Anal. Chem. 2001, 73, 1336-1344
Concepts and Preliminary Observations on the Triple-Dimensional Analysis of Complex Volatile Samples by Using GC×GC-TOFMS Robert Shellie, Philip Marriott,* and Paul Morrison
Chromatography and Molecular Separations Group, Department of Applied Chemistry, Royal Melbourne Institute of Technology, GPO Box 25476V, Melbourne, Victoria 3001, Australia
The high-resolution two-dimensional comprehensive gas chromatography (GC×GC) separation of a complex sample of an essential oil is reported, with tentative identification of selected separated components provided by time-offlight mass spectrometry (TOFMS). The GC×GC technique allows orthogonal separation mechanisms on the two columns to achieve separation of components that otherwise are unresolved on a single column, as is demonstrated for the pairs of components borneol and terpinen-4-ol, and cis-caryophyllene and β-farnesene. Peak compression and a short second column used in GC×GC lead to generation of fast second-dimension GC peaks and higher detection sensitivity, by about 25 times, as compared to conventional GC elution. This allows many more compounds to be recognized when using the GC×GC approach. Additionally, rapid mass spectral methods are required if accurate data and reliable searchable spectra are to be obtained for the fast peaks; this is achieved with TOFMS. This leads to a three-dimensional analytical technique. Application of the technique to the complex essential oil sample containing a range of chemical compound classes shows that superior separation and more accurate peak assignment results. Once peaks are identified within the two-dimensional separation space, it is conceivable that mass spectrometry might no longer be required for the routine analysis of such samples, instead relying on the precision of flame ionization detection to give quantitative analysis; however, the support of mass spectral characterization will be invaluable in validating the GC×GC approach.
1. INTRODUCTION Contemporary high-resolution separation of volatile and semivolatile compounds normally employs the technique of capillary gas chromatography/mass spectrometry (GC/MS).1 The basic method has not changed greatly over the last 20 years, although there have been improvements in some of the mass spectral approaches, such as automated peak detection and library assign* To whom correspondence should be addressed: E-mail: philip.marriott@ rmit.edu.au. (1) Ragunathan, N.; Krock, K. A.; Klawin, C.; Saaki T. A.; Wilkins, C. L. J. Chromatogr. A 1999, 856, 349.
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ment of peak identity. More recently, MS/MS techniques have also provided improved component identification and sensitivity, especially for the limited spectral fragmentation information produced with soft ionization methods; liquid chromatographyelectrospray ionization MS and related techniques benefit from such approaches.2,3 The use of the mass spectrometer to provide the unique identity of components that overlap on the GC column and, hence, neither can be quantitated nor their structures assigned in the absence of mass spectral data highlights the limitation of single-column capillary gas chromatography, especially when analyzing complex mixtures. Davis4 has described the GC separation problem on a statistical basis, in which the probability of overlapping peaks will be common in all but the simplest mixtures, and has recently shown in subsequent work that the need for a two-dimensional separation exists5 if improved component separation is required. Giddings has defined the problem in general terms of dimensionality in the separation process,6 especially concluding that a method that comprises two separation dimensions that allow orthogonal separation (where the chemical processes occurring in each dimension and, hence, the separation achieved between sample components is based on different interaction mechanisms) will provide superior component discrimination. It can be said that the capacity of the separation system has been increased. It is widely acknowledged that multidimensional gas chromatography can deliver improved component separation. There have been numerous reviews and books devoted to the subject of multidimensional gas chromatography (MDGC). Schomburg has demonstrated the technical implementation of MDGC.7 As recently as 1999, Bertsch8 commented that even though the power of MDGC had been appreciated and commercial systems were available the realization of useful and readily employed MDGC systems was lacking. The crux of the problem is that conventional MDGC applies the two-column separation advantage only to limited regions of a sample, that is, where the “heartcuts” are (2) Kienhuis, P. G. M.; Geerdink, R. B. Trends Anal. Chem. 2000, 19, 460. (3) Volmer, D. A.; Vollmer, D. L.; Wilkes, J. G. LC-GC Asia Pac. 1998, 1, 44. (4) Davis, J. M. In Statistical Theories of Peak Overlap in Chromatography; Brown, P. R., Grushka, E., Eds., Chromatogr. Sci. Series, Vol. 34; Marcel Dekker: New York,1974. (5) Davis, J. M.; Samuel, C. J. High Resolut. Chromatogr. 2000, 23, 235. (6) Giddings, J. C. J. Chromatogr. 1995, 705, 3. (7) Schomburg, G. J. Chromatogr. 1995, 703, 309. (8) Bertsch, W. J. High Resolut. Chromatogr. 1999, 22, 647. 10.1021/ac000987n CCC: $20.00
© 2001 American Chemical Society Published on Web 02/10/2001
conducted. The hyphenated technique of MDGC/MS will again provide more identification of components, (see, for example, the analysis of chiral components of a fruit extract9), but the overall separation improvement is still limited to the relatively small capacity increase of the MDGC process over that of the single column analysis. For a sample of greater complexity that requires MDGC to be applied over the total sample elution, a new approach to the MDGC experiment is demanded. The answer is reasonably logical, but took some years to come to fruition. Jorgensen and Bushey showed10 that the direct combination of different high performance liquid chromatography (HPLC) modes (e.g., size exclusion and reversed-phase) in which the second dimension separation is conducted on a much faster time scale than that of the primary dimension allows a comprehensive separation, with solutes spread across the total separation space. The key to the GC application of a comprehensive chromatography analysis was the ability to rapidly pulse segments of effluent from column 1 to column 2.11 Phillips proposed a heated zone between the two columns,12 subsequently using a rotating heated sweeper to compress and deliver zones of solutes to the second, fast analysis column.13 Recently, an issue of the Journal of High-Resolution Chromatography [Vol 23 (2000)] was dedicated to the memory of Phillips and the contributions he made to this area, covering many areas of comprehensive gas chromatography technology. The present group14-16 applied a cryogenic method to achieve the same effect. The two most complex sample types reported to date using GC×GC are petroleum-derived samples17 and vetiver essential oil,18 although the advantages of GC×GC still enhance the analysis of mixtures such as semi-volatile aromatics19 and sterols.20 While GC×GC provides unsurpassed resolution capability, the most pressing need is to prove that the implied complexity of a mixture is represented by the multitude of peaks spread around the 2-D space. This may be viewed as validation of the power of the technique. If each separated peak can be positively identified and, thus, should be reported, quantitated, and taken into consideration in the analysis, then the technique can be justified to analysts. If, moreover, the technique is simple to implement, then all laboratories should be able to enjoy the advantages of the technique. Thus mass spectrometry, the most recognized spectroscopic tool for identification of GC separated components, is required for the detection step in GC×GC. Because pulsed peaks may be of the order of 150 ms wide at half-width (depending upon the column length of the second column and the carrier gas flow velocity and temperature used), conventional quadrupole instruments are unsuited to the task. Time-of-flight (TOFMS) (9) Full, G.; Winterhalter, P.; Schmidt, G.; Herion, P.; Schreier, P. J. High Resolut. Chromatogr. 1993, 16, 642. (10) Bushey, M. M.; Jorgensen, J. W. Anal. Chem. 1990, 62, 161. (11) Phillips, J. B.; Beens, J. J. Chromatogr. A 1999, 856, 331. (12) Phillips, J. B.; Ledford, E. B. Field Anal. Chem. Tech. 1996, 1, 23. (13) Phillips, J. B.; Venkatramani, C. J. J. Microcolumn Sep. 1993, 5, 511. (14) Marriott, P. J.; Kinghorn, R. M. Anal. Chem. 1997, 69, 2582. (15) Kinghorn, R. M.; Marriott, P. J. J. High Resolut. Chromatogr. 1999, 22, 235. (16) Kinghorn, R. M. PhD. Dissertation, Royal Melbourne Institute of Technology, 2000. (17) Beens, J. PhD. Dissertation, University of Amsterdam, 1998. (18) Marriott, P.; Shellie, R.; Fergeus, J.; Ong, R.; Morrison, P. Flavour Fragr. J., 2000, 15, 225. (19) Marriott, P. J.; Kinghorn, R. M.; Ong, R.; Morrison, P.; Haglund, P.; Harju, M. J. High Resolut. Chromatogr. 2000, 23, 253. (20) Truong, T.; Marriott, P.; Porter, N. J. A.O.A.C. Int. 2001, in press.
instruments possess the necessary speed of spectral acquisition to give g50 spectra/second, and thus, ∼10 spectra/peak. Mass spectrometry using a quadrupole instrument has been reported for GC×GC,21 but the analysis had to be slowed excessively to get just one reasonable scan across the peaks. The general attributes of TOFMS have been reported by Guilhaus.22,23 Although this technology is not new and is wellestablished for instruments such as the MALDI-TOF method, the coupling of GC methods to TOFMS instruments is only very recent. Guilhaus has reviewed the limited prior work in this area,24 although the need to adequately match the mass spectral capabilities with the chromatographic performance has been recognized for some time,25 and in that review, the limitation of speed in the TOFMS detection was attributed to data handling considerations. The primary applications proposed appear to be for fast GC, for which the number of peaks produced per time is increased over conventional GC methods.26 This allows faster turnaround of sample analysis, but can only be realized in a practical sense if data analysis is also performed fast. Hence, automated reporting, peak deconvolution (fast elution methods would normally decrease the solute separation), library searching, and other data processing functions must be completed within the time scale of the GC/ MS run. Recently, Sacks and co-workers reported the use of fast GC/MS with pressure tuning to increase the separation of target solutes in various synthetic mixtures.27 This same technology was applied to a test mixture of gasoline-range hydrocarbons.28 Also at Pittcon 2000, two papers on the three-dimensional technique of GC×GC-TOFMS were presented by the Centres for Disease Control that discussed fundamental considerations29 and its application to endocrine disruptor analysis.30 In the present study, reduction in total analysis time is not an object of the study. However it should be recognized that the greater peak selectivity of the GC×GC experiment could only be achieved (if indeed it could be achieved at all) in a single-column GC analysis of significantly greater time. Hence, with respect to equivalent peak capacity, the present analysis is a fast analysis, although the primary column is essentially operated under conventional conditions of 3 °C/min temperature programming with a 30m × 0.25 mm ID column. An alternative view of the GC×GC method here is that the second column is, in effect, acting as a detection step for the solutes eluting from the first column. Its detection mechanism is merely a separation process, which happens to be selected according to orthogonal separation principles, and the TOFMS is a mass-selective response as a third dimension. (21) Frysinger, G. S.; Gaines, R. B. J. High Resolut. Chromatogr. 1999, 22, 251. (22) Coles, J. N.; Guilhaus, M. Trends Anal. Chem. 1993, 12, 203. (23) Guilhaus, M. J. Mass Spectrom. 1995, 30, 1519. (24) Guilhaus, M.; Selby, D.; Mlynski, V. Mass Spectrom. Rev. 2000, 19, 65. (25) Holland, J. F.; Enke, C. G.; Allison, J.; Stults, J. T.; Pinkston, J. D.; Newcomoe, B.; Watson, J. T. Anal. Chem. 1983, 55, 997A. (26) van Ysacker, P.; Guilhaus, M.; Roach, L.; Mlynsky, V.; Janssen, H.-G.; Leclercq, P. A.; Cramers, C. A. Proceedings of the 18th International Symposium of Capillary Chromatography, Riva del Garda, Italy, May 2024, 1996; p 1496. (27) Roberts, G.; Sacks, R. Abstract No. 265, Pittcon 2000, New Orleans, U.S.A., March 12-17, 2000; Veriotti, T.; Sacks, R. Abstract No. 268, Pittcon 2000, New Orleans, March 12-17, 2000. (28) Veriotti, T.; Sacks, R. Anal. Chem. 2000, 72, 3063. (29) Dimandja, J.-M.; Grainger, J.; Patterson, D. Abstract No. 267, Pittcon 2000, New Orleans, March 12-17, 2000. (30) Dimandja, J.-M.; Clouden, G. C.; Colon, I.; Grainger, J.; Patterson, D. Abstract No. 1192, Pittcon 2000, New Orleans, March 12-17, 2000.
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This paper reports the three-dimensional GC×GC-TOFMS analysis of a lavender sample to demonstrate the analytical power of the technique to provide improved separation and identification of major and minor components of the essential oil. 2. EXPERIMENTAL SECTION 2.1 Gas Chromatography System. A model 6890GC (Agilent Technologies, Burwood, Australia) with Chemstation data system was used in these studies, either as a stand-alone unit with flame ionization detection (where the FID was operated at a data collection frequency of 100 Hz) or interfaced to the mass spectrometer. The Chemstation event control is used to instruct the modulation control system to commence modulation at a precise time. 2.2 Column Set. The same column combination found to be suited to essential oil work previously31 was used for the present study. In the GC/MS setup, the second column was inserted through the heated interface, held at 250 °C, with ∼ 27 cm of the column located in this transfer region. The primary column was a 30 m × 0.25 mm ID × 0.25 µm film thickness (df) BPX5-coated column (5% phenyl; low polarity), directly coupled to a 2.0 m × 0.1 mm ID × 0.1 µm df BP20-coated column (poly(ethylene glycol); polar column) second dimension. Both columns were from SGE International (Ringwood, Australia). The GC was operated under temperature program conditions, from 60 to 240 °C at a temperature program rate of 3.0 °C min-1. These conditions were chosen as standard for most of our studies to allow direct comparison with literature data.32 Split injection was employed, with a split ratio of ∼50:1. 2.3 Cryogenic Modulator. The GC×GC experiment is achieved here by use of a cryogenic modulator as described elsewhere,33 with the pneumatic system outlined therein replaced by a motor drive. The first demonstration of the principles of cryogenic modulation was reported in 1997,14 and a summary of its unique capabilities was recently outlined.34 The present unit, the LMCS Everest model, was from Chromatography Concepts (Doncaster, Australia). The cryogenic system was retrofitted to the 6890GC. It was operated under modulation timing of 4-s cycle time and 0.5-s hold time in the release position. The CO2 cryogen coolant maintained the temperature of the modulation trap to at least 100 °C below the prevailing oven temperature. Figure 1 illustrates a schematic illustration of the instrumental setup. 2.4 Time-of-Flight Mass Spectrometry. A Pegasus II TOFMS instrument (LECO Corp., St Josephs, MI) was used to acquire mass spectral data, using 70 eV electron impact ionization. The transfer interface temperature was 245 °C. The mass range collected was from 45 to 250 m/z, with 50 spectra/s transferred to the data station. Although 5000 spectra/s are acquired, these are processed by sum averaging 100 transients into one spectrum prior to submitting to file. Data were recorded and analyzed using the software provided with the Pegasus instrument. Most often the m/z 93 ion data, diagnostic for many of the terpene compounds of interest in these samples, were exported for chromatogram (31) Shellie, R.; Marriott, P.; Cornwell, C.; Morrison, P. J. High Resolut. Chromatogr. 2000, 23, 554. (32) Adams, R. P. Identification of Essential Oils by Ion Trap Mass Spectrometry; Academic Press: New York, 1989. (33) Kinghorn, R. M.; Marriott, P. J.; Dawes, P. A. J. High Resolut. Chromatogr. 2000, 23, 245. (34) Marriott, P.; Kinghorn, R. M. LC-GC Eur. 2000, 13, 428.
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Figure 1. Schematic diagram of the GC×GC-TOFMS system. GC components: 1, autoinjector; 2, autosampler tray; 3, modulation device; 4, moveable cryotrap; D1, first-dimension gas chromatography column; D2, second-dimension gas chromatography column; 5, heated reentrant interface region. TOFMS components: 6, ionizing filament, electron flux, and collector; 7, push plate; 8, ion optics; 9, beam of ionized molecules; 10, reflectron; 11, multichannel plate detector.
plotting. This served to make data processing faster. This instrument is capable of unit m/z resolution only. The NIST library provided with the instrument was used for spectral searching. 2.5 Data Conversion. Either Chemstation data (100 Hz), or TOFMS data (50 Hz) may be exported in ascii file format (*.csv files) for subsequent data display. They comprise 2 or 3 columns of data for GC-FID and GC-TOFMS, respectively. To generate the 2-D separation space display, a conversion program has been written to take the desired time-response data and generate a matrix based on the modulation frequency and sampling rate. The two sets of data (original exported file and the matrix file) were read into either Origin (Microcal Software, Northampton, MA) or Transform (Fortner Research), respectively, to present “normal” or 2-D chromatographic traces. Mass spectral data may be exported in a variety of data types (total ion counts or selected ion response) to give different chromatographic interpretations of the results. 2.6 Samples. The whole essential oil was provided by Australian Botanical Products (Hallam, Victoria, Australia) and was used as received. GC×GC-FID results for a number of essential oil samples have been reported recently.18,31 3. RESULTS AND DISCUSSION 3.1 Description of the Three-Dimensional Analysis System. Conventional multidimensional GC separations (effectively limited to two separation dimensions) involve heartcutting sections of a first-dimension separation to a second column, where in a subsequent elution step, greater resolution of previously poorly resolved components is achieved. The standard approach is to use two different phase types to effect this condition where the retention mechanism of each column involves different solutestationary phase interactions. Maximizing the multidimensional separation advantage over a complete chromatographic analysis is elegantly invoked in the comprehensive GC×GC technique. Because the rapid, repetitive heartcut process is immediately followed by fast GC analysis at the prevailing oven temperature, the orthogonality of the two columns’ separation mechanisms should be ensured, and the second column’s ability to provide resolution of components, therefore, maximized. Figure 2 illustrates the rapid 2-D analysis of pulsed
Figure 2. Schematic diagram illustrating the procedure for acquisition of comprehensive gas chromatography data. The first dimension (1D) is temperature programmed 50 to 240 °C; the modulator results in rapid delivery of pulsed bands to the second dimension (2-D) every 4 s. Thus, the second dimension acts as a fast analyzer of the peaks arriving at the end of the first dimension. The oven temperature increments are ∼0.2 °C during the 4-s interval of elution on the second column.
packets of solute as the primary separation (1-D) proceeds. The temperature increment during the 2-D analysis will be about 0.2 °C, over a 4-s elution time at an oven temperature program rate of 3 °C/min. The first column acts precisely as a conventional capillary GC column. Over the 63-min analysis time, there will be a maximum of some 945 second dimension-pulsed analyses. The system used in the present study comprises two independent gas chromatographic dimensions (D1 and D2, Figure 1) based on a comprehensive gas chromatography process. The third dimension is provided by mass spectral acquisition of spectra on a time frame compatible with the fast GC elution on D2. TOFMS is the most viable technology available to give this rapid spectrumacquisition capability. A multidimensional system provides additional data where the dimensions are orthogonal; that is, the dimensions must give independent response information. Although GC/MS is accepted as a typical example of a twodimensional instrumental analysis system, it has been demonstrated that the combination of GC with GC in the comprehensive separation step prior to MS is capable of independent separation mechanisms such that the GC×GC column coupling does constitute orthogonal separate dimensions. Thus, the orthogonal two-dimensional GC/MS experiment can be extended to a threedimensional experiment of GC×GC-MS in which a twodimensional GC process is finished with mass spectral signal acquisition of components which have been better separated on the two columns. Such a system has a number of advantages, primarily for complex mixture analyses when multiple overlapping solutes will reduce the effectiveness of mass spectral analysis or quality of mass spectra if only a single column is employed in GC/MS. The MS identification capacity can also provide a more informative interpretation of the identities of components where they are separated in the second dimension and, therefore, provides a basis for making conclusions on the system performance of the coupled column experiment. The three-dimensional analysis system, comprising twodimensional separation and a subsequent third dimension of spectral identification, is most suitably developed in the form of comprehensive gas chromatography with time-of-flight mass spectrometry. Every solute is subjected to the dual separation mechanisms of the coupled columns with neighboring compo-
nents resolved to the greatest extent and rapid scanning of the mass spectrometer, yielding sufficient spectra to define the peak contour when data are converted to matrix format. The ultimate aim will clearly be to temporally resolve all components, with the spectrometer only required for its identification capabilities. This is a significant change to the role of the mass spectrometer when used in a single-dimensional separation GC/MS system, where the MS both gives identification and also, importantly, provides a degree of specificity to allow overlapping components to be separately recognized. Thus, in a sample such as kerosene there will be considerable overlap of different classes of compounds, but extracted ion chromatograms allow, for instance, the branched alkanes and mono- and diaromatics to be drawn out of the spectral data by choosing appropriate diagnostic ions. The mass spectrum at any particular scan will still clearly be a composite of the unresolved components at that point, and the use of off-peak background subtraction will not completely correct for the overlapping components. This means that mass spectra must be compromised to some extent, although its wide acceptance testifies to the value inherent in the GC/MS method. By contrast, in the GC×GC-TOFMS system, there will be many more completely resolved components, and in these cases there is less of a requirement to resort to methods that can give a screen or deconvolution of overlapping components. Additionally, background subtraction will be a simpler process, because in many cases, spectral interferences do not have to be subtracted from overlapping peaks, but it is the spectrometer background which is subtracted. In a study of atmospheric organic analysis by using GC×GC-FID,35 it was demonstrated that the chemical baseline causing an elevated detector response above the true baseline can lead to considerable uncertainty in estimation of total chemical load of a sample in single-column analysis of complex mixtures. The high resolution power and 2-D separation space means that it is relatively easy to locate a background that just constitutes detector baseline. This will mean also that spectra should be “cleaner” and more precisely represent the true component, and this should mean fundamentally better library matching. 3.2 Sequential Pulses of Modulated Components, and Resolution of Peaks in 2-D. The GC×GC technique relies on pulsing the effluent from the first column to the second column, with usually about 5 pulses/peak as the desired frequency. Coeluting peaks from the first column are simultaneously collected and pulsed together to the second column, and provided the selectivity of the second column permits, they will then be separated as very sharp individual peaks. Given that the second column is operated under fast elution conditions, the pulses appear at the end of the second column with peak half-widths of the order of a few tenths of a second. For FID, a detection frequency of 50-100 Hz is typically used. To obtain reliable peak profiles with MS detection, a similar sampling frequency is required. Such fast spectral acquisition is available with TOF technology. The data report from the GC×GC-TOFMS experiment reveals groups of pulsed peaks with the same library identification, corresponding to the series of pulses for the one component. These will be separated by a time interval of 4 s, due to the 0.25 (35) Lewis, A. C.; Carslaw, N.; Marriott, P. J.; Kinghorn, R. M.; Morrison, P.; Lee, A. L.; Bartle, K. D.; Pilling, M. J. Nature 2000, 405, 778.
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Figure 3. A: Pulsed series of peaks obtained at the end of the second dimension for a single first-dimension chromatographic peak, denoted compound X. B: Two overlapping peaks, X and Y, from the first column form an envelope profile of pulsed peaks at the end of the second column. Provided the stationary phase shows adequate selectivity to separate the two pulsed components, we get resolution of the two species as shown, and hence, interweaving of the two resolved pulsed bands.
s-1 modulation frequency. Where overlapping peaks from the first column occur, then the result will be interleaved pulses of peaks, provided they are resolved on D2, alternatively identifiable by their library matches. Figure 3A illustrates the hypothetical interpretation of such situations for a single peak; in this case, there are nine separate pulses shown. It is possible to overlay onto the pulsed peak profile the nonmodulated peak distribution (shown by the dotted line in Figure 3B), which in the present case is simply obtained by not turning on the cryofluid and not operating the modulating trap. Generally, there is excellent correspondence of these two profiles, although the pulsed peaks have substantially greater response heights due to the zone compression effect of the modulator. The total peak area is conserved for the nonmodulated single peak and the pulsed series of peaks. A case of a multiple (two) peak overlap (Figure 3B) shows that two components which would be poorly resolved on the first dimension (shown as the broad broken lines) are pulsed into slices and completely resolved on the second column with interleaved peak pulses. The two components are denoted X and Y, respectively. 3.3 GC×GC-TOFMS of Essential Oil. In the GC×GCTOFMS experiment, a data report at the conclusion of the analysis 1340
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should identify the individual pulses as their appropriate identities at the respective retention times. Thus, for Figure 3B, nine separate peaks identified by the characteristic mass spectra of compound X and nine for compound Y are expected. The actual number reported will be determined by sensitivity considerations, especially for the minor pulsed peaks at the extremities of the band. Table 1 illustrates a summary of the data report for the analysis of a lavender essential oil sample, in which peaks identified as the same component are grouped together. Some compounds were either not identified in the library matching process, or were not included in the automatic report. The original data stream may be converted following procedures outlined elsewhere18 into matrix format to generate a 2-D separation space. The 2-D plot will be constructed with axes of first-dimension retention (1tR) and second-dimension peak position (2tR). The latter of these might not strictly be retention on the second column, depending on the phenomenon of peak “wrap-around”, where peaks take longer than the modulation time to traverse the second column. Figure 4A is a chromatogram of the lavender pulseddata stream from which the data in Table 1 were generated. Converting the pulsed data from Figure 4A into matrix format produces the 2-D separation space seen in Figure 5. The components at a retention time of 1270-1290 s (Table 1; components 21, 22) coelute on the first column. The 2-D space shows that their contour peaks are resolved in the second dimension; they are identified by library matching as borneol and terpinen-4-ol, with the former eluting slightly before the latter on the first column. Figure 4B,C is an expansion of selected regions of interest of Figure 4A. The smaller magnitude of the peak pulses at the periphery of the chromatographic peak means that librarymatch quality deteriorates for these peaks, but the major pulse at the peak maximum is better matched. Because the GC×GC technique gives greater peak sensitivity than the corresponding normal capillary GC result, then for a given injected amount, the match quality will be better for the GC×GC result. Indeed, it is possible to get matches in the GC×GC method where no peak was seen in the normal GC analysis. More abundant components consist of a greater number of pulses above the nominal detection or identification limit of the mass spectrometer. This will also depend on the relative mass spectral properties (ion abundances) that affect the library search quality. Thus, column 2 in Table 1 shows that different components have a different number of individual peaks that have separately listed peak maxima. For instance, minor component 6 gave only one identified peak at 801.83 s, whereas 1,8-cineole (component 10) gave three peaks at 856.59-, 860.71-, and 864.55-s retention time (differing by the 4-s modulation frequency) that were identified by the library search routine as being this compound. The major component linalyl acetate (component 25) was positively matched in 6 sequential pulses, being at retention times of 1426.4, 1430.3, 1434.1, 1438.2, 1442.3 and 1446.2 s. Again, an approximately 4-s interval is seen between each successive linalyl acetate pulse, which corresponds to the modulation frequency. It can also be seen in Figure 5 that the major component has a broader peak representation in the 2-D plot, because the contours are drawn at a selected peak height response and the more abundant component will give a wider peak at a given response height.
Table 1. Library Matches for Pulsed Chromatographic Result Shown in Figure 5.a peakb
retention time (s)c
identityd
matche
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
619.25, 623.23 671.67, 675.63 691.39, 695.41, 699.97, 704.01 763.77, 767.63, 771.57 787.91, 791.87 801.83 817.73, 821.57 824.41, 828.53, 832.47 836.19, 840.11, 844.19 856.59, 860.71, 864.55 906.37, 910.23 945.77, 949.73 954.87, 958.77 958.59, 962.39 970.71, 974.49 980.55, 984.59 986.79, 990.55, 994.35 1025.9, 1029.7, 1033.9, 1037.3 1095.6, 1099.3 1201.4, 1205.2 1273.5, 1277.5, 1281.2, 1285.2 1279.1, 1282.8, 1286.8, 1290.9 1327.2, 1331.1, 1335.0 1412.1, 1415.9 1426.4, 1430.3, 1434.1, 1438.2, 1442.3, 1446.2 1522.6, 1526.3, 1530.4, 1534.4 1554, 1557.8, 1561.9 1731.7, 1735.4, 1739.1 1787.2, 1791.3 1794.4, 1798.3 1894.7, 1898.7, 1902.6, 1906.7
899 851 883 867 785 868 861 895 857 887
32 33 34 35 36 37 38
1915.3, 1919.1, 1923.1 1926.7, 1930.7, 1934.5 1963.1, 1967.3, 1971.1, 1975.0, 1979.0 2075.7, 2079.6, 2083.5 2151.9, 2155.7, 2159.6 2342.6, 2346.9, 2351.0 2478, 2481.5
camphene R-thujene R-pinene sabinene carene 3,7,7-trimethyl-1,3,5-cycloheptatrienef β-pinene limonene β-phellandrene 1,8-cineole unknown 1 linalool oxide cis-sabinene hydrate p-cymene γ-terpinene terpinolene 3,7,7-trimethyl-bicyclo[4.1.0]hept-4-ene-3-oelf linalool 1,2,3,4-tetramethyl benzenef camphor borneolg terpinen-4-olg R-terpineol naphthalene linalyl acetate unknown 2 bornyl acetate neryl acetate geranyl acetate germacrene D 1,7-dimethyl-7-(4-methyl-3-pentenyl)tricyclo[2.1.1.0(2,6)]heptanef cis-caryophylleneg β-farneseneg R-farnesene germacrene A germacrene B caryophyllene oxide cadinol
862 815 904 785 568 872 890 680 912 846 864 830 770 880 828 864 889 759 852 863 885 899 855 846 825 762
a Identifications were based on the NIST library matches and retention time data derived from known elution patterns of the essential oil components, as reported in Adams’ text.32 Because each chromatographic peak consists of a series of pulses, the library will give a series of matches for these related pulses. The number of successful matches will be determined in part by the intensities of the series of pulses that are above a nominal threshold allowing adequate library identification. Note that a comprehensive terpenes library was not available for this study, and so this limits the number of positive identifications b Peak numbering identification as in Figure 5. c Retention time as given in the peak table listing in the post-analysis report. d Peak identified by library and reference data. e Peak match is a similarity index given by the mass spectrometry software, and is reported for the best match found from among the pulsed peaks for the compound. f Compounds were given by the library match only, and could not be confirmed by Adams’ data. g Compounds are pairs of overlapping components.
The GC-TOFMS analysis of the same sample at the same concentration is shown in Figure 6. In this case, the same column set was used, but the cryofluid was not supplied to the modulator. The result indicates that the sensitivity is much worse for the nonmodulated case, as is well-established now for comprehensive GC that incorporates zone compression between the two dimensions. Thus, component 32 (cis-caryophyllene) has a maximum relative response of 50 000 in the GC×GC experiment, but only 2000 in the normal GC experiment. This analysis is able to provide neither the degree of component sensitivity provided by GC×GCTOFMS nor the quality of spectral matching for the same amount of sample introduced to the column. The expanded peak inset indicates that peak widths are about 5 s, as compared to
