Art ides Anal. Chem. 1994,66, 425-430
Multidimensional Gas Chromatography Coupled with Infrared and Mass Spectrometry for Analysis of Eucalyptus Essential Oils Kevin A. Krock, N. Ragunathan, and Charles L. Wilkins' Department of Chemistry, University of California, Riverside, Riverside, California 9252 1-0403
A parallel cryogenic trapping multidimensional gas chromatography /Fourier transform infrared spectrometry/mass spectrometry combination (MDGC/FT-IR/MS) is evaluated for analysis of essential oils. As a demonstration of the value of this analytical approach, analyses of several essential oil samples are reported. The method developed to differentiate these oils is quite general and can be applied equally well to analysis of various other complex mixtures. By use of a multiport manual valve system in conjunction with multiple parallel cryogenic traps, it is demonstrated that three-stage or higher order gas chromatographic separations (CC") are practical, providing the expected improvements in separation efficiency needed for accurate qualitative analysis. Chromatographic and spectroscopic data obtained by analysisof two authentic and one known adulterated Eucalyptus australiana oil samples are utilized as benchmarks for analysis of a fourth sample which is suspected of being adulterated and a fifth sample from an unknown source. It is concluded that the presence of either camphor or a combination of a-thujene, decane, sabinene, 8-phellandrene, and y-terpinene is indicative of eucalyptus oil adulteration. Gas chromatography has been the analytical tool of choice for separation and identification of essential oil components for many years. Traditionally, oil analysis has been performed mainly with flame ionization detection (FID) or with mass spectral detection.' However, there are several potential difficulties with both of these methods. When FID detectors are employed, retention times from separation on two different polarity columns are used to identify various components, requiring that retention times of authentic standards must be known for chromatography on both columns for each component of interest. Furthermore, because FID detectors are nonspecific, it is difficult to deal with component overlap. ~~
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(1) Chang, S. K. C.; Rayas-Duarte, P.; Holm,E.; McDonald, C. Anal. Chem. 1993, 65, 334R.
0003-2700/94/0366-0425$04.50/0 0 1994 Amerlcan Chemical Society
Mass spectrometry (MS) offers many advantages over FID detection with respect to specificity. Unfortunately, because most essential oils contain significant numbers of compounds which are isomers of one another, their positive identification using only mass spectral detection is often precluded. A wellknown method of overcoming this difficulty is to incorporate infrared (IR) spectroscopy in the analytical system. Use of this complementary detector, in addition to mass spectrometry, greatly enhances the reliability of component identification.2" However, the accuracy of the identification step is still very much dependent upon the purity of the compounds resulting from a chromatographic separation. With mixtures containing as many components as an essential oil, it is virtually certain that components will be incompletely separated using a single stage of ~eparation.~ An appealing potential solution to this dilemma is the use of multidimensional gas chromatography (MDGC),*y9 as reported here. With MDGC, it should be possible to conveniently achieve much greater separation efficienciesthan is practical for a single-stage separation. When this methodology is coupled with infrared and mass spectral detection, the accuracy of qualitative analysis is limited by the quality and size of spectral databases available, rather than by an inability to obtain spectra of pure mixture components.10 (2) Wilkins. C. L. Science 1983, 222, 291-296. (3) Wilkins, C. L.; Giss, G. N.; White, R. L.; Brissey, G.M.; Onyiriuka, E. C. Anal. Chem. 1982,54, 2260-2264. (4) Cooper, J. R.; Bowater, I. C.; Wilkins, C. L. Anal. Chem. 1986, 58, 2791-
2796.
( 5 ) Gurka, D. F.; Titus, R. Anal. Chem. 1986,58, 2189-2194. (6) Olsen, E. S.; Diehl. J. W. J. Chromatogr. 1989, 468, 309-317. (7) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418-424. (8) Giddings,J. C. HRCCC, J.High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10, 319-323. (9) Bertch, W. Multidimensional Chromatography Techniquesand Applications; Cortes, H. J., Ed.; Chromatographic Science Series; Marcel Dekkcr: New York and Basel, 1990, Vol. 50, pp 74-174. (10) Krock, K. A.; Ragunathan, N.; Klawun,C.;Sasaki,T.; Wilkins, C. L. Analyst, in press.
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A demanding application which is a worthwhile test of the MDGC/IR/MS combination is differentiation of adulterated essential oils from naturally occurring oils. In this context, an adulterated oil is one which is derived from a starting oil (presumably less expensive), altered to mimic another more expensive oil. Alternatively, synthetically produced major components (adulterants) are used to dilute the natural oils and increase profitability. However, although the adulterated materials are less expensive to produce than the natural oils, their chemical properties (e.g., taste, smell, stability, etc.) may be inferior to the natural products they replace. For eucalyptus oil, in the past, testing was performed by simply measuring the optical rotation of a sample. For example, natural Eucalyptus australiana oil typically has an optical rotation of approximately +3.6O while adulterated samples usually have negative rotations. However, in the recent past, those producing substitutes have become more sophisticated and have added optically active compounds to mimic the rotation of the authentic eucalyptus oil, rendering the optical rotation assay ineffectual. Because the adulteration additives are very similar to the components of natural oils, this modification is difficult to assess by GC with an FID or MS. A possible method allowing analysis with GC/MS would be to use chiral columns and analyze the enantiomeric ratios.” However, this method is not general and is suitable for target analysis only. Fortunately, in some adulteration cases, the materials that are altered to simulate natural oils contain some remnants of their true origin, but these traces are typically masked by high concentrations of compounds that are found in the natural oil. As a consequence, these mixtures present a difficult analytical problem, because relatively minor components are masked by the presence of the chemically very similar major components. In the research presented here, combined parallel cryogenic trapping MDGC/IR/MS is investigated as a possible means of differentiating between natural and adulterated essential oils. Specifically, known samples of two natural eucalyptus oils and an adulterated sample derived from camphor oil were analyzed. Compounds present in the latter sample but not in the known natural samples were identified as “marker” compounds indicative of the origin (natural or synthetic) of an oil. Using these compounds as references, a suspected adulterated oil and an oil of unknown origin were analyzed. The results described here demonstrate the power of multidimensional separations with spectroscopic detection and suggest the wide potential applicability of this approach to complex mixture analysis.
EXPERIMENTAL SECTION Instrumentation. A commercially available HewlettPackard (HP) GC/IR/MS system was modified for multidimensional gas chromatography. The system consists of a H P 5890 Series I1 gas chromatograph coupled in parallel with an H P 5965B infrared detector (IRD) and a H P 5970B mass selective detector (MSD). A schematic diagram of the instrument is shown in Figure 1. Details of the instrument modifications are described elsewhere.*J2J3 Effluent output (1 1) Mosandl, A.; Schubcrt, V. J. Essenf. Oil Res. 1990,2, 121-132. (12) Ragunathan, N.; Krock, K. A.; Wilkins, C. L. Anal. Chem. 1993,65, 1012-
1016. K. A.; Ragunathan, 1012-1016.
(13) Krock,
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N.; Wilkins, C. L. J . Chromarogr. 1993, 645,
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& l
MS / B
Vent
Figure 1. Schematic diagram of the parallel Cryogenic trap MWC/ FT-IR/MS system: (A) Rt,-1701 Intermediate polarity precolumn; (B) HP 59708 MSD; (C) 59658 I R D (D) four-port, two-way valve (300 O C maximumtemperahue);(E)extemalmakeup gas;(F)six-port eekctkn valve (300 O C maximum temperature); (0)stainless steel cryogenic traps; (ti) three-port, two-way valve (300 O C maximum temperature); (I) Stabllwax polar analytical column; (J) Rt,-5 nonpolar analytical column. Table 1. 011 Samples Exampkd In Thk Study
sample
source
optical rotation’ (deg)
A
natural eucalyptus oil natural eucalyptus oil known adulterated oil suspected adulterated oil eucalyptus oil of unknown origin
+3.1 +3.6 -1.5 +3.1 +3.6
B C D
E
0 Measured with a Perkin-Elmer Model 241 polarimeter, using the 589-nm sodium Dline.
from the lightpipe infrared detector is either vented or collected into one of the six parallel cryogenic traps. For second and higher stages of analysis, trapped effluents are released from the selected trap to one of the analytical columns by turning off the liquid nitrogen flow to that trap. Preceding any higher order separations, a first-stage analysis is carried out and examined to determine the MDGC strategy. In the present configuration, the valve switching required for multistage experiments is accomplished manually. Using the arrangement depicted in Figure 1, the parallel cryogenic trapping multidimensional system does not interfere with normal operation of the GC/IRD/MSD system and both infrared and mass spectral data are obtainable for eluting components resulting from any stage of G C separation. Samples. The five eucalyptus oil samples analyzed by MDGC/IR/MS in this study are listed in Table 1, together with their optical rotations. All samples were obtained from Berj6 (Bloomfield, NJ). Chromatography. For separations, injections were 0.1-pL splitless injections followed by an injection port purge 55 s after injection. Three columns were used in the chromatograph: one precolumn and two analytical columns. The precolumn was a Restek (Bellefonte, PA) Rt,-1701 intermediate polarity column (30 m X 0.32 mm, 1.0 mm film thickness), and the two analytical columns were Restek (Bellefonte, PA) polar Stabilwax and nonpolar Rt,-5 columns (both 30 m X 0.32 mm, 1.0 mm film thickness). The temperature was programmed to hold the columns at 50 OC for 2 min, then to increase at 4 OC/min to 200 OC, followed
Table 2. Component IdentHlcatlons Supported by Both infrared and Mass Spectra
peak no.
peak ID
peak no.
peak ID
peak no.
peak ID
1 2 3 4 5 6 7 8 9
a-pinene l,&cineole terpinolene p-cymenene linalool fenchyl alcohol trans-pinocarveol 4-terpineol a-terpineol
10
geraniol a-terpinenyl acetate aromadendrene camphor a-thujene camphene sabinene @-pinene @-myrcene
19 20 21 22 23 24 25 26 27
decane a-phellandrene 3-carene a-terpinene limonene y -terpinene carvomenthene @-phellandrene p-cymene
11
12 13 14 15 16 17 18
by an additional 10-min period at the maximum temperature. Linear carrier gas flow velocities used in all parts of the study were -30 cm/s. Each sample was separated on each of the three stationary phases by utilizing a looping sequence to retrap allof thecolumn effluents from a particular injection following detection, and then each of the chromatograms was grouped by stationary-phase separation and compared against one another. Differences between the natural oils and the known adulterated sample were spectrally identified using multidimensional GC as appropriate to validate the identifications. Then, the remaining two unknown samples were analyzed in order to ascertain the presence or absence of the marker compounds. Spectrometry. Infrared spectra using the HP 5965B were collected at a rate of 10 scans/spectrum with 16-cm-l optical resolution, which corresponds to 1 spectrum/s. Infrared reconstructed chromatograms were produced using a secondderivative selective wavelength reconstruction between 3 100 and 2800 cm-l. Mass spectra recorded on the H P 5970B were performed in full-scan mode scanning between 20 and 275 D, corresponding to an acquisition rate of 1.3 spectra/
-
2.0EIB E.EE+B
3
4 L.
A
.
9
7
P
1'
Time (min.)
Figure 2. Final portion of the precolumn chromatogram for each of the samples. Asterisks mark the largest differencesbetweenthe natural and adulterated oils. The early part of the chromatogram Is not Included because of a large dynamic range disparity between It and the portion plotted: (A)known naturaloil; (9)knownnaturaloil; (C)knownadutterated 011; (D) suspected adulterated oil; (E) oil of unknown origin. All chromatograms are plotted on the same ordinate scale. Peak numbers refer to component identifications In Table 2.
S.
Infrared and Mass Spectral Libraries and Searches. Two infrared vapor-phase libraries were used in the identification process. The first was the 3054-spectrum Hewlett Packard version of the EPA vapor-phase library, and the other was a 2020-spectrum Hewlett Packard flavor and fragrance library. Both libraries represented the spectra with 16-cm-l resolution. The Hewlett Packard version of the NIST (formally NBS) mass spectral library was used for mass spectral identifications. The library contained -42 000 spectra organized for probability-based matching (PBM). Spectral library searches were performed using the standard HP ChemStation search routines using default search strategy values. Identifications of several of the components presented in Figures 2-7 are presented in Table 2. Identifications listed are supported by both infrared and mass spectra. RESULTS AND DISCUSSION Initial separation of the samples on the various stationary phases provided a very rapid assay of the number of components in each of the oils. Figure 2 shows the corresponding portions of a segment of the chromatogram for each of the samples. Examination of partial chromatograms of the natural oils (A and B in Figure 2) reveals some differences between the two natural oils, but it has been well established in the literature that components and component concentrations can vary
considerably with several variables such as time of year, geographic location, and tree type.14 Upon comparing the partial chromatogram of the known adulterated sample (C) with those of the natural oil samples (A and B), two areas of difference (indicated with asterisks) were identified. A third area of difference was identified at retention times between 14 and 18 min (not shown in Figure 2). These differences were identified either by obvious differencesin the number of peaks or by comparing the infrared and mass spectra of peaks with similar retention times. As an example of the latter, the peaks located at -27.0 min in parts A and B of Figure 2 have spectra that match that of 4-terpineol very well, but the material eluting with the same retention time in Figure 2C (indicated with an asterisk) has IR and mass spectra indicative of a mixture. Therefore, in a separate experiment, a series of heart cuts were trapped for further MDGC analysis (cut 1, 14.0-18.0 min; cut 2, 22.223.8 min; cut 3, 26.8-28.0 min). Figure 3 is an expandedscale comparison (showing more detail) of the portions of the Figure 2 first-stage chromatograms selected for heart cutting. Materials eluting during each of the indicated time intervals were trapped in separate traps for all three samples. Subsequently, each trapped heart cut was independently reinjected onto one of the analytical columns. In each case, the total (14) Simmons, D.; Parsons, R. F. Biochem. S p t . Ecol. 1987, 15, 209-215.
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cut #3
28.5
21.0
2e.a
L1.5
21.5
23.0
11.6
z4.0
2.1.5
21.0
Time (min.)
Flgure 3. Chromatograms from the precolumn showing detail of the areas heart cut for multidimensionalSeparation: (A) known natural oil; (B) known natural oil; (C) known adulterated oil. Peak numbers refer to component identificationsin Table 2. Ordinateaxes are scaled 200% relative to Figure 2.
I . 0Et6 8 . BECB
25,s
26.0
26.5
27.B
27.5
2
26.8
tB.5
2¶.0
29.1
30.0
,.5E*S 1. 0ccs
5 . BE+, 0.0EIB
Frequency (cm-1)
50
I 00
150
m/z
Flgure 4. Spectra obtained from the peak indicated in the precolumn chromatogram of the known adulterated oil. Spectraldata suggest this is an overlapping peak: (A) TIC for the known adulterated oil. Ordinate axis is scaled 100% relativeto Figure 2; (B) infraredspectrum obtained from averaging over the Indicated peak; (C) mass spectrum obtained from averagingover indicatedpeak. Peak numbers refer to component identifications In Table 2.
sample emerging from the infrared lightpipe was retrapped in an empty cryogenic trap and subsequently injected onto the other analytical column of different polarity for an independent second-stage analysis. Thus, with a single sample injection into the GC, three heart cuts could be collected and subjected to second-stage separations on both polar and nonpolar analytical columns. Obviously, a similar procedure would permit multiple-stage GC separation, if fewer original heart cuts were examined. Figures 4 and 5 provide an example of the value of multidimensional separations in relation to spectral and identification quality. Figure 4A shows the precolumn chromatogram of the known adulterated oil (sample C). Because the infrared and mass spectra of the component marked with an asteriskdid not match those of the substances eluting at the corresponding times for the authentic samples (samples A and B), this material was suspected as a possible 428
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(A) TIC of the heart cut separated usingthe nonpolar column. Ordinate axis is scaled 2000 % relativeto Figure2; (e)infrared and mass spectra obtained from peak 13; (C) infrared and mass spectra obtained from peak 8.
adulterant. Parts B and C of Figure 4 are the infrared and mass spectra, obtained by averaging spectra across the indicated peak. Spectral library searches on these spectra did not provide any close library matches. Careful examination of the infrared spectrum in Figure 4B shows two minor 0-H stretch bands at 3500 and 3650 cm-l typical of alcohols. The strong C=O stretch at 1790 cm-l is indicative of a ketone. However, none of the library spectra matched this unusual pattern of bands or any of the other band shapes, providing further evidence that there might be more than one compound present. The mass spectrum in Figure 4C shows some evidence of a mixture of two components. For example, the peaks with m / z 152 and 154 could be Ma+for two different compounds. The m / z 154 ion loses HzO and CH3 to form ions with m/z 136 and 139, respectively. These losses are typical of an oxygenated terpene, most likely an alcohol. The m / z 152 ion loses CH3 to produce the m / z 137 fragment ion, but there is no indication of loss of H20, which is typical of terpene ketones. The mass assignments are supported by the infrared spectral features, but the identification of the two components is still not complete. Figure SA is the secondary chromatogram of the peak labeled in Figure 4A, using the nonpolar analytical column. It is seen to consist of a mixture of at least two compounds (labeled 8 and 13 in Figure 5A). Parts B and C of Figure 5 are the infrared and mass spectra for the peaks labeled 13 and 8, respectively. Spectral library searches of these two sets of spectra provided convincing identifications of peak 13 as camphor and peak 8 as 4-terpineol. Similar secondstage separations were carried out on the other heart cuts as well. After comparison of the secondary separation peak heights in Figure 5A to the primary separation peak height in Figure 4A (labeled with an asterisk), it is apparent that there is a loss of sample. Additionally, less significant sample loss can be identified by comparing the peak heights of the primary separation of cut 1 in Figure 3A-C and the peak heights of the secondary separations of cut 1 in Figure 6A-C. One of the known and constant sources of the sample loss is the split
-
.
1 DCCS
1
15
20
23
..sltS m...L*m
3.,+'1B 24
A
Time (min.)
Figure 6. TIC of heart cut 1 for each of the five samples separated using the nonpolar column. Asterisks represent presence of a marker compound. Ordinate axes are scaled 200% relative to Figure 2. Peak numbers refer to component Identifications in Table 2.
1,
1111
Heam;; #
'
I - ' '
'*
Time (min 1
Flgure 7. TICS of the known adulterated sample: (A) precoiumn chromatogram: (B) chromatogramresultingfrom secondary separation of heart cut 1 (same as Figure 6C); (C) chromatogram resulting from tertiary Separation of the heart cut from the secondary separation (17.0 min to 18.5 min). Ordinate axes are scaled 200% relative to Flgure 2. Peak numbers refer to component identifications in Table 2.
of the sample between the two detectors because detection occurs before trapping. However, the split ratio between the IRD and MSD does not account for the varying losses between these figures. The sources of additional sample loss have not been identified at this time, but these unknown factors are presently under examination. Double heart cutting is also a powerful tool for complex mixture analysis, as has previously been demonstrated.15 An example from the present analysis, where a tertiary separation proved necessary, is summarized in Figures 6 and 7. Figure 6 contains partial second-stage separations of cut 1 for each of the five samples, separated on the nonpolar analytical column. There are clear differences between these five chromatograms. Those peaks appearing only in the adulterated oils (marked with asterisks) are considered potential marker compounds and were identified by library searches utilizing the IR and MS spectra obtained at those retention ( 1 5 ) Wright, D. W.; Mahler, K. 0.;Ballard, L. B. J . Chromatogr. Sci. 1986, 24, 60-65.
times. The difference between the known and suspected adulterated oils is in the relative quantities of the components indicated with asterisks and in the peaks eluting at -17.5 min. In order to investigate the latter difference, a tertiary separation on the polar column was carried out by cryogenically trapping the section eluting from 17.0 to 18.5 min in the nonpolar column secondary separation for the known adulterated oil. Figure 7 shows partial chromatograms for the initial separation on the intermediate polarity precolumn (A), the second-stage separation using the nonpolar analytical column (B), and the third-stage separation using the polar analytical column (C), plotted on the same ordinate scale. The apparent loss of sample from cycle to cycle in Figure 7 is primarily due to the selection of progressively smaller peaks for subsequent reseparation rather than actual sample loss due to the split between the IRD and MSD. In parts A and B of Figure 7, the regions heart cut for subsequent further separation are indicated. The third-stage separation clearly reveals at least five components, positively identified by their infrared and mass spectra as carvomenthene, a-terpenene, limonene, P-phellandrene, and p-cymene. In addition to demonstrating the enhanced separating capability of MDGC, this experiment also shows that sample looping or recycling can be carried out with reasonable efficiency. In fact, other experiments, not included in this study, suggest that looping of sections can be carried out for at least 10 cycles. Following completion of the separations, the spectroscopic data were examined for differences between all of the components in each of the three heart cuts. From these comparisons, the following compounds were identified as adulteration markers for the eucalyptus oils under consideration: camphor, a-thujene, decane, sabinene, 0-phellandrene, and y-terpinene. Because adulterated eucalyptus oil typically derives from natural camphor oil, camphor is expected to be, and is, the main marker compound. Upon completion of the identification of the markers, the unknowns were analyzed for the presence or absence of those compounds. In the analysis of the unknowns, the presence of camphor in any concentration was taken to be prima facie evidence of adulteration, and the absence of camphor but the presence of others was considered a strong indication of adulteration of the oi1.16 Examination of the unknowns (samples D and E) led to the conclusion that the suspected adulterated oil (D) is indeed an adulterated oil, while the other unknown sample (E) is a true eucalyptus oil sample. Sample D contained a sizable amount of camphor as well as small amounts of the other marker compounds. However, sample E contained none of the marker compounds, within the limits of detection. As mentioned in the introduction, the components present in any particular sample of eucalyptus oil depend on the specific tree, the geographic location, and the season in which the trees were harvested, and it could be argued that the compounds identified here as marker compounds were just the result of a lack of sufficiently representative set of samples. However, most of the marker compounds identified here do not appear in the oil of any of the different eucalyptus trees which have been Sabinene and y-terpinene appear in some ~~~
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(16) Barton, A. F. M.; Tjandra, J.; Nicholas, P. G.J . Agric. Food Chem. 1989, 37, 1253-1257. (17) Hedges, L. M.; Wilkins, C. L. J. Chromatogr. Sci. 1991, 29, 345-350.
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types of trees in trace levels, but the natural oils examined in this study were from the E. australiana family. Previous research shows that these compounds are not present in this 0i1.17
CONCLUSIONS Analysis of a variety of authentic and simulated eucalyptus oils by means of parallel cryogenic trapping MDGC/FTIR/MS shows that such instrumentation is useful for complex mixture analysis. It has been demonstrated that multidimensional GC of materials separated as a result of a singlesample injection is possible using as many as three different GC columns. As shown here, three-stage gas chromatography (GC3) is useful for the analysis of several heart cut sections of a chromatogram or for analyzing a complete sample using three independent columns. The latter possibility allows quick screening of samples using a single instrument for component (18) Canigueral, S.; Vila, R.; Iglesias, J.; Bellakhdar, J.; Idrissi, A. I. J . Essent. Oil Res. 1992, 4, 543-545. (19) Singh, A. K.; Gupta, K. C.; Brophy, J. J. J . Essent. OilRes. 1991,3,449450. (20) Brophy, J. J.; Lassak, E. V. Fluuour Frugrunce J. 1991, 6, 265-269. (21) Brophy, J. J.; Boland, D. J. FIuuour Fragrunce J . 1990, 2, 87-90. (22) Dellacassa, E.; Menendez, P.; Moyna, P.;Soler, E. Fluuour Fragrance J . 1990, 5, 91-95. (23) Erazo, S.;Busto, C.;Erazo, A. M.; Rivas, J.; Zollner, 0.;Cruzat, C.;Gonzalez, J. PIuntes Med. Phytother. 1990, 24, 248-257. (24) Weston, R. Phytochemistry 1984, 23, 1943-1945.
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separation and identification while the former technique allows one to carry out detailed analysis of small subsets of a mixture. In the present study, preliminary screening of the data collected from different oils in various columns was done manually. Such an approach to data analysis of complex mixtures with a system such as that described here poses a considerable problem because such a large amount of data is generated. An obvious extension of the methodology is to place the entire process, including the necessary valve switching and experiment sequencing, under computer control. When this is combined with more effective multispectral data interpretation algorithms, including both spectral and multicolumn retention time information, a practical and very general mixture analysis system will be possible. Thus, multidimensional GC/IR/ MS appears to have great potential for further development, a project we are currently pursuing.
ACKNOWLEDGMENT The authors gratefully acknowledge the National Science Foundation for financial support (CHE-92-01277) and Marc Parilli of the Berj6 Co. for the eucalyptus oil samples. Received for review August 11, 1993. Accepted November 19, 1993." a
Abstract published in Aduunce ACS Absrrucrs, December 15, 1993.
