Complexometric Titration of Urinary Calcium and Magnesium CLAUDE L. YARBRO and ROBERT L. GOLBY Deparfrnenf o f Biochemistry and Nufrifion, University o f Norfh Carolina, Chapel Hill,
b Urinary calcium is determined by addition of an excess of standard ethylene glycol bis-(0-aminoethyl ether)-N,N’-tetraacetic acid and backtitrating with calcium, using Calcon as an indicator. Calcium plus magnesium may b e determined by addition of an excess of (ethylenedinitri1o)tetraacetic acid, followed by back-titrating with calcium using Eriochrome as an indicator. The phosphate and citrate normally present in urine do not interfere. Protein when present in amounts greater than 1 mg. per ml. interferes; it may be removed b y precipitating with trichloroacetic acid. A single determination using this method requires 5 minutes as compared with 2 to 24 hours for other methods.
T
rapid, accurate method for the determination of calcium in urine saves considerable time over methods based on the isolation of calcium oxalate (1, 3, 8). Wilson (9) determined calcium in urine by titration with disodium (ethylenedinitri1o)tetraacetate (EDTA), using ammonium purpurate (murexide) as an indicator after first removing phosphate. This indicator has certain disadvantages: The pink to purple color change is difficult to judge and, even where photometric titrations are used (6), the rapid destruction of the dye is a cause of concern. Although calcium may be determined in the presence of phosphate by back-titration procedures utilizing an excess of EDTA ( d ) , the presence of magnesium interferes with such procedures, even though it is first precipitated as the hydroxide (excess EDTA will dissolve magnesium hydroxide). However, EDTA can be used for the determination of calcium plus magnesium in urine by backtitration. Hildebrand and Reilley (6) found that 1 - (2 - hydroxy - 1 - naphthylazo) - 2naphthol-4-sulfonic acid (Calcon) gives a very sharp color change from pink to blue when calcium is titrated a t p H 12.5 with EDTA. Golby, Hildebrand, and Reilley (4) used this indicator and reagent to titrate calcium in serum directly. Although Calcon overcomes the objections associated with murexide as an indicator, the presence of magnesium in urine still constitutes a problem. HIS
504
ANALYTICAL CHEMISTRY
N. C.
Schmid and Reilley ( 7 ) used ethylene glycol bis-(p-aminoethyl ether)-AV,N’tetraacetic acid (EGTA) to determine calcium in the presence of magnesium by a coulometric titration. Because this reaction seemed to be selective for calcium it appeared that ethylene glycol bis-(p-aminoethyl ether)-N,N’-tetraacetic acid could determine calcium in urine in the presence of both magnesium and phosphate by back-titration. DETERMINATION
OF
CALCIUM
Standard calcium, 0.100OOM. Dry primary standard grade calcium carbonate to a constant weight, and dissolve in the minimal quantity of liL‘ hydrochloric acid with heating to ensure complete solution and evolution of carbon dioxide. Dilute to the appropriate volume. Ethylene glycol bis-(8-aminoethyl ether)-N,N’-tetraacetic acid, 0.0lAf (EGTA, available from Geigy Industrial Chemicals as CHEL DE). Dissolve approximately 0.85 gram in the minimal quantity of 2M sodium hydroxide and dilute to 250 ml. Standardize against 0.1OOOOM calcium, using Calcon as an indicator. Sodium hydroxide, 26f. Potassium cvanide. 0.7M. Calcium indicator.’ Dissolve 150 mg. of Calcon (J. T. Baker Chemical Co.) in 100 ml. of methanol. Syringe microburet (Micro-Metric Instrument Co.), calibrated to deliver 0.5 ~ 1per . scale division. Procedure. To 1 ml. of urine (calcium concentration, 1 t o 8 millimoles per liter) in a 30-ml. beaker, add 1 ml. of 0.7M potassium cyanide from a buret. Pipet in succession 1 ml. of standard 0.01M EGTA and 2 ml. of 2M sodium hydroxide into the above mixture, and dilute to approximately 15 ml. with distilled mater. Add 5 drops of calcium indicator just prior to titration to obtain a blue to green color, depending upon the amount of pigment in the urine. Titrate the solution to the first appearance of pink color, using standard 0.1M calcium solution delivered from the syringe microburet. Stir the solution continuously during the titration with an electric stirrer fitted with a small paddle. Reagents and Apparatus.
I
DETERMINATION OF CALCIUM PLUS MAGNESIUM
Reagents and Apparatus.
ard calcium, 0 . l O O O O M .
Stand-
(Ethylenedinitri1o)tetraacetic acid, 0.015M. Dissolve approximately 1.1 grams of disodium (ethylenedinitri1o)tetraacetate dihydrate (J. T. Baker Chemical Co.) in 200 ml. of water and standardize against 0.1N calcium, using Calcon as an indicator. Potassium cyanide, 0.7M. -4mmonia buffer. Dissolve 67.5 grams of ammonium chloride in 570 ml. of concentrated ammonium hydroxide and make up to 1 liter with distilled water. Magnesium plus calcium indicator. Dissolve 1 gram of Eriochrome Black T (Matheson, Coleman & Bell) in 25 ml. of distilled water, add 1 ml. of 0.5M sodium carbonate, and dilute to 100 ml. with isopropyl alcohol. Filter through a Buchner funnel and store in the cold. Syringe microburet. Procedure. This procedure is essentially a modification of the Wilson method (9). To l ml. of urine (Ca Mg concentration 3 to 10 millimoles per liter) in a 30-ml. beaker, add 1 ml. 0.7M potassium cyanide from a buret, and then in succession add 1 ml. of standard 0.0151M EDTA and 2 ml. of ammonia buffer. Dilute the solution to approximately 15 ml. with distilled water. Add 2 drops of magnesium plus calcium indicator to give a blue to green color, depending upon the amount of pigment in the urine. Titrate the solution to the first appearance of winered color, using standard 0.1M calcium solution as described above. Both procedures may be adapted for use Tvith a 5-ml. buret calibrated in 0.02 or 0.01 ml. If this adaptation is used, the standard calcium solution should be 0.0100011.f and a quantity of EDTA (for Ca hIg) or ethylene glycol bis-(p-aminoethyl ether)-A-,h”-tetraacetic acid (for Ca) should be added to the urine R-hich will give a back-titration of 1 to 3 ml.
+
+
RESULTS
The method was first tested on n-ater solutions containing known amounts of calcium and magnesium. The total concentration of calcium plus magnesium was of the order of O.OlM, with a calcium to magnesium molar ratio varying from 0.1 to 9 (Table I). The maximum deviation of calcium found from that taken was 3.6%, with an average of 2.3%. For calcium plus magnesium the maximum deviation was 1.1%, with an average of 0.5%. Where the molar ratio of Water Solutions.
calcium to magnesium is greater than 2 , the reliability of determination of magnesium by difference decreases rapidly. Recoveries of Calcium and Magnesium f r o m Urine. The recovery of calcium added t o urine ranged from 98 t o 104%, with a n average of l O l % , a n d for magnesium, t h e recoveries ranged from 95 t o 1047,, with a n average of 100% (Table 11). Tests f o r Interference in Urine.
T h e effect of added citrate and phosphate on t h e determination of calcium a n d calcium plus magnesium in urine was tested (Table 111). Addition of phosphate up t o 0.05 millimole per ml. of urine had no effect upon the determination of calcium plus magnesium. However, added phosphate a t this upper Icvel (0.05 millimole per ml. of urine) produced interference in the determination of calcium, in one case giving a value 13% below that obtained n itli the urine alone. Because the level of phosphate occurring in urine is in the range of 12 to 30 millimoles per liter and the amount of added phosphate n hich caused interference was approsiniately t n ice that normally found in urine, no interference may be expected from the phosphate normally occurring in urine. Levels of added citrate u p to 0.01 millimole per ml. of urine had no effect on either determination. As protein (usually albumin) may appear in the urine in various pathological conditions, the effect of added serum albumin upon the determination of calcium and of cnlciuni plus magnesium in urine was evaluated. Although Golby, Hildebrand, and Reilley (4) have determined calcium in serum by direct titration without removing proteins, 10 mg. serum albumin per ml. of urine in these back-titration procedures made the determination of calcium or magnesium plus calcium impossible. At protein levels of 1 mg. per nil. of urine or less, no interference was noted. I n the present procedure, the protein is apparently competing with the indicator for the calcium titrant; the protein was removed from a n y urine sample which gave a n immediate positive test for protein when acidified Kith acetic acid and heated. The following procedure for its removal may be used:
To 5 ml. of urine in a 15-ml. centrifuge tube, add 5 ml. of 5y0 trichloroacetic acid, mix well, and allow to stand for several minutes. After centrifugation a t 1500 r.p.m. for 10 minutes, take 2-ml. aliquots of the supernatant solution (equivalent to 1 ml. of urine) for analysis. h-eutralize the trichloroacetic acid present with 0.2 ml. of 2-11 sodium hydroxide and analyze the re sulting mixture by the above procedures. I n other tests on the methods presented, trace metals (principally iron)
Table I.
Determination of Calcium and Calcium plus Magnesium in Water Solutions
Ca
hk Taken, bimolesj L.
Taken, mmoles/ 1. 8.65 i.69 6.73 4.80 2.88 1.92 0.96
1.01
2.03 3.05 5.09 i.13 8.14 9.16
Table II.
Ca
Found, mmoles/ 1. 8.91
Ca
14
21 22 23
+2.7 -1.4
"3.6 +1.0
+O.l
0.0 -0.2
-0.9
1.
70
0.77 1.82
-23.7 -10.3
2.92
-4.3
4.94 7.08
-2.0
-0.7
7.96
-1.1 -1.0
-2.2 -1.2
9.05
+
big Found, Recovered ________ hlg Recovered mmoles/ Mmoles/ mmoles/ mmoles/ cCI 1. L. 1. /a 1. /C 5.44 8.68 ... ... ... 18.44 9.73 ioi:2 15.17 5.40 18.92 ... 10124 10h:5 10.24 18.58 4.80 lb0:O 5.06 99.4 6.59 10.65 ... . . . . 20.34 9.75 ioi:4 , . . ... 16.34 6.68 20.59 ... ... 0.94 97.6 20 51 5 01 104 4 5 06 99 4 11.60 4 91 2 92 100 3 12 56 14 76 9 61 10 28 101 0 2 96 15 19 7 84 14 56 4 72 98 3 4 83 95 3 3 31 4 93 12 81 14 89 9 50 98 8 3 27 15 40 10 4i 102.8 8 27 15 04 4 95 103 1 5 31 104 3 Ca
Calcium __ Found, Dev.,
+
Calcium Magnesium Found, Dev., mmoles/l. %
mmoles/l.
70
43 5 50 5 43 5 36 5.35
+24 00 -13 - 1.5
8 86 8 87 8 77 8 70
++ 3.9 2.7 - 0.6 + 0.2
10 6.5
5
0.00 20,oo 50.00 0.00 0.00
0.00 0.00 0.00 3.00 10.00
6.85 6.77 6.55 6.60
0.00 20.00 50.00 0.00 0.00
0.00 0.00 0.00 3.00 10.00
2.92 2.98 2.83 2.89 2.86
0.00 20.00 50.00 0.00 0.00
0.00 0.00 0.00 3.00 10.00
3.31 3.34 2.88 3.11 3.24
6.59
8 68
$2 1 1-2 2 +10
+o. 2
10.86 10.68 10.55
+i.g $0.9 $0.3 -0.9
+' 2.0 - 3.1
4.91 4.91 5.05 4.72 4.62
... 0.0 $2.8 -3.9 -5.9
+' 0.9 -13.0
4.93 5.03 4.91 5.04 5.10
10,75
- 1.0 - 2.1
- 6.0 - 2.1
+2:0
-0.4 $2.2 +3.4
Comparison of Proposed Method with Other Methods
Present Method Ca, Ca big, mmoles/l. mmoles/l. 6.59 10.6-5 8.94 15.45 4.09 7.55 5.18 8.39 8.15 11.88 6.25 11.71 3.51 7.94
+
20
9.73 9.78 9.87 9.92 9.95 10.02
+1.9
%
Effect of Added Phosphate and Citrate on Determination o f Calcium and Magnesium in Urine
Table IV.
12 17 19
$0.2
+2.9
Found,
Citrate Phosphate Added, Added, Sample Xfmoles/L. Mmoles/L. 11 0 00 0 00 20 00 0 00 50 00 0 00 0 00 3 00 0.00 10.00
13
Dev.,
1. 9.68
.1k Found, mmolesj Dev.,
Recovery of Added Calcium and Magnesium from Urine
Mg
12
70
+ Mg
Found, mmoles/
1-3.0
7.91 6.86 4.03 2.84 1.99 0.97
Added, Added, Sam- mmoles/ nimoles/ ple L. 1. 11 0.00 0.00 0.00 9.61 10.18 0.00 5.09 4.80 12 0.00 0.00 0.00 9.61 10.18 0.00 5 09 4.80 13 0 00 0 00 0 00 9 61 10 18 0 00 5 09 4 80 14 0 00 0 00 0 00 9 61 10 18 0 00 5 09 4 80 Table 111.
Dev.,
Method of Wilson (9) Ca, Ca hfg, mmolesjl. mmoles/l. 10.22 6.86 15.91 8.79 7.72 3.85 4.92 8.46 7.73 11.33 11.70 6.13 3.62 7.72
+
Method of Shohl and pedley (8), Ca,
?rfmoles/L. 6.57 8.80 4.14 5.16 7.41 6.06
3.89 ~~
VOL. 30, NO.
4, APRIL 1958
505
present in urine interfered slightly. This interference was removed by the addition of cyanide as described in the experimental procedure. Comparison w i t h Other Methods.
Calcium was determined in seven different samples of urine by t h e present method, by t h e complexometric method of Wlson (9),and by the oxalate precipitation method of Shohl and Pedley (8). I n Table IV, the vaIues for calcium obtained by the present procedure compare favorably with the other methods, as do the values for calcium plus magnesium with the Wilson procedure (9). The method of Sholil and Pedley requires approxiniately 24 hours for completion and the procedure of Kilson require 2 t o 3 hours. Precision of Method. To calculate the standard deviation of these procedures, calciuni and calcium plus magnesium were determined on 20
individual aliquots of the same urine. The magnesium content of the urine was obtained by difference between the calcium and the calcium plus magnesium determination. The results of this experiment were (in millimoles per liter): Ca = 8.41 =t0.032; Ca hIg = 14.44 =t0.033; AIg = 6.03 =k 0.177. Twenty triplicate titrations of urinary calcium where the amount of calcium varied from 2.23 to 8.94 millimoles per liter agreed within i 2.9%.
+
ACKNOWLEDGMENT
The authors n-ish to acknowledge the technical assistance of Thomas Austin. They also express their appreciation to Geigy Industrial Chemicals for supplying saniples of EGTA used in this experiment.
LITERATURE CITED
(1) Berger, E., Clin. Chem. 1 , 249 (1955). (2) Brunisholz, G., Genton, M., Plattner. E., Helv. Chim. $eta 36, 782 (1953), (3) Fiske, C. H., Logan, M. A , , J . B i d . Chem. 93, 211 (1931). (4) Golby, R. L., Hildebrand, G. P.,
Reilley, C. N., J . Lab. Clin. M e d . 50. 498 11957). (5) Hildebrand, G.' P., Reilley, C. S . , A h A L . CHEK 29, 258 (1957). (6) Horner, I\-.H., J . Lab. Clin.M e d . 45, 4.51 14.5.5) 951 i (1955).
((7) 7 ) sei;; Schmid, R. W., Reilley, C. K., AXAL. CHEU.29, 264 (1957). (8) Shohl, A. T., Pedley, Pt F. G., J . Bio2.
Chem. 50. ,537 t(1922). L H L L I. (9) W&on, A,' A , , ' J . Comp. Pathol. Therap. 63, 294 (1953).
RECEIVED for review July 29, 1957. Accepted November 26, 1957. Supported in part by research grant (PHS8-248) from the National Institute of ilrthritis and Metabolic Diseases, Sational Institutes of Health, Public Health Service.
Infrared identification of Disaccharides JONATHAN W. WHITE, Jr., C.
R.
EDDY, JEANNE PETTY, and NANCY HOBAN
Eastern Regional Research laboratory, Philadelphia, Pa.
b The value of infrared spectra for the identification of amorphous disaccharides and their acetates, b y comparison with spectra of known disaccharides and their acetates, i s demonstrated. Infrared spectra of ten amorphous disaccharides of Dglucose, of D-glucose and D-fructose, and of their 0-octaacetates are presented over the range of 650 to 1500 cm.J Potassium bromide disks were used. All spectra differ in sufficient detail to allow differentiation among closely related disaccharides.
A
chromatographic analysis has been of inestimable value in separation of mixtures of sugars, their identification by chromatographic evidence alone is not conclusive. Relative rates of migration on paper or column, together with color reactions v i t h spray reagents, only contribute to identification. Physical properties of isolated samples or derivatives are also required. Heretofore, crystallization of the sugar or derivative has been considered imperative before an identification is unequivocal. Sugar samples isolated by chroniatographic means may be so small that i t is practically impossible to crystallize LTHOUGH
506
ANALYTICAL CHEMISTRY
them. Hence, melting point, x-ray diffraction, and crystallographic study are eliminated as tools for identification. Determination of optical rotation does not require crystalline samples, but values may become only approxiniations as sample weights decline into the lower milligram range. The infrared absorption spectrum of a compound is increasingly used for its identification and analysis. As a unique physical property that is not primarily dependent on crystallization, it offers a most useful criterion for identification of disaccharides. Infrared spectra of all types of carbohydrates have been published. I n the first of a series of papers on this subject, Barker and his colleagues (5) stated that determinations of infrared spectra offer poll-erful means for the comparison of supposedly identical samples of carbohydrates. They examined the spectra of a variety of carbohydrates and their derivatives, but have not published them in sufficient detail for comparison purposes. They noted that spectra in the 730- to 960cm.-' range allow assignment to the CYor p-series of D-glucopyranoses, Khistler and House (20) have also reported that certain regions of absorption are characteristic of the configuration of the anomeric carbon.
Kuhn (13) has published the spectra of 79 carbohydrates and derivatives over the range 8.0 to 15.0 microns. Of these, only t n o are of sugars included here. Kuhn's spectra were determined with an amount of snniple in the beam too small to permit maximum utility of the curres for comparison purposes. Infrared spectra of a large number of sugar acetatrs and related compounds have been determined a t the National Bureau of Standards (12). Crystalline materials were used but the spectra were all determined in solution. The pressed-disk technique is useful when water-soluble materials are examined. It also prrmits the use, where necessary, of less-than-milligram samples ( 1 ) . Some difficulties have been experienced in comparing disk spectra with those obtained from mulls (d, 3, 8). Barker et al. (6) have ascribed the changes they previously (3)noted during aging of disks containing certain carbohydrates to the relatively high moisture content of potassium bromide disks. They did not find any such changes in any of the three disaccharides included in their study. The mlue of infrared spectra in identification of larger molecules such as trisaccharides was cited by Whiffen (19), n h o noted that identification by melting point and rotation
