Figure 3. Maxima of a charge-transfer complex between tetracyanoethylene and a compound containing two olefin types Abscissa, wavelength (mp) Ordinate, intensity (absorbance)
case there is presumably less steric inhibition of hyperconjugation. Other more subtle effects such as cis-trans isomerism have not been considered. I n applying the present complexing technique, cognizance must be taken of interfering substances such as conjugated olefins and aromatics which not only form complexes absorbing a t similar wavelengths, but which also may react irreversibly with TCNE, usually via a Diels-Alder reaction. Preliminary consideration of the quantitative aspect of this method suggests that for analytical purposes an empirical determination of an apparent absorptivity for each olefin type, such as carried out by Long and Neuzil (6), is more desirable than the experimentally complicated, Benesi-Hildebrand ( I ) technique, ACKNOWLEDGMENT
The author thanks F. T. Weiss of these laboratories for his help and suggestions during the course of this
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work and also M. A. Muhs and A. G. Polgar for supplying the olefins used in the present study. LITERATURE CITED
(1) Benesi, H., Hildebrand, J., J . Am. Chem. SOC.71, 2703 (1949). (2) Chapman, J., Parker, A , , J . Chem. SOC.1961,2075.
13) Ham. J.. Platt. J.. McConnell. H.. ' C h e k Phys. 19, 1301 (1951).
>.
(4) Heilbronner, E., Helr. Chim. Acta. 36. 1121 11953).
(5) Long, D., X'euzil, R., ANAL. CHEX 27, 1110 (1955). (6) Merrifield, R., Phillips, W,,J . Am. Chem. SOC.80, 2778 (1958). (7) Mulliken, R., Ibid, 811 (1952). (8) Turner, D., J . Chem. SOC.1959, 30.
RALPHH. BAUER Shell Development Co. Emeryville, Calif. RECEIVEDfor review August 7 , 1962. hccepted October 25, 1962.
Programmed Cryogenic Temperature Gas Chromatography Applied to the Separation of Complex Mixtures SIR: We report the development of a technique for separating multicomponent mistures of highly volatile compounds by programmed temperature gas chromatography in the low temperature range-Le., below room temperatureand describe its application to the analysis of some comples mixtures of volatile compounds isolated from foodstuffs. The technique was developed in this laboratory about two years ago for separating volatile flavor and odor components in irradiated meat and has since been applied successfully to a variety of complex mixtures isolated from foodstuffs such as coffee and fish. The method has been described previously at meetings in Boston (8, 9). Subsequently, an application of the technique (to studies of the volatile components isolated from haddock) was 110
ANALYTICAL CHEMISTRY
reported from this laboratory by Mendelsohn, Steinberg, and Merritt ( 7 ) and again by hlerritt and Nendelsohn ( 1 2 ) . Recently, Baumann, Klaver, and Johnson (3) have described the application of a similar technique to the separation of mistures of C1 to C4 paraffins and olefins, and C1 to C16 n-paraffins. It is the purpose of this report to record in the permanent literature some of the advantages to be gained by employing PTGC in the cryogenic temperature range by describing the results of some studies which have been made in this field. EXPERIMENTAL
I n the early studies cryogenic temperature programs were obtained by a simple procedure. An appropriate Ushaped, glass gas chromatography column was placed within a cylindrical glass jacket. A suitable coolant--e.g.,
a mixture of dry ice in ethanol, temperature about -80' C.-was prepared in a Dewar flask and then, just prior to the start of the chromatogram, was poured into the jacket surrounding the column. Because the coolant jacket \\-as not insulated, the temperature of the column started to rise a t a nearly uniform rate almost immediately. The temperature was measured by a thermometer in the coolant jacket which was read through the outside of the glass. When the temperature of the column had risen to -65' C., the sample mas injected onto the column, or as was more frequently the case, was swept onto the column from a gas trap (6)with carrier gas. Elution proceeded as the temperature rose. -4schematic diagram of the apparatus is shown in Figure 1. The temperature programs obtained in this way are nearly linear at a rate of about 3' C. per minute in the range from -65' to -20' C., but as the
cryogenic temperature gas chromatographs will be given in a forthcoming manuscript,
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RESULTS AND DISCUSSION
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6 A S SAMPLING DEVICE
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Figure 1. Apparatus for programmed cryogenic temperature gas chromatography
temperature approaches ambient, the rate tends to decrease and the program becomes nonlinear from -20" C. to ambient. h typical temperature program is shown on the abscissa of Figure 2b. The reproducibility of the programs is quitc good for a given set up of the coolant jacket and column and varies but slightly for normal changes in ambient temperature. The departure of the program from linearity in the upper range is of little consequence where separation is the prime concern, but, for applications of the technique where the values of retention volumes are important, a more sophisticated apparatus than that described herein was required. Such an apparatus has been designed and constructed to obtain dual column, functional group, retention volume constants (IS, 14) in the cryogenic temperature range. This apparatus employs a column chamber which is initially cooled to a cryogenic starting temperature by a stream of refrigerated air. Starting temperatures from - 196' to -80" C. have been obtained by the use of various coolants t o refrigerate the air stream. Linear temperature programs have been achieved a t various rates from 2" to 10' C. per minute by means of a heater wire installed in the column chamber. The programs have been controlled both manually and automatically. Moreover, with the heated column chamber, the temperature range may be extended from subambient to superambient. For example, a typical program frequently employed has been from -70" to
+loo" c.
Programmed cryogenic temperature gas chromatography has been studied using a variety of packed '/r-inch and 1/8-inch and capillary 0.01-inch columns and employing argon ionization detectors as well as both conventional katharometers and microvolume thermal conductivity cells. Operating parameters such as stationary liquid phases,
flow rates, etc., have also been studied. The following column liquids are satisfactory: squalane, P,P'-oxydipropionitrile, Carbowax 4000, and Silicone SF-96. Base line drift can be adjusted to a negligible amount by proper compensation of carrier flow characteristics. The sensitivity of response of argon ionization detectors is enhanced from 15- to 20-fold when the carrier gas is cooled before entering the detector. Data for the operation of programmed
yl: 0 -65
The investigation of flavor and odor components isolated from food involves the separation and identification of very small amounts of substances in mixtures containing from 20 to perhaps 40 or 50 components. The successful analysis of such mixtures employing low temperature, high vacuum, fractional distillation methods for separation, followed by mass spectrometric identification, has been previously reported ( 4 , 1 O , I I ) ; but gas chromatographic methods afford the most satisfactory means of achieving a higher degree of separation of such mixtures. Gas chromatographic separations have indeed been used by a number of workers ( 1 , 2, 6, 16, 16), but the chromatograms have usually been obtained a t low-i.e., ambient or near ambient-isothermal column temperatures. I n our experience chromatograms obtained at room temperature failed to show adequate separation of multicomponent mixtures of very volatile substances. Figures 2a and 3a are typical examples of room temperature chromatograms of a volatile component sample isolated from irradiated ground beef. The chromatogram shown in Figure 2a is for a typically nonpolar
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Figure 2. Gas chromatographic separation of the volatile components isolated from irradiated ground beef Room temperature; about 27' C. Temperature programmed nonlinearly from -65' C. to ambient 5% (wt./wt.) squalane on 80- to 100-mesh firebrick, 6 ft. X in. RaPesargon ionization detector Sample size1 about 1 0 - 8 mole of gas a. b.
VOL. 35, NO. 1, JANUARY 1963
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Figure 3. Gas chromatographic separation of the volatile components isolated from irradiated ground beef a. Room temperature: about 27' C. C. to ambient b. Temperature programmed nonlinearly from -65' 5% (wt./wt.) P,P'-oxydipropionitrile on 80- to 1 00-mesh firebrick, 6 ft.
X ' I t in. RaZz6argon Ionization detector Sample size: about 1 0-3 mole of gas
liquid phase-Le., squalane-whereas that shown in Figure 3a is for a typically polar liquid phase-Le., P,P'-oxydipropionitrile. Obviously, this separation cannot be enhanced by a choice of the column liquid. Some attempts to improve the separability by employing isothermal chromatography a t cryogenic temperatures-e.g., - 80' C.-resulted in failure. Fewer peaks were observed than a t room temperature. When the columns were programmed from -65°C. to room temperature, however, a startling improvement in separability was obtained. The effect was the same whether a polar or a nonpolar liquid was employed on the column. Typical programmed cryogenic temperature chromatograms of irradiated meat volatiles are shown in Figures 2b and 3b. The efficacy of programmed cryogenic temperature separations is quite general and has been widely used by us in a variety of food flavor and odor studies. One of the most difficult mixtures encountered in such work is that obtained in the study of coffee aroma. The remarkable effectiveness in separation obtained by cryogenic programming is illustrated by the chromatograms shown in Figure 4. Other applications have been reported previously, (7, 12). 112
ANALYTICAL CHEMISTRY
Figure 4. Gas chromatographic separation of the volatile components isolated from ground roasted coffee beans Room temperature: about 27' C. Temperature programmed nonlinearly from - 6 5 ' C. to ambient 5% (wt./wt.) &P'-oxydipropionitrile on 80- to 1 00-mesh firebrick, 6 ft. X '/4 in. RaZz6argon ionization detector Sample size: about 1 0-3 mole of gas a. b.
-4s a method of separation, the absolute values of retention volume, their reproducibility, and the linearity of the program are unimportant. Eluted components, monitored by an appropriate detector, can be cold trapped in the usual way and identified by mass spectrometry or infrared spectrophotometry. The volatile components of a sample of irradiated meat were thus determined by subsequent mass spectrometry. Moreover, the technique of cryogenically programming a chromatography column has recently been employed in conjunction with the simultaneous analysis of the effluent gas stream with a time-of-flight mass spectrometer. I n these applications the use of the coolant jacket apparatus has served admirably well and offers the utmost in simplicity. For those gas chromatographic applications which depend on the accurate measurement of retention volume, an apparatus capable of providing a reproducible and preferably linear temperature program is required. Such a n apparatus has been devised and used to establish the validity of dual column retention volume constants ( I S , 14) in the cryogenic temperature range. Cryogenic temperature programs have
also been employed for the separation of fixed gases or very volatile substances at extremely low starting temperatures. For example, carbon dioxide, hydrogen sulfide, and methane have been separated by programming in the range from -160' to -80' C. The details of the various applications of programmed cryogenic temperature gas chromatography, the equipment devised, and a study of a number of operating parameters will be described in a series of forthcoming papers. LITERATURE CITED
M.L., Driscoll, J. L., McCarthy, A. I., J . Food Sci. 2 6 , 163 i 1961). (2) Bailey, 9. D., Mitchell, D. G., Bazinet, M. L., Weurman, C., Ibid., 27, 165 (1962). (3) Baumann, F., Klaver, R. F., Johnson, J. F., paper presented at the Fourth International Gas Chromatography Symposium, June 13-16, 1962, Hamburg, Germany (preprints of papers DD. 227-36). (4) 'Bazinet, M. L., Merritt, C., Jr., Anal. Chem. 34, 1143 (1962). ( 5 ) Bazinet, M. L., Walsh, J. T., Rev. Sci. Instr. 31, 346 (1960). (6) Issenberg, P., Wick, E. L., J . Agr. Food Chem., in press. (1) Bailey, S.D., Bazinet, - I
(7) Mendelsohn, J. M., Steinberg, AI., Merritt, C., Jr., paper presented a t the 22nd Meeting of the Institute of Food Technologists, June 10, 1962, Miami. (8) Merritt, C., Jr., New England Chromatography Society, Oct. 9, 1961, Boston, Mass. (9) Merritt, C., Jr., Third Symposium on Electron Beam Processes, March 24. 1961, Boston, Mass. (Published in the Proceedings, Bakish, R., editor, hlloyd Electronics Corp., 1961, p. 226). (10) Merritt, C., Jr., Bazinet, 11. L.,
Sullivan, J. H., Robertson, D. H., J . Aar. Food Chem.. in Dress. (11)”Merritt, C., ’ Jr.; Bresnick, S. R., Bazinet, M.L., Walsh, J . T., Angelini, P., 7, 784 (1959). (12) Merritt, C., Jr., Mendelsohn, J. M., Paper presented a t the 142 National Meetiniz. ACS Div. of Aer. and Food Chem.,-Sept. 12, 1962, ,
