3,3’-DMNB in the starting material. The 3,4’-dimethyl isomer, described in this study, was not reported. A UV characterization of 2’,3-DMAB has been reported from work on the labeling of the amine with tritium by catalytic exchange (10). It is now recognized that the characterizations previously reported utilized a mixture of methyl isomers. The tritiated 2’,3-DMBA from exchange labeling was purified from radioactive impurities by partition chromatography and was shown to be free of other methyl isomers by the methods described here. The availability of pure 2,3’-DMNB now provides a tool for further investigations with this drug on the mechanism of the chemical induction of colonic tumors. Nitro-substituted aromatic compounds have been suggested as possible precursors for carcinogenic compounds in vivo (11). It has been shown that N-hydroxy compounds can be esterified in vivo with sulfate or other anions. The resulting
(10) M . W. Noall, J. Label. Compounds, 5, 192 (1969). ( 1 1 ) J . H. Weisberger, Cancer, 28,60 (1971).
ester has an increased carcinogenic activity (12). In this series of compounds, only one brief sentence in the literature states that 3,3’-DMBA has carcinogenic activity, probably similar to that of 2’,3-DMBA ( 1 ) . The 3,4’-isomer has not been reported. Thus, 2’,3-DMNB and 2’,3DMAB are compounds of prime interest for biochemical study and are now available free of other methyl positional isomers. The biological role of other positional isomers of this series remains to be determined.
ACKNOWLEDGMENT The authors thank Kenneth J. Lyman for skilled technical assistance in the determination of the GC-MS data. Received for review June 27, 1972. Accepted December 8, 1972. This work was supported in part by grants from the National Institutes of Health (GM-13901) and The Robert A. Welch Foundation (Q-125). D.M.D. is a Fellow of the Intra-Science Research Foundation, 1971-75. ( 1 2 ) J R DeBaun, J Y R Science 167, 184 (1970)
Smith, E
C
Miller
and J
A
Miller
On-the-Fly Gas Chromatography-Infrared Spectrometry Using a Cholesteric Liquid Crystal-Eff bent Interface John 0 . Lephardt and Bernard J. Bulkin’ Department of Chemistry, Hunter College of the City University of New York, 695 Park Avenue, New York, N. Y. 10021
On-the-fly infrared spectra of organic vapors in quantities as small as 5 0 p g have been obtained using a cholesteric liquid crystal-effluent interface. The liquid crystal fractionates sample from carrier gas. The technique yields spectra which are solution rather than gas phase spectra, eliminating problems of rotational structure and vaporliquid frequency shifts. The system can also be used for on-the-fly spectra of organic vapors in an air stream, with comparable efficiency. A technique for extending the system to accommodate higher boiling materials is described.
A wide variety of techniques for obtaining infrared spectra of gas chromatograph effluents have been described in the literature (1). Each of these may be designated as either a trapping or an on-the-fly technique. While trapping methods potentially offer the maximum sensitivity for infrared analysis (if 100% of the effluent can be transferred to the infrared cell), they have disadvantages which limit their utility. The trapped effluent can be concentrated in the IR cell allowing small quantities (-0.1 pg) to be measured. However, the time required to trap an effluent, transfer it to a suitable IR cell, and record the infrared spectrum can result in considerable delay between 1
To whom correspondence should b e addressed
(1)
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Ancillary Techniques of Gas Chromatography L S Ettre and W H McFadden Ed , Wiley-lnterscience, New York, N Y , 1969
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
exit of the effluent from the GC and availability of a plotted spectrum for analysis and decision making. There are thus many applications where an on-the-fly analysis is required even if this means increasing sample size. The development of rapid scanning dispersive spectrometers and interferometric Fourier spectrometers led to development of on-the-fly GCIR techniques. These systems offer approximately real time spectral analysis but have their own limitations. Operating on the effluents as they exit from the gas chromatograph, these techniques must record spectra of dilute solutions of sample in a carrier gas. In addition, they must be able to generate correct relative spectral intensities for a sample which varies in concentration with time. The result of these sampling problems is a minimum sensitivity of only 10-50 pg (2). Additional problems associated with on-the-fly GCIR techniques are the gaseous phase of the sample and the determination of when the sample is in the infrared cell. A spectrum recorded on a gas phase sample will contain rotational structure which distinguishes it from its liquid phase counterpart. The gas phase spectrum often shows frequency shifts for some bands of up to 20 cm-I compared to the liquid spectrum. These differences from liquid spectra can hamper the identification of materials if comparison with a liquid phase reference spectrum is used. Determination of when a sample enters an on-the-fly GCIR cell is generally made for a particular set of experi(2) J 0 Lephardt. Ph D Thesis. University of Maryland. 1972
mental conditions. A time delay between the maximum sample concentration as recorded by the GC detector and arrival of the maximum concentration in the IR cell results from the transfer line connecting the devices. This delay time varies with flow rate and must be determined accurately if spectra are to be recorded on effluents with GC bandwidths of a few seconds. In this paper, we describe a GCIR system which combines features of trapping and on-the-fly techniques. The heart of the system is a cholesteric liquid crystal film. As will be seen, the effluent-film interface operates on the same principle as the gas chromatograph that precedes it. On an efficient gas chromatograph column, the sample components may be described as bands moving down the liquid phase. If a thin film of a liquid phase material is coated on an infrared transmitting window, the film will act as a short column for effluents flowing over it. Each effluent will be adsorbed as it exits the GC, producing a zone that moves across the film. If the film transmits sufficient energy in the infrared, a spectrum of each effluent in solution in the film may be recorded as its zone traverses the film. Subsequently, the flow of pure carrier gas regenerates the pure film. The utilization of a thin film to fractionate GC effluents from the carrier gas enables their concentration as with trapping techniques but permits their return to the carrier gas after traversing the film. Provided the film can only support a zone equal to the half-band width of the effluents, no degradation of the chromatographic resolution should occur using such a cell. The concentration of effluents in a thin film offers several advantages over a gas cell for the measurement of IR spectra. An efficient gas cell for recording spectra of the effluents as dilute solutions in the carrier gas must have two features-long path length and small volume. These two criteria make it difficult to obtain high optical throughput (2,570 is a typical figure). A thin film cell can be assembled with throughput over much of the infrared spectral range exceeding 80%. This throughput advantage results in an approximately 3x advantage in signal-tonoise ratio in recorded spectra. Since the amount of scale expansion possible and the ultimate sensitivity is proportional to the signal-to-noise ratio of the spectra obtained, the thin film cell can offer an advantage in sensitivity. Another advantage of the thin film GCIR cell over a gas cell is the absence of rotational structure in the spectrum. This feature permits more reliable comparison of recorded spectra with collections of reference spectra (most of which are liquid phase, solution, or KBr pellet spectra). With proper selection of the film. spectral frequencies virtually unshifted from those of the neat liquid can be obtained. Clearly the choice of film material is crucial to success of this technique. The material must meet the following requirements: 1. It must efficiently trap a wide variety of organic compounds in a nonselective fashion, while having no affinity for helium. It would be desirable for the film to fractionate organic compounds from air as well; 2. The material must be chemically inert for general applications; 3. The material should either be able to operate over a wide temperature range, or a number of easily interchangeable materials for different GC operating conditions should be available (see also an alternative approach to this requirement discussed below); 4. Moderate film thicknesses (approx. 50 1) should have high infrared transmission over the region from 4000-400 cm-1; 5. The film must regenerate quickly in the flow of carrier gas. Certain cholesteric liquid crystals can meet all of these requirements, while providing additional advantages as
Figure 1. Liquid crystal film GCIR cell
well. It has been known for many years that these materials have a bulk interaction with organic vapors which causes a shift in their birefringence. Preliminary investigations in this laboratory showed that for approximately 50 organic compounds with varying functional groups, the infrared spectrum in the cholesteric solvent was identical (within 2 c m - l ) to that of the spectrum of the pure liquid (3). This was true for a variety of cholesteric mixtures as well. Of the solutes examined, only CBr4 reacted. Cholesterics are available over a wide temperature range. For some, the cholesteric phase exists for only one degree or less; for others, it is quite broad ( 4 ) . Many seem to interact with organic vapors to produce changes in birefringence color. Because, from the viewpoint of molecular vibrations, the cholesteric materials are primarily hydrocarbon, the infrared spectra contain few intense absorption bands outside the C-H region. They are thus quite applicable to the studies proposed. Use of certain cholesteric liquid crystalline materials as the thin film of a GCIR cell offers one additional capability not available with gas cell techniques. Use of such a material as the thin film permits the adsorption of an effluent to be detected by monitoring of the film's color. A shift in the color of the film can 'be used to trigger the start of infrared scanning. Since the sample to be measured triggers the start of spectral data collection, delay time measurements required with gas cells can be eliminated with a cholesteric liquid GCIR system. In this paper the results of using one such cholesteric system, 8070 cholesteryl nonanoate-20% cholesteryl propionate, are described. Use of the system to obtain on-thefly spectra of GC effluents as small as 50 pg is presented. as well as results of on-the-fly identification of materials in an air stream. Possibilities for improvements in the system are also described.
EXPERIMENTAL The GCIR cell employed is illustrated in Figure 1. The cell geometry is that of a standard fixed thickness infrared liquid cell. A Swagelok fitting is built into the cell for connection to a heated transfer line extending from the gas chromatograph exit port. Nichrome heating wire (ln/ft) was imbedded in epoxy around the cell frame t o heat the cell. The heater was supplied by a Variac stepped down through a 6.3-V filament transformer, ( 3 ) B. J. Bulkin and K . Krishnan, Abstracts of t h e 162nd ACS National Meeting, Washington, D.C.. 1971. ( 4 ) G. W. G r ay, "Molecular Structure and t h e Properties of L i q u i d Crystals," Academic Press, N e w Y o r k , N.Y., 1962.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
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Figure 2. Infrared spectrum of 80/20 cholesteryl nonanoate/cholesteryI propionate (by weight) liquid crystalline film Triggering of the start of spectral scanning was done manually on the teletype. A time delay associated with the transfer line between the gas chromatograph and the liquid crystal film cell was determined relative to the gas chromatograph’s detector recorder display. By initiating scanning a t a series of delays relative to the GC detector maximum response for sucessive 1-pl injections of trichloroethylene, the time delay for one column flow rate was determined. Provided the transfer line was operated above the boiling point of all the effluents, the delay time was inversely related to the flow rate. N o advantage has yet been taken of the cholesteric color change for triggering, however this work is in progress. The cholesteric system used is one which provides particularly brilliant colors. The gas chromatograph used for the GCIR measurements was a Carle “Basic” gas chromatograph equipped with 8-ft x Y&-in. SF96 15%, on 60-80 mesh chromosorb stainless steel columns. Xo effort was made to optimize the chromatographic conditions employed as principal concern was the infrared spectra. The technique does not appear to require any particular nonstandard GC features although a constant temperature effluent stream is important. The material used for the thin film was a mixture consisting of 8070 cholesteryl nonanoate and 2 0 2 cholesteryl propionate. The esters were Eastman reagent chemicals and were used without further purification. Weighed quantities of the esters were dissolved in petroleum ether and films were prepared by evaporation of the solvent from a drop of solution placed on the cell window.
RESULTS AND DISCUSSION
Figure 3. Scale-expanded 4 c m - resolution spectra recorded on-the-fly (16/64 scans) for effluents of 1 wl injections of a ) toluene, b ) chloroform, c) 1,2-dichloroethane, and d ) CCll using t h e liquid crystal GCIR cell Spectra were recorded a t a mirror retardation sufficient to yield 4 cm-I resolution using a Digilab FTS-14 Fourier Transform
Spectrometer operating between 400 cm-1 and 1000 c m - l . The interferometer was repetitively scanned eight to sixteen times during an on-the-fly run, and interferograms coadded into computer memory. A single scan takes approximately 1 second, the transformation approximately 15 seconds, and plotting approximately 30 seconds. Nothing in the experimental technique restricts operation to the 400 cm-1 to 1000 cm-l spectral range. However the particular interferometer used was equipped for a 1000 cm-I higher frequency limit. A stored reference mode was employed where 4-10 times the number of scans to be employed for measuring the effluents were collected, transformed. and stored in the reference file. Ratioing sample spectra against this reference spectrum ensured that the ratio spectra obtained were sample spectrum noise limited. The Digilab software automatically provides for proper scaling of the reference spectrum. 708
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
Figure 2 shows the infrared s p e c t r u m ( P e r k i n - E l m e r 521) of the 80% cholesteryl nonanoate-20% cholesteryl p r o p i o n a t e m i x t u r e between 4000 and 400 cm-’. It c a n be s e e n that t h e m i x t u r e has fair t r a n s m i s s i o n properties t h r o u g h o u t m o s t of the infrared region. E x c e p t i o n s t o t h i s generalization are t h e C-H s t r e t c h i n g region and part of t h e C-0 region. B e c a u s e of i n s t r u m e n t a l l i m i t a t i o n s in o u r laboratory, t h i s film is u s e d only between 1000 a n d 400 c m - I . However, i t is clearly useful over a wider r a n g e w i t h a standard interferometric Spectrometer (e.g., Digilab F T S - 1 4 ) . Figure 3 shows on-the-fly infrared s p e c t r a of 1-pl inject i o n s of various organic c o m p o u n d s o b t a i n e d u s i n g the liqu i d crystal interface. T h e s e c o m p o u n d s are a b i a s e d set of s a m p l e s i n that all e x h i b i t m e d i u m - s t r o n g absorption bands in the 500-1000 cm-1 region. It should b e n o t e d , however, that t h e m a j o r i t y of s t r o n g g r o u p frequencies i n organic c o m p o u n d s occurs outside t h i s range. F o r a conv e n t i o n a l Fourier t r a n s f o r m s p e c t r o m e t e r , most organic c o m p o u n d s s h o u l d give c o m p a r a b l y sensitive s p e c t r a . The s p e c t r a of F i g u r e 3 all s h o w excellent signal-tonoise ratio. T h e y also are liquid p h a s e s p e c t r a w i t h o u t freq u e n c y s h i f t s or r o t a t i o n a l s t r u c t u r e . A s p e c t r u m of a p p r o x i m a t e l y 50 pg of trichloroethylene recorded on-the-fly u s i n g t h e liquid c r y s t a l GCIR cell is s h o w n i n F i g u r e 4. T h i s s p e c t r u m is at the lower l i m i t of
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Figure 6. Spectra of nitrobenzene recorded using the liquid crystal GClR cell. a , immediately upon entrance of the effluent 93.$
500
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Scale-expanded spectrum of approximately 50 pg of trichloroethylene recorded on-the-fly (16/64 scans) at 4 cm-’ resolution using the liquid crystal GClR cell Figure 4.
T
801 a.
R
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Figure 5 . Infrared spectra of trichloroethyiene. a , 1 pl effluent recorded on-the-fly (16/64 scans) in the gas phase: b , 1 pI effluent recorded on-the-fly (16/64 scans) using the liquid crystal GClR cell; and c, capillary film of neat liquid between K B r plates
distinguishability for identification as trichloroethylene. One can set standards of minimum sensitivity based on signal-to-noise ratio arguments, but these will not be considered here. To illustrate the combined effects of the liquid crystal film on the effluent a spectrum of a 1-p1 injection of trichloroethylene recorded on-the-fly (16 scans) using the liquid crystal GCIR cell with the cholesteric film removed is shown in Figure 5 . An on-the-fly spectrum of a 1-pl in-
into the cell (16/64 scans scale expanded) and b , approximately one-half hour later (16/64 scans) scale expanded. The apparent absorbances below the base line in spectrum a are indicative of surface effects
jection (16 scans) obtained with the film present as well as a spectrum of a capillary film of pure liquid C2HC13 are included for comparison. The presence of P and R rotational bands in the gas phase spectrum and their absence in the thin film spectrum is notable. Careful measurement also shows that the Q branches of the gas phase spectrum occur at different frequencies from the liquid spectrum minima. As expected, the cholesteric film shows substantial enhancement of intensity over the gas phase spectrum. Comparison with the neat liquid indicates that the film has adsorbed more than 50% of the sample during the scans. The cholesteric region of an 80:20 mixture of cholesteryl nonanoate and cholesteryl propionate occurs between 45 and 47 “C. The effect of effluent boiling point on the adsorption on and desorption from the film poses limitations on the range of usefulness of a liquid crystal GCIR system. Experiments were performed to determine the feasibility of an alternative approach t o recording high boiling materials. When 1 p1 of nitrobenzene (bp 220 “C) was injected onto the column, the presence of the effluent in the film (as evidenced by the color) was observable for an extended period. Using the IR cell, spectra were recorded as a 1-p1 effluent entered the cell and approximately one half hour later; these are shown in Figure 6. Surface effects are observable in the first spectrum suggestive of condensation but are absent in the later spectrum. These are never seen for low boiling materials. The later spectrum is typical of a solution spectrum of nitrobenzene. To remove all of the nitrobenzene from the film required in excess of 1 hour of passing pure helium at low flow rates over the film as well as heating the cell above the cholesteric-isotropic transition temperature. Using a system where a “T” connector replaces the gas chromatograph, and using air in place of helium with a high flow rate (10 ml/sec), the spectrum of a 1-p1 injection of nitrobenzene shown in Figure i a was obtained. Upon completion of the plotting of this spectrum (approximately 1 minute after injection), another spectrum was recorded (Figure 7 b ) . As can be seen, the film has completely regenerated. At these high flow rates, spectra of low boiling materials could not be obtained as the sample was probably only in contact with the film for a fracANALYTICAL CHEMISTRY. VOL.
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Figure 7. Spectra of nitrobenzene ( 1 1 1 ) injected into an air stream and recorded on-the-fly using the liquid crystal GClR cell (16/64 scans). 1-11 injection: a , recorded immediately; b , recorded 11/2 minutes later after completion of plotting of spec-
ZT
trum
GC E F F LUENTS
AUXILLARY GAS SUPPLY
Figure 9. Spectra of 1-11 injections of various amines into an airstream recorded on-the-fly using the liquid crystal GClR cell. a, rn-chloroaniline, bp 230 "C;b, o-toluidene, bp 200 "C; c, ochloroaniline,bp 209 "C; and d , N,N-dimethylaniline,bp 192 "C.
RESTRICTOR
Figure 8. Schematic of system for extending the upper temperature limit of operation of liquid crystal GClR cell. A Venturi-type valve restricts flow of added gas back into the column of the GC
tion of a scan. An arrangement which produces a higher carrier gas:sample ratio for high boiling materials is shown in Figure 8. Such an arrangement extends the useable temperature range of a liquid crystal GCIR system, without degrading GC performance. The systems described can work with carrier gases other than helium. We have used the high flow rate system to obtain on-the-fly spectra of some amines in an air stream. The cholesteric film seems to have no strong interaction with the air stream, establishing a stable color close to that in helium. Figure 9 shows the spectra of some amines which were injected into an air stream through a "T" connector fitted with a septum. This system should be quite useful for systems where components in air must be detected and identified. An additional modification of the liquid crystal GCIR system is possible to increase the sensitivity further. If the film is coated onto an ATR plate, multiple passes through the sample could be achieved with corresponding multiplication of the sample absorbance and higher sensitivity. Use of such a configuration would require films with thickness less than 101 to ensure that the penetration depth extends to the surface of the film and all adsorbed species are seen.
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CONCLUSIONS The liquid crystal GCIR system offers some distinct advantages over a gas phase system for on-the-fly GCIR operation. With the liquid crystal system, the spectra obtained are solution spectra and more readily comparable with reference spectra. Color shifts occurring on adsorption and desorption of the effluents onto the liquid crystal film can be monitored to trigger infrared scanning and eliminate delay time errors associated with gas cell operation. Sensitivities of the two techniques are comparable for the described results, but selection of another material for the thin film (most liquid phase materials are good contenders) may permit more concentration of the effluents in the film, with correspondingly higher sensitivity. Use of an ATR system could also enhance sensitivity by multiplying the absorbance by the number of reflections. A disadvantage of liquid crystal GCIR system is its limited temperature range with a particular liquid crystal. One can, however, extend the range by changing the carrier gas to effluent ratio for high boiling materials. Further work on these and other related systems is in progress. Received for review August 30, 1972. Accepted November 30, 1972. This work was supported by a grant from the U S . Army Research Office-Durham. Purchase of the Digilab FTS-14 was made possible by a grant from the National Science Foundation.
