concentrations. This is not true of other oxidizing agents such as Fe(III), Cr(VI), V(V),and Ce(1V). I n addition to the great effect of temperature and concentration on the rate of oxidation of 8-aminoquinoline when these agents are used, there are also different pH requirements for the oxidation. All of these factors tend to decrease the interference of these materials in the manganese determination. The results of an interference study to determine the limiting concentration of other oxidizing agents in the manganese determination are given in Table I. Limiting concentration is defined here as the concentration needed to cause an error of 0.010 in the measured absorbance of manganese. I n addition to the interfering ions, all solutions contained 11.O pg. of manganese.
Because the method developed will be particularly useful for small amounts of manganese it should find great utility for manganese trace analysis in water. Therefore, in addition to other oxidizing agents, many ions present in natural waters were also checked as interferences. The addition of 25 p.p.m. of C U + ~ , Zn+2, or K + to 0.22 p.p.m. Mn(I1) caused no error. Results were not affected by 200 p.p.m. C a f 2 and Mg +z* The method was checked by preparing three synthetic samples. Results are given in Table 11. LITERATURE CITED
( I ) Bankavskis, J., Latvijas PSR Zinatnu Akad. Vestis 9 , 115 (1955). ( 2 ) Cunningham, T. R., Coltman, R. W., Znd. Eng. Chem. 16, 58 (1924).
( 3 ) Forman, L., J . Am. W a t e r U'orks Assoc. 21, 1212 (1929). (. 4 .) Gates. E. M.. Ellis. G. H.. J . R i d . Chem. 168, 537' ( 1 9 4 i ) . 15) Gustin, V. K . , Sweet. T. R.. A N A L . CHEM.35, 44 (1963) ( 6 ) Zbid., p. 1395. ( 7 ) Kolthoff, J. RI., Sandell, E. B., "Textbook of Quantitative Inorganic Analysis," 3rd ed., p. 564, Rlacmillan, New York, 1952. ( 8 ) Prodinger, JT.) Jfikrochemie 36, 580 (1951). ( 9 ) Wiese, A. C., Johnson, B. C., J . B i d . Chem. 127, 203 (1939). 1-AUGHN K. GUSTIN'
THOMAS R. SWEET ,McPherson Chemical Laboratory The Ohio State University Columbus IO, Ohio Present address: Corning Glass Works, Corning, N. Y . m'ork taken in part from the Ph.D. dissertation of Vaughn K. Gustin, The Ohio State University, Columbus 10, Ohio, 1963.
Spectrophotometric Determination of Calcium in Milk Using 2,2 '-(E th a ned iy Iid ene d in itrilo)d iphe no I [Gl y oxa I Bis (2 - hy d roxya n iI)] SIR: Because of the wide distribution of calcium in nature, a great variety of procedures have been developed to measure it. Accompanying metals, however, often interfere with the chemical reactions involved in measuring calcium, so various means must be used to circumvent the interference. Calcium which plays a significant role in the physical and chemical properties of milk, must be measured accurately on a routine basis. Several reported methods are based on titration (ethylenedinitri1o)tetraacetate with (EDT.1) (1, 8, 9, If) and various indicators, but orthophosphate ions present in the milk may interfere with the end point ( 2 , 5, 6). Several methods (3, 7 , 10, fa) reported more recently are based on the color produced with 2,2'-(ethanediylidenedinitri1o)diphenol [glyoxal bis(2hydroxyanil) (GBH*\)], a Schiff base, where a red innercomplex salt is formed. Interference by other salts or ions can be blocked easily to give a specific test for calcium ( 3 ) . This paper presents a method for microestimation of calcium in milk that has the advantages of a colorimetric procedure. I t requires only a small sample and avoids the difficulty of determining an exact end point. These modifications are believed to have considerably improved Kerr's method (7') and yielded a convenient procedure adaptable to biological materials.
DB. Absorbance of the red calcium complex was measured with a Model D U Beckman spectrophotometer, using 1-cm. matched, square cells in each case. The pH values of buffers and reaction mixtures were checked with a pH meter. Reagents. STANDARD CALcIuhi SOLUTION. Dissolve 2.4972 grams of reagent grade calcium carbonate, dried a t 110" C., in concentrated hydrochloric acid. Make to 1 liter with deionized water. This solution contains 1.00 mg. of calcium per ml. ( 6 ) . GLYOXAL BlS (2-HYDROXYANIL) SOLUTION, 0.5%. Dissolve 0.5 gram of Fisher G-147 glyoxal bis(2-hydroxyanil) in 100 ml. of rediqtilled methanol ( 7 ) .
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EXPERIMENTAL
500 550 WAVELENGTH (mp)
Apparatus. The initial spectrum was determined with a Beckman recording spectrophotometer, Model
Figure I . Absorption spectrum of the reaction product of calcium and glyoxal bis2-hydroxyanil
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ANALYTICAL CHEMISTRY
600
TRIS BUFFER SOLUTIOKWITH PH 12.7. To 1 liter of 0.1M tris(hydroxymethylaminomet,hane) is added 100 ml. of 10% potassium hydroxide. AMMONIUMOXALATE-OXALICACID SOLUTIOK WITH PH 5 (4). Dissolve 27 grams of ammonium oxalate and 1.26 grams of crystalline oxalic acid and dilute to 2 liters with deionized water. All other materials were prepared directly from reagent-grade chemicals, except' that the methanol and et,hanol (95%) were freshly distilled before use. Procedure. h 2-ml. sample (concentrations of 0.40 to 4.00 mg. per ml. of calcium) is pipetted into 50-ml. round-bottomed centrifuge tube, and 10 ml. of ammonium oxalate-oxalic acid solution is added after the pH is adjusted to greater than 5 with 1Oyo potassium hydroxide, using 1 drop of Congo red as an indicator. The tubes are capped and allowed to stand for 1 hour. At the end of the timed period the tubes are placed in a centrifuge and spun for 15 minutes a t 3,000 r.p.m. The supernatant is carefully poured off. The precipitate is dissolved in I S HC1 (about 2 ml.). The contents are quantitatively transferred and diluted to give a calcium concentration within the desired range. For a skim milk sample, the precipitate is transferred to a 200-ml. volumetric flask and brought to ~ o l u m ewith deionized water. After a further 1 : l dilution, a 10-ml. sample is pipetted into a 125-ml. Erlenmeyer flask, and 5 ml. of buffer (tris/ KOH at pH 12.T) is added. The blank is 10 nil. of deionized water and 5 ml. of buffer (tris '%OH a t pH 12.7). The 0.5 ml. of color reagent, glyoxal bis(2hydrosyanil), and 10 ml. of ethanol are added in that order, with mixing, before proceeding t o the next flask. Absorbance was measured after a 10-minute color development period.
RESULTS A N D DISCUSSION
Optimum Wavelength for M e a surement of Light Absorbance. Light absorbance of t h e calcium-glyoxal bis(2-hydroxyanil) complex was studied over a wide range of the recording spectrophotometer, and then in more detail on the Beckman DU model. Absorbance of the color complex is maximum a t 524 mp, as shown in Figure 1. Effect of Solvent System. Kerr ( 7 ) obtained best color stabilit,y with a mixture of ethanol and n-butyl alcohol, but ethanol and methanol also gave good stability. X study of these solvent systems showed that ethanol alone was suit'able (F'igure a), but is was essent,ial to have the solvmts freshly distilled. Spurious resulk mere attributed to impure ethanol. Stability of the Color Complex. There is a gradual decrease in absorbance x i t h time, but t h e reading is relatively stable between 4 a n d 20 minutes (Figure 3). It is recommended t h a t the sample be read a t 10 minutes. Effect of pH. T h e cola,, complex was found t o be most stable between pH 12.5 and 13.0. Uniform results required close control of pH. Therefore more buffer and slightly higher p H were used than in Kerr's method. Calibration Curve and Precision. T o construct a standard curve and determine the precision of the procedure, the absorbance produced a t six different concentrations of calcium WVBS determined 16 times. Table I shows the precision as measured by
standard deviation from the mean for each concentration. Recovery of Added Calcium. Recoveries of calcium were good when skim milk was measured before and after addition of 0.16 t o 8.00 mg. of calcium per ml. (Table 11). T h e recovery experiments also show t h a t the procedure compares favorably with the E D T A method (4) in sensitivity.
Calcium added, mg./ml. 0 0.16 0.80 1.60 2 40 3.20 4.00 8 00
GBHA method Calcium found, Recovery, mg. /ml. % 1.40 ... 1.50 96.2 2.17 98.6 2.93 97.7 3.74 98.4 4.52 98.3 5.37 99.4 8.87 94.4
Effect of Phosphate. Small amounts of added phosphate had a marked effect on color development. At low levels (1 pg.) of calcium as little as 2 fig. per ml. of phosphate interfere. The interference is more pronounced at higher levels of calcium. I n the procedure, interference by phosphate is eliminated by precipitation of the calcium with oxalate prior to color development. I n the absence of phosphate interference, the oxalate precipitation step may be eliminated. Effect of Magnesium. Magnesium interferes with color d e w l o p m e n t only
0.8 0.7
0.6
98
Method
Std. dev. f O 004 f 0 008 f O 004 f 0 007 f O 02 f O 03
Table II. Comparison of Accuracy of GBHA Spectrophotometric Method and EDTA Titration Method for Determination of Calcium in Skim Milk
0.9
.600
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Table 1. Precision of calcium taken, Absorbance pg./ml a t 524 mp 1 0 164 2 0 322 3 0 496 5 0 801 7 1 13 10 1 64
t
0.4 I
EDTA method Calcium found, Recovery, mg./ml. 70 1.27 . . . 1.42 99.3 2.00 96.6 2.76 96.2 3.52 95.9 4.34 97.1 5.02 95.3 7.74 83.5
when it reaches a concentration above 40 hg. per ml. The procedure is applicable to other biological materials as well as milk because magnesium interferes only at high concentrations and phosphate interference can be eliminated. The influence of other metals and ions has been investigated and eliminated by Goldstein (3)and Ken*( 7 ) . LITERATURE CITED
( 1 ) Diehl, H., Ellingboe, J., ANAL. CHEM.
32, 1120 (1960). (2) Gehrke, C. LT.] Affsprung, H. E., Yung, C. L., Ibid., 26, 1944 (1954). (3) Goldstein, I)., Stark-%Iayer,C., Anal. Chin!. .4cta 19, 437 (1958). (4) Henly, A. A., Snunders, R. A,, Analyst 83, 584 (1958). ( 5 ) Jcnness, It., ANAL. CHEM. 2 5 , 966 (1953). (6) Kamal, T. H., A g r . Food Chem. 8, 156 (1960). ( 7 ) Kerr, J. R . W., Analyst 8 5 , 867 (1060). (8) Lindstrom. F.. Diehl. H.. ANAL. CHEM.32. 1123 (1960) (9) Toribara, T. Y . , K'oval, L., Talanta 7, 248 (1961). (10) Umland, F., Merkenstnck, IC, z. Anal. Cheni. 176, 96 (1960). (11 ) Van Schouwenburg, J. Ch. ANAL. CHEM.32, i o 9 (1960). (12'1 Williams. K. T.. IVilsnn., .J. R - -. . , A N A L .C H E ~33, . 244 (1961). THOMAS .4. KICKERSOX EDWIN E. TOOR RE ARTHUR A. Z I M M E R ~ ~
0.3
~
0.9 0.1
C U N C t N I K A IlUN WF C A L L I U M ( r / m l )
Figure 2. Effect of solvent system on standard curve 0 Ethanol-butanol ( 1 : I ) solvent of Kerr ( 7 ) 0
A
Ethanol (95%) Methanol Methanol-butanol ( 1 : 1 )
5 10 15 PO 95 30 35 TIME (Minuter)
Figure 3. Effect of time on color intensity 0-0 Ethanol solvent 0- 0 Ethonol-butanol of Kerr (7)
solvent
Department of Food Scienre :tnd Technology University of California Davis, Calif. Present address: Whittier School, Whittier, Calif.
High
O N Eof the authors (A.A.Z.) is indebted to t,he Sational Science Foundation for :i research participation grant ((;20100). VOL. 36, NO. 8, JULY 1964
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