Table I. Crystal Violet Solvent ExtractionSpectrophotometricDetermination of Br04- Ion in 6oCoY-Ray Irradiated CsBrOa Time of Sample irradiation, hrs [Br03-],M lo3 AbSobsd Abscor r a [Br04-],M 106 1-1 20.5 1.03 0.652 0.645 2.36 1-2 20.5 1.04 0.650 0.638 2.29 1-av ... ... .*. 2.33 i.0.03 2- 1 44.5 1.04 0.795 0.783 3.84 2-2 44.5 1.05 0.802 0.788 3.89 ... ... 2-av ... 3.87 =t0.02 3-1 65.1 0.995 0.774 0.775 3.75 3-2 65.1 1.10 0.854 0.826 4.30 3-av ... ... ... 4.03 2r 0.27 4- 1 90.5 1.02 0.802 0.796 3.98 90.5 1.03 0.813 0.806 4.08 4-2 4-av ... ... 4.03 Ilt 0.05 5-1 103 1.oo 0.795 0.795 3.97 5-2 103 1.01 0.792 0.789 3.90 5-av ... ... ... 3.94 i 0.04 6-1 164 1.oo 0.798 0.798 4.00 6-2 164 1.01 0.778 0.776 3.76 ... ... ... 6-av 3.88 i: 0.12 7-1 234 0.995 0.776 0.777 3.77 7-2 234 0.992 0.758 0.760 3.59 7-av ... ... ... 3.68 i 0.08 The Absoorrdata were obtained by normalizing the measured absorbance to the value one would obtain for a 1.00 X 10-3M Br03- solution. All correction factors, except with sample (3-2), were 50.014 absorbance unit. The correction factors were calculated from the data in curve 2, Figure 3, and are considered valid because the BrOa ion absorbance was independent of the Br03- ion concentration (see curve 3, Figure 3). Once the absorbance has been normalized, the Br04- ion concentration could be determined. I
.
.
0
analyzed for perbromate ion without a preliminary reduction of these species.
ported above were performed by Marion Ferguson of the Oak Ridge National Laboratory Analytical Chemistry Division.
ACKNOWLEDGMENT The authors thank E. H. Appelman of the Argonne National Laboratory for the gift of the 0.2M K B r 0 4 solution used in the initial phases of this investigation. The quantitative flame spectrophotometric analyses for potassium re-
RECEIVED for review September 29, 1969. Accepted November 24, 1969. Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corp.
Microdetermination of Chromium in Biological Materials by Gas Chromatography John Savory, Paul Mushak, F. William Sunderman, Jr.,l Richard H. Estes, and Norris 0. Roszel Pathology Department, University of Florida College of Medicine, Gainesville, Fla. 32601 MEASUREMENTS OF LEVELS of chromium in biological materials are important from both a toxicological aspect, and from the functioning of chromium as a trace element essential to normal processes in man. The occurrence, functioning, and measurement of chromium in biological systems has been reviewed recently by Mertz (1). Gas-liquid chromatography (GLC) has been used for the detection of metals as the &diketone chelates and extensive studies have been made of the basic problems involved in these Present address, McCook Teaching Hospital, University of Connecticut Medical School, Hartford, Conn. 06112 (1) W. Mertz, Phys. Reu., 49, 163 (1969). 294
measurements (2, 3). Many workers (4-8) studying the fluoro-P-diketonates have employed the electron capture detector because of its exceptional sensitivity to halogenated (2) R. W. Moshier and R. E. Sievers, “Gas Chromatography of Metal Chelates,” Pergamon Press, Oxford, 1965, and references cited therein. (3) W. E. Ross and R. E. Sievers, Tuluntu, 15, 87 (1968), and references cited therin. (4) D. K. Albert, ANAL.CHEM.,36, 2034 (1964). (5) W. D. Ross, ibid., 35, 1596 (1963). and G. Wheeler, J ~ .jbjd,, , 37, 598, (6) W. D. Ross, R, E. sievers, (1965). (7) W. D. Ross and G. Wheeler, Jr., ibid., 36,266 (1964). (8) R. E. Sievers, J. W. Connolly, and W. D. Ross, J. Gus Chromutog., 5, 241 (1967).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
organic compounds. Gas chromatographic detection of metal P-diketonates has been adapted to a number of specific quantitative analytical methods (3, 9-17). Preliminary reports (18, 19) from the authors' laboratory outlined a procedure for the gas chromatographic determination of chromium in serum. This present report establishes optimum conditions for the formation from serum and urine of chromium trifluoroacetylacetonate [Cr(TFA)3] and quantitation of this chelate by GLC. EXPERIMENTAL
Apparatus. Acid digestion and chelation-extraction procedures were carried out in 1-dram vials (A. H. Thomas Co.). The polyethylene cap liners were removed and the caps placed in refluxing benzene for 2 hours to remove organic compounds which might interfere with subsequent gas chromatographic determinations. Digestions were carried out using a thermostatically controlled aluminum heating block, and the chelation of chromium from the digest was performed using a heated shaking block. Both of these heating units have been described previously (19). A HewlettPackard model 402 gas chromatograph with a 'jaNi electron capture detector was employed with the following operation conditions: column, glass, 10 ft X 0.25-in. i.d., packed with 5 x QF-1 on WAW chromosorb 60-80 mesh pretreated with dimethyldisilazane; carrier gas, argon (95 z)-methane (5 column flow, 80 ml per min., column temperature, 150 "C.; preheater temperature, 175 "C. ; detector temperature, 200 "C.; detector, 63Ni electron capture in the pulse mode with a pulse interval setting of 150. Reagents. The acid digestion mixture consisted of concd H N 0 3 , 60% HC104, and concd H2S04 (3:3:1). All concentrated acids were of ultra high purity (Aristar, British Drug Houses, Ltd. Poole, England). The trifluoroacetylacetone (Peninsular Chemical Research Inc., Gainesville, Fla.) was distilled through a 6-inch Vigreux column. A fraction bp 107 "C, was collected and stored at 4 "C. A, standard solution containing 20 mg of chromium per 100 ml was prepared by dissolving 960 mg of CrK(S04)2.12H20 in 500 ml of water. This solution was further diluted to yield working standards. Chromium trifluoroacetylacetonate was synthesized (20) and appropriate standards were prepared by dissolving the pure compound in Nanograde benzene (Mallinckrodt Chemical Works, St. Louis, Mo.). Procedure. Blood was collected in acid-washed plastic syringes using sterile aluminum needles, and urine was collected in acid-washed bottles. To 1-dram vials were added 1 glass microbead and 0.2 ml of acid digestion mixture. Into the vials were transferred 0.2 ml of serum or urine, 0.2
z);
(9) G. P. Morie and T. R. Sweet, ANAL.CHEM., 37, 1552 (1965). (10) G. P. Morie and T. R. Sweet, Anal. Chim. Acta, 34, 314 (1966). (11) R. W. Moshier and J. E. Schwarberg, Talanfa, 13, 445 (1966). (12) W. D. Ross and R. E. Sievers, 6th International Symposium on Gas Chromatography - . and Associated Techniques, Rome, Italy, Sept. 22, 1966 (13) . , C. Gentv. C. Houin. and R. Schott. 7th International Svmposium on- Gas Chromatography and Its Exploitation, Copenhagen, Denmark, June 1968. (14) W. D. Ross and R. E. Sievers, 156th National Meeting, American Chemical Society, Atlantic City, N. J., Sept. 9, 1968 (15) W. D. Ross and R. E. Sievers, ANAL.CHEM., 41, 1109 (1969). (16) M. L. Taylor, E. L. Arnold, and R. E. Sievers, Anal. Lett.,
i, 735 (196s). (17) M. H. Noweir and J. Cholak, Environ. Sci. Technol.,. 3,. 927 ' (1969). (18) J. Savory, P. Mushak, N. 0. Roszel, and F. W. Sunderman, Jr., Fed. Proc., 27, 777, #3154 (1968) (19) J. Savory, P. Mushak, and F. W. Sunderman, Jr., Aduan. Chromafog., 181-186 (1969). (20) R. C. Fay and T. S . Piper, J. Am. Chem. SOC.,85, 500 (1963).
ml of chromium standard solution, or 0.2 ml of distilled demineralized water (reagent blanks). The vials were placed in the heating block adjusted to 190 "C for serum or 200 "C for urine, and the digestions were carried out by the method reported previously by this laboratory (19). The remainder of the procedure involving extraction of the chromium as Cr(TFA), from the acid digest was also as described previously (19). This extraction was carried out using a benzene solution of trifluoroacetylacetone. Three microliters of the benzene extract containing the Cr(TFA), were injected onto the column of the gas chromatograph. Cr(TFA), trans isomer emerged from the chromatographic column after a retention time of 10 minutes and the cis isomer emerged after 12 minutes. There was a ratio of 4 :1 in the amount of trans to cis isomer formed. The total area under the peaks of both trans- and cis-Cr(TFA), were measured by means of a disc integrator, and the concentration of chromium was computed from a calibration curve prepared via analysis of standard chromium solutions. RESULTS AND DISCUSSION
In order to achieve quantitative isolation of chromium in serum via the chelate Cr(TFA)3, it was first necessary to destroy completely all organic compounds present in the sample. No Cr(TFA)3 could be extracted from untreated serum or urine, or from a protein-free filtrate of serum or urine. The wet ashing technique used in the present procedure produced total degradation of organic matter in serum and urine. The presence of protein fragments in the digest was determined by applying samples of the digest to thin-layer chromatographic plates, developing, and spraying with a ninhydrin solution. The acid digestion described in this paper gave clear thin-layer chromatograms. Furthermore, where incomplete ashing was evident from ninhydrin positive thin-layer chromatograms, virtually no Cr(TFAI3 could be formed from the acid digest. Conditions for the digestion were studied to provide optimum yields of Cr(TFA)3 with gas chromatograms devoid of peaks other than solvent, chelating agent, and chelate. Digestion temperatures of 190 "C for serum and 200 "C for urine provided these optimum conditions. Slightly lower digestion temperatures provided good yields of C I ~ T F A but ) ~ gave gas chromatograms containing several unidentified peaks which interfered with the resolution of the Cr(TFA), peaks. Results obtained from serum using a digestion temperature of 180 "C were reported previously (19). Maximum extraction of chromium(II1) from the acid digest was achieved at a pH of 5.8-6.2, thus confirming work carried out by Scribner et a!. (21). Benzene was used as the solvent for extraction since it gave very little response to the electron capture detector. In addition, an ultrapure grade of benzene is available commercially. Removal of unreacted trifluoroacetylacetone from the benzene extract was accomplished by a sodium hydroxide wash as described by Ross and Sievers (3). This step is important since the electron capture detector is extremely sensitive to trifluoroacetylacetone. Reaction conditions for the formation of Cr(TFA)3 from aqueous chromium(lI1) solutions were studied to achieve optimum yields of the chelate. Analyses of undigested 100 pg per 100 ml aqueous standard solutions of chromium were made while the heated shaking apparatus was maintained at 25,40,55, and 70 "C. Triplicate measurements were made at each temperature using an extraction time of 30 minutes. Maximum yield of Cr(TFA)g was obtained at 70" with 30%, ~~
(21) W. G. Scribner, M. J. Borchers, and W. J. Treat, ANAL. CHEM.,38, 1779 (1966).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
295
GAS CHROMATOGRAMS OF Cr(TFA),
nI
Serum A Trans Cr(TFA1, B Cis Cr(TFA13
I\
STANDARD
A
I7
E
Authentic
Figure 1. Chromatograms of trans- and cis-Cr(TFA)a from digested serum, digested standard solution of chromium(III), and a solution of authentic Cr(TFA), in benzene w
130
p:
2 120 w u
i
lI0
z 100
;; 90
0
p:
0 VI
m
80
a
-
v 70
2
/-
60
-- 5c a
E
--2
40-
3
30-
R e g r e s s i o n Line
= 0.901
I a 205 0 10p: z
0
0
0
Ib
I
I
I
I
I
I
40 50 d0 iU 80 90 100 110 C H R O M I U M ( p g i 1O O r n 1 ) G A S C H R O M A T O G R A P H I C PROCEDU.RE
x)
30
I
120
Figure 2. Comparison of chromium concentration measurements in 16 serum samples by the gas chromatographic and atomic absorption spectrometric procedures indicates calculated regression line - represents theoretical relationship of x = y
---
296
Table I. Recovery of Chromium Added to Serum and Urine Number Chromium, ( p g per 100 rnl) of Specimens analyses Added Found Serum 5 80 7 8 . 2 1 6.1 13 100 Serum 92.5 =k 7.6 8 100 Urine 94.2 f 7 . 1
Recovery, 98 i 7.6 92.5 1 7.6 94.2 1 7.1
30%, and 65% of the maximum yield a t 25, 40, and 55 "C, respectively. The optimum times for reacting the standard chromium solutions in the shaker were ascertained by varying the duration of shaking while keeping the temperature constant a t 70". The yield of Cr(TFA), achieved a maximum after 45 minutes with 70 and 85 of maximum after 15 and 30 minutes, respectively. Yields of Cr(TFA)3 from aqueous solutions of chromium (111) were determined by comparing gas chromatogram peak heights with standard solutions of authentic Cr(TFA),. Yields in the 10-100 pg per 100 ml range averaged 8 2 x . The deviation from 100% may be attributed to the volatility of CrC13.6H20formed during the digestion step. Losses due to this factor were reduced by avoiding overheating of the digestion mixture. A calibration curve of total peak area of cis- and trunsCr(TFA), us. chromium concentration was linear up to 100 pg of chromium per 100 ml. Chromium standard solutions used for purposes of this calibration were carried through the entire procedure, including the acid digestion. Chromatograms obtained from digested serum, standard, and authentic Cr(TFA), synthesized in our laboratory are shown in Figure 1. Gas chromatographic conditions were selected to achieve isolation of cis- and trans-Cr(TFA), from other peaks obtained from extracted serum and urine samples. The identity of the Cr(TFA), peaks in chromatograms from serum and urine was ascertained by comparison of retention times of the assigned peaks with authentic samples of trans- (and cis-) Cr(TFA)3 under varying conditions of column temperature, carrier gas flowrate, and column packing; and by addition of a solution of authentic Cr(TFA), isomer mixture to each of the benzene solutions of the serum and urine extracts. Measurements of chromium(II1) recovery were performed by additions of the chromium standard solution to individual serum and urine samples. As indicated in Table I, recoveries of chromium were essentially quantitative. Several anions and cations were tested for interference in the determination of chromium. These ions were added to a standard solution containing 100 pg of chromium per 100 ml and the solutions were carried through the acid digestion and gas chromatographic procedure. The ions were tested for interference at the following concentrations : arsenic, lead, mercury, lithium, manganese, and strontium-50 pg per 100 ml; barium, copper, nickel, and zinc-100 pg per 100 ml; potassium-40 mg per 100 ml; calcium-10 mg per 100 ml; phosphorus (as HzP04-)--5 mg per 100 ml. None of these ions produced alterations in the yield of Cr(TFA), obtained from the standard chromium solution. Determinations of serum and urine chromium levels by GLC were compared with measurements by the atomic absorption procedure of Feldman et al. (22). The comparison data for 16 serum samples are given in Figure 2. The slope of (22) F. J. Feldman, E. C . Knoblock, and W. C . Purdy, Anal. Chim. Acta, 38, 489 (1967).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
Sample Serum Urine
Table 11. Precision of Duplicate Measurements of Serum and Urine Chromium Chromium concentrations, pg Cr per 100 ml N ~ of, Std dev of samples Range Mean duplicate analyses 20 4-124 44.7 h3.0 20 4-196 36.5 k2.9
the regression line (m)was 0.901 with a standard error of estimate (S) of 8.2 pg per 100 ml and a correlation coefficient (R)of 0.963. A similar comparison of 8 urine samples was made giving m = 1.043, S = 5.8 pg per 100 ml, and R = 0.923. Gas chromatography offers a potential advantage of greater sensitivity over atomic absorption. In the present study, the limit of sensitivity for the detection of chromium was 0.03 picogram injected onto the column of the gas chromatograph. This amount, which gave a peak height of 5 % above the base line, is equivalent to 0,001 pg of chromium per 100 ml of final benzene solution. In comparison, according to Feldman and Purdy (23) the limit of sensitivity of atomic absorption is 0.6 pg of chromium per 100 ml in methyl isobutyl ketone solution. In order to validate the method further, an experiment was carried out in which 4 rabbits were injected subcutaneously with 200 mg of chromium as an aqueous solution of Na2Cr207. Two control rabbits, not treated with Na2Cr2O7were also used in this study. Blood was obtained from all rabbits at regular intervals and all urine was collected. Chromium determinations were performed on all samples in duplicate. The serum chromium level of 4 pg per 100 ml prior to treatment rose to 43 pg per 100 ml immediately following the injection and gradu-
(23) F. J. Feldman and W. C . Purdy, Anal. Chim. Acta, 33, 273 (1965).
Rel. std. dev., 6.7 7.9
ally fell to 12 pg per 100 ml after four days. The total chromium excreted in the urine was 300 pg per 24 hours during the first day and dropped to 102,47, and 10 pg per 24 hours during the second, third, and fourth days, respectively. The chromium values determined in this experiment included both chromium(V1) and chromium(III), since the method included treatment of the acid digest with sodium sulfite to quantitatively convert all chromium(V1) to chromium(II1). The reproducibility of the method was evaluated by performing duplicate analyses on the serum and urine samples and this precision is summarized in Table 11. ACKNOWLEDGMENT
The assistance of W. Mertz and J. T. Piechocki in performing the atomic absorption measurements, and the technical assistance of D. S . Lindberg and L. G. Schrader is acknowledged and appreciated. RECEIVED for review August 1,1969. Accepted November 24, 1969. Supported by U. S. Atomic Energy Commission Grant AT-(404-3461, American Cancer Society Grant E374B, American Cancer Society Institutional Grant ACS-IN62H, and by Public Health Service Research Grant (National Cancer Institute) CA-98783-02.
Determination of Relative Rates by DifferentialThermal Analysis 1,ynn J. Taylor and S a n d r a W. Watson Okemos Research Laboratory, Owens-Illinois, Inc., Okemos, Mich. 48864
PREVIOUS STUDIES (1-4) have demonstrated that differential thermal analysis (DTA) can be employed for the determination of rate constants and activation energies. We report an extension of these methods which makes it possible to determine the relative rates of reaction of different reactive compositions under comparable conditions. According to the treatment of Borchardt and Daniels ( I ) , the expression for the reaction rate corresponding to a point on the DTA thermogram is
( 1 ) H. J. Borchardt and F. Daniels, J. Am. Chem. Soc., 79, 41
(1957).
(2) H. J. Borchardt, J. Inorg. Nucl. Chem., 12, 252 (1960). (3) G. 0. Pilovan. I. D. Ryabchikov, and 0. S. Novikova. Nature. 212, 1229 (i966). (4) A. V. Santoro, E. J. Barrett. and H. W. Hoyer, J. Am. Chem. Soc.. 89, 4545 (1967).
where n is the number of moles of reactant present at time t , no is the number of moles of reactant initially present, K is a heat-transfer coefficient characteristic of the apparatus, A is the total area under the curve, C, is the sample heat capacity, and AT is the differential temperature. At this point, the quantity of reactant remaining is
where a is the area swept out by the curve between the start of the reaction and time t . Ordinarily, the first term within the brackets in Equation 1 is much smaller than the second ( I , 2 ) ; we have been able to verify this by examination of experimental data obtained from an epoxy-amine reaction. If we neglect the first term, Equation l reduces to
_ -dn_ -_ _noAT dt
A
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
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