study. Practically no differences have been observed between the SOs concentrations measured by a gas chromatograph equipped with FPD and values obtained directly with FPD for total gaseous sulfur ( 4 ) for measurements performed in Cincinnati, Ohio, or Raleigh, North Carolina. Figure 5 compares responses of the gas chromatographic system and the FPD in the analyses of samples of ambient air. This study indicated that gaseous sulfur in the Cincinnati atmosphere is largely SO2. This gas chromatographic system will be used to survey the atmosphere in several urban areas and in areas near kraft mill activities to determine the ratios of SOsto total atmospheric gaseous sulfur. Studies to date show the gas chromatographic system to be potentially the most reliable method for measuring ambient concentrations of hydrogen sulfide and sulfur dioxide. The system
produces a specific quantitative response to SOz and H2S since the FPD has at least a 10,OOO:l rejection ratio between sulfur and nonsulfur compounds and the elution order of these compounds is such that no other known or imaginable sulfur compound in the atmosphere could be eluted before or with these species. ACKNOWLEDGMENT The authors are grateful for the assistance of Ralph Baumgardner, who assisted in calibration studies.
RECEIVED for review February 3, 1971. Accepted March 12, 1971. Mention of commercial products does not imply endorsement by the Air Pollution Control Office or the Environmental Protection Agency.
Determination by Gas-Liquid Chromatography of Physiological Levels of Chromium in Biological Tissues Glenn H. Booth, Jr., and William J. Darby Department of Biochemistry, Dicision of Nutrition, Vanderbilt University School of Medicine, Nashville, Tenn. 37203 A gas-liquid chromatographic method for the analysis of chromium in complex biological tissues such as liver is presented. Following wet digestion of tissue, chromium is chelated with trifluoroacetylacetone and this complex analyzed on a gas-liquid chromatograph equipped with electron capture detectors. Minimal detectable concentration of Cr in liver is less than 20 ng Cr/g tissue. Recovery of added inorganic chromium or %r from liver averaged 95%. This analytical method is applicable to a variety of tissues and permits determination of Cr in tissues at levels encountered in physiological and deficiency states.
CHROMIUM is a trace element of potentially great physiologic and nutritional interest. A recent review by W. Mertz ( I ) summarizes current knowledge of the biological effects of this element. Additional insight into the metabolism of chromium requires a method which will determine Cr over the range of 10 to 200 ng Cr/g tissue (10-200 ppb) and which is free of interference from other constituents of tissues. Gas-liquid chromatographic determination of volatile metal chelates of Cr, Al, Fe, Be, and Cu has been used successfully for inorganic analysis (2-8). The potential specificity and sensitivity of gas-liquid chromatography (GLC) makes it the method of choice for analysis of many trace metals in biological tissue. GLC methods for the determination of chromium in serum and urine and in whole blood and plasma
have been published by Savory et al. (9, IO) and Hansen et a/. (11), respectively. The analysis of complex biological materials such as liver, kidney, spleen, and diet which contain high concentrations of many interfering metals and other substances has proved a much more difficult task than that of blood, serum, and urine and to date has not been possible by GLC methodology. Moreover, the requisite sensitivity for analysis of chromium in tissues in physiologic and deficiency states have not been achieved. Sensitivity is adequate for purified standards with detection limits in the subpicogram region; however, published GLC methods do not have the sensitivity required to analyze tissues for chromium at levels in the physiologic range of 10 to 50 ng Cr/g tissue (10-50 ppb) in blood, serum, or more complex tissue. The method proposed is a modification of the procedure of Savory (9, IO) which permits analysis of diverse biological materials including liver, fat, plasma, and diet, and increases detection limits of chromium in tissues to values below 20 ng Cr/g tissue (20 ppb). Sensitivity is adequate for the analysis of Cr in tissues in physiologic and deficiency states. This GLC method meets the need for a versatile, specific, sensitive assay for chromium to further understanding of the metabolic role of this trace element. EXPERIMENTAL
(1) W. Mertz, Physiol. Rec., 49, 163 (1969). (2) R. W. Moshier and R. E. Severs, “Gas Chromatography of Metal Chelates,” tst ed., Pergamon Press, Oxford, England, 1965. (3) R. E. Sievers, B. W. Ponder, M. L. Morris, and R. W. Moshier, Inorg. Chern., 2, 693 (1963). (4) W. D. Ross, ANAL.CHEM.,35, 1596(1963). (5) W. D. Ross and G. Wheeler, Jr., ibid., 36, 266 (1964). (6) W. D. Ross, R. E. Sievers, and G. Wheeler, Jr., ibid., 37, 598 (1965). (7) R. E. Sievers, J. W. Connolly, and W. D. Ross, J. Gas Chromatogr., 5 , 241 (1967). (8) W. D. Ross and R. E. Sievers, ANAL.CHEM., 41,1109 (1969).
Apparatus. A Varian Aerograph Model 1840 Dual Column Gas Liquid Chromatograph with dual +jaNi electron capture detectors was used for analysis. Operating conditions were: 10 f t X 2 mm glass columns packed with 3x (9) J. Savory, P. Mushak, and F. W. Sunderman, Jr., J. Chromarogr. Sci., 7, 674 (1969). (10) J. Savory, P. Mushak, F. W. Sunderman, Jr., R . H. Estes, and N. 0. Roszel, ANAL.CHEM.,42, 294 (1970). (11) L. C. Hansen, W. G. Scribner, T. W. Gilbert, and R. E. Sievers, ibid., 43, 349 (1971). ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
831
Tris-(l,l,l-tr~uoro-2,4-pentanediono)-chro~um(III) (Cr(TFA)$ was synthesized and purified by the method of Fay and Piper (12, 13). Purification was accomplished by chromatography on alumina and by fivefold recrystallization from hot heplane-benzene.
W v)
z
0
n cn
PROCEDURE
U
Determination of Chromium Levels. Into a 30-ml Kjeldahl vycor flask were placed: the sample to be analyzed, 10 ml of acid digestion mixture, and a glass boiling stick. All flasks were boiled until the residual volume was approximately 1 ml H 8 0 4 (30 to 45 minutes). The contents were transferred quantitatively with 10 ml of deionized water to 50-ml borosilicate glass round-bottom centrifuge tubes with ground glass joints; 4.0 ml of concd NH4OH were added to each tube. The pH was adjusted to 5.8-6.1 using H S 0 4 (Ultrex) and NH4OH (Aristar) with the addition of approximately 5 ml deionized water to dissolve the precipitate present. After the addition to each tube of 10 ml of 2 M acetate buffer, pH 6.0, and 2.0 ml of a 4.1M TFA solution in benzene (1 :1 v/v), the tubes were tightly capped with polyethylene stoppers and incubated with shaking for one hour at 70 "C. Tubes were cooled to room temperature, centrifuged 20 minutes at 2,400 rpm, and the aqueous layer was aspirated and discarded. Twenty milliliters of 1.ON NaOH were added to each tube, the tubes manually shaken for two minutes, centrifuged at 1,800 rpm for five minutes, and the aqueous layer aspirated and discarded. This NaOH washing procedure, which removed free TFA and many of the less stable metal chelates, was performed a total of 4 times. Thirtynine milliliters of benzene were added to each tube and the tubes shaken. Two microliters of this 1:40 dilution were then injected into the GLC for analysis. Use of a 10-pl syringe permitted bracketing the 2 - 4 sample (fore and aft) with 2 pl of benzene for more reproducible injections. Standard solutions made from purified synthetic Cr(TFA)3 were analyzed daily before and after experimental samples to construct the standard curve for determining absolute chromium content. Each experimental sample was analyzed by GLC a total of five times and the average value taken as the Cr content of that sample. Under the conditions of analysis, the first Cr(TFA)8 peak (trans isomer) emerged with a retention time of 5.6 minutes. The relative retention time of trans-Cr(TFA)3/lindane was 0.141. Time requjred between injections was approximately 15 minutes (Figure 1). Peak height measurement of the first Cr(TFA)3peak (trans isomer) was used for quantitative determinations.
W
U
C r ( TFA 1
2 0
(misomer
W IW
Cr (TFA) 3
n
-
0
I
2 3 4 5
6 7 8
9 1011 1 2 1 3 1 4 1 5
TIME ( m i n u t e s )
Figure 1. Typical gas chromatogram of Cr analysis of liver sample
I 4W
Cr (TFA12
358
20 IO
'0
50 100 150 200 250 300 350 400 450 500 550
m/e Figure 2. GLC-mass spectrograph of peak from purified synthetic Cr(TFA)3 OV-225 on Gas Chrom Q, 100-120 mesh (Applied Science Lab.); prepurified NP carrier gas; flow 30 ml/min; column temperature, 143 "C; injector temperature, 185 "C; detector temperature, 245 "C; detector potential, 90 volts, dc. An LKB gas chromatograph-mass spectrometer Type 9000 equipped with a 10 ft X 4 mm glass column packed with 5 % QF-1 on H.P. Chromasorb W (AW-DMCS), 80/100 mesh (Applied Science Lab.) operated at 70 mev was used for GLC-MS determinations. jlCr was determined using a Nuclear Chicago Model 4998 gas radiochromatography counting system and Nuclear Chicago scaler Model 8725 with well type NaI crystal. Visible and ultraviolet spectra were obtained with a Beckman DB scanning spectrophotometer. All glassware was washed in a solution of 1 "03:3 HCl:4 H 2 0and rinsed with deionized water. Reagents. The acid digestion mixture was a 6: 3 :1 (v/v/v) mixture of concd H N 0 3 (Aristar grade, Gallard-Schlesinger, British Drug House), 7 3 z HClOI, concd H2S04 (both and Ultrex Ultrex grade, J. T. Baker). Aristar grade ",OH grade acetic acid were used. Trifluoroacetylactone (TFA) (Pierce Chemical) was glassdistilled daily through a 10-inch Vigreux column packed with glass wool to obtain the fraction boiling at 107 "C. Pesticide-grade benzene (Fisher) was used. A chromium stock standard of 100 pg Cr/ml H20 was prepared from ACS certified potassium dichromate (KzCrzO7) (Fisher). Chromium working standards of 1 pg Cr/ml and 10 ng Cr/ml were prepared fresh daily from the stock standard for use in recovery experiments. 51Cr (61.2 mCi/mg) as CrC13 was purchased from New England Nuclear. 832
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
RESULTS
All standard curves for determining the absolute chromium content of samples were constructed using purified synthetic Cr(TFA)3. The synthesis and purification of this compound by the method of Fay and Piper (12, 13) yielded sharp crystals which melted cleanly at 161.5-162.5 OC, well above the 155 "C reported in the literature (13). Ultraviolet and visible spectra matched reported values closely. Analysis by GLC equipped with an electron capture detector showed only one pair of peaks. [Cr(TFA)3 occurs as cis and trans isomers.] Analysis by GLC-mass spectrometry produced the mass spectrum shown in Figure 2, which was identical for both peaks. There was a molecular ion at m/e 511, the molecular weight of Cr(TFA)3. The most abundant ion was at mje 358, corre(12) R. C. Fay and T. S. Piper, J . Amer. Chem. SOC.,84, 2302 (1962). (13) Ibid.,85, 500 (1963).
sponding to Cr(TFA)2. Prominent peaks were also seen at m/e 205 and 43, corresponding to Cr(TFA) and C H a C k O + , respectively. The identity of these fragments was confirmed by the presence of a characteristic isotopic abundance pattern for chromium in the appropriate peaks and by analogy to mass spectra of other chromium complexes which also show sequential loss of chelating units (14-16). This mass spectrum, its molecular ion and fragmentation pattern, and the other physical and chemical data clearly established that the two peaks seen in GLC analysis were the cis and trans isomers of Cr(TFA)3 and that the crystallized product was pure. Cr(TFA)3 as normally formed and which has not been recrystallized exists as 80% tran~-Cr(TFA)~ and 20% cisCr(TFA)3. The purification and recrystallization process used for preparation of Cr(TFA)3 standards yielded predominantly trans-Cr(TFA)(; the crystals were 96 rrunsCI-(TFA)~and 4 % ~ i s - c r ( T F A )as ~ determined by integration of GLC peak areas. The Cr(TFA)3 standard curves, then, represent mainly tr~ns-Cr(TFA)~.To determine Cr content of a sample the peak height of the trans isomer was measured and the amount of tr~ns-Cr(TFA)~ present quantitated by comparison with the purified synthetic Cr(TFA)3 standard curve. This was then multiplied by the conversion factor, 1.20, to convert hans-Cr(TFA)3 to ci~,trans-Cr(TFA)~ which then represented the total Cr contained in the sample. Solutions of the purified synthetic Cr(TFA)3 were analyzed on a GLC equipped with an electron capture (EC) detector and standard curves were constructed (Figure 3). The range from 25 pg to 250 pg Cr(TFA)3 (2.5-25 pg Cr) was used for tissue analysis. Since progressive deviation in detector response was observed with the 63NiEC detector above 250 pg Cr(TFA)3, all tissue samples were diluted so that the chromium concentration fell within the above limits, The minimum detectable amount of Cr(TFA)3 (peak height 3 times noise level) was 2.5 pg (0.25 pg Cr). To determine the minimal detectable quantity of chromium in tissue and to calculate the recovery of added chromium from tissue, the following experiment was performed. Livers were removed from normal 500-g chow fed rats, pooled, and homogenized. Aliquots of this liver homogenate were added to four groups of Kjeldahl flasks (5 flasks/group, 0.9 g liver/ flask) for Cr analysis. To one group no inorganic Cr was added. To the other three groups, 20 ng, 50 ng, and 100 ng of inorganic chromium were added to each flask in the respective groups. A fifth set of 5 flasks contained no sample and served as blanks. To all flasks, acid digestion mixture was added and all flasks were carried through the analytical procedure as per the experimental section. Each sample was analyzed on the GLC five times and absolute Cr content determined from the purified synthetic Cr(TFA)3 standard curves. The results of this study are shown in Table I. These results demonstrate that Cr determination in liver is possible below the 20 ng Cr/g liver level to approximately the 10 ng Cr/g level. The recovery of added chromium averaged 95%. Apparently none of the substances contained in a tissue even so complex as liver interferes with this analysis. Analyses have also been done on epididymal fat pads, serum, and diet and no interference was experienced. The minimal amount of Cr detectable by this method is (14) C. G. MacDonald and J. S. Shannon, Aust. J . Chem., 19, 1545 (1966).
(15) B. R. Kowalski, T. L. Isenhour, and R. E. Sievers, ANAL. CHEM., 41, 998 (1969). (16) J. L. Booker, T. L. Isenhour, and R. E. Sievers, ibid.,p 1705.
600 700
c
E 500E
400-
k.
300Y
2 a
2001001
1
1
0 50 100 150
250
375
500
Cr (TFA), INJECTED ( p g ) Figure 3. Standard curve of purified synthetic Cr(TFA)3
Table I. Recovery of Inorganic Cr from Liver. Cr reTotal CrfJ Tissue Covered content i Crc plus Cr std dev added Cr added Sample Blank 70 ng i 10 91 ng i- 14 21 ng Liver Liver + 20 ng Cr 112 ng i- 27 42 ng __ 21 ng
+ 50 ng Cr Liver + 100 ng Cr
Liver
133 ng f 19
63 ng
188 ng f 36
118 ng
20 ng 42 ng 50 ng 91 ng -100 ng
a To aliquots of rat liver homogenate (0.9 g) were added zero, 20 ng, 50 ng, and 100 ng Cr standards. Blank contained no tissue. All samples were analyzed for chromium by the procedure as described. Absolute amount of chromium recovered was determined from standard curves of purified synthetic Cr(TFA)?. Mean of 5 replicate samples, each analyzed on GLC 5 times. Tissue Cr is total Cr content minus Cr content of blank.
Table 11. 51CrRecovery Sample to which slCr added
Stage of procedure analyzed Hz0 Acid digestion Extraction by TFA Buffer, 4M TFA HzO Complete analysis Complete analysis Serum, 2.0 ml Liver homogenate, 0 . 9 g Complete analysis
Recovery of original counts,
109, 106 99, 98 85, 113, 119 93, 95
88, 104
approximately 10 ng. The amount of tissue analyzed is limited only by the digestability in 10 ml of acid digestion mixture. These two factors define the minimum Cr tissue concentration determinable for a given tissue. Standard deviations for the five replicate aliquots of blanks, liver, liver 20 ng Cr, liver 50 ng Cr, and liver 100 ng Cr are shown in Table I. The average standard deviation for all samples was 21.5 ng with an average relative standard deviation of 17.5z. Radiotracer 51Cr studies were also used to measure chromium recoveries (Table 11). plCr was not necessary for the Cr analysis but was used merely as another method of measuring recoveries.) 51Cr recovery was calculated following acid digestion, following extraction into the organic benzeneTFA phase, and at the end of the complete procedure. These recoveries following each stage of the procedure
+
+
+
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1 9 7 1
833
averaged 104%, W r was added to aliquots of serum (2 ml) and to liver homogenate aliquots (0.9 g), processed as described in the experimental section, and 51Cr recovery determined. These recoveries of 51Crfrom tissues averaged 95 %. Since the procedure of four washings with 1 N NaOH was rather rigorous, a standard solution of crystallized Cr(TFA)3 was carried through the clean-up procedure to determine any losses. No loss of Cr(TFA)3 was detected within the accuracy of the method. The original valence state of the Cr to be analyzed does not affect the analysis. Standards of either Cr(V1) or Cu(II1) were carried through the complete analysis and found to give identical results. To determine if species of chromium chelates other than Cr(TFA)3 were being formed in the analysis process, 51Cr which had been carried through the complete analysis was analyzed on a GLC. The effluent stream was split between a continuous flow radiomonitor and a flame ionization detector. Only one pair of radioactive peaks was seen and the retention times of these peaks corresponded to those of purified synthetic Cr(TFA)3. The combination of the four NaOH washes together with use of a 10-ft 3 OV-225 column produced a relatively clean chromatogram as seen in Figure 1. N o peaks overlapping the Cr(TFA)a trans isomer peak were seen. Other unidentified peaks did appear occasionally but these varied from sample to sample and never presented an analytical problem. DISCUSSION
The methods for Cr analysis in serum, blood, and urine recently published by J. Savory et al. (9, IO) and L. Hansen et al. (11) utilize the high resolution of gas-liquid chromatography to achieve specificity for Cr by separating its chelates from the other metal chelates present. They also both utilize the great sensitivity of the GLC to make possible detection of the extremely small quantities of chromium present in these tissues. The analysis of more complex biological tissues such as liver has proved a more difficult problem than blood, serum,
and urine. Liver contains more different metals and higher concentrations of metals than do the simpler tissues. In gas chromatographic analysis, many of these such as iron, copper, cobalt, and nickel, tend to emerge shortly after the solvent peak in large trailing peaks. These are probably thermal decomposition products and they obscure any Cr peaks present. Therefore, to analyze Cr in liver, it was necessary to improve resolution through more rigorous chemical purification of the chelation products and through GLC column materials with different retention properties. For the study of chromium tissue levels in physiological and deficiency states, it is necessary to measure Cr levels below 50 ppb (50 ng/g) This value had been the highest sensitivity achieved for tissue Cr using GLC methodology reported to date. The sensitivity for detection of pure Cr(TFA)3 standards is far in excess of this 50 ppb tissue Cr limit; however, the same sensitivity has not been possible for Cr contained in tissues. The method presented herein permits analysis of Cr concentrations in tissue of less than 20 ng Cr/g tissue. This permits the determination of levels of tissue Cr in physiological and deficiency states and, therefore, should provide a valuable tool for study of the metabolism of this element. ACKNOWLEDGMENT
C. Wetter and B. Fox were responsible for the GLC-MS determinations. J. T. Watson assisted with the mass spectra interpretations. J. Coniglio and A. Schulert provided fruitful discussion and suggestions. RECEIVED for review October 2, 1970. Accepted March 2, 1971. From a thesis to be submitted to the Graduate Faculty of Vanderbilt University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry. This work was supported in part by USPHS Grant No. AM 05441 and Vivian B. Allan M.D.-Ph.D. Fellowship. The LKB GLC-MS was purchased from funds supplied by NSF Grant No. GU-2057. The Varian Aerograph GLC was purchased in part from funds supplied by USPHS Grant No. ES00267.
Assay of Phenols and Arylamines via Oxidative Coupling David N. Kramer and Lucio U. Tolentino Physical Research Laboratory, Research Laboratories, Edgewood Arsenal, Md. 21010 Assay procedures are described for phenols and arylamines involving oxidative coupling with N,N-dimethylp-phenylenediamine to form the highly colored indaniline and indamine dyes, respectively. A potassium ferricyanide-sodium dichromate solution was found to be the best reagent for the oxidation of N,N-dimethyl-pphenylenediamine. The optimum conditions for maximum dye formation were at pH 9.5 for phenols and pH 6.1 for arylamines at 25 O C . The phenols and arylamines are assayable in the 10-6 to molar range with a relative standard deviation of 0.01 to 0.02.
AMONG THE MANY VARIED methods reported and in use for the assay of phenolic and arylamines compounds, the oxidative coupling procedure, employing N,N-dialkyl-o-phenylenediamine to form the highly colored indaniline and indamine dyes has been cursorily explored. Morita and Kogure ( I ) reported (1) Y.Morita and Y . Kogure, Nippon Kagaku Zasshi, 86 (1) 82 (1965). C . A,, 63 14064h. 834
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
a procedure for the determination of phenylenediamine isomers involving an oxidative coupling with phenol or a-naphtho1 using potassium ferricyanide to produce the colored indamine. The procedure required an extraction with chloroform, Camber (2) determined ketosteroid salicyloylhydrazones by oxidative coupling of N N-diethyl-p-phenylenediamine using a periodate oxidant. He utilized potassium ferricyanide oxidant in the estimation of phenols. He also investigated the use of silver nitrate and mercuric chloride as oxidants. The indaniline dyes, which have importance in the photographic industry, have been thoroughly investigated with respect to the mechanism of their formation (3),chromogeni(2) B. Camber, Nature, 175, 1085 (1955). (3) P. W. Vittum and G . H. Brown, J . Amer. Chem. SOC.,68, 2235 (1946).
