0
0 I00
200
300
400
500
Nanograms arsenic
Figure 3.
Arsenic standard curve
The sensitivity of the procedure described in this paper is compared with two other AAS methods in Figure 4. The double beam flameless method ( 7 ) is approximately three times more sensitive than the deuterium-corrected hydrogen flame technique (11). The concentrating effect of the liquid nitrogen trap increases the sensitivity of the single beam HZflame procedure until it is comparable with the flameless technique over the critical 0 to 100-ng range.
RESULTS AND DISCUSSION Quantitative recovery of nanogram amounts of added arsenic from whole blood has been demonstrated. Similar results were obtained with other biological samples, including milk, hair, and tissue. These results are in preparation for publication (13). The single beam AAS arsine response is essentially linear to 100 ng of arsenic, and a useable curve to 500 ng can be plotted. The minimum detection level is about 5 ng arsenic. The use of a 2-mV recorder scale gives a 5X expansion of absorbance over the normal 10-mV output. With this Orheim, L. Lippman, C. J Johnson, H . H . Bovee, "Lead and Arsenic Levels of Dairy Cattle in Proximity to a Copper Smelter," in preparation.
(13) R M .
IO0
200
300
Nanograms arsenic
Figure 4. Comparison of sensitivity with other A A S methods 0 ,By flameless AAS ( 7 ) ; 0 , By single beam argon/hydrogen-entrained air AAS; A , By double beam argon/hydrogen-entrained air AAS, deuterium correction ( 17 )
increased sensitivity, flame noise becomes a critical factor a t the arsenic wavelength of 1937 A. To reduce this problem, a 20-mesh Tyler standard screen sieve (20-cm diameter) was centered over the flame. A 10-inch 3-sided chimney of sheet metal was designed to shield, additionally, the front and sides above the hydrogen flame from air currents. The burner undercarriage was surrounded with asbestos cloth. With the added shielding, flame noise was reduced to 0.005 absorbance unit. The applicability of this technique to other hydrides is being investigated. Received for review August 27, 1973. Accepted January 14, 1974.
Gas Chromatographic Determination of Barbiturates after Extractive Methylation in Carbon Disulfide Hans Ehrsson' Pharmaceutical Centre, Sodersjukhuset, 100 64 Stockholm 38, Sweden
Determination of barbiturates by gas chromatography is complicated by adsorption to the chromatographic support ( I ) . Methylation has in several cases been used to overcome this problem. The reaction has been performed prior to the gas chromatographic procedure using dimethyl sulfate (2, 3 ) , diazomethane ( 4 ) , and methyl iodide ( 5 ) Present address, Department of Pharmacology, Medical University of South Carolina, Charleston, S.C. 29401 (1) B. J. Gudzinowicz and S. J. Clark, J. Gas Chromatogr., 3, 147 (1965), (2) E. Mary Baylis, D E. Fry, and V. Marks, Clin. Chim. Acta, 30, 93 (1970). (3) J. T. Stewart, G . B. Duke, and J. E. Willcox. Anal. Lett., 2, 449 (1969). (4) J. G . H . Cook. C. Riley, R . F . N u n n . and D. E. Budgen, J. Chromatogr., 6, 182 (1961). (5) W. Dunges and E. Bergheim-lrps. Ana/. Lett., 6, 185 (1973).
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 7 , JUNE 1974
or in the injector of the gas chromatograph by flash methylation with tetramethylammonium (6) or trimethylanilinium ( 7 ) hydroxides as reagents. Both techniques have certain disadvantages. The former requires, in general. long reaction times. and with the latter there is a risk of incomplete methylation (8) and decomposition by the strongly alkaline reagent solutions (9, I O ) . In both techniques, such solvents have been used that large tailing fronts are obtained and the sensitivity of the flame ionization detector cannot normally be utilized to its full extent. Alkylation of acids by an extractive technique was used (6) G . W. Stevenson, Anal. Chem., 38, 1948 (1966) (7) E. Brochmann-Hanssen and T . 0 . Oke, J. Pharm. Sci.. 58, 370 (1969). (6) H . J. Kupferberg, Ciin. Chim. Acta, 29, 283 (1970). (9) J C Van Meter and H . W . Gillen, Clin. Chem., 19, 359 (1973). (10) R . J Perchalski and B. J. Wilder, Clin. Chem., 19, 788 (1973).
Table I. Methylation of Phenobarbital with 0.1M Tetrapentylammonium as Counter Iona Reaction time, min
Yield of dimethylphenobarbital, (A
42 72
5 15 30
94
60
103
A
Organic phase: Carbon disulfide, methyl iodide 10 i.1. Aqueous phase: 0.1M tetrapentylammonium in phosphate buffer, pH 10 (i. = 0.2); phenobarbital, 0.1 m g . Phase volumes: 0.5 ml; Temperature: 25 " C . The yields were determined by GLC with FID and expressed with dimethyl-phenoharbital (synthesized according t o the Experimental section) as reference. "
initially by Brandstrom and Junggren (11) for preparative organic purposes. The acids are extracted as ion pairs from a water phase to an organic phase having a poorly solvating ability consequently giving them a high reactivity in nucleophilic displacement reactions. The technique has lately been applied for the gas chromatographic determination of carboxylic acids ( I Z ) , phenols ( 1 3 ) , chlorthalidone ( 1 4 ) , and nitrazepam (15). Methylene chloride or benzene have been used as organic solvents in these determinations. The disturbances from the solvent can be decreased by use of carbon disulfide which has a very low flame ionization detector response (16). It gives a small solvent front and enables determinations with short retention times. This shortens the time of analysis and gives an increased sensitivity (17).
EXPERIMENTAL Apparatus. The gas chromatograph was a Varian 1700 equipped with flame ionization detectors. The column was of stainless steel (150 X 0.2 cm i.d.) and packed with 3% SE-30 on 100/120 mesh Varaport 30. Nitrogen flow was 30 ml/min; hydrogen flow. 30 ml/min: and air flow. 300 ml/min. Reagents. Barbiturates were of pharmacopoeial grade. Methyl iodide was obtained from E. Merck AG, Darmstadt. Tetrabutylammonium (TBA) and tetrahexylammonium (THA) hydrogen sulfate were delivered by. AB Hassle, Molndal, Sweden. Tetrapentylammonium (TPeA) hydroxide was prepared from the iodide by shaking with silver oxide (18). All solvents were of analytical grade. Preparation of Milligram Amounts of Dimethyl-Phenobarbital a n d Dimethyl-Pentobarbital. Fifty mg of the barbiturate dissolved in 25 ml of 0.02M THA hydrogen sulfate in phosphate buffer, pH 10 ( p = 1) was mixed with 10 ml of carbon disulfide and 0.5 ml of methyl iodide. The mixture was shaken a t room temperature for 10 min. The organic phase was separated, extracted with distilled water, and evaporated to dryness. Hexane and distilled water were added to the residue. After a thorough shaking of the mixture, the organic phase was evaporated to dryness. The residue was re-crystallized twice from ethanol/water. The IR spectra were in accordance with those reported for N,N'-dimethyl-phenobarbital and N,N'-dimethyl-pentobarbital, respectively (19). Dimethyl-phenobarbital had m.pt. 87 "C. Dimethyl-pentobarbital was a n oil. Methylation of Phenobarbital at the Microgram Level. Phenobarbital, 2.5 pg, was mixed with 0.5 ml of 0.01M THA hydrogen sulfate in phosphate buffer, p H 10 ( p = 11, 0.5 ml of carbon ( 1 1 ) A. Brandstrom and U. Junggren, Acta Chem. Scand., 23, 2204 (1969). (12) H. Ehrsson, Acta Pharm. Suecica, 8, 113 (1971). (13) H. Brotell, H. Ehrsson, and 0. Gyllenhaal, J. Chromatogr., 78, 293 (1973). (14) M. Ervik and K . Gustavii, Anal. Chem., 46,39 (1974). (15) H. Ehrsson and A. Tilly, Anal. L e t t , 6,197 (1973). (16) 8.L. Walker, J. Gas Chromatogr., 4,384 (1966). (17) R . P W. Scott. D. W. J Blackburn, and T. Wilkins, J. Gas Chromatogr., 5, 183 (1967). (18) K . Gustavii and G . Schill, Acta Pharm. Suecica, 3, 259 (1966). (19)J. M Manson and J A. R . Cloutier, Appl. Spectros., 15, 77 (1961).
2
L
min
Figure 1. I n t e r f e r e n c e b y t e t r a h e x y l a m m o n i u m i o d i d e in t h e determination of phenobarbital Organic phase: Carbon disulfide, Methyl iodide 10 pl, Aqueous phase: Phenobarbital 0.1 mg, Phosphate buffer, pH 10 ( p = 0.2)with 0.01Mtetrahexylammonium hydrogen sulfate. Phase volumes: 0 . 5 mi. Reaction time: 5 min at 25 "C. 1.0 pl of the organic phase is injected. A = dimethyl-phenobarbital. Chromatographic conditions: Column temperature: 160 "C; Injector temperature: 275 "C; Detector temperature: 210 'C; Sensitivity setting: 16 X lo-"
1 5
10
min
Figure 2. C h r o m a t o g r a m f r o m a r e a c t i o n m i x t u r e w i t h 5 dimethyl-phenobarbital
g/gl of
For derivative preparation, see Experimental section. Injected amount:
3.5 pI containing 5 ng/pl of dimethyl-phenobarbital. Chromatographic conditions: Column temperature: 150 "C: Injector temperature: 175 "C; Detector temperature: 200 "C; Sensitivity setting: 16 X lo-'*. The dotted curve illustrates the solvent front after injection of 3.0 pI of methylene chloride using the same chromatographic conditions ANALYTICAL CHEMISTRY, VOL
46, NO. 7. JUNE 1974
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disulfide, and 10 pl of methyl iodide. The mixture was shaken for 5 min at room temperature and centrifuged. About 3-4 pl of the organic phase were injected into the gas chromatograph.
RESULTS AND DISCUSSION Reaction Conditions. Phenobarbital and pentobarbital were chosen as model compounds in this study because of their frequent use and great difference in lipophilic character (20). The methylation was performed at p H 10 giving optimal conditions for the ion pair extraction of these barbiturates (21). The methylation rate of phenobarbital in carbon disulfide with 0.1M TPeA as counter ion is demonstrated in Table I. A quantitative derivatization is obtained in about 1 hr. The reaction takes place in the organic phase, and its rate is influenced by the degree of extraction, which depends on the polarity of the ion pair and the concentration of the counter ion (12). With TBA (0.1M) a yield of 98% of phenobarbital is extracted to the organic phase (conditions as in Table I, no methyl iodide). Pentobarbital was quantitatively methylated in 15 min with 0.1M TPeA. The higher reaction rate of pentobarbital compared to phenobarbital is probably attributed to its more lipophilic character, cf. (22). The phenobarbital and pentobarbital derivatives have partition coefficients > 100 between carbon disulfide and (20) D. J. Lamb and L. E. Harris, J . Amer. Pharm. Ass., Sci. Ed., 49, 583 (1960) (21) K . 0. Borg and G . Schill, Acta Pharm. Suecica, 5, 323 (1968). (22) K . Gustavii, Acta Pharm. Suecica, 4, 233 (1967).
water which means that >99% of the derivatives is present in the organic phase (equal phase volumes). Interference from Tetraalkylammonium Iodide. Figure 1 gives a chromatogram obtained after injection of a reaction mixture of dimethyl-phenobarbital with THA as counter ion (injector temperature 275 "C). The dimethylphenobarbital peak is preceded by a large tailing peak originating from the reagent. Iodide is formed by the reaction and is extracted as tetraalkylammonium ion pairs to the organic phase. The tetraalkyl ammonium iodide decomposes a t high injector temperatures to the corresponding trialkylamine which gives rise to the interfering peak (23, 24). It could be completely eliminated by lowering the injector temperature to 180 "C or by extracting the organic phase with an aqueous solution of silver sulfate. The sulfate salt was preferred because of the low extraction constants of sulfate ion pairs (25). Determination at the Microgram Level. A chromatogram obtained after injection of a reaction mixture of 5 ng/p1 of phenobarbital as the dimethyl derivative is given in Figure 2. Compare the tailing solvent front of methylene chloride chromatographed under the same conditions. ACKNOWLEDGMENT I am indebted to Gbran Schill for valuable criticism of the manuscript and to Barbro Naslund for drawing the figures. Received for review October 23, 1973. Accepted February 8, 1974. (23) J MacGee and K G Allen, Anal Chem 42, 1672 (1970) (24) D R Matthews. W D Shults, and J A Dean, Anal Lett, 6, 513 (1973) (25) A Tilly ActaPharm Suecica, I O . 111 (1973)
Sensitive Gas Chromatographic Determination of Cyanide James C. Valentour, Vijay Aggarwal, and Irving Sunshine Cuyahoga County Coroner's Office, 2121 Adelbert Road, Cleveland, Ohio 44106, and The Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44 106
The extreme toxicity of cyanide compels sensitive methods for its detection and determination. Reviews ( I , 2) of the methods for the determination of cyanide indicate that numerous approaches are used. Among these, gas chromatography (3, 4 ) has not been applied successfully to the determination of microgram or submicrogram quantities of cyanide. This paper reports a sensitive gas chromatographic procedure for the determination of cyanide in biological specimens based upon its conversion to cyanogen chloride using chloramine-?' (sodium p-toluene sulfonchloramide) (5, 6). The cyanogen chloride thus formed is extracted with hexane and injected into a gas chromatograph equipped with an electron capture detector.
( 1 ) L. S. Bark and H. G . Higson. Analyst (London).88, 751 (1963). (2) D . B. Easty. W. J. Blaedel, and L . Anderson, Ana/. Chem.. 43, 509 (1971) . (3) K . G . Woolmington, J . Appl. Chem.. 11, 114 (1961). ( 4 ) C . R . Schneider a n d H . Freund. Ana/. Chem., 34, 69 (1962). (5) J. Epstein, Ana/. Chem., 19, 272 (1947) (6) M. Feldstein and N . C. Klendshoj, J. Lab. Ciin. Med., 44, 166 (1954).
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EXPERIMENTAL Apparatus. The gas chromatograph used was a Hewlett-Packard Model 5713 equipped with a 63Ni pulsed linearized detector. A &foot long, Y4-inch 0.d. stainless steel column packed with 7% Halcomid M-18 on Anakrom ABS 90-110 mesh was employed. The flow rate of 5% methane in argon was 40 ml per minute and the oven was maintained at 55 "C. A 1-millivolt strip chart recorder and a Perkin-Elmer P E P data reduction system were used for measuring retention times and areas. Reagents. Spectrograde hexane was used. The other reagents were reagent grade. The chloramine-T solution was prepared by dissolving 250 mg of the salt in water and diluting to 100 ml. Procedure. The reagents and solutions to be analyzed were kept in an ice bath, and only one sample was processed at a time. These precautions minimized small, but measurable, losses of the volatile cyanogen chloride. Cyanide was separated from blood, urine, (diluted) gastric contents, and aqueous solutions by a generally accepted technique using microdiffusion cells containing 0.1N sodium hydroxide in the central reservoir (6). After separating the cyanide from the specimen by this technique, 1.00 ml of the sodium hydroxide was placed in a screw-cap culture tube. To this, 2.00 ml of hexane, 2.0 ml of 1.OM sodium dihydrogen phosphate, and 1.0 ml of the chloramine-T solution were added. The tube was capped, briefly shaken, and replaced in an ice bath. After approximately 5 min-
