Flame Photometric Determiriation of Cakiwrn in Biological Material Effect of Low Level Impurities from Calcium Oxalate Precipitation T. Y. TORIBARA, PRISCILLA A. DEWEY, and HUBER WAI!I.IER Department o f Radiation Biology, School of Medicine and Denfidry, Universify o f Rochesfer, Rochester, N. Y.
A routine procedure for the determination of calcium in the serum from abnormal and normal human patients has been devised. The calcium, separated as the oxalate from a deproteinized solution, is measured in a flame photometer. The flame emission is affected by small quantities of ammonium salts which might b e carried from the separation procedure and by the type of acid used to dissolve the calcium oxalate. Small losses of calcium are attributed to the solubility of calcium oxalate. By a standardization of procedure, all the variables can b e combined into a single correction factor.
F
of the ultrafilterability of calcium in the serum of human patients, it was desired to measure calcium with a maximum of accuracy on a minimum of sample. A comparison was first made between the direct titration of pure calcium solutions with ethylenediaminetetraacetate and the flame photometric method. The titration method, using either visual or photometric end points, gave results with a spread of 10% from the true values. The flame photometric method, on the other hand, gave results which were all within 2% of the true values on similar samples. Because the use of pure calcium solutions for flame photometry gives the maximum sensitivity, it was decided to isolate the calcium as the oxalate from all samples prior to analysis. This eliminated possible difficulties in the determination of calcium in sera obtained from human patients nith disturbances in phosphorus and protein levels. An additional source of difficulty was eliminated by deproteinizing the sera before precipitation of the calcium oxalate. I n checking the proposed procedure with known quantities of calcium, it was noted that the flame emission was not as great as would be expected from a solution of essentially pure calcium oxalate dissolved in dilute hydrochloric acid. Further investigaOR THE STUDY
540
e
ANALYTICAL CHEMISTRY
tions showed that miall concentrations of certain impurities introduced during the preparation of serum samples for calcium determination may have pronounced as well as u iexpected effects on the flame emission of calcium. EQUIPMENT AND MATERIALS
The instrument u,;ed was a Weichselbaum-Varney flr,me photometer equipped with an RCA 1P21 photomultiplier tube. T i e wave length selected was 620 mp. A stock solution of calcium n-as made to contain 1.000 mg. of calcium per ml. by dissolving 2.4970 grams of Iceland spar in a minimurr volume of dilute hydrochloric acid and diluting to 1 liter n-ith distilled rrater. Further dilutions were made from this stock solution to obtain the standarc solutions for the flame photometer. Reagent grade chemicals mere used to inake other solutionri. The ammonium salt solutions used in this study were made by weighing out the solids according to the formulas given on the bottle. Hydrochloric, nitric, and perchloric acid solutions mere made by dilution of the concentrated acids with no further standardization. PR0CEI)URE
Separation and Determination of Serum Calcium. Pi?et 2 ml. of serum into a 10-nil. volunetric flask. Add 6 ml. of 10% trichlcroacetic acid and dilute to the niailc with distilled water. Shake thoroughly and transfer niost of the contents t o a 15-ml. conical centrifuge tube. Spin a t 2000 r.p.m. for 10 minute:; t o separate the protein. Pipet 5 ml. of supernatant liquid into a 15-ml. conical centrifuge tube, add 1 drop of bromcresol green and 1 ml. of 4y0 arimonium oxalate. Then add nmmoniuni hydroxide dropwise until the indiator turns blue. Place in an ice bath or refrigerator for 1 hour or more. Spin a t 2000 r.p.m. for 10 minutes and remove the supernatant liquid by suction using a capillary tube with a cuned tip. (Tilt the centrifuge tube after most of the supernatant liquid has been removed.) Wash
the precipitate with 1 ml. of 1% ammonium oxalate and repeat the ceiitrifugation procedure. Dissolve the precipitate by adding 0.5 ml. of 0.514' hydrochloric acid and placing the tube in a beaker of hot water. Transfer the calcium solution to a 25-m1. volumetric flask to which 0.5 ml. of 1% Sterox (A. S. Aloe Co., St. Louis, 1x0.) has been added. Dilute to the mark with distilled water. Measure in the flame photometer using a calcium solution containing 4 p.p.m. to set the instrument a t SO and a solution containing Sterox but no calcium to set the zero reading. Solutions of 100 y of calcium precipitated as the oxalate in the above manner and treated as described gave an average reading of 73.8. The amount of calcium in the serum is calculated as folloms: (reading) X 10 = mg. Ca/100 ml. of serum 73.8
Flame Photometer Reading. The technique involved .in operating a flame photometer IS a somewhat arbitrary one. Within certain limits a number of different results can be obtained depending on when the meter is read. For many solutions the reading quickly attains a maximum value when the solution is first introduced into the flame, then gradually drifts as aspiration is continued and assumes a steady position which may be 10% or more lower than the initial reading. Other solutions show no such drift but quickly rise to a steady value. Consistent readings may be obtained only by the proper standardization of manipulations, such as introduction of solution into the flame, duration of aspiration into the flame, and duration of interval between successive introductions into the flame. The meter reading requiring the least standardization of technique and, therefore, the one least subject to personal variation, was found to be the lowest value. The possibility of drift in the instrument from the time i t was standardized until the sample mas read was reduced by standardization of the instrument before and after the sample was read. Only when the standard solution gave the same reading before and after the reading of the sample mas the sample reading recorded.
Table 1.
(Solutions contained 4 p p m . of calcium) % Emission of Solutions Containing HC1 HNOa HC104 98.4 101.7 100 97.8 103.7 114 95 104 119 95.6 106.6 120 95.3 105.3 120
hlolarity 0.001 0.004 0.01 0.04 0.10
Table
SHhC1 0 0 0 0 0 0 0 0.0001 0.0001 0.0002
0.0002 0.0002 0.0002
Effect of Acids on Calcium Emission
II.
Normality H,C,O, (XH4)GOr 0 0 0 0 0 0.0002 0.0002 0 0.0002 0.0002 0 0 0.0002
0.0002 0.0009
0.0018 0.003G 0.009 0
0 0 0 0 0 0 0
Samples of 100 y of calcium carried through tlie serum calcium precipitation procedure gave significantly less emission tban did standard solutions containing corresponding quantities of calcium diluted with water. I n investigating the iollowing factors that contribute to lower ilanie emission for calcium, the same pipet was used to measure all the samples, and deterniinations.were made .In duplicate.
a b c
d
105 107
Effect of Ammonium and Oxalate on Calcium Emission (Solutions contained 4 p.p.m. of calcium)
VARIABLES AFFECTING FLAME EMISSION
$ample
HzCzO4 100
Treatment for 25-hI1. yo of Final Volume Standard Dilution with water 100 Precipitation as oxal91 ate Dilution with 0.OliV 9G HC1 Dilution with HC1 93 and ammonium oxalate
Snniple c was diluted to 25 ml. with distilled water and sufficient hydrochloric acid to make the final acid concentration 0.01N. Sample d mas diluted to 25 ‘ml. with distilled water, She same amount of acid as c, and an amount of amnionium oxalate equivalent to the calcium present. These results indicate that hydrochloric acid and ammoniuni oxalate depress the flame emission of calciuni appreciably, and also that less than 100% of the calcium is precipitated with oxalate. Influence of Different Acids. Because the calcium oxalate precipitate must be dissolved in acid for the flame photometric determination, i t 5s importaiit to know the effect of the
HC1 0.01 0.01 0.01 0.01 0.01 0 0.01 0 0 0 0
0.01 0.01
%
Emission 91.3 S3.8 83.4 86.3 100
100 96.4 100
100 93.4 93.8 90
90.6
acid on the emis$ion. The effect of adding different quantities of acid t o the same quantity of calcium is shown in Table I. The results, expressed as per cent of the net reading of a solution containing 4 p.p.m. of calcium with no excess acid added, are soniewhat deceiving. Hydrochloric, nitric, and perchloric acids up t o 0 . l U give no emission a t 620 mp, but a 0.lili oxalic acid solution gave an emission 19yoof the emission of a 4 p.p.m. calcium solution. The values shown in the table are net values. Of especial interest is perchloric acid, which increases the calcium emission markedly although the acid itself gives no emission. The solutions used for Aame photometry are about 0.01M in acid, and it can be seen that such a concentration has imparted a near-maximum effect. Influence of Ammonium Salts. Because the calcium is precipitated with ammonium oxalate and the precipit a t e is washed with dilute ammonium oxalate, it is important to know the effect of any ammonium oxalateremaining with the precipitate. T o eliminate the possibility of precipitation of the calcium when the ammonium oxalate was added, each of these solutions was made 0.01M in hydrochloric acid. The results (Table 11) demonstrate that the effect of ammonium oxalate is not only pronounced a t low concentrations, but also goes through a maximum (as evidenced by a minimum emission) as the concentration of ammonium oxalate is increased, The data in the second part of the table may be used to break up the effect
of aminonium oxalate into the effects of the separate ions. The oxalate has no effect, as exactly the same results are obtained in the presence or absence of oxalic acid. This attributes the properties affecting flame emission of ammonium oxalate to the ammonium ion. These data also show that a t an ammonium chloride concentration of 0.0002Ar and a hydrochloric acid concentration of O.O1hr, the effects are additive. Further [studies were made on the effect of the ammonium ion by using different concentrations of the chloride and the nitrate vith and without acid present (T:tble 111). The effects are similar to those observed for ammonium oxalate, the greatest being observed near O.OO1hr. The effect of 0.01N acid is interesting. From the data in Table 11, i t appeared that the depressing effects of ammonium chloride and hydrochloric acid were additive. However, as the ammonium concentration is increased, the effect of the same quantity oi acid changes from one of depression to elevation, and finally to no effecl, when tlie acid and ammonium salts are present in equivalent amounts. Although hydrochloric and nitric acidri were shown to have small but opposite effects when added to calcium solutions, both acids acted similarly in the presence of animonium salts, CALCIUM RECOVERY BY OXALATE PRECIPITATION
Solubiliiy Losses. I n the usual micromethods for the determiiiation of calcium, it is assumed t h a t oxalate completely precipitates all the calcium (6). The present work indicates that less than 100% of the calcium is precipitated as the oxalate. The existence and extent of solubility losses x-ere shown by the following two experiments. The substances which affect the flame emir sion mere removed after the calcium o:ralate mas precipitated, and then the calcium content was measured by flame photometer. Two separate runs were made in which eight samples containing 100 y of calcium mere precipitated v-ith ammonium oxalate in Vycor centrifuge tubes. After the usual centrifugation and washing, four of the samples \\ere placed in the muffle furnace a t 500’ C. to convert the oxalate to carbonate. The carbonate mas dissolved in hydrochloric acid, the excess acid mas removed by evaporation to dryness, and the remaining calcium chloride was diluted to 25 ml. The other four samples Tvere treated in the usual manner of dissolving the calcium oxalate in 0.01N hydrochloric acid. In the two runs the calcium oxalate solutions gave averages of 91.0 and 92.5% emission 8,s compared to 96.3 and 96.0% emission for tlie ignited precipitates. VOL. 29, NO. 4, APRIL 1957
541
Table 111.
Normality, NHaClor NHaN03 0.0001 0.0002 0.0004 0.0008
0.001
0.0012
Effect of Ammonium Salts on Calcium Emission
(Solutions contained 4 p.p.ni. of calcium) % Emission of Solution Containing ”IC1 “,NO3 T\”,Cl 0.01N HCl T\TH4h’03 0.01N “03 100 ... 99.4 96.9 93.8 90.0 91.9 87.5 82.1 80.0 84.3 84.4 80.6 81.9 ... 81 .o
...
78.8
79.4 86.3 100
0.010
86.3 100
0.100
110
0,004
0.040
107
0
ANALYTICAL
CHEMISTRY
(“03)
82.2 (HCl)
...
...
88.8 94.4 102 103
...
Need for Protein Removal. The presence of protein partially prevents the depression of the flame emission of calcium caused by phosphate (1). For studies on sera from patients with abnormal protein and phosphorus values, these variables were eliniinated completely by routinely separating the calcium by precipitation as the oxalate. However, in carrying out this separation, other studies (2) indicated that the presence of protein in sonie aged sera from norinal individuals could interfere with the precipi-
82:2
79.4
...
The final solutions of the ignited precipitates should contain only calciuni chloride comparable to the standard solutions. The lower results indicate that tlie calcium was not precipitated completely by the oxalate. Precipitations rrere made with the conventional ammonium oxalate solution and one that was presaturated with calcium oxalate. In the usual precipitation of calcium, 5 ml. of a deproteinized solution containing the equivalent of 1 nil. of serum (approximately 100 y calcium) is mixed with 1 ml. of a 4% ammonium oxalate solution. This represents an excess of oxalate of more than one hundredfold. It was found that doubling the quantity of oxalate made no measurable difference in the calcium recoveries. I n order to show the effect of using a presaturated ammoriiuni oxalate solution, the volume of the calcium solution was reduced markedly, and the amount of animonium oxalate solution was increased. A quantity of 100 y of calciuni in 1 ml. n-as mixed with 2 ml. of 4% ammoniuni oxalate solution. The precipitates were centrifuged, washed, separated again by centrifugation, and dissolved in 0.01N hydrochloric acid for flame photometric determination. I n a coniparison run the average emission for four samples precipitated by tlie usual 4% ammonium oxalate solution was 91.9% as compared to an average emission of 93.7% for a like number of samples precipitated nith a presaturated ammonium oxalate solution. The data indicate that these conditions may decrease solubility losses, but they would not be practical for routine purposes.
542
+
+
90.0 94.4 101 103
tation of calcium n hen ainnionium oxalate was added di-ectly to sera. The present studies sho s that erratic results may also be obtained by precipitating calciuni in fresh sera obtained from patients with grossly abnormal serum protein values. In Table IV arc shown a series of calcium determinations on the sera of a patient rvith 130eck’s sarcoidosis. The direct precipit2,tion mas carried out by another laboratory which used the indirect nieasurement of calcium by titration of the oxalate. The precipitation of calcium oxalate from the trichloroacetic acid filtrate (proteinfree solutions) of the serum was carried out in this laboratory, and the calcium was measured directly by flame photometer. In the tima interval studied, the patient was urlder treatment for reduction of his calcium level, and tlie clinical laboratory results would indicate success except for the inexplicable rise shown by the last two samples. Concurrent studies of another nature in this laboratory 011 the same patient indicated that the calcium level during the same interval never changed. The higher rejults mal be explained by possible contamination of the precipitate with magnesiuin oxalate (6) or other reducible niai,erial, and the lorn results by incomplcte precipitation.
‘Table IV. Compariwn of Results from Different Laboratories Using Different Procedures
Date 1/20 1/24 1/28
2/1 2/2
2/3 2/7 3/ 2
Mg. Ca/100 11.11. Direct pptn. Pptn. from mith oxalate motein-free titration + solution 13 5
12 9
10 8
9 2
11 6 11 4
11.3
11.2 11.3
11.3
To obtain a better comparison between the two methods of precipitating the calcium, the sera from trvo abnormal patients were studied in this laboratory. The results are shown in Table V. I n all cases the calcium was determined directly by flame photometer after solution of the calcium oxalate. J was the same patient with Boeck’s sarcoidosis, and the results on his sera show that direct precipitation can give erratic values which tend to be lo~v. Based on the present studies, the routine procedure for the determination of calcium includes a protein precipitation with trichloroacetic acid followed by precipitation of the calcium with animonium oxalate. DISCUSSION
Certain variables must be controlled in the flame photometric determination of calcium, even if the calcium is first separated by precipitation as the oxalate. Of interest are tlie effects of certain compounds on the emission of calcium, especially when these conipounds by themselves have no emission a t the wasre length employed.
Table
V.
Patient
Effect of Protein on Procedure
ME. Ca/100 M1. Pptn. from Direct protein-free PPtn. solution
Acids have been reported previously (3) to have no effect below 0.02N for sulfuric and nitric acids, and no effect below 1 . O N for hydrochloric acid. Other workers (4) have reported that higher intensity readings could be obtained when carbonates were dissolved in perchloric acid rather than nitric or hydrochloric acids, but no quantitative data were given. Although the results of Table I indicate that measurably different results would be obtained if nitric were substituted for hydrochloric acid in the solution of calcium oxalate, the results of Table I11 indicate that small quantities of ammonium salts have a decided modifying effect on the acids. To check these observations, 18 samples containing 100 y of calcium each were precipitated with ammonium oxalate and treated in the usual manner. The six samples dissolved in 0.01N hydrochloric acid gave an average emission of 90,6% of a standard sample compared to 91.5% for samples dissolved in nitric acid and 113.5’% for samples dissolved in 0.0lAT perchloric acid. Apparently, sufficient ammo-
niuni salts remained in the precipitates to modify the effects of the acids so that little difference existed between hydrochloric and nitric acids. The magnitude of the difference between perchloric and other acid solutions is striking. The effectiveness of small conceiitrations of ammonium salts indicates the importance of thoroughly removing the wash solution after the final centrifugation of the calcium oxalate precipitate. A volume of 0.04 ml. (about 2 drops) of the 1% ammonium oxalate solution diluted to 25 ml. (the final volume for flame photometry) results in a solution 0.0002N in ammonium ion. Previous work ( 1 ) stated that amnioniuni ion (from 1.8 to 36 p.p.m.) had no effect on the calcium emission of a diluted serum to which sufficient sodium had been added to bring the level to 5 nieq. per liter. I n the same work, samples in which the calcium was first separated as the oxalate were prepared for flame photometric analysis by the addition of 5 nieq. per liter of sodium in order to use the same standard solutions, and no unusual effects were observed. Apparently the modifying effects of tlie various other ions and protein in the diluted serum and the large amount of added sodium in the separated calcium oxalate solution masked the phenomena noted in this work, where the system was kept as
pure as possible. Magnesium in quantities up to 10 times the calcium content had no effect in the present work. The data clearly show that, during the preparation of serum samples for the flame photometric determination of calcium, certain unavoidable errors are introduced which affect the total flame emission. Froin a practical standpoint i t is possible to make a simple correction which consolidates both the solubility loss and the effect of ionic impurities. Two standards each containing 100 y of calcium are included with each group of sera or ultrafiltrates and carried through the precipitation and separation procedures. The flame photometer is standardized with a solution prepared by diluting the standard calcium solution to a concentration of 4 p.p.m. with distilled water. For 35 of the 100-7 samples treated exactly the sanie as the sera, the flame photometer readings were 92.3 f L7Y0 of the reading obtained from the standard solution. The true calcium contents of the sera and ultrafiltrates can then be calculated from the flame photometric value multiplied by 1.083. This method provides an exact standard for comparison because each solution is essentially pure calcium oxalate dissolved in the same quantity of hydrochloric acid. Although small quantities of several
ions added to a pure system produced unusual and appreciable effects, tlie nonadditive nature of these effects made i t difficult to predict the net result of a mixture of different ions on the flame emission of calcium. By keeping the additions to the solutions for flame photometry to a minimum, a maximum sensitivity to small changes in calcium concentration mas obtained and unnecessary complications were avoided. The technique of using a spectroscopic buffer was considered but not used, and only the acid necessary to dissolvc the calcium oxalate was added. ILITERATURE CITED
(1) Chen, P . S., Jr., Toribnrn, T. Y., AXAL.CHEM.25, 1642 (1953). (2) Ibid., 26, 1987 (1054). (3) Fearless Camera Corp., Scientific Instrument Division, Los Angeles, Calif., Bull. 151-A. (4)Hinsvark, 0. N., Wittwer, S. H., Sell, €3. YI., ASAL. CHEM. 25, 320 (1953). (5) Kirk, P. L., “Quantitative Ultramicroanalysis,” p. 152, TT7iley, NC\T York, 1950. (6) Smith, R . G., Craig, P., Bird, E. J., Boyle, A. J., Iseri, L. T., Jacobson, S. D., Myers, G. B., Ant. J. Clin. Pathol. 20, 2G3 (1950).
RECEIVED for review August 13, 1956. Accepted December 5, 1956. Work performed under contract with the U. S. Atomic Energy Commission a t the University of Rochester Atomic Energy Project, Rochester, N. Y.
Volumetric Determination of Sulfate by Titration of Excess Lead Nitrate with Potassium Chromate Using Siloxene Indicator FREDERIC KENNY, R. B. KURTZ, INGE BECK, and IRENA LUKOSEVICIUS Department of Chemisfry, Hunter College, New York,
,Sulfur in solution a s sulfate is precipitated with a large excess of carefully measured standard lead nitrate solution. T h e excess lead is then titrated in a dark chamber with standard potassium chromate. T h e sudden change in potential at the end point causes siloxene indicator to emit light, which is measured by a multiplier photometer. This new method makes possible the accurate determination of sulfur in less than 2.5 hours. Unknown samples, differing in sulfur content from the standard samples by as much as loyo,have yielded good results. Larger differences have not yet been investigated.
T
N. Y.
gravimetric determination of sulfur as barium sulfate is time-consuming and often subject to considerable error because of coprecipitation and loss of precipitate. In the method discussed here, the sulfate to be determined is precipitated as lead sulfate by the addition of a measured amount (known to be in excess) of standard lead nitrate solution. The excess lead is then titrated in a dark chamber (8, 10) with standard potassium chromate. The end point is reached when the lead has been quantitatively precipitated. The sudden increase of chromate in the solution a t this point brings about an increase in HE CONVENTIONAL
the potential, which causes the siloxeiie indicator (9, 11) to emit light. The light is measured with a photometer and thus marks the end point of the titration. Chromate concentration and pH are factors in de1;erniining potential. For the quantitative precipitation of lead, the desirable pH range is 1.9 to 3.0 (11). A pH of about 2.85 was used in this work. PREPARATION AND PROPERTIES INDICATOR
OF
A comparison of photometer readings obt.ained in preliminary experiments VOL. 29, NIO. 4, APRIL 1957
543
