column is ready for another run the next morning. For less critical separations the flow rate can be speeded up, as discussed by Schroeder and Robberson ( 7 ) . The instrument can also be used to produce other types of gradients. The gradient generating system consists of a closed mixing vessel of constant size. Consequently, the formula of Drake (3) for such a system can be used to compute the various concentration parameters. The gradient can be calculated in successive portions, depending on its shape and the amount of solenoid valves available. If needed, stepwise elution can also be programmed. I n this case, the mixing vessel must be substituOed for a capillary tube having as many capillary inlet,s as there are to be steps in the elution proIf automatic regeneration gram. should also be desired, the regeneration buffer can t,hen be fed directly into the capillary collecting tube or a t some other point closer to the top of the column. Timers are commercially availablee.g. from Giannini Controls Co., Cramer Division, Centerbrook, Conn., Type 511 or 521, which could be adapted to this instrument whenever construction of the described timer should not be possible.
However, in most of these designs the sequence of timing impulses is not generated by a stepping switch, but by a battery of microswitches each activated by its own timing cam mounted on the timing motor axis. Therefore, considerable care must be taken to adjust the timing cams so that no overlap or dead time is produced between two buffers. Such timers are less readily adapted to produce other gradients because it is considerably more complicated to change all the timing cam settings. Changing the timing intervals during a run would also be difficult. ACKNOWLEDGMENTS
We thank W. A. Schroeder for a gift of the peptide mixture from bovine liver catalase, for the use of some of the facilities of his laboratory, especially the Technicon AutoAnalyzer, and for his advice during this investigation. We would also like t o thank Heinz FraenkelConrat for a gift of the tryptic peptides from tobacco mosaic virus. We are greatly indebted to E. Carver Jewett for his expert technical assistance in designing and constructing the timer.
LITERATURE CITED
(1) Brusca, D. R., Gawienowski, A. M., J. Chromatog. 14, 502 (1964). (2) Catravas, G. N., ANAL. CHEM.36,
1146 (1964). (3) Drake, B., Arkiv Kemi 8 , 1 (1955). (4) Funatsu, G., Biochemistru 3, 1351 (1964). (5) Peterson, E. A., Sober, H. A,, “A Laboratory Manual of Analytical Methods of Protein Chemistry,” Vol. I, p. 88, P. Alexander, R. J. Block, Eds., Pergamon Press, New York, 1960. (6) Schroeder, W. A,, Jones, R. T., Cormick, J., McCalla, K., ANAL.CHEM. 34, 1570 (1962). (7) Schroeder, W. A,, Robberson, B. ANAL.CHEM.37, 1583 (1965). (8) Schroeder, W. A., Shelton, J. R., Shelton, J. B., Olson, B. M., Biochim. Biophys. Acta 89,47 (1964). WILFRIED A. ROMBAUTS A. RAFTERY MICHAEL Division of Chemistry and Chemical Engineering California Institute of Technology Pasadena, Calif. 91109 INVESTIGATION supported in part by the Arthur Amos Noyes Research Fund and in part by a grant (HE-02558) from the National Institutes of Health, United States Public Health Service.
Analysis of Cigarette Smoke Fraction by Combined Gas Chromatography-Infrared Spectrophotometry SIR: Recently, an instrument was described (4, 7 ) which is claimed to eliminate the need for manual transfer of collected gas chromatographic peaks to infrared absorption cells. The instrument permits the shunting of an eluting peak directly into a gas absorption cell and provides a rapid (45 seconds) scanning of the infrared spectrum of the sample from 2.5 to 15.0 microns. The utility of the equipment was demonstrated experimentally by separation and spectral analysis of components in simple synthetic mixtures of compounds ( 7 ) . As with many instruments, such separations do not prove the value of the equipment when used in studies on complex mixtures such as natural products. T o determine such value, we have used the instrument to study components in an ether codistillate of cigarette smoke condensate. Details of the performance of the equipment and on the possible identities of some components in the codistil1at.e are presented herein. EXPERIMENTAL
Sample Preparation. Smoke condensate (980 grams) was partitioned between 4 liters of ether and 4 liters of 1N aqueous NaOH. The alkaline layer was washed with an additional 1614
ANALYTICAL CHEMISTRY
4 liters of ether, and all ether solutions were combined. Bases were removed from the combined ether solutions with two successive extractions (3.6 liters each of 0.2N HC1). The resulting ether solution of neutrals was washed free of mineral acid with small portions of water, dried over Na2S04, and the ether slowly distilled. The distillate, which gradually assumed a light yellow color during collection, was then concentrated on Stedman and spinning band columns to a volume of 5 ml. as previously described (6). Gas Chromatography. Usually, 100-pl. aliquots of the concentrated distillate were sufficient for both gas chromatography and infrared spectral analysis of individual peaks. The gas chromatographic portion of the instrument was a Loenco Model 70 (no endorsement implied) equipped with a thermal conductivity detector and fitted with dual columns (10 feet X 0.25-inch 0.d.) packed with 20% Apiezon L on Chromosorb W. Operating conditions were as follows: column temperature, 55’ C. for 20 minutes, then programmed a t 2’ per minute to 100’ C.; flow rate, 60 ml. per minute of helium; injector temperature, 200’ C.; detector temperature, 250’ C. Infrared Spectral Analysis. Using the Wilks chromatograph spectrograph, selected gas chromatographic peaks were shunted into the infrared absorption cell heated to 200’ C.
-
Between successive samples the cell was flushed with carrier gas by opening the entrance and exit valves. Infrared spectra of authentic compounds were obtained in the same manner as the unknowns in the mixture. RESULTS AND DISCUSSION
The gas chromatogram of the ether codistillate and tentative identifications of some components are included in Figure 1. Peaks were identified by infrared spectral characteristics and gas chromatographic retention times. The infrared spectra of some peaks indicated the presence of more than one component, as would be expected in a complex mixture of this type. Although the infrared spectra were somewhat lacking in resolution, they were of sufficient value when used in conjunction with retention data to provide further evidence of peak identities. For example, the spectra of peaks 4 and 5 were indicative of simple aldehydes. A study of the elution pattern of a homologous series of authentic normal aldehydes showed that the peaks in question elute between n-butyraldehyde and n-valeraldehyde. Therefore, peaks 4 and 5 appeared to be branched chain aldehydes and probably CS compounds.
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Figure 1 .
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Chromatogram of ether codistillate of cigarette smoke condensate 1. 3.
4. 5. 6. 12. 16. 17. 18. 19.
Acetaldehyde Ethyl acetate Isovaleraldehyde a-Methylbutyraldehyde n-Propyl methyl ketone Toluene Ethylbenzene and others m-Xylene Styrene, o-xylene, and others Dipentene
Additional studies with authentic compounds showed that peaks 4 and 5 corresponded to isovaleraldehyde and a-methylbutyraldehyde. The spectrum corresponding to peak 6 resembled that of methyl ethyl ketone, a known smoke component. However, the retention time of authentic methyl ethyl ketone did not match the unknown and further st,udies with authentic ketones showed that peak 6 was identical to n-propyl methyl ketone in retention time and infrared spectral characteristics. The spectrum of peak 6 was distinctly different from that of diethyl
ketone which had a similar retention time. The spectrum of peak 18 (Figure 2) resembled that of styrene, another known smoke component, except for a strong band just below 6 microns and a weak band just below 14 microns. Also, the retention time of authentic styrene was identical with that of peak 18. However, o-xylene eluted in t,he same time and the weak absorption just below 14 microns could be attributed to a relatively small amount of this component. The presence of carbonyl absorp-
tion in peak 18 indicated the presence of a t least one other component. The retention time and infrared spectrum (Figure 3) of the major peak in the chromatogram (peak 19) were identical with those of dipentene, a common constituent of tobacco smoke. These examples illustrate the utility of this instrument for separating complex mixtures of natural products. Even with only partial chromatographic resolution-e.g., peaks 4 and 5 in Figure 1-useful spectra can be obtained for some components. Compounds with a wide range of boiling points can be determined easily (peak 1, acetaldehyde, b.p. 21' C., peak 19, dipentene, b.p. 178' C.) on a single injection of the sample. However, the infrared spectra of fairly broad peaks cannot be obtained because of the low concentrations in the carrier gas-e.g., spectra of the unnumbered peaks eluting between styrene and dipentene. Although the instrument yields a small spectral readout (21 X 17 em.), suitable modification to improve this characteristic could be made easily. Interestingly, several of the compounds identified in the ether codistillate have also been found in the methanol codistillate of cigarette (1-3) and cigar (6) smoke condensate. These compounds include toluene, ethylbenzene, m-xylene, o-xylene, styrene, and dipentene. Other aromatic hydrocarbons, 2,5-dimethylfuranJ and a vinylcyclohexene reported in the methanol codistillate were not found in the present study. One hydrocarbon in the methanol codistillate, p-xylene, eluted with m-xylene under the chromatographic conditions used here and may, therefore, be present in the codistillate in amounts too small to detect. One of the components in the ether codistillate has no6 been previously reported in cigarette smoke: a-methylbutyraldehyde. 1
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WAVELENGTH, MICRONS
WAVELENGTH, MICRONS
Figure 2. Infrared spectrum of peak 18 containing styrene and other compounds
Figure 3. Infrared spectra of peak 19 from cigarette smoke fraction ( A ) and authentic dipetene (e)
VOL. 37, NO.
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Although infrared spectra were also obtained for peaks 2 , 7 to 11,14, and 15, the characteristics were not sufficiently distinct to claim tentative identification.
(2) Johnstone, R. A. W., Quan, P. M., J . Chem. SOC.1963, p. 2221. (3) Johnstone, R. A. W., Quan, P. M., Carruthers, W., Nature 195, 1267 (1962). (4) Kelliher, J. M., Brown, R. A., Abstracts, p. 28, Eastern Analytical Sym-
posium, New York City, November
LITERATURE CITED
(1) Cook, J. W., Johnst'one, R. A. W., Quan, P. M., Israel J. Chem. 1 , 356 (1963).
1964.
(5)-0sman, S., Barson, J., Tobacco Sci.
8, 158 (1964). (6) Stedman, R. L., Miller, R. L., J . Chromalog. 11, 409 (1963).
(7) Wilks, P. A., Jr., Brown, R. A., ANAL. CHEM.36, 1896 (1964).
IRWIN SCHMELTZ C. D. STILLS W. J. CHAMBERLAIN R. L. STEDMAN Eastern Utilization Research and Development Division Agricultural Research Service U. S. Department of Agriculture Philadelphia, Pa. 19118
Determination of Mercury in Wheat and Tobacco Leaf by Neutron Activation Analysis Using Mercury-197 and a Simple Exchange Separation SIR: We report a method for determination of mercury by neutron activation analysis which is more rapid and potentially more sensitive than methods previously described (3, 4,6, 9-11). None of the other methods appears suitable for a rapid and routine analytical procedure. In this study, a mercury exchange method was developed and applied to the separation of mercury from irradiated samples of wheat and tobacco. The method is highly selective so that further purification is not required. EXPERIMENTAL
Apparatus. Samples were counted using a R I D L 200 channel analyzer equipped with a 2- X 2-inch NaI(T1) well type detector (1- X 11/2-inch well size). The reflux condenser used was a Friedrichs Drip Tip type T 24/40; total length, 350 mm. A standard solution of mercury was prepared by dissolving a known weight of mercuric oxide in reagent grade concentrated nitric acid and diluting with demineralized water to give 48.2 pg. per ml. of mercury in a final concentration of O.1N nitric acid. The ground wheat used was supplied by the U. S. Food and Drug Administration. Tobacco leaf was obtained from the College of Agriculture, University of Maryland; the leaf was dried in an oven a t 45' C. for two weeks prior to analysis. Sample Irradiation. A weighed sample was transferred to a polyethylene vial in accordance with the procedure of Kim and Meinke (5). Mercury evaporation (9, 11) was minimized by coating the vials with paraffin, and the temperature of the reactor a t the loading site was maintained a t less than 30' C. ( 1 ) . The vials were positioned side by side within a polyethylene bottle along bith a vial containing the standard mercuric nitrate solution and irradiated in the University of Maryland reactor. The peak thermal neutron flux at the sample position was 1.4 X 10" neutron cm.-* set.-'
1616
ANALYTICAL CHEMISTRY
Rocedure. The irradiated sample was transferred to a 50-ml. round bottomed distilling flask containing 3 ml. of concentrated nitric acid. About 50 pg. of mercury carrier were added and the flask was connected to a reflux condenser. The sample was refluxed for 20 minutes using a Glas-Col heater. A 1- to 1.5-ml. portion of concentrated sulfuric acid was added through the side arm of the condenser, and heating was continued for an additional 25 minutes. The flask was cooled with an ice bath, and disconnected from the condenser. The outside of the flask was warmed to about 80" C. to eliminate nitrogen dioxide and was then replaced in the ice bat#h. The solution was diluted with 2 ml. of water and about 5 ml. of concentrated ammonium hydroxide were added dropwise until a solution with a pH of 1 to 2 was obtained. The solution was transferred to a 30-ml. Boston bottle and 0.5 gram of ammonium bromide was added; the bottle was shaken to dissolve the salt. A 0.050-ml. portion of triple-distilled mercury was added with a microliter pipet; the bottle was capped and shaken for 5 miputes. The mercury droplet was separated from the solution by passing the mixture through a fritted glass disk. The mercury was washed with water and acetone, and the droplet was transferred to a Lusteroid centrifuge tube (1 X 3.5 inches) for counting purposes. The mercury droplet was dissolved by adding 6 to 7 drops of concentrated nitric acid; volume was made to 10 ml. with water and counted in the spectrometer. The 68- to 77-k.e.v. photopeak region was measured to determine the mercury con tent .
Table 1. Activation Analysis of Tobacco Sample for Mercury Wt. of Level of
sample, gram
Mercury found, gram
Hg in sample, p.p.m.
0.543 0.560 0.602
0.21 X 0.26 X loeB 0.27 X lod6
0.39 0.46 0.45 0.43 f 0,03"
0
Table 11. Activation Analysis of Wheat Sample for Mercury
Wt. of sample irradiated, gram
Level of Mercury Hg in found, gram sample, p.p.m.
0.634 0.779 0.872 0.738 0.872
5.9 X 5 . 5 X loT8 5.8 X 4.8 X 6 . 0 X lod8
5
0.093 0.071 0.066 0.065 0.069 0.073 zt 0.010"
Standard deviation.
Table 111. Activation Analysis of Ground Wheat Samples (0.1 p.p.m. of Hg added) Wt. of
sample irradiated, gram
0.6798 0.7102 0.9010
RESULTS AND DISCUSSION a
Results obtained from the neutron activat,ion analysis of tobacco and wheat samples are summarized in Tables I and 11, respectively. Table I11 shows analytical data for the wheat sample (original Hg content, 0.073 p.p.m.) with 0.10 p.p.m. of Hg added as N-(ethylmercury) p-toluene sulfonanilide; the
Standard deviation.
Level of Hg in sample found 0.16 0.17 0.18 0.17 f O.0lo
Standard deviation.
expected value of 0.17 p.p.m. was obtained. Other investigators suggested that the loss of mercury during acid digestion could be considerable (8, 9). This
