Table I. Absorbance as a F u n c t i o n of Cysteine (for S a m p l e Less R e a g e n t B l a n k ) Amount of cysteine monohydrate, fig 25 ml’”
10.9 21.8 54.6 109.3 132.1 54.5d
Absorbance a t 562 nm Observedh
0.070 0.134 0.340 0.690 0.810 0.337
CalculatedC
I 0.004 + 0.003
0.069 0.137
0.003
0.337
0.003 I!= 0 . 0 0 3 I0 . 0 0 3
0.699
I!=
+
0.817 0.349
“ T h e amount c cysteine monohydrate correspon to the values termined by the other analytical methods. Absorbance was determined on 25 ml of final solution containing 6 pmoles of iron(II1) and 15 pmoles of Ferrozine, and 3 ml of monocbloroacetate buffer a t pH 3.2. ‘ Calculated values are based on the molar absorptivity of 28000M-’crn-1 for iron(I1)-Ferrozine chelate and the assumption that cysteine is oxidized to cystine. Solution contains a mixture of various amino acids In the amount 100 times that of cysteine.
Some of this interference can be overcome by the addition of an additional amount of Ferrozine. Cysteine in the amounts as low as 10 pg and as high as 150 18/25 ml have been determined using 1-cm cells as summarized in Table I. The relative precision changes from 4% a t 20 pg to 1%a t 5 0 - ~ glevels. A small negative deviation from Beer’s law is observed a t concentrations higher than 125 1g/25 ml. This may be due to the incompleteness of the oxidation or side reaction of oxygen with cysteine. Spectrophotometric results indicate that cysteine is oxidized to cystine. The existence of cystine had been confirmed qualitatively.
Received for review October 9, 1973. Accepted February 27. 1974.
Determination of Chromium in Biological Samples Using Chemiluminescence Raymond T. Li and David M. Hercules Department of Chemistry, University of Georgia, Athens, Ga. 30602
The importance of chromium and its function in biological systems have been extensively reviewed by Mertz (1). Quantitative measurement of chromium in various animal tissues and in a large variety of foods has been done by Schroeder et al. ( 2 ) . They reported that the chromium concentration in human tissue differs with the geographic origin of the samples. Generally, they found higher Cr levels in males than females; for example in human serum: 520 ppb for males and 170 ppb for females. Analysis of chromium in human serum has been investigated by various authors (3-11). Recently, Seitz et al. (12) reported a chemiluminescence method for the analysis of chromium in water. The method is based on the light emission catalyzed by chromium(II1) in the luminol-hydrogen peroxide reaction. The method is sensitive for chromium in the sub-partsper-billion range, and is specific for chromium when EDTA is added to the sample prior to analysis, in order to mask interference from other metal ions. (1) W .
Mertz. Physiol. Rev.. 49, 163 (1969)
(2) H . A . Schroeder, J. J. Balassa. and I . H. Tipton, J. Chronic Dis..
(3) (4) (5)
(6) (7) (8) (9) (10) (11) (12)
916
15, 941 (1962). H . P. Yule, Anal. Chern.. 37, 129 (1965). E. R. Kowalski, T. L. Isenhour, and R. E. Sievers, Anal. Chern.. 41, 998 (1969). L. C. Hansen, W . G . Scribner, T. W . Gilbert, and R. E. Sievers, Anal. Chern., 43, 349 (1971) G. W. Dickinson and V . A. Fassel. Anal. Chern., 41, 1021 (1969). G. Tessari and G . Torei, Talanta. 19, 1059 (1972). C . E. Champion, G . Marinenko, J. K . Taylor, and W. E. Schmidt, Anal. Chern., 42, 1210 (1970) E. A. Solov’ev, E. A . Bozhevol’nov, A. I . Sukhanovakoya, G . P. Tikhonov, and Y. V. Golubev, Zh. Anal. Khirn.. 25, 1342 (1970) F . J. Feldman, E. C. Knoblock, and W. P u r d y , Anal. Chim. Acta. 38, 489 (1967). I . W. F . Davidson and W. L. Secrest. Anal. Chern.. 44, 1808 11972) > - -, W. R. Seitz, W. W. Suydam, and D. M . Hercules, Anal. Chern.. 44. 957 (1972).
A N A L Y T I C A L C H E M I S T R Y , VOL
46, N O . 7, J U N E 1974
This article reports the application of chemiluminescence to the determination of chromium in biological samples as part of a larger program of applying chemiluminescence methods to biological systems. We used NBS standard reference materials: orchard leaves (SRM 1571) and bovine liver (SRM 1577), to check the validity of the method. Both materials have no “certified” value reported for Cr; SRM 1571 has an uncertified value of 2.3 ppm. Chromium analyses were done on human serum samples and compared with literature data. Pooled serum samples were analyzed by chemiluminescence, atomic absorption, and colorimetric methods, to compare the three methods.
EXPERIMENTAL Apparatus. The apparatus used for chemiluminescence was t h a t described by Seitz e t al. (12). A Cary Model 15 recording spectrophotometer was used for the colorimetric method. A Perkin-Elmer Model 305 B double beam atomic absorption spectrometer equipped with a heated graphite atomizer HGA 70 was used for atomic absorption measurements. The chromium hollow cathode lamp was operated a t 25 milliamperes throughout the entire analysis. U’et ashing of samples was carried out using a n aluminum heating block. This block had nine holes, each 211, inches deep, to accommodate 18- X 150-mm Pyrex test tubes. A hole of equal depth but smaller diameter was drilled to fit a 400 “C thermometer so that the temperature during the ashing could be followed. The block was checked for uniformity of heating. Heating uniformity is considered under “Results and Discussion.” Reagents. Cltrex grade sulfuric acid and nitric acid (J. T. Baker Co.), and reagent grade perchloric acid (Matheson, Coleman and Bell) were used for wet ashing. Disodium ethylenediamine tetraacetate, boric acid, potassium hydroxide, and hydrogen peroxide (3%) were Fisher Scientific certified grade. The sodiu m luminol salt was prepared from luminol (Aldrich Chemical Co.) and reagent grade sodium hydroxide (Fisher Scientific Co.) ( 1 3 ) . The salt was twice recrystallized from water. Standard (13) E. H . SOC..
Huntress, L. N. 56, 241 (1934).
Stanley, and A. S. Parker, J. Arner. Chern.
chromium(II1) solutions were prepared by dilution of 0.1000M chromium(II1) nitrate. For the colorimetric method, potassium permanganate, sodium dihydrogen phosphate monohydrate, phthalic anhydride, and sodium azide were all Fisher Scientific certified grade. 1,5-Diphenylcarbohydrazide was "Baker Analyzed" reagent. The detailed procedure was that of Saltzman (14). For the atomic absorption method, methyl isobutyl ketone was Fisher Scientific certified grade. Hydrochloric acid was DuPont reagent grade. All the reagents and standard solutions were made u p with deionized water from a Continental Water Conditioning Company's deionization system. Standard reference materials were obtained from the National Bureau of Standards. Procedures. Wet ashing techniques used to dest,roy organic material in biological samples for trace metal analysis have been thoroughly reviewed by Smith (15), Middleton and Stuckey (16, 17), and Gorsuch (18). The method used in this work was Gorsuch's method modified by Agterdenbos et al. (19); except that the ashing temperature was at no time higher than 230 "C. The ashing solution was HN03:H2S04:HC104, 3 : l : l . For the decomposition of organics in orchard leaves a 5:2 mixture of "03 and HC104 was used because of calcium in the sample. Samples for ashing were dried, weighed, and placed in 18- X 150-mm test tubes. Generally 1 ml of acid mixture was used for 1 gram of sample. The samples and the blanks were placed in the aluminum block and heated to the desired temperature. A glass bumping stick was inserted in each of the sample tubes. Triplicate sample and blank solutions were ashed each time. The amount of sample used was ea. 0.25 gram. In the comparison of methods, a larger sample was required; a total of 15-20 grams of pooled human serum was ashed and then made up to 50 ml with deionized water in a volumetric flask. One ml was pipetted out for the chemiluminescence method, 5-ml samples were used for atomic absorption, and the remainder for the colorimetric analysis. In the experiments on comparison of dry and wet ashing using orchard leaves as samples, the dry ashing method of Thiers (20) was followed. The laboratory glassware a t the earlier stages of this work was cleaned by boiling the aqua regia for 1 to 2 hours, soaked overnight, washed at least three times with deionized water, and then dried overnight in an oven at 110 "C. Later, commercial Xochromix (Godax Laboratories) was used in place of aqua regia. For the chemiluminescence method, after a sample had been wet ashed, it was transferred to a 250-ml polyethylene plastic bottle. The pH of the solution was adjusted to approximately 4.5 with 1M KOH. From this point on, the detailed experimental procedure was the same as that described by Seitz et al. ( 1 2 ) .Absolute signal levels from blanks were not recorded. The modified technique, involving heating of the sample loop to ensure specificity, was used. In comparison of chemiluminescence, atomic absorption and colorimetric methods, the atomic absorption methods of Feldman et al. (10) and Davidson and Secrest ( 2 1 ) were used. Briefly, both methods require wet decomposition of the sample. However, in Feldman's technique, the chromium was oxidized with lO-lM KNn04 to chromium(VI), followed by methyl isobutyl ketone extraction. In Davidson's technique, the sample was analyzed directly after wet decomposition. The two gave similar results, Davidson's method was adopted as a matter of personal choice. In the colorimetric method, Saltsman's procedure was used. The serum sample, after wet decomposition, was oxidized by 1 0 - I M K M n 0 4 to chromium(V1). The excess MnO4 was destroyed by dropwise addition of 5% NaN3; 2 ml of 4M phosphate buffer was added before the addition of diphenylcarbazide to minimize interference of other metal ions. The absorbance was measured at 540 nm .
RESULTS AND DISCUSSION Both wet and dry ashing are widely used for the decomposition of organic matter in samples for trace metal anal(14) (15) (16) (17) (18)
B. E. Saltzman, Anal Chem., 24, 1016 (1952). G . F. Smith, Anal. Chim. Acta. 8, 397 (1953). G . Middleton and R. E. Stuckey, Analyst (London).78, 532 (1953). G . Middleton and R . E. Stuckey, Analyst (London).79, 138 (1954). T. T. Gorsuch "The Destruction of Organic Matter," Pergamon
Press, Oxford, England, 1970. Agterdenbos, L. van Brockhaven, B. A . H. G . Jutte. and J. Schuring, Talanta. 19, 341 (1972). (20) R. E. Thiers in "Methods of Biochemical Analysis," D. Glick, E d . , Interscience. New York. N Y . , 1954, Vol. V , p 289. (19) J.
Table I. Chemiluminescence Determination of Chromium i n Orchard Leaves after Wet and Dry Ashing Sample
Dry, ppm
Wet, ppm
1 2 3 4 5
2.2 2.1 1.9 1.9 2.0 f = 2.0 Z t 1
f
=
2.8 2.3 2.4 2.1 2.6 2.4 i3
Table 11. Recoveries of Standard Cr (111) after Wet Ashing Sample
Cr(II1) added, ppb
Cr(II1) recovered, ppb
1
10.4 10.4 10.4 10.4 10.4
11.4 9.9 9.9 9.9 10.4 f = 10.3 % 0 . 5
2 3 4 5
6o
r
c
z W
30
2
4
6
CHROMIUM ADDED (M x 10')
Figure 1. Calibration curves
using the standard addition method
-___
Standard addition into a blank. Addition made after ashing. - - - Standard addition with orchard leaves. Addition made before ashing
ysis. In a collaborative study of wet and dry ashing, Kowalczuk (21) reported that in six feed samples analyzed by ten different laboratories for trace metal ions using atomic absorption there was no significant difference between the results obtained by the two ashing methods; however, the relative standard deviations were high. In the present work, both dry and wet ashing techniques were tested t o determine if they were suitable for Cr analysis by chemiluminescence. Results of this study are presented in Table I. The data indicate that there may be loss of chromium during dry ashing. Therefore, the wet ashing procedure was adopted. Table I also indicates that the precision of the wet ashing technique was ca. *lo%. Uneven heating in the aluminum block was thought to be the problem. Thermometers were used to monitor each of the sample positions in the aluminum block. Below 190 "C, the samples were heated uniformly to =k1 "C. However, a t temperatures higher than 190 "C uneven heating occurred, as much as 15 "C difference between locations in the block. Only those holes in the heating block were used that were within *3 "C of the indicated temperature. Recovery of chromium after wet ashing was checked by adding known spikes of chromium to blanks and putting them through the wet ashing procedure. The results are ( 2 1 ) J. Kowalczuk, J . A s s . Offic.Anal. Chem., 53, 926 (1970) ANALYTICAL CHEMISTRY, VOL
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Table 111. Determination of Cr(II1) i n Orchard Leaves (SRM 1571) Cr(II1) found, ppm
f
Table V. Determination of Cr(II1) i n Human Serum Samples
Cr(II1) reported by NBS
2.2 2.1 2.4 2.3 2.4 2.5 2.6 2.2 2.1 2.5 2.6 2.4 2.6 2.3 2.3 2 . 4 i 0.13
2.3
(Neutron Activation Analysis)
163 235 112 145 208 126 88 186 245 60 40
=k ?C
i
i =k
i
+
i
i ?C
i
Pooled relative standard deviation
15 10 4 14 9 10 6 6 8 6 2
=
6.1%
Table VI. Comparison of Methods on the Determination of Cr(II1) i n Pooled Human Seruma
Table IV. Determination of CrIIII) i n Bovine Liver (SRM 1577)
Sample
1 2 3 4 5
Cr(II1) found, ppm
f =
0.82 0.86 0.82 0.74 0.94 0.90 0.80 0.88 1.01 0.96 0.87 i 0.06
T>2lO0C
T,lenol Orange. Fifty mg of xylenol orange, 9 6 7 ~purity (BDH Chemicals Ltd., Poole. England) was dissolved in 100 ml of 0.01M perchloric acid. Chondroitin Sulfate C. A solution containing 1 mg of chondroitin sulfate C (Sigma Chemical Co., St. Louis, Mo.) per ml of distilled water was prepared. Procedure. To a solution ( 2 ml) containing 2 mg of glycosaminoglycans, 2 ml of scandium chloride solution and 0.3 ml of 0 . 1 M (1) (2) (3) (4)
A Dische, J. Brol. Chem.. 167, 189 (1947). L. A . Elson and W . T. J. Morgan, Biochem J . , 27, 1824 (1973). A . H . Wardt and G . A . Michos. Ana/. Biochem , 51, 274 (1973). S. S. Berman, G . R . Duval, and D. S Russell, A n a / . Chem.. 35, 1392 (1963)
sodium hydroxide were added. The mixture was stirred vigorously and centrifuged. The supernatant was discarded, and the precipitate was washed twice with 5 ml distilled water and centrifuged. The precipitate was dissolved in 4 ml of 0.1M HCIOI, stirred, and diluted with 4 ml distilled water. Samples (0.4 ml) were diluted with 10 ml of 0.01M perchloric acid, and 0.8 ml of xylenol orange solution was added. The solution was stirred and its optical density a t 553 n m cs. a reagent blank was determined. Standardization of Scandium Chloride Solution. Scandium chloride solution was standardized us. EDTA solution by spectrophotometric titration at p H 3.5 using Muroxide as indicator ( 5 ) . The EDTA solution in turn was standardized us. zinc chloride solution in acetate buffer of p H 5 with xylenol orange as indicator (6). Effect of Acidity. Quantitative precipitation of glycosaminoglycans was obtained in the p H range of 3.5-5.0 as determined by the negative carbazole reaction of the supernatant. Low results were obtained when the p H was less than 3.5. At pH greater than 5.0, the precipitate undergoes hydrolysis and basic salts are precipitated. Chondroitin Sulfate C Standard Curve. Increasing amounts of chondroitin sulfate C were precipitated as described and the amount of scandium in the precipitate was determined spectrophotometrically. Except for amounts of chondroitin sulfate below 0.05 mg, the optical density of the red color is proportional to the concentration of chondroitin sulfate C. Composition of the Precipitate. Samples of hyaluronic acid and chondroitin sulfate A were precipitated with scandium chloride and the amount of scandium in each precipitate was determined. Results are given in Table I. From these data, the precipitates were found to contain 1 mole and 0.5 mole of scandium per disaccharide unit of chondroitin sulfate and hyaluronic acid, respectively. Because of the difference in the composition of these precipitates, the results of the analysis of samples containing both components should be expressed in terms of the glycosaminoglycans used as standard. Effect of Other Substances. The effect on the analysis, of substances which are commonly present in glycosaminoglycan preparations, is shown in Table 11. (5) S. P. Sangal. Mikrochem. J 9, 38 (1965). (6) J. Korbl and R . Pribil, ChemistAnaiysf,45, 102 (1956). ANALYTICAL CHEMISTRY, VOL
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