ANALYTICAL CHEMISTRY
1642 of certain cations makes the end point slightly less vivid and may therefore reduce the accuracy slightly. For example, if aluminum is present, the color change a t the end point is from red to pale orange; in the absence of aluminum a pink to yellow change is observed, Table I1 gives typical data obtained in the titration of thorium. Data for the recovery of thorium by mesityl oxide extraction and Versene titration are given in Table 111. LITERATURE CITED
(1) Banks, C. V., and Diehl, ANAL.CHEM.,19, 222 (1947).
(2) Blaedel, W. J., and Malmstadt, H. V., Ihid., 23, 471 (1951). (3) Deshmukh, G. S., and Swamy, L. K., Ihid., 24, 218 (1952). (4) Goetz, C. A., Loomis, T. C., and Diehl, H., Ihid., 22, 798 (1950).
Gooch, F. A., and Kobayashi, Am. J. Sci., 45, 227 (1918). Gordon, L., and Stine, C. R., B N ~ LCHEM., . 25, 192 (1943). Haar, K. ter, A n a l y s t , 77, 859 (1952). Levine, H., and Grimaldi, F. S., Atomic Energy Commission, AECD-3186 (February 1950). Marton, G. R., Atomic Energy Commission, Rept. MC-113, (Feb. 20, 1945). hloeller, T., and Fritz, h’. D., - 1 s . i L . CHEM.,20, 1055 (1948). Smales, A. A , . and Airey, L., A t o m i c Energy Research Establishment, Harwell, England, C/M 131. RECEIVED for review January 26, 1953. Accepted July 16, 1953. Presented a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, ?darch 1953. Contribution No. 260 from the Institute for Atomic Research and Department of Chemistry, Iowa State College, Ames. Iowa. Work was performed in the Ames Laboratory of the Atomic Energy Commission.
Determination of Calcium in Biological Material by Flame Photometry P. S. CHEN, JR., AND T. Y. TORIBARA School of Medicine and Dentistry, University of Rochester, Rochester, N . Y . Calcium determinations in biological material by flame photometry at 620 mp are convenient, rapid, and reliable. Phosphate suppresses the calcium emission, and protein partially prevents the action of phosphate. Studies to determine when correction must be made for phosphate inhibition are reported. Procedures are given for determination of calcium in blood serum, serum ultrafiltrate, and urine.
T
HE use of flame photometry for the clinical determination of sodium and potassium has become common practice. This technique, however, has not yet displaced the classical methods for estimating calcium by oxalate precipitation. The emission spectrum of calcium is relatively weak and therefore is especially subject to interference by the presence of strong emitters such as sodium or potassium. In addition, certain anions such as phosphate (4,7 ) lower the intensity of the emission spectrum. I n the present investigation, a number of the variables encountered in the determination of calcium in biological samples have been studied in detail. With certain precautions, the flame photometric method has proved convenient, rapid, and reliable. METHODS, MATERIALS. AND EQUIPMEYT
The instrument used was a Weichselbaum-Varney flame photometer equipped with an RCA 1P 21 photomultiplier tube and supplied with city gas passed through two ballast tanks to minimize fluctuations in pressure. Although highest readings were obtained at 556 mpLIthe background was also high and more reproducible results could be obtained a t 620 mp. Even a t this wave length the sensitivity was sufficient to permit the direct determination of calcium in diluted serum and urine, obviating time-consuming procedures such as protein precipitation, precipitate washing, and acid-ashing. In determining calcium, the meter is adjusted to zero after a solution containing all the ions except calcium has been placed in the atomizer. The 100% adjustment is made using a solution lvith a known calcium content-e.g., 2 p.p.m.-and selecting the proper sensitivity range. The unknown solution is then placed in the atomizer, and the per cent of the 100% value read on themeter-e.g., 75% of 2 p.p.m. = 1.5 p.p.m.-gives the calcium content. The authors’ experience with calcium has been that the scale is linear with respect to the calcium content of the solution when all other variables are held constant. In the case of an ion that did not give a linear response, it would be neces-
sary to construct a calibration curve by using a number of solutions of different known ion content. Inorganic phosphate in trichloroacetate filtrates and total phosphate in wet-ashed (concentrated sulfuric acid and 30% hydrogen peroxide) specimens were determined by the method of Fiske and Subbarow ( 3 ) . The ultrafiltrate of serum was prepared using the apparatus of Toribara (6). EFFECT OF ALKALI CATIOKS
Sodium. As shown in Figure 1, both the blank and sample readings increase linearly with increasing sodium concentration. Therefore, for routine analyses, the sodium level must be maintained a t some fixed value. Weichselbaum (1, 2 ) has suggested a level of 5 meq. of sodium per liter and this concentration is conveniently and accurately attained by addition of sodium chloride to 1 to 100 dilutions of blood and serum ultrafiltrate, on the assumption that the original samples contain 150 meq. per liter. This assumption introduces negligible error. With urine and other specimens containing variable amounts of sodium, preliminary determinations of this alkali must be made to determine the quantity of sodium chloride to be added to give a final dilution of 5 meq. per liter. Potassium. The effect on a calcium solution is shown in Figure 2. The potassium emission a t 620 nip is 2.0 times that of an equivalent amount of sodium when no calcium is present. I n 1 to 100 dilutions of blood serum, or serum ultrafiltrate, so little potassium is present (0.04 to 0.06 meq. pry liter) that the determination of calcium is not seriouslv affected. However, the concentration of potassium in urine is sufficiently high and variable to be of significance, The correction factor may be determined on the basis of an equivalent sodium effect by multiplying the potassium content by 2.0. EFFECT OF PHOSPHORUS
The depression of the emission spectrum of calcium by the presence of inorganic phosphate has been reported (4, 7 ) . This inter-
V O L U M E 2 5 , NO. 11, N O V E M B E R 1 9 5 3 Table I.
Effect of Phosphate on Serum and Ultrafiltrate
Apparent Calcium, P.P.M.a (1: 100 Dilution) Direct Dilution6 Oxalate PrecipitateC Serum Ultrafiltrate Serum Ultrafiltrate 0 1 03 0 54 1 01 0 64 0 85 0 52 1 68 1 02 0 51 0 85 0 54 0 80 0 54 16 8 a Read against standards rwntaining no phosphate. 5 Diluted serum already contained 2.08 p.p.m. total and 0.48 p.p.m. inorganic phosphorus. Diluted ultrafiltrate contained 0.54 p.p.m. total and 0.49 p.p.m. inorganic phosphorus. C Oxalate precipitate contained no phosphate except that which y a s added.
Phos horus Adged, P.P.M.
ference has been confirmed, but the action of phosphate has proved to be complex and is modified by a number of factors. Representative data are presented in Figure 3. The addition of increasing amounts of inorganic phosphate to a solution of calcium chloride (1 p.p.m. of calcium) in distilled water gave decreasing emission readings and the maximum inhibition of about 25% was reached a t a level of about 1 p.p.m. of phosphorus (calciumphosphorus ratio of 0.775). Similarly, the addition of ester phosphate (sodium @'-glycerophosphate) also reduced the reading gwen by 1 p.p.m. of calcium. but greater quantities of organic phosphorus were required to produce both just detectable and also maximal spectral inhibition. In biological solutions, the presence of protein prevents, in part, the inhibitory action of phosphate. Therefore, an extremely large amount of phosphate must be added to serum to cause any depression of the calcium emission, whereas the absence of protein in ultrafiltrate allows maximum suppression with the phosphate normally present (no further depression is caused by addition of phosphate). This is shown in Table I, where a comparison is made between calcium isolated from the ultrafiltrate by alkaline precipitation of the oxalate and that determined directly, In urine, too, there is sufficient inorganic phosphate to give maximal suppression of readings, as shown by the following experiment. A urine sample diluted 1 t o 100 contained 2.25 meq. per liter of sodium, 0.95 meq. per liter of potassium (total alkali effect in terms of sodium = 2.25 2 X 0.95), and 7.3 p.p.m. of phogphorus. Bfter the sodium effect had been adjusted to 5 meq. per liter, uncorrected analysis, using pure solutions as standards, a calcium content of 175 p.,p.m. of undiluted urine was given. When the calcium was precipitated by the addition of ammonium oxalate, dissolved, and then determined by the flame photometer. a value of 236 p.p.m. of undiluted urine was obtained. The calcium emission in the urine sample was depressed 26%, the same depression obtained when phosphate is added to a solution of calcium.
+
The partial prevention of phosphate suppression by protein as shown in Table I and Figure 3 was further demonstrated. In
1643
Table I1 are the results obtained before and after precipitation of the proteins by trichloroacetic acid. Here, removal of protein resulted in phosphate inhibition and much lower readings. Insignificant amounts of calcium were carried down in the protein precipitate. The effect of trichloroacetic acid, in the concentrations used, on the spectrum of a calcium chloride solution was negligible.
Z -
I
5
60-
-I
3 w l-
0
40-
a
-1
0
K w
20
INORGANIC PHOSPHATE
0 GLYCEROPHOSPHATE
-
BLOOD
A SERUM
SERUM ULTRAFILTRATE
I
4
0 $.I
0.3
3.0 6.0
0.6 1.0 PHOSPHORUS
IO
30
p.p,m.
Figure 3. Effect of Phosphorus on Calcium Emission at 620 M p under Various Conditions
The reverse experiment xxs also conducted. By shaking 25 ml. of serum for 3 days with the sodium form of a cationic exchange resin, Dowex 50, the calcium content was reduced to 8 p.p.m. Some of the serum was also treated with ammonium oxalate to remove calcium. The samples for analysis were 1 to 100 dilutions of these sera containing 1 p.p.m. of added calcium, and 0,0.84, or 1.68 p.p.m. of added phosphorus. In all cases, the flame photometer indicated a calcium content of 1.OB p.p.m., showing that serum prot,ein prevents the spectral inhibition of phosphate. -4s a confirmatory test, a known protein mas added to solutions of calcium and phosphorus. A 4% aqueous solution of Armour's crystallized bovine albumin, treated with ammonium oxalate, contained 6 p.p.m. of calcium. Samples for analysis were 1 to 100 dilutions of this solution containing added calcium and phosphorus. The r e s u h , given in Table 111, again show the protection against phosphate inhibition given by protein. NONINTERFERISG IONS
Table IV shows the noninterfering ions. I n concentrations up to 20 times that present in serum diluted 1 t o 100, they produce no detect,able effect on t.he calcium emission. The first figure represents an approximation of the concentrations most likely to be encountered in routine analyses. 9
RECOMMENDED ANALYTICAL
ANALYTICAL CHEMISTRY
1644 Table 11. Effect of Protein Removal Apparent Calcium, P.P.M. (1:100 Dilution) Serum Trichloroaoetjc filtrate Trich1oroacet;o filtrate Trichloroacetic filtrate Trichloroacetic filtrate Trichloroacetic filtrate Ashed protein precipitate
1.00 0.72 0.72 0.72 0.72 0.72 0,008
++ 2.1 p.p.m. P 8.4 p.p.m. P + 16.8 p.p.m. P + 33.6 p.p.m. P
Table 111. Effect of Albumin and Phosphorus Phosphorus, P.P.M.
Apparent Calcium, P.P.M. With albumin No albumin
0 1.68 3.36
1.10 1.00 0.98
1.00 0.80 0.80
with distilled water. Dilute aliquots of this solution 1 to 10 or 1 to 20 for flame photometric readings, making certain that all final dilutions contain 5 meq. of sodium per liter (10% calcium Sterox 2). Method 2. If only a small amount of sample is available, or if sodium and potassium as well as calcium are to be determined, an alternative method can be used. In this procedure acidified diluted urine is read directly on the flame photometer. First determine the sodium and potassium concentrations of the acidified urine. Calculate the milliequivalents per liter of sodium and potassium present when the urine is diluted 1 to 100 or 1 to 200. Using one of these dilutions, add sufficient calcium Sterox 2 to bring the final concentration of sodium plus 2.0 times potassium to 5 meq. per liter. Compare on the flame photometer against standards containing 1 p.p.m. of phosphorus or read against standards containing no phosphorus and correct by a predetermined phosphate quenching curve similar to Figure 3. DISCUSSION
Table IV.
Noninterfering Ions Ion, P.P.M.
Oxalate Sulfate Ammonium Bicarbonate
0.05- 1.0 0.3 - 6.0 1.8 - 36 6.0 -120
predetermined factors or the addition of phosphate to comparable standards. Calcium in urine is most conveniently determined by first isolating as the oxalate to eliminate all interferences and correction factors. Reagents. Stock Calcium, 1.00 mg. per ml. Dissolve 2.4970 grams of Iceland spar in a minimum volume of dilute hydrochloric acid and dilute to 1000 ml. with distilled water. Substock Calcium. Dilute 10 ml. of stock solution to 1000 ml. The substock calcium concentration is 10 p.p.m. Sodium Chloride, 100 me per liter. Dissolve 5.845 grams of dried reagent grade sodium %loride and dilute to 1000 ml. of solution. Sterox. Dissolve 5 grams of Sterox SE (Monsanto) in 500 ml. of water. A touch of caprylic alcohol on the end of a stirring rod can be used to break up the foam incurred in preparing this solution or any of the subsequent solutions. Calcium Standards. In 500-ml. volumetric flasks, place 25 ml. of sodium chloride solution 10 ml. of Sterox solution, and calcium substock to obtain on dilution the desired calcium concentration. Working concentrations are 0 to 2 p.p.m. of calcium. Prepare similar phosphate-containing standards by effecting a 1 p.p.m. concentration of phosphorus with diammonium hydrogen phosphate. Calcium Sterox. Dissolve 5.117 grams of dried reagent grade sodium chloride in a 500-ml. volumetric flask with 5 grams of Sterox and dilute to volume. One milliliter of this solution diluted to 50 ml. (2% solution) has a concentration of 3.5 meq. per liter of sodium. Calcium Sterox 2. Dilute 143 ml. of the above solution to 500 ml. with distilled water. A tenfold dilution of this solution has a concentration of 5.0 meq. per liter of sodium. Procedure. Blood Serum. Dilute 0.5 ml. of serum and 1 ml. of calcium Sterox to 50 ml. Read against standards Containing no phosphorus. Serum Ultrafiltrate. Dilute the same as serum. but read against the standards containing phosphorus (1 p.p.m.). An alternative method is to read the diluted ultrafiltrate against the working standards used in the serum analysis and then correct for phosphate quenching, using a predetermined phosphate quenchin curve similar to Figure 3. The reading will be raised about 2 5 k after correction. URINE,Method 1. Place 1 to 5 nil. of acidified urine (add concentrated hydrochloric acid to a yellow color of bromocresol green) in a 15-ml. conical centrifuge tube. Add 2 ml. of saturated ammonium oxalate and ammonia until a green color is obtained and allow to precipitate for several hours. Centrifuge and carefully aspirate off the supernatant liquid. Wash the precipitate once by adding 2 ml. of 2% ammonia, followed by centrifugation and careful aspiration. Dissolve the precipitate in a few drops of dilute hydrochloric acid and transfer to a volumetric flask. The size depends on the amount of precipitate. Usually 10 times the volume of the urine sample is a good first approximation. Wash all the dissolved calcium carefully into the volumetric flask and dilute to volume
.
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The direct determination of the calcium content of diluted serum and serum ultrafiltrate requires only a small (0.5-ml.) sample. The potassium content of the serum is so low that it doeq not lead to serious errors (about +2%) and the sodium concentration is constant enough to allow easy compensation. Because of the great variability of concentrations of sodium and potassium in urine and the availability of large amounts of sample, LMethod 1, involving a preliminary oxalate precipitation, is preferred to the direct dilution technique. This step removes the interfering sodium, potassium, and phosphate ions. While it has been shown spectrographically ( 6 ) that ordinarily about 10 to 15% of the oxalate precipitated at pH 5 is due to magnesium, the presence of magnesium will not interfere with the flame photometric procedure. Extensive washing of the precipitate is unnecessary, as oxalate does not interfere. As long as all the calcium is precipitated, dissolved, and quantitatively transferred, the results will be accurate. The analysis of specimens of urine containing albumin or protein would be difficult to evaluate if the direct method were used and, in such a case, the oxalate precipitation method would be almost mandatory. For precise measurements, making frequent alternating readings of standard and unknown d l give a precision of &lyoas claimed by Weichselbaum (1) for this instrument. In routine laboratory analysis =!=3to 4% is easily obtained. The accuracy of the method is of the same order as the precision, as it is a direct comparison method in which readings of an unknown are compared with a standard calcium solution. Despite the interference problems encountered in the flame photometric determination of calcium, the procedure still remains rapid, accurate, and sensitive. In many respects flame photometry surpasses the conventional methods of calcium determination. ACKNOWLEDGMENT
The writers wish to acknowledge the assistance of W. F. Xeuman in the organization of this work. LITERATURE CITED
(1) Fearless Camera Corp., Scientific Instrument Division, Los Angeles, Calif., Bull. 151-A. ( 2 ) Fearless Camera Corp., Los Angeles, Calif., “WeichselbauniVarney Cniversal Spectrophotometer Manual of Operation
and Maintenance.” (3) Fiske, C. H.. and Subbarow. Y.. J . B i d . Chem., 66, 375 (1925). (4)Mosher, R. E., Itano, AI., Boyle, A. J., Myers, G. B., and Iseri. L. T., Am. J . Clin. Pathol., 21,75 (1951). (5) Smith, R. G., Craig, P., Bird, E. J., Boyle, A. J., Iseri, L. T., Jacobson, S. D., and Myers, G. B., Ibid., 20,263 (1950). (6) Toribara, T. Y.,AXAL.CHEM.,25, 1286 (1953). (7) Zak, B., XIosher, R. E., and Boyle, A. J.. Am. 6.C l i n . Pathol., 23, 60 (1953). RECEIVED for review April 28, 1953. Accepted August 14, 1953. Based on work performed under contract with the United States Atomic Energy Commission a t the University of Rochester Atomic Energy Project, Rochester, N. Y.
