept covered with a watch-glass to prevent spat- tering. Three per cent ammonium oxalate is then added, a few drops at a time, to the boiling solution, waiting after each addition until the result- ing precipitate has become coarsely crystalline. The amount of ammonium oxalate to be added depends on the amount of calcium in solution. Twice the amount necessary to combine with all the calcium is sufficient. After the calcium oxalate has become coarsely crystalline and has settled to the bottom of the flask, it should be frequently stirred up in the liquid to pre- vent the latter from boiling over suddenly. After the precipitate has be- come crystalline and has settled, the solution is allowed to cool to room temperature. When cold, 8 cc. of 20 per cent sodium acetate solution are added to it slowly and with constant stirring. The solution is then allowed to stand in a cool place from four to eighteen hours, filtered cold and washed free of chlorides with cold 1 per cent ammonium oxalate solution. The pre- cipitate is allowed to dry and then incinerated with the filter paper in a plati- num crucible. It is finally heated in a blast lamp to constant weight. Details for the determination of magnesium need not be given here.

The points investigated were the following: (1) The possible substitution of ordinary concentrated hydrochloric acid for 4 hydrochloric acid; the possible omission (2) of ammonium oxa- late; (3) of oxalic acid; (4) of heating; (5) of standing over night; (6) the possible substitution of shaking for either heating or stand- ing over night; (7) the determination of the calcium oxalate by titration with potassium permanganate; (8) the separation and washing of the calcium oxalate by means of the centrifuge instead of by filtration.

Various combinations of these changes were tried. Each change in detail was considered with respect to accuracy, simplicity and rapidity.

A. GRAVIMETRIC DETERMINATIONS.

1. Use of concentrated hydrochloric acid instead of + hydrochloric acid. A number of determinations on different samples of acid showed that ten drops of concentrated hydrochloric acid (sp. gr., 1.20) from a 5 cc. pipette are practically equivalent to 10 cc. of ; hydrochloric acid. Since good check results were always obtained in a large number of determinations in which concen-

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Francis H. McCrudden

trated hydrochloric acid was used instead of $ acid, this modi- fication of the procedure was adopted and used throughout in the analyses reported in this paper.

A solution of calcium chloride was prepared and the calcium in 50 cc. determined by the standard method already described with the following results.’

0.A o.& 0.436

101 102

0.1434 0.1435

The solutions filtered clear and no precipitate formed in the fil- trate on standing. This solution was used in the subsequent analyses numbered 4-15 and 100-110.

2. Omission of boiling and omission of oxalic acid. Analyses numbered 100 and lOOa, were carried out just as were analyses 1, 2 and 3, except that no oxalic acid was added and the solution was not boiled. Nos. 1OOb and 100~ were carried out in the same way except that they were shaken ten minutes after the addition of the sodium acetate instead of standing over night.

100 1OOa 1OOb 1000

0.134!2 0.1203 0.1416 0.1433

These results are very low, partly due to the passage of the very fine precipitate through the filter paper. The next analyses were designed to determine whether the low results were due to the omission of the oxalic acid, of boiling, or of both.

3. Omission of oxalic acid. These analyses were carried out just as were Nos. 1, 2 and 3, except that the addition of oxalic acid was omitted and an equivalent amount of ammonium oxa- late added instead.

0.14432 Oh32 104 104s 105 1058

0.1433 0.1433 0.1430 0.1434

The results are only slightly low, so that the omission of oxalic acid alone does not account for the low results of analyses Nos. 100 and 100a. The precipitate is so fine, however, that it is trouble- some to wash it out of the flask and it clogs the filter paper so

1Certain observations were checked by independent determinations by 5 second chemist (Miss Fales). These check analyses are designated by numbers above 100.

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190 Determination of Calcium

that the liquid filters very slowly. Sometimes a slight cloudiness is observed in the filtrate; this in time settles out a little and can be seen when the filtrate is shaken. This is probably due to the fraction of a milligram of calcium oxalate which passes through the paper and is responsible for the slightly low results. The fineness of the precipitate can be accounted for by the omission of the oxalic acid. Calcium oxalate is somewhat soluble in an acid solution so that when oxalic acid is,added a part only of the calcium oxalate is precipitated. Boiling makes the precipitate still more soluble. It is a well-known fact that if a crystalline precipitate is somewhat soluble, the crystals grow larger-the smaller crystals going into solution, the larger crystals growing by accretion. And so, in the case of the calcium oxalate, diges- tion in the hot acid solution gives the precipitate an opportunity to form in large crystals, and when the ammonium oxalate and sodium acetate are subsequently added the rest of the calcium oxalate comes down and precipitates partly, at any rate, on the large crystals. The digestion over night brings about further growth of the large crystals at the expense of the fine precipitate.

4. Omission of boiling. Two analyses were carried out just as were Nos. 1, 2 and 3, except that the solution was not heated, precipitation taking place in the cold. The results were:

CL25 0.:420

Although low, they are not so low as the results of Nos. 100 and 100a. The low results of Nos. 100 and 1OOa are therefore not due to omission of either oxalic acid or of boiling alone but to the omis- sion of both. The precipitate is so fine that some may pass through the filter. A filtrate which is apparently practically clear at first may show a fine precipitate on standing over night. Fil- tration is slow for the fine precipitate soon clogs the paper. The fineness of the precipitate is accounted for by omission of diges- tion in the hot liquid as explained above. One advantage in the omission of heating was noted; viz: the precipitate does not stick to the sides of the flask and hence less water is necessary in trans- ferring it to the filter paper.

5. Substitution of shaking for standing over night. Four analyses were carried out as were Nos. 1, 2 and 3, except that the

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Francis H. McCrudden 191

flasks were closed with rubber stoppers and shaken for ten minutes after the addition of sodium acetate instead of being allowed to stand over night.

O&36 o&5 109 110

0.1434 0.1436

6. Shalcing instead of boiling before addition of sodium acetate. When the procedure used in Nos. 1, 2 and 3 was modified to the extent of shaking for ten minutes after the addition of ammonium oxalate and again for ten minutes after the addition of sodium acetate, boiling after the addition of both oxalic acid and ammo- nium oxalate being omitted, it was found thatthe precipitatewas fairly coarsely crystalline, filtration was easy and no precipitate appeared in the filtrate upon standing. Assuming that the results would have been correct, the precipitates were not ignited and weighed.

7. Omission of boiling; shaking instead of standing over night. In analyses which were carried out exactly as were Nos. 6 and 7, (i.e., boiling omitted) except that after the addition of sodium acetate the flasks were closed with rubber stoppers, the solution shaken for ten minutes and filtered immediately instead of after standing over night, a very fine precipitate passed through the paper as in analyses Nos. 6 and 7. For this reasonthe analyses were not finished.

In analyses in which both boiling and standing over night were omitted, the flasks being stoppered and the solutions shaken after the addition of the oxalic acid, after the addition of the ammonium oxalate and again after the addition of the sodium acetate, it was found that the precipitate was coarsely crystalline, filtration was easy and the filtrate remained clear upon standing. Assuming that the results would have been correct, the analyses were not finished. One advantage in shaking as compared with heating is that the precipitate does not adhere to the sides of the flask and hence is easily washed on to the filter. It is a question, however, which is the simpler technique.

The same result was noted in analyses in which boiling and standing over night were omitted, the flasks being shaken for ten minutes twice, viz: after addition of oxalic acid and after addition of sodium acetate:

O&4 O&5

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192 Determination of Calcium

The results are correct, precipitates coarsely crystalline, filtration easy, the filtrates remaining clear on standing. The precipitate does not adhere to the sides of the flask.

The results’ thus far outlined indicate that, in carrying out the method, 10 drops of concentrated hydrochloric acid may be used instead of 10 cc. of $ acid; that the oxalic acid should not be omit- ted; that boiling may be omitted if the solution is shaken for ten minutes after the addition of the oxalic acid; and that standing over night may be omitted if shaking for ten minutes after the addition of the sodium acetate be substituted.

B. TITRATION WITH POTASSIUM PERMANQANATE.

The next experiments were designed to determine whether the calcium could be accurately determined by titration of the oxalate with potassium permanganate. This method is sometimes recom- mended, but as ordinarily carried out it can not give absolutely accurate results. It has been shown by Richards2 and myseW that if the calcium oxalate precipitate be washed with distilled water, some dissolves, making the results low. If the pre- cipitate be washed with dilute ammonium oxalate solution in the manner described, the ammonium oxalate clinging to the pre- cipitate will react with the permanganate and the result will be high. It seemed probable, however, that if the precipitate were washed free of impurities with dilute ammonium oxalate and then washed a few times with very small amounts of cold distilled water, allowing thorough draining after each washing, these errors could be minimized. Accordingly, three sets of analyses were made as follows :

1. Calcium oxalate was precipitated, filtered and washed with dilute ammonium oxalate in the usual manner. The precipitate was then titrated with potassium permanganate to determine how much too high washing with ammonium oxalate makes the result.

‘These conclusions are based not only upon results given here, but also upon similar results of a number of other experiments in which calcium oxa- late was precipitated in these various ways in preparation for the volu- metric determinations. The results given above suffice to bring out the points desired.

2 PTOC. of the Amer. Acad. of Arts and Sciences, xxxvi, p. 277, 1901. ‘Lot. cit.

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Francis H. McCrudden I93

2. Calcium oxalate, precipitated and filtered as before, was washed with cold distilled water. Titration with permanganate indicated the extent of loss caused by the solubility of calcium oxalate in distilled water.

3. Calcium oxalate, precipitated and filtered as before, was washed free from chlorides with 0.5 per cent ammonium oxalate solution. This was followed by washing three times with cold distilled water, each time using only enough to fill the funnel one-half to two-thirds full and allowing thorough draining each time.

For these analyses a new solution of calcium chloride was used. Three determinations of the calcium in 50 cc. by the standard method yielded the following:

0400 o&4 o.lYo2

The results of the three sets of analyses outlined above were as follows :

1. Washing with 0.6 per cent ammonium ozalate.

0445 0.254 2. Washing with distilled water.1

o.& o.& 3. Washing with ammonium oxalate and then with diistilled water.

0202 0.2:501

These results show that calcium may be determined by titra- tion of the oxalate with potassium permanganate if washing with dilute ammonium oxalate is followed by washing three or four times with cold distilled water.

C. EFFECT OF UBIC ACID.

In consideripg the applicability of the method for the estimation of cal- cium in the urine, the possible effect of the presence of uric acid had to be

‘It was noted in these and other analyses that the size of the crystals makes as great or greater difference than the nature of the washing fluid. If the precipitate is coarsely crystalline, even cold distilled water alone may be used for washing. If the precipitate is very finely crystalline, even 0.5 per cent ammonium oxalate carries a little into solution which precipi- tates out later.

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Determination of Calcium

determined. When calcium is precipitated as oxalate in neutral or slightly acid solution and allowed to stand over night, uric acid may be precipitated. Such contamination of the precipitate would not affect the accuracy of the gravimetric method, since the precipitate is ignited before weighing. It would, however, interfere with the volumetric method. The possible pre- vention of the precipitation of uric acid will be discussed later. On the assumption that it is precipitated, as a means of getting rid of it I have thought of washing the calcium oxalate with sodium hydroxide solution. It was found, however, that even fairly strong sodium hydroxide does not easily dissolve the uric acid contaminating a calcium oxalateprecipitate; furthermore, even dilute sodium hydroxide dissolves some of the calcium oxalate. 1 have concluded, therefore, that if calcium oxalate, precipitated from urine, is contaminated with uric acid, as indicated by a reddish color of the precipitate, the calcium should not be determined by titration.

D. USE OF THE CENTRIFUGE.

A number of experiments were undertaken to determine whether it might not be of advantage to use the centrifuge in separating and washing the precipitated calcium oxalate instead of filtering in the usual way. They showed that while accurate results may be obtained by its use, the work involved and time consumed is greater than when filtration is employed.

E. USE OF GOOCH CRUCIBLE OR HARDENED FILTER POINT AND SUCTION.

When Gooch crucibles were tried, it was found that thin layers of abes- tos did not retain the precipitate. Where layers thick enough to retain the precipitate were used, they soon clogged up and prevented passage of fluid.

F. EFFECT OF PHOSPHATES,MAGNESIUM AND SMALL AMOUNTS OF IRON UPON THE RESULTS.

In order to convince myself that the presence of magnesium, phosphates or iron would not affect the accuracy of the calcium determination if boil- ing and standing over night were omitted, the following analyses were made. Nos. 70 and 71 were carried out on a pure calcium chloride solution, Nos. 72 and 73 on the same solution to which were added equivalent quantities of magnesium chloride and sodium phosphate, together with a little ferric ammonium sulphate.

70 0.1377 0.1377 0.1376 0.1377

PART II. THE DETERMINATION OF CALCIUM IN URINE.

The method which has been described in Part I, when applied to the estimation of calcium in urine, gave accurate results. Cer- tain steps in it were, however, found to be unnecessary, even inad-

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Francis H. McCrudden 195

visable, owing partly to the small amounts of calcium, partly to the character of the urine.

Fresh urine from a number of subjects was mixed and the cal- cium content in 200 cc. estimated by the method described last year. The results follow:

1. Omission of ammonium oxalate. Two analyses in which the addition of ammonium oxalate was omitted showed that this reagent was superfluous in the estimation of calcium in urine.

2. Shaking instead of standing over night. It has been shown in Part I that shaking of the solution may be substituted for standing over night when there is considerable calcium present. The fine precipitate which comes down on shaking after the addi- tion of sodium acetate has in that case a large amount of coarsely granular precipitate to which to attach itself. In the case of urine the amount of precipitate that comes down before add- ing sodium acetate is small. Hence shaking for ten minutes is insufficient to give a coarsely granular precipitate. In two analyses, in which this modification was introduced, the calcium oxalate was so finely divided that some passed through the filter. The analyses therefore were not finished.

3. Effect of bo&ng the solution. In all these analyses filtra- tion was slow and washing of the precipitate difficult because the filter became clogged. When boiling was omitted this was not the case. Boiling seems to precipitate a very small quantity of sticky, slimy substance, possibly mucus, which coats the cal- cium oxalate precipitate and the filter paper, impeding filtration. Boiling also seemed to prevent the formation of a coarse precipi- tate, for when the urine was not boiled the precipitate wascoarse enough to be retained by the filter.

It was noted, further, that, if the urine is boiled and then allowed to stand as usual over night, the calcium oxalate precipi- tate is occasionally contaminated with uric acid. In no case did this occur when boiling was omitted. While it is difficult to assign

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196 Determination of Calcium

a reason for this, the point is of interest, for the calcium can be determined by titration with permanganate if it can be precipi- tated free from uric acid.

4. Shaking instead of boiling. Analyses carried out as directed in my previous paper, except that, instead of boiling the urine, it was vigorously shaken in a stoppered flask, gave good results. Filtration was easy and the filtrate remained clear.

&?42 O.&I

5. Shaking instead of boiling: omission of ammonium oxalate. In the analyses, the results of which follow, vigorous shaking was substituted for boiling and the addition of ammonium oxalate was omitted. The results are good, filtration was easy and the filtrate remained clear.

dir42 O.&I

6. Omission of boiling and of ammonium oxalate: shaking instead of standing over night. In the analyses following, a mixed urine was used which gave 0.0275 gram of calcium oxide in 200 cc. by the standard method. It was found that shaking could not be substituted for standing over night if the urine was boiled, because the fine precipitate so obtained passed through the filter. This was not the case when the urine was not boiled as the results of analyses 15 and 16 show.

o.&* OS&

Filtration was easy and the filtrate remained clear on standing. 7. Urine not boiled or shaken. In the case of pure solutions,

in which calcium is fairly concentrated, it has been shown that either boiling or shaking is essential. Neither is essential in the case of urine as is shown by the results of analyses 17- 20 in which both boiling and shaking were omitted. In 17 and 18 ammonium oxalate was added; not in 19 and 20. In all, the urine was allowed to stand over night, after the addition of sodium acetate.

cl.& o.& O.& o.c%l

Filtration was more rapid in those analyses in which ammonium oxalate was omitted.

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Francis H. McCrudden 197

In the analysis of urine, as in the case of pure solutions, the use of hardened filter points or of Gooch crucibles and suction was found to be impracticable. Concerning titration with perman- ganate, the considerations previously outlined are applicable. It is advantageous to use filtered urine for the analysis, and in urines which contain small amounts of calcium, litmus paper should be used in adjusting the reaction in the first step of the analysis.

SUMMARY.

I. For the determination of calcium in pure solutions, which may contain also magnesium, phosphates and small amounts of iron, or in the ash of food or feces, the best procedures as developed in this work are as follows:

SOLUTIONS REQUIRED : 2.5 per cent oxalic acid; 3 per cent ammonium oxalate; 20 per cent sodium acetate.

1. The solution is brought toavolume of 75 to 150 cc. Concen- trated ammonia water is added drop by drop till the solution is just alkaline, as shown either by the appearance of a precipitate of calcium and magnesium phosphates or by the use of an indicator (aliaarin red or litmus paper).

2. Concentrated hydrochloric acid is added drop by drop until the solution is just acid, as shown either by the disappearance of the precipitate or by an indicator. In the presence of iron an indi- cator must be used.

3. Ten drops of concentrated hydrochloric acid (sp. gr., 1.20), approximately equivalent to 10 cc. of ; HCl, are added.

4. Ten cubic centimeters of 2.5 per cent oxalic acid are added. 5. Either of two procedures may here be used: a. The solution is boiled until the precipitated calcium oxa-

late is coarsely crystalline,1 and then an excess of 3 per cent am- monium oxalate is slowly added to the boiling solution and the boiling continued until the precipitate is coarsely crystalline.2

b. The flask, closed with a rubber stopper, is shaken vigorously for ten minutes. An excess of 3 per cent ammonium oxalate is then added.

‘If but little calcium is present, nothing precipitates at this point. *If but little calcium is present, it is not necessary to add oxalate.

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198 Determination of Calcium

6. The solution is cooled to room temperature and 8 cc. of 20 per cent sodium acetate added. (In case of ash of feces, add 15 CC.)

7. The solution may either be (a) allowed to stand over night or (b) stoppered and vigorously shaken for ten minutes.

8. The calcium oxalate is filtered off on a small ash-free paper and washed free from chlorides with 0.5 per cent ammonium oxa- late solution.

9. Either of two procedures may next be followed: a. The precipitate and filter are dried, burned in a platinum

crucible to calcium oxide and brought to constant weight by heat- ing in a blast-lamp.

b. The precipitate is washed three times with cold distilled water, filling the filter about two-thirds full and allowing it to drain completely before adding more. A hole is made in the paper and the calcium oxalate washed into the flask. The volume of the fluid is brought up to about 50 cc. and 10 cc. of concentrated sulphuric acid are added. The oxalate is titrated immediately with standard potassium permanganate.

Determination of magnesium in the filtrate may be carried out as described in my previous paper. Or the filtrate may be evapor- ated to dryness in a porcelain dish after the addition of nitric acid and the residue heated over a free flame until the ammonium salts are destroyed and the residue fuses. After cooling, the resi- due is taken up in water and a little hydrochloric acid, and the magnesium precipitated as usual. Another simple method of destroying the organic matter is to add 3 to 5 cc. of concentrated sulphuric acid to the filtrate and evaporate in a small Kjeldahl flask until the water is removed. The sulphuric acid may then be neutralized with ammonium carbonate and the magnesium precipitated as usual.

In the analysis of ash of feces, carried out by the usual method the calcium oxalate is apt to precipitate in a very finely divided condition. Filtration is slow and loss may result on this account. This difficulty may be obviated as has been indicated, by the addi- tion of 15 cc. instead of 8 cc. of sodium acetate. I have shown that, if the solution is kept cool, no phosphate or magnesium is brought down by this excess of sodium acetate.

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Francis H. McCrudden 199

II. For the determination of calcium in urine, the following method is recommended:

SOLUTIONS NEEDED: 2.5 per cent oxalic acid; 20 per cent sodium acetate.

1. If the urine is alkaline it is made neutral or slightly acid. 2. The neutral or slightly acid urine is filtered. 3. Two hundred cubic centimeters of urine are used for analy-

sis. If only faintly acid to litmus paper, 10 drops of concentrated hydrochloric acid are added.

If the urine is strongly acid, it may be made just alkaline with ammonia and then just acid with hydrochloric acid. If the urine is turbid or contains little calcium, litmus paper should be used in determining these changes; otherwise, the appearance and dii- appearance of a precipitate of phosphates are sufhcientlyaccurate indicators. Ten drops of concentrated hydrochloric acid (sp. gr., 1.20) are then added.

4. Ten cubic centimeters of 2.5 per cent oxalic acid are next added.

5. Eight cubic centimeters of 20 per cent sodium acetate are added.

6. The urine is either allowed to stand over night at room tem- perature or is shaken vigorously for ten minutes.

7. The calcium oxalate is filtered, and washed free from chlor- ides with 0.5 per cent ammonium oxalate solution.

8. a. The precipitate may then be dried, ignited, heated in a blast-lamp to constant weight and weighed as calcium oxide.

b. If free from uric acid, the calcium oxalate may be washed three times with distilled water and estimated by titration with permanganate as described in the outline of the method for pure solutions.

Magnesium is determined in the filtrate just as in the case of pure solutions.

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Francis H. McCruddenURINE

DETERMINATION OF CALCIUM IN AND PHOSPHATES: THE

IN THE PRESENCE OF MAGNESIUM THE DETERMINATION OF CALCIUM

1911, 10:187-199.J. Biol. Chem. 

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