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Multidimensional GC For Qualitative IR and MS of Mixtures
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t is now common, following gas chromatographic separation, to use IR or MS separately or in combination for qualitative analysis of volatile mixtures. One premise of this approach is the assumption that regardless of the detector used, it is being presented with pure mixture components. This assumption is particularly important when computer-readable library search algorithms are used, because these methods rely on matching IR or mass spectra with those from spectral library databases as the separation proceeds. Obviously, if eluting gas chromatographic peaks are composed of unresolved components, there is a reduced likelihood of correctly identifying the major constituents and relatively little chance that minor constituents will be detected or recognized. Because of the sample size
Charles L. Wilkins University of California-Riverside 0003 - 2700/94/0366 -295A/$04.50/0 © 1994 American Chemical Society
of GC by Davis and Giddings (2) revealed Multidimensional that even for single-stage separations of moderately complex mixtures (e.g., a 50chromatography component mixture separated by a colwith a peak capacity [2] of 100 combined with highly umn peaks), no more than 37% of the theoretinumber of peaks would be observed. specific detection can cal Furthermore, only 18% of the components emerge as pure peaks. achieve the resolution would The authors concluded that the gravity of the peak overlap problem on crowded necessary for chromatograms appears to be underestimated in most work, and that seeking sysseparation of complex tems of higher resolution—which would be substantially less crowded—deserves volatile mixtures wider attention. They suggested that mul-
required for joint IR and mass spectral analysis, use of the highest resolution capillary GC columns is not practical, and alternative strategies for high-performance GC are essential. Several years ago a statistical analysis
tidimensional chromatography offered a potentially promising solution to this dilemma; indeed, in subsequent years several chromatographers devoted a good deal of attention to multidimensional chromatography (3). In this Report I will review the theory of multidimensional chromatography and illustrate the applicability
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of such methodology for GC applications in which multispectral detectors are used. Theory As originally developed by Giddings, the theory of multistage separations (4) pre dicts that the single-stage separation of even a moderately complex mixture is unlikely to be used successfully for resolv ing the components and that most chro matographic peaks will contain more than one component. Simple calculations show that suffi cient improvement of peak capacity using a single-stage separation is impractical (5). However, for multidimensional sepa rations in which more than one chromato graphic column is used, the peak capacity increases in a multiplicative way (6), and simulation studies suggest that the capac ity of the second dimension of a 2D sepa ration need not be very large to obtain a high-resolution separation (7). Briefly, the basis of this theory is the assumption that a mixture elution profile is Poissonian (2) and that mixture compo nents will arrange themselves randomly along some axis x, which represents (for example) chromatographic retention time. These assumptions lead to an analyt ically useful expression of the relationship between the number of peaks p, observed in a chromatogram, the expected compo nent number m, and the column peak ca pacity « c
ties (6) and that the use of multidimen sional gas chromatography (MDGC) holds promise for achieving greatly im proved separations.
Davis (8) recently generalized Roach's original theory (9), which described the overlap of circles in a 2D plane, to an «dimensional space appropriate for applica tion to «-dimensional separations, such as those discussed here. Davis concludes that this theory quantifies the incredible power of 3D and 4D separations. The present state of the art of a variety of mul tidimensional separation systems was re cently reviewed [10), and (judging from the applications demonstrated thus far) there is little doubt that further develop ment of such methods would significantly extend analytical chemists' ability to sepa rate complex mixtures. In its simplest form, an MDGC experi ment involves isolation of the components contained in a small segment or segments of a complex chromatogram generated by a first-stage gas chromatographic column (the precolumn) in an intermediate cryo genic trap. The trapped chromatographic
effluents are then separated by using a second chromatographic column (the ana lytical column) of different selectivity from the precolumn (11). A number of researchers have demon strated the potential of MDGC for the analysis of complex mixtures (12-16). In addition, the Siemens Sichromat 2 is now available. This instrument, based on the pioneering work of Schomburg, Husmann, and Weeke (17,18), has a precol umn followed by a single valveless switch that can be used for heart-cutting precol umn components to route them to a sec ond analytical column. However, this method and implementation of MDGC, in which a single intermediate cryogenic trap is used, share certain disadvantages. First, a single-stage approach requires η sequential injections to trap η sequential heart cuts, with each injection and trap ping followed by second-stage analysis of the particular heart cut. Thus analysis time is extended by a factor nt, where t is the time to inject, trap, and analyze any one of the » heart cuts. A second disadvantage is that the switching does not provide a way to en rich minor components, and the single-
The maximum of this function occurs when the expected number of compo nents matches the peak capacity, leading to the prediction that, with random spac ing, one can never see more than 37% of the peaks theoretically possible with uni form spacing.
(*frc)— = e_1 = ° · 3 6 7 9
(2)
Still more pertinent, in the present con text, is the corollary that the maximum number of single-component peaks (s) would be 18% of the peak capacity, even under the most favorable circumstances.
However, theory predicts that, in the limit, peak capacity for a two-stage separa tion increases approximately as the prod uct of the individual column peak capaci 296 A
Figure 1 . 2D GC/MS of lacquer thinner. (a) Total ion chromatogram after low-resolution first-stage analysis showing the area heart cut. (b) Total ion chromatogram of the heart cut after higher resolution second-stage analysis. (Adapted with permission from Reference 24.)
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trap method does not provide a means for parallel enrichment of more than one minor component at a time from different segments of a complex chromatogram, where minor component samples from multiple injections are combined. Finally, switching and single-trap methods make it inconvenient or impossible to carry out multiple-column multidimensional separations (GC") via sample looping or recycling. These shortcomings prompted the investigation of multiple parallel intermediate cryogenic trapping as another way to analyze complex mixtures (14,15). Instrumentation for MDGC Following the early demonstration of 2D GC by Simmons and Snyder in 1958 (19), the majority of MDGC applications reported in the literature use only two columns. Although Simmons and Snyder suggested the obvious extension to the use of multiple columns, most researchers have not pursued multidimensional separations. The commercial instrument mentioned above was interfaced with a Hewlett Packard mass-selective detector and a Mattson Cryolect matrix isolation FT-IR spectrometer in the first demonstration of 2D GC/MS/IR (20). In another case, the Siemens 2D gas chromatograph
was interfaced with an isotope ratio mass spectrometer to permit analysis of heart cuts purified via the 2D GC procedure (21). Other researchers have reported similar systems in which a Perkin Elmer gas chromatograph was interfaced with an ion trap mass spectrometer (22) and modified Hewlett Packard gas chromatographs were interfaced with low-resolution mass spectrometers (23,24). Figure 1 shows a 2D GC/MS separation in which a single heart cut from a lacquer thinner with four identifiable peaks after a low-resolution first-stage analysis had at least 13 components, all of which could be identified following higher resolution second-stage analysis (24). Other researchers have developed 2D GC approaches using parallel stages of GC analysis on two separate columns interfaced with a common detector (25). In all of these examples, both serial and parallel, excellent reproducibility and increased separation efficiencies were achieved. In fact, Gupta and Nikelly (25) suggest that 2D GC is the method of choice for mixture analysis when a more specific GC/MS method is not available (23). These recent results, as well as other successful applications of 2D GC to a variety of analytical problems, are further evidence that the earlier suggestions
Figure 2. Six-trap two-column MDGC experiment.
of the possible value of higher dimensional GC (1,2,16) are well-founded. Applications of multidimensional GC As discussed in a recent review (26), the idea of multidimensionality has been associated with various GC techniques. In the present context, 2D GC refers to a gas chromatographic separation in which portions of an initial chromatogram (heart cuts) are subjected to a second stage of GC analysis, using a column of different polarity. In turn, a 3D analysis would be one in which a third stage of GC analysis is applied to selected heart cuts of the second-stage chromatogram, and so on. Obviously, the dimensions in question are information vectors rather than physical dimensions and could be as large as desired or practical. Using this definition of multidimensional GC, in the limit, would extend it to the very interesting multiplex technique of Phillips and co-workers (27, 28), in which all mixture components are passed through both a rapid GC precolumn and a serially connected analytical GC column, a mode of analysis termed "comprehensive 2D GC" (29). In recent years there has been a significant increase in reports of applications of 2D GC in a variety of analytical contexts, including petrochemical, environmental, flavor and fragrance, and biomedical analyses. Most of these were based on use of, at most, a single trapping device, and a great many involved serial GC with no intermediate trapping (30-36). Multiple parallel cryogenic traps also have been used to allow multiple heart cuts during a single chromatographic separation (37). Parallel cryogenic trapping A parallel cryogenic trapping experiment is depicted in Figure 2. In this hypothetical separation, six different portions of the primary column chromatogram, each corresponding to the components eluting in two or three chromatographic regions, are trapped. Then the contents of each trap are injected sequentially into an analytical column with separation characteristics different from those of the primary column, resulting in the six second-stage chromatograms shown on the left. Although columns with similar medium resolution are used in practical sys-
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terns, more peaks will typically appear in the secondary chromatogram than in the portion of the original chromatogram selected for heart cut analysis. To illustrate the idea, use of the same second-stage column is implied; however, because the secondary analysis is carried out sequentially, different analytical columns could be used for each of the six trapped sam-
ples, if there were suitable valves and switching. In principle, simultaneous second-stage analysis would be possible. If higher second-stage resolution is necessary, often it can be obtained by shortening the heart cut times for trapping (e.g., trap 10-20-s segments to capture fewer mixture components in each trap). Alternatively, 1-3-min segments
Time (min) Figure 3. Multidimensional GC/IR/MS. (a) Schematic diagram of the seven-trap system, (b) Analysis of an unleaded gasoline sample showing the first 20 min of the Gram-Schmidt reconstructed total IR response chromatogram from precolumn separation and the mass spectral total ion chromatograms of the five heart cuts from the analytical column. The heart cuts were released to the analytical column from cryogenic traps in the sequence in which they were collected. (Adapted with permission from Reference 15.) 298 A
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could be trapped to speed analysis and to collect more components per heart cut. Shorter heart cut time intervals will result in improved component resolution in the secondary separations because the number of components injected into the second-stage column will be reduced, and it is more likely that they will successfully separate. As mentioned above, an additional advantage of a parallel trapping system for the enrichment of minor components is that one can simultaneously enrich different segments of the precolumn chromatogram, followed by a multidimensional chromatographic separation. Furthermore, stages of GC analysis other than the two depicted in Figure 2 (GC) could be used. Thus the enriched sections could be analyzed with various parallel analytical columns following a single enrichment step to improve the overall separation and identification capacity. The obvious outcome of this procedure is that a considerable amount of time can be saved in determining minor components in a complex mixture. From an information standpoint, each column used will provide an additional retention time parameter for each separated mixture component. For routine MDGC analysis, the GC" technique will allow a large number of iterations for a given heart cut section through the use of various parallel analytical columns. For example, it is possible to re-trap the effluent emerging from the FT-IR lightpipe because the IR measurement is nondestructive. Samples from the precolumn or from analytical columns are split in parallel to the IR detector and the mass spectrometer (with the greatest percentage of the sample in each case routed to the IR detector), and the portion analyzed by the IR detector is recycled for further evaluation. Significant reductions in time for complex mixture analysis can be achieved, permitting spectroscopic measurements of a chosen chromatographic segment many times over, with separations carried out on columns of different selectivity. Multiple parallel trap MDGC Some of the 2D GC results discussed above establish that cryogenic trapping does not appreciably reduce chromatographic resolution for subsequent GC
analysis (i.e., minimal peak broadening is observed) and that retention times are quite reproducible. Equally important, the anticipated benefits of MDGC have been demonstrated. For example, heart cuts of
a first-stage separation containing 7,3,12, and 12 peaks, respectively, produced second-stage chromatograms with 16,17,30, and 30 peaks (24). Thus the original 34 peaks were further separated into a total
F i g u r e 4 . C o m b i n e d M D G C / M S / I R of a f o u r - c o m p o n e n t m o d e l m i x t u r e . (a) Primary separation using an intermediate polarity column, (b) Mass spectrum of the material indicated by the asterisk, (c) IR spectrum of the material indicated by the asterisk, (d) Secondary separation of the material indicated by the asterisk, (e) Mass and IR spectra for the material from peaks 1, 2, and 3 after the secondary separation.
Analytical
of almost three times as many components. For any trapping system approach to multidimensional GC, obvious concerns include reproducibility of adjusted peak retention times, sample losses during recycling procedures, and quantitative reproducibility of chromatograms. In one test of the parallel cryogenic trapping concept, a two-trap manually operated valve-based system was fabricated and tested by analyzing unleaded gasoline samples (14). In another study (15), a manually controlled seven-trap cryogenic trapping system, shown in Figure 3a, was fabricated and retention time reproducibility quantitatively evaluated for corresponding heart cuts from multiple injections of unleaded gasoline. This arrangement allows great flexibility. For example, a single-stage GC separation with mass spectrometric and IR detection can be accomplished by setting appropriate valves so that none of the effluent emerging from the FT-IR is routed into any of the cryogenic traps, but merely bypasses the traps to the vent. Alternatively, the eight-port valve can be set to trap up to seven different heart cuts; and the second eight-port valve can be used, together with two port valves, to route any selected heart cut into either of the two analytical columns for second-stage separation, followed by mass spectral and IR analysis. In the retention time trials, a fivecomponent second-stage fraction showed a standard deviation of the adjusted retention times of 0.05 min, whereas an eightcomponent second-stage chromatogram had a standard deviation of 0.02 min. Figure 3b shows the chromatograms obtained for one of these analyses, where five heart cuts were trapped and further separated following a single sample injection. Because of the excellent reproducibility, the use of this technique makes it possible to analyze complex mixtures without sacrificing dynamic range and to take advantage of multiple retention times on different columns in qualitative analysis applications. These characteristics seem ideally matched with the requirements of environmental, biological, essential oil, and petroleum chemistry. More important, such a system provides the flexibility to take advantage of the information-rich mass spectrometric and IR detectors at each stage of the analysis.
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REPORT MDGC/IR/MS analysis The power of a MDGC/IR/MS analytical system is best illustrated by specific examples. Figure 4a shows the chromatogram resulting from a single-stage separation of a component model mixture using the intermediate polarity column (see Fig-
ure 3). Figures 4b and 4c show the mass and IR spectra for the peak marked with the asterisk. After a second injection of the sample, the material eluting at the retention time of the second peak was cryogenically trapped and subjected to second-stage analysis using a higher po-
A.
Figure 5. Combined MD GC/MS/IR of eucalyptus oil. Partial first-stage chromatograms of (a) authentic eucalyptus oil and (b) adulterated eucalyptus oil. (c) IR and mass spectra for materials indicated with asterisks in (a) and (b).
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larity column. Figure 4d shows the resulting chromatogram, which reveals three components, in addition to residual component 1 carried over in the cryogenic trapping process. Figure 4e contains the mass and IR spectra of peak 1 (1,3-dichlorobenzene),peak2 (y-terpinene), and peak 3 (undecane). The use of 2D GC thus allows the unambiguous identification of the mixture components, which would have been impossible using a single stage of GC separation in which only composite mass and IR spectra are obtained. Figure 5 shows a comparison of partial single-stage chromatograms of a known natural eucalyptus oil sample (Figure 5a) and an adulterated oil sample (Figure 5b) (37). Judging by the peak shapes and retention times, one would be tempted to conclude that the peaks marked with an asterisk result from the same compound. However, as is evident in Figure 5c, the mass and IR spectra show significant differences; thus, one or both may contain unresolved components. Second-stage analysis of the asterisklabeled material in Figure 5a reveals that it is a single pure component, 4-terpineol. On the other hand, second-stage separation of the corresponding material from Figure 5b yielded the chromatogram in Figure 6a, revealing the presence of at least three components. Figures 6b and 6c show the mass and IR spectra for peak 1, identified as the adulterant, camphor, and peak 2, which is identified as 4-terpineol. Here, too, use of 2D GC combined with IR and MS detection makes possible unambiguous identification of materials that might have been erroneously identified through single-stage GC analysis. Conclusions There is no question that qualitative and quantitative analyses of complex volatile mixtures continue to be challenging problems. Because of the sample size requirements for spectroscopic detectors, the highest resolution columns cannot be used for mixture separation when such detectors are used. However, it is important to achieve high-resolution separations that are consistent with the sample limitations. If information-rich spectroscopic detection methods are to be used most effectively in combination with GC, it is
Figure 6. Second-stage analysis of the adulterated eucalyptus oil. (a) Total ion chromatogram. (b) IR and mass spectra of material from peak 1. (c) IR and mass spectra of material from peak 2.
(6) Giddings, J. C.J. High Résolut. Chromatogr. Chromatogr. Commun. 1987,10, 319. (7) Shi, W.; Davis, J. M. Anal. Chem. 1993, 65,482. (8) Davis, J. M.Anal. Chem. 1993, 65,2014. (9) Roach, S. A. The Theory of Random Clumping; Methuen and Co. Ltd.: London, 1968. (10) Cortes,N.J./. Chromatogr. 1992,626,3. (11) Bertsch, W. In Chromatographic Science Series, Vol. 50, Multidimensional Chromatography Techniques and Applications; Cortes, H. J., Ed.; Marcel Dekker: New York and Basel, 1990; pp. 74-144. (12) Himberg, H.; Sippola, E.; Riekkola, M. L /. Microcolumn Sep. 1989,1,271. Partial support from the National Science Foun(13) Bertsch, W.J. High Résolut. Chromatogr. dation under grant CHE-92-01277 during the Chromatogr. Commun. 1 9 7 8 , 1 , 289. preparation of this article is gratefully acknowl(14) Ragunathan,N.;Krock,KA.;Wilkins, edged. Thanks also are due to Kevin Krock for C. L Anal. Chem. 1993, 65,1012. his assistance in literature review and prepara(15) Krock, Κ A; Ragunathan, N.; Wilkins, tion of the figures. C. L./. Chromatogr. 1993, 645,153. (16) Jennings, W. G.; Settlage, J. Α.; Miller, References R J.; Raabe, W. G.J. Chromatogr. 1979, 186,189. (1) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983,55,418. (17) Schomburg, G.; Husmann, H.; Weeke, F. (2) Giddings, J. C.Anal. Chem. 1967,39, /. Chromatogr. 1974,99, 63. 1027. (18) Schomburg, G.; Husmann, H.; Weeke, F. (3) Multidimensional Chromatography: Tech/. Chromatogr. 1975,112,205. niques and Applications; Cortes, H. J., Ed.; (19) Simmons, M. C; Snyder, L R Anal. Marcel Dekker: New York, 1990. Chem. 1958,30,32. (4) Giddings, J. C. Anal. Chem. 1967,39, (20) Rodriguez, P. A; Eddy, C. L; Marcott, C; 1027. Fey, M. L; Anast, J. L.J. Microcolumn (5) Davis, J. M./. High Résolut. Chromatogr. Sep. 1991,3,289. 1991,14,501. (21) Nitz, S.; Weiureich, B.; Drawert, F.J. High
essential that the uniquely powerful approach of multidimensional GC be combined with the highly specific detection methods now available. The potential efficiency and flexibility that could be achieved through the use of multiple cryogenic trap systems offer one possible solution to this analytical problem. Recent results suggest that practical implementation of such approaches are well within the reach of current technology.
Résolut. Chromatogr. 1992,15,387. (22) Johnson, G. L.; Tipler, A; Crowshaw, O.J. High Résolut. Chromatogr. 1990,13,130. (23) Lamparski, L. L; Nestrick, T. J.; Janson, D.; Wilson, G. Chemosphere 1990,20, 635. (24) Krock, Κ A; Wilkins, C. L. Anal. Chim. Acta 1993,277,381. (25) Gupta, P. K; Nikelly, J. G. Anal. Chem. 1991, 63,1264. (26) Himberg, K; Sippola, E.; Riekkola, M-L. /. Microcolumn Sep. 1989,1,6. (27) Liu, Z.; Zhang, M.; Phillips, J. B.J. Chro matogr. Set. 1990,28, 567. (28) Mitra, S.; Phillips, J. B. Anal. Instrum. 1989,18,127. (29) Liu, Z.; Phillips,J. B.J. Chromatogr. Sa. 1991,29,227. (30) Szakasits, J. J.; Robinson, R E. Anal. Chem. 1991, 63,114. (31) Borg-Karlson, A-K; Lindstrom, M.; Norin, T.; Persson, M.; Valterova, I. Acta Chem. Scand. 1993, 47,138. (32) Mosandl, A; Fischer, K; Hener, U.; Kresi, P.; Rettinger, K; Schubert, V.; Schmarr, H-G./. Agric. Food Chem. 1991,39, 1131. (33) Karl, V.; Schmarr, H-G.; Mosandl, A / . Chromatogr. 1991,587,347. (34) Herraiz, M.; Reglero, G.; Herraiz, T ; Loy ola, E.J. Agric. Food Chem. 1990,38, 1540. (35) Wong, B.; Castellanos, M.J. Chromatogr. 1989,495,21. (36) Sonesson, A; Larsson, L.; Andersson, R; Adner, N.; Tranberg, K-GJ. Clin. Micro biol. 1990,28,1163. (37) Ragunathan, N.; Krock, K; Wilkins, C. L. Anal. Chem. 1994,66,425.
Charles L. Wilkins receivedhL· B.S. degree from Chapman College in 1961 and hL· Ph.D. in organic chemhtryfrom the Uni versity of Georgia in 1966. Following a postdoctoral appointment at the University of California, Berkeley, he joined the faculty of the University of Nebraska-Lincoln, where he remained until 1976. Since 1981 he has been a professor ofchemutry at the University of California, Riverside, where he L· director of the Analytical Chemtory Instrumentation Facility. HL· research fo cuses on FTMS and computer-assKted chemical analysL·, including development of integrated multepectral analysL· systems. He can be contacted at the Department of Chemtory, University of California-River side, Riverside, CA 92521.
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