Rubeanic Acid for Determination of Copper in Human Serum D. S.
McCANN, PATRICIA BURCAR, and A. J. BOYLE
Department o f Chemistry, Wayne State University, Detroit,
b Copper in biological tissue or enzyme preparations is determined with rubeanic acid after digestion with a nitric-perchloric acid mixture. The method is free from extraction or transfer procedures and is satisfactory for as little as 0.75 ‘y of copper on a routine basis.
Mich.
Table 1.
Sample 2 ml. serum
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determination of trace quantities of copper is of real interest to a diversity of analytical chemists dealing with organic, material. Clinical chemists are frequently concerned with copper levels of serum. Biochemists use copper concentration to follow the purification of certain copper-bearing enzymes. The dairy and fat industries must keep copper contamination within very narrow limits, bwause it catalyzes the development of rancidity. Rribeanic acid has been used as an organic reagent for the colorimetric determination of copper in magnesium alloys for a number of years. Its use for the quantitative estimation of copper in biological tissues was suggested by Willard, Mosher, and Boyle (‘7). This paper describes a procedure used in this laboratory for the estimation of copper in biological material with this reagent. Through the early fifties the clinical methods for the estimation of copper usually employed diethyldithiocarbamate. One of the most carefully worked out is described by Gubler et al. (3). However, the method requires considerable skill on the part of the technician to yield reproducible results. A number of factors create interferences in the color development, so that blank readings and corrections must be made on each serum sample. If the copper concentration is rather high, the color is stable for only one-half hour. I n the 1950’s a series of phenanthroline derivatives for copper analysis was developed by Smith and his colleagues (2, 4-6). Their use in the determination of copper in serum has been described recently (8). An exEellent review of available methods for the determination of trace amounts of copper was published by Borchardt and Butler in 1957 ( 1 ) . These authors considered some ten possible reagents for the HE
Copper Additions to Human Serum
Absorbance 0.270 0.270 0.268 0.268 0.350 0,340 0.345 0.345 0.347 0.347 0.415 0.420 0.418 0.412 AV.
Std. dev.
Per sample 3.60 3.60
3.57 3.57 4.67 4.53 4.60 4.60 4.62
4.62 5.53 5.60 5.58 5.50
cu, Y
In 2 . 0 ml:
serum 3
fin
3 60
3.57 3.57 3.67 3.53 3.60 3.60 3.62 3.62 3.53 3.60 3.58 3.50 3.58 10.04
Dev. 0.02 0.02 0.01 0 01
0.09 0.05 0.02 0.02 0.04
0.04 0.05 0.02 0.00
0.08
quantitative estimation of copper in paper and paper products and chose bathocuproine (2,9-dimethyl-4,7-diphenyl-1,lO-phenanthroline) as most advantageous for their purposes. They eliminated rubeanic acid from their considerations because of the insolubility of its copper complex in organic solvents. The method outlined here for serum employs a wet digestion procedure in which the material is taken to dryness. Interference from iron is eliminated by complexation with malonic acid. I n these circumstances extraction of the copper is unnecessary and the rubeanic acid-copper complex may be developed in aqueous solution.
Malonic Acid Solution. Dissolve 10 grams of reagent grade malonic acid in 100 nil. of deionized water. Neutralize with concentrated ammonium hydrovide to a faint odor of ammonia and dilute with deionized water to 500 ml. Standard Copper Solution. Dissolve 50 mg. of copper wire (electrolytic) in 10 ml. of concentrated nitric acid, warming to speed solution. Add this solution to 500 ml. of water containing 10 grams of dibasic sodium phosphate, and dilute t o 1 liter with deionized water. Dilute 4 ml. of this stock solution to 100 ml. with deionized water to yield a working standard containing 2 y of copper per ml.
REAGENTS
PROCEDURE
Digestion Mixture. Mix 1 volume of doubly distilled 72% perchloric acid with 9 volumes of concentrated nitric acid. Buffer Color Reagent. Dissolve 300 grams of C.P. sodium acetate trih y d G t e in 500 ml. of water and filter through an open-textured filter paper. T o this add 280 ml. of reagent grade glacial acetic acid. Dissolve 200 mg. of gum arabic in 20 ml. of mater. Dissolve 100 mg. of rubeanic acid in 20 ml. of ethyl alcohol, warming if necessary. Add both to the buffer mixture and dilute to 1 liter with deionized water. Under refrigeration this reagent is stable for months.
Pipet 2.0-ml. samples of serum into 1 X 6 inch test tubes. Add 4.0 ml. of acid digestion mixture to each tube and place them in an aluminum heating block, 3 X 3 X 12 inches, which has been preheated on a medium hot plate to approximately 120’ C. (The block contains 13 holes, l’/a inches in diameter and 21/2 inches deep to accommodate 12 samples and one blank, so that multiple samples may be run singly or in duplicate without individual attention. A thermometer well, 21/, inches deep, is situated a t one end of the block, so that the temperature may be noted a t any time.) Switch the hot plate to high and evaporate samples to incipient VOL. 32, NO. 4, APRIL 1960
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dryness (complete dryness is not necessary), which requires approximately 3 hours and a final temperature of 260’ to 290’ C. Remove the digestion tubes from the block and cool. Wash down the wall of each test tube with 1.0 ml. of malonic acid solution and warm momentarily in boiling water t o ensure complete solution of the residue. Cool the tubes, add 3.0 ml. of buffer color reagent to each, and mix by swirling. The final pH of the mixture will be 4.2 to 4.3. Carry a digestion blank through the process with each batch of samples. After 30 minutes, measure the absorbance of the samples on a suitable spectrophotometer a t 385 mp (a Beckman DU was used to determine the point of maximum absorption of the complex and a Bausch &: Lomb Spectronic 20, for the preparation of Table I ; the latter is used for all copper determinations in this laboratory). Prepare a standard curve covering the range of 0 to 8 y1using the digestion procedure describcd. Prepare a new curve whenever n m stock reagent is prepared. RESULTS A N D DISCUSSION
Table I illustrates the reproducibility
that may be expected for the estimation of copper in human serum with rubeanic acid. The color complex has a molar absorptivity of 15,600, obeys Beer’s law over the range of 0 to 8 y, and is stable for several hours. llalonic acid is used in the procedure to avoid any possible error due to iron interference. (Iron in serum is often variable because of red cell hemolysis.) The absorbance of the blank in this procedure is negligible. The method has been used on aortic tissue and enzyme preparations and therefore appears suitable for most biological preparations, if 0.75 y of copper is present in the sample (absorbance. 0.055). Prior to using the aluminum block and test tubes for the digestion procedure, 25-ml. Erlenmeyer flasks were employed. While precision and accuracy were similar to those experienced with the present procedure, a t least 4.5 hours mere required for sample digestion. The digestion procedure is extremely smooth, which is not alnays the case in the wet ashing of organic material. Virtually no attention is required during the process. Khether the
sample is carried to incipient dryness or complete dryness is optional. Final dilution of the sample n-ith malonic acid and buffer color reagent is done in the digestion tube, which eliminates sample transfer. LITERATURE CITED
(1) Borchardt, L. G., Butler, J. P., ANAL.CHEM.29,414-19 (1957). ( 2 ) Diehl, Harvey, Smith, G. F., “The
Copper Reagents: Cuproine, Seocuproine, Bathocuproine,” G. Frederick Smith Chemical Co., Columbus, Ohio,
1958. ( 3 ) Gubler, C. J., Lahey, 11. E., Ashen-
brucker, H., Cartwright, G. E., Win-
trobe, iLI. &I.,J. B i d . Chem. 196,209-20 (1952). ( 4 j Smith, G. F., JIcCurdy, W. H., Jr., AKAL.CHEM. 24,371-3 (1952). ( 5 ) Smith, G. F., Kilkins, D. H , Zbzd., 25,510-11 (1953). (\6 ,) Wlkins. D. H.. Smith. G. F.. Anal. Chim. A d a 9 , 538’(1953). ( 7 ) Willard, H. H., Mosher, R. E., Bovle, A. J., - 1 x . 4 ~ .CHEK 21, 598-9 (1949). ( 8 ) Zak, B., Clin. Chim. .icta 3, 328 (1958). RECEIVEDfor review October 15, 1959. Accepted January 11, 1960. Kork supported by grants from the llichigan Heart Association and the Sational Institutes of Health.
Potassium Cobaltinitrite as a Precipitation Form for Cobalt Using Radiocobalt Tracer DARNELL SALYER’ and THOMAS R. SWEET Department o f Chemistry, McPherson Chemical laboratory, The Ohio State University, Columbus
b Very little information appears in the literature concerning the rate of precipitation of cobalt by potassium nitrite. The rate of precipitation was studied under widely differing conditions of volume, concentration of cobalt and reagents, temperature, and presence of other materials. The extent of precipitation was followed by the use of radiocobalt. Under suitable conditions, precipitation of cobalt as cobaltinitrite is quantitative in much less than the usually recommended 24 hours or overnight period.
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precipitation of cobalt as yellow potassium cobaltinitrite was reported more than 100 years ago by Fischer ( 2 ) and Saint-Evre (4). This reaction has been used principally for the separation of cobalt from nickel. HE
Many modifications or variations of Fischer’s original method have appeared. Most standard tests give a procedure very similar to Brunck’s modification (1). Kallmann (S) made a number of important observations concerning the interference difficulties usually encountered and suggested modifications which resulted in a considerably improved method. The rate of precipitation of tripotassium cobaltihexanitrite has received very little attention by previous n-orkers. When indicating the time required for quantitative predipitation, the usual statements found in the literature vary from about 4 to 24 hours. Other references to rate indicate observed trends, such as the fact that volume is an important factor in the time needed for complete precipitation. MEASUREMENT OF RATES OF PRECIPITATION
Present address, Eastern Kentucky State College, Richmond, Ky. 1
548
ANALYTICAL CHEMISTRY
The extent of precipitation with time
IO, Ohio
was measured as the follon-ing factors were varied: volume, temperature, amount of cobalt, excess of potassium nitrite, and presence of other material during separations. The effect of time of standing on the amount of cobalt precipitated was noted as each factor n-as varied, the other factors being held constant. The use of radiocobalt tracer facilitated this study greatly, as the amount of cobalt that remained unprecipitated at a given time could be determined by measuring the activity of a portion of the supernatant liquid. At a given time after the addition of the reagent, approsimately 15 to 30 ml. of supernatant liquid n-ere filtered off and a 10- or 25-nil. portion was taken for activity measurements. By comparing the activity of this portion with the total activity added, and by using the known total volume of the reaction mixture, the per cent cohalt remaining in the solution. and hence
