Spectrophotometric Determination of Calcium ROBERT E. SCOTT
AND
C. R. JOHNSON, University of Texas, Austin, Tex. It secms iricvitable a t present that calcium must be precipitated in an insoluble compound prior to practical colorimetric determination. However, this step has several advantages, especially when calcium oxalate is the precipitate. This compound precipitates quantitatively in coarse, easily washed, slowly soluble crystals of definite composition a t pH 3.7 or less (8),and many interferences may be removed by a single precipitation. For use in combination with oxalate in a colorimetric method, permanganate is at least as good as any other reagent. Thus, in the present work calcium was precipitated as oxalate and determined by several variations as permanganate.
Simple and rapid methods for dotermping calcium b y precipitation as oxalate and colorimetric measurement as permanganate are especially suited for use with a spectrophotometer or photoelectric colorimeter. O n c e a calibration curve i s obtained b y adapting one of the methods to a particular instrument, only one or two easily prepared and very stable standard solutions and a few common reagents are required. While the methods d o not avoid precipitation of calcium oxalate, the end point of the analytical reaction is reached automatically. In one method additional speed is gained b y avoiding solution of the precipitated oxalate, in another, filtration, washing, and solution of the precipitate are avoided. A summary of calibration data shows that precision is satisfactory. Run-of-the-mill results are in good agreement with official and standard methods in determination of calcium in waters, brines, food concentrates, baking powders, magnesium concentrates and cell feeds, and chemical reagents.
APPARATUS AND REAGENTS
A Coleman lModel 10-S-30 spectrophotometer with matched square cuvettes 1.308 cm. in depth was used for most of the transmittance measurements, which were made between 28’ and 32’ C., with water as reference liquid. Other instruments were used for comparisons and checks. Volumetric measurement? in the calibrations were made with calibrated 5-ml. and 50-ml. burets; in the analyses, transfer pipets were used. A Beckman Model G meter was used for pH measurements. In method 2 an IEC clinical centrifuge was used. REAGENT1. The standard oxalate solution containing the equivalent of 1.000 mg. of manganese per ml. was prepared by making 5.738 grams of reagent grade oxalic acid dihydrate or 6.099 grams of pure sodium oxalate to 1 liter with distilled water. REAGENT 2. The standard 0.0910 N permanganate solution .contained 2.877 grams of potassium permanganate per liter. After standardization against reagent 1 it was adjusted when necessary to contain 1.000 mg. of manganese per ml. Stock solutions of reagents 1 and 2, prepared in 2- to 3-liter quantities, were found to keep their titer to 1 part in 1000 for over 3 months, when stored in the dark, with occasional use. BUFFERS. The acetate buffer of pH 4.7 contained 68.1 g r a m of sodium acetate trihydrate and 31.3 ml. of 16 N acetic acid per liter. The formate buffer of pH 3.7 contained 31.5 grams of ammonium formate and 20.8 ml. of 24 N formic acid per liter. For method 3a a set of reagents similar to those used by McComas and Rieman (8) was made up: 0.5 M oxalic acid, 1.0 M formic acid, and 1.0 ;If ammonium formate. ACIDSAND BASES. Hydrochloric acid, 6 N , sulfuric acid, 36 N and the volatile acids used in the buffers were purified by distillation for one set of reagents. Other sets prepared with commercial acids of good quality. gave equally satisfactory results in many comparisons. Commercial 36 N sulfuric acid after dilution to 18 N was treated with permanganate to the first faint permanent pink. Not more than 2 mg. of manganese er liter should be nc!ded to the 18 N acid. ,Carbonate-free 20 $sodium hydroxide was made by saturating distilled water with the reagent and allowing t,he sodium carbonate to settle out; 0.1 N a n d 1 N solutions of sulfuric acid and sodium hydroxide were used for pH adjuntments. SALTSOLUTIONS. Saturated ammonium oxalate solution wa;prepared from C . P . crystals. Standard calcium solutions were prepared in 3-liter quantities. Calcium sulfate solutions were made from C.P. material. The calcium content was found as permanganate and calcium carbonate, and the solutions were diluted to contain 0.500 mg. of calcium per ml. Calcium chloride solutions were prepared from salt twice crystallized and centrifuged. The calcium content was found as permanganate, calcium carbonate, and silver chloride, and the solutions were adjusted to contain 1.000 mg. of calcium per ml.
C
OLORIMETRIC determinations of calcium have been based upon final precipitation of the element as alizarinate (7)’ phosphate (2, 3, 6, fd), tungstate (1 f ) , 8-hydroxyquinolate (14, calcium potassium nickelonitrite (fO),oxalate (9, IS), and picrolonate (1 ), The colorimetric comparisons have been made, respectively, with alizarin, molybdenum blue, titanous chloride, Folin’s reagent, the green nitrite-antipyrine color, ferric thiocyanate or iodine, and the red color obtained by treating picrolonic acid with bromine and alkali. While some of these determinations have been widely used ab colorimetric methods for the special purposes for wi.ich they were developed, their general limitations are not very well established. Few of them have been studied spectrophotometrically or adapted for spectrophotometric use. I t was the purpose of the present investigation to develop and test the accuracy of some rapid and reasonably precise spectrophotometric methods, applicable to the determination of small and moderate quantities of calcium. No attempt was made to include the range of microanalysis, for which practically all thr methods cited above were designed. Well adapted as some of these methods may be for microdetermination of calcium, certain faults make them unsuitable for rapid and accurate spectrophotometrlc determination of larger quantities of the element. Some utilize expensive or un9table reagents, double precipitations, or chromogenic agents producing colors which are inhibited, retarded in development, or altered by numerous interfering substances. Others require a multiplicity of reagents, or employ very indirect equivalent relations. Even the micromethods, most frequently used in clinical work, seem to have little promise as general spectrophotometrir methods-for example, the color developed in the calcium phosphatemolybdenum blue procedure is a function of a t least seven variables other than the concentration of calcium, which is found indirectly as phosphorus in calcium phosphate. This latter substance has a somewhat indefinite composition, depending on the conditions under which it is prebipitated. Moreover, it must be formed at a pH between 7 and 12, a requirement unfavorable for the separation of interfering substances commonly occurring with calcium. A recently published method (13) depends upon the extremely indirect relation of calcium to iodine through oxalate and ceric sulfate, and requires ten reagent solutions. The otherwise simple method of Laidlaw and Payne ( 7 ) requires a double precipitation. In general, the micromethods are indirect and timeconsuming, and the special techniques used in handling extremely small quantities of material often lack precision, although their arruracy may be adequate, even if low.
RAPIDCHECKSFOR REAGENTS.One or more of the following procedures may be used for checking the standard oxalate and permanganate solutions (reagents 1 and 2). Measure 4.000 ml. of reagent 1 into a 50-ml. beaker and add 10 ml. of water and 4 ml. of 18 N sulfuric acid. Titrate, either with or without heating the solution, with reagent 2, adding it from a calibrated 5-ml. buret to the first permanent pink. Dissolve 0.2000 gram of pure, dry sodium oxalate in 100 ml. of 1 N sulfuric acid, heat, and titrate with reagent 2 from a 50-ml. buret. Weigh out 1.200 grams of Mohr’s salt, ferrous ammonium sul504
505
ANALYTICAL EDITION
August, 1945
fate hexahydrate, of known factor purity, dissolve in 50 ml. of 1 N sulfuric acid, add 5 ml. of 85% phosphoric acid, and titrate with reagent 2. CHOICE
Table II.
OF W A V E L E N G T H F O R MEASUREMENTS
According to Hantzsch and Clark ( 4 ) dilute permanganate solutions have sharp transmittance minima a t 526.7 and 546.3 mp. In the graphs given by Kasline and Mellon (6) the minima seem to be a t about 526 and 546 mp; these values were also found in this laboratory with a Coleman 10-5-5 double monochromator spectrophotometer. A GE recording spectrophotoheter (10 mp slit) showed minima a t 526 and 544 mp. A Coleman 104-30 double monochromator spectrophotometer showed a single minimum at 529 mp. Any of these wave lengths may be used for spectrophotometric determination of permanganate. For photoelectric colorimetry a yellow-green filter with transmittance maximum a t 530 mp is suitable. Transmittance-concentration measurements made with the Coleman 10-5-30 instrument a t 529 mp with potassium permanganate solutions containing between 0.200 and 2.000 mg. of manganese per 100 ml. showed that Beer’s law holds precisely over this range, the following linear equation applying: C = (-2.512 loglo T
+ 5.018)/1
C is in milligrams of manganese per 100 ml., T is the transmittance in per cent, and .? is the cuvette depth in centimeters. This equation was useful as a fourth means of checking reagent concentrationp rapidly. It cannot be used to check calibration curves obtained by subtraction of permanganate with measured amounts of oxalate or calcium oxalate in sulfuric acid solution. The equilibria established in such systems involve ions intermediate between permanganate and manganous ion, and linear relations cease t o hold. However, the redox buffers comprising the analytical systems used in the present work are stable during the specified period of use, and calibration curves obtained with known amounts of calcium are highly reproducible.
Total Calcium
a b
Calcium as Permanganate by Method 3 Transmittances with Various Instruments and Wave Lengths Coleman Coleman Coleman 10-5-30 10-5-5 10-5-5 529 muo 526 mub 646 mu)
Mu.
%
%
%
0.100 0.800 0.600 1,000 1.60 2.20 2.80 3.40
10.0 11.9
8.0 10.2
9.0 11.1
Medians from eight sets of observations. From smooth curvea drawn through plotted medians.
ing to 100 ml. To eliminate constant errors, four complete sets of reagents from different sources were used in the calibrations. The median transmittances found in about 400 calibration tests are given in Tables I and 11. During the calibrations, methods 1 and 2 were studied with harticular regard to the deviations that might be expected in measurements made with reagents from various commercial sources. Each “average deviation” in Table I is based on 8 t o 10 test systems made with standard calcium sulfate solutions, and consists of the conventional “average deviation of a single observation” multiplied by 100 and divided by the corresponding median. The deviations show that good precision is obtained by these methods even with commercial reagents. Calibrations by method 3 were used more especially to compare transmittance values found with different instruments, wave lengths, and slit widths, the cuvette depth being held constant a t 1.308 cm. The reproducibility of the bow-shaped calibration curves obtained in all cases was further established b> the fact that they could be duplicated with test solutions made by mixing calculated amounts of permanganate and sodium oxalate or permanganate and manganous sulfate. SPECTROPHOTOMETRIC M E T H O D S F O R C A L C I U M
Table I.
Calcium as Permanganate
By Method 1 Median Total transAverage calcium mittance deviation
.vu. 0.100 0.300 0.600 1.000 1.60 2.20 2.80 3.40
% 97.7 93.4 84.9 70.8 47.9 30.5 18.9 11.2
B y Method 2 Median Total transAverage calcium mittance deviation
%
Mu.
%
%
0.31 0.35 0.35 0.40 0.52 0.48 0.95 1.5
0,500 1 ,000 2.00 3.00 4.00 5.00 6.00 7.00
94.5 87.5 70.0 51.0 35.1 23.9 15.9 10.2
0.40 0.46 0.49 0.51 0.57 0.71 0.85 0.74
SUMMARY
OF C A L I B R A T I O N S
Preliminary to the calibrations, tests were made to establish for 0.8- t o 2.5-mg. quantities of calcium the conditions for quantitative precipitation which also met the requirements of the spectrophotometric measurements. Precipitation of these quantities of calcium either by 2 ml. of reagent 1 from a volume of 2 to 8 ml., or by 1 ml. of saturated ammonium oxalate aolution from a volume of 4 to 40 ml., was essentially complete a t pH 3.7, 4.7, or 8.0 without buffers, or a t pH 3.7 and 4.7 with 1 ml. of formate or acetate buffer, respectively. The trend and terminals of the extrapolated calibration curves subsequently obtained under the above allowable conditions offered further evidence of completeness of precipitation over an even wider range of calcium concentrations than that studied in the preliminary tests. Calibrations were made by the methods descrilyd herewith for analyses, except that the sample solutions contained known quantities of calcium. Readings were always taken between 5 and 30 minutes after adding permanganate to the test solutions aild mak-
PREPARATION OF SAMPLE SOLUTIONS.Samples for analysis may be taken directly from solutions containing mainly calcium chloride or sulfate, and from most natural waters. However, if the chloride concentration is high, spectrophotometric methods 1 and 2 cannot be used. In general, the aliquot portions finally taken for analysis must be free from high concentrations of oxidizing or reducing substances and interfering ions-i.e., metals forming insoluble oxalates or anions forming insoluble calcium salts. Any significant quantities of such substances present in the original sample should be removed by the usual methods. The spectrophotometric methods described below are designed for an accuracy equal t o that of the best colorimetric analyses; the limiting accuracy attainable by refinements of technique and use of the best instruments is perhaps about 0.3%. Two insidious errors to be avoided are the use of formate buffers in methods 1 and 2, and neglect of the adherent layer of calcium oxalate, not removable by mere rinsing, which forms on electrodes and stirrer during unnecessarily protracted pH adjustments. After each precipitation of calcium oxalate, the system should be digested a t least 10 minutes a t about 90” C., then 30 minutes or more a t room temperature. METHOD 1. Run a n aliquot portion containing about 2 mg. of calcium into a 50-ml. beaker and add 1 ml. of acetate buffer and 2.00 ml. of reagent 1. Adjust the pH to 4.7 for maximum bufTering capacity; a lower pH may be preferable if certain interferences are present. Digest, reducing the volume to between 2 and 8 ml., then cool. Filter the supernatant liquid through a 15-ml. Pyrex filtering crucible of medium porosity and wash the precipitate and beaker with three 2-ml. portions of water. Collect the filtrate and washings in a 100-ml. flask under a bell jar with top and side tubulations for the crucible holder and suction tube, respectively. Add
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I N D U S T R I A L A N D ENGINEERING CHEMISTRY
10 ml. of 18 sulfuric acid and 2.00 ml. of reagent 2. Wait until the color change has occurred, then dilute to 100 ml. and measure the transmittance of a portion of the solution a t an appropriate wave length within 20 minutes. Read the result of the analysis from a calibration curve. Method 1 may be checked by determining the precipitate on the filter by method 3. METHOD 2. Adjust the pH of the sample solution to 4.7 before dilution t o final volume. Run an aliquot portion containing 2 to 4 mg. of calcium into a 50-ml. volumetric flask; add 1 ml. of acetate buffer and 4.00 ml. of reagent 1. Digest for 10 minutes on a hot plate, with occasional swirling, then cool and digest for 30 minutes a t room temperature. I n the present work the volume a t this stage was always between 8 and 20 ml. Make the system up to 50.0 ml. with distilled water, mix quickly, then pour the bulk of the mixture into a 50-ml. tube and centrifuge for 10 minutes a t about 3000 r.p.m. Pipet 25.0 ml. of the clear supernatant liquid into a 100-ml. flask, add 10 ml. of 18 N sulfuric acid and 2.00 ml. of reagent 2, and make to 100 ml. Complete the analysis as in method 1. METHOD3. Pipet an aliquot portion containing about 1.7 mg. of calcium into a 50-ml. beaker and add 1 ml. each of formate buffer and saturated ammonium oxalate solution. Adjust the pH to 3.7. Digest a t about 95" C., reducing the volume to about 20 ml. Cool, then digest a t room temperature for a t least 30 minutes. Pour the supernatant liquid through a 15-ml. Pyrex crucible of medium porosity and wash the precipitate with four 1- to 2ml. portions of water. Rinse the upper inside wall of the crucible with short spurts from a wash bottle. Remove the crucible from the holder and rinse off the outside and bottom thoroughly. Using the tubulated bell jar described above, put two 10-ml. portions of hot 9 N sulfuric acid slowly, with stirring, through the beaker and crucible and coIlect the solution and two 5-ml. water washings in a 100-ml. volumetric flask. Add 2.00 ml. of reagent 2 and complete the analysis as in method 1. METHOD3a. The range and precision of the above methods may be increased by such modifications as the following, adapted from McComas and Rieman ( 8 ) :
Table 111.
Vol. 17, No. 8
Comparisons with Official and Standard Methods
Sample Analyzed
Potential Interferences New in Original Material Method P.p.m.
Calcium Found in Sample Other New methmethod odr P.p.m. P.p.m
Water 7465
35
6.9
Water 5818
3)
24
27
Water 5817
3
35
34
Water 6130
1 2 3
51 52 53 61 57 59 (420) (361) 332 330 334
62
Water 5811
T.S., 417; Mg, 34; SO,, 103
Sea water 7037
T.S., 27, 900; .Mg, 1060: SOI, 2200
Water 5909
T.S., 6230; M g , 183; so4, 2115; C1. 1320
Effluent 7036
T.S 10200. Mg 165. so,, ioo;' CI. Bo6of HCOJ, 646
1
2 3 3 3s 1b 26
3b 3 38
3b 1
3 3a 1b
3b
Yo M Cln solution
Ce%feed Dry whole milk Spinach trate
concen-
MgC11,34 MgC12, 73 P,0.70; Fe,
