GC GC GC GC
Comprehensive 2-D GC provides high-performance separations in terms of selectivity, sensitivity, speed, and structure. Jean-Marie D. Dimandja Spelman College
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he circumstances surrounding the development of the key concepts in comprehensive 2-D GC are worth telling, for they testify to the powers of observation that have sparked the birth of many a novel technology. On a cold winter morning about 20 years ago, John B. Phillips, then a chemistry professor at Southern Illinois University, was warming up his car when he noticed his rear-window defogger wasn’t doing its job. Later that day, he went to the local auto parts store to purchase a defogger repair kit, which contained a bottle of electrically conductive paint (copper in ethyl acetate solvent) and a small brush with which to apply the paint to the problem area to reestablish electrical contact. But when Phillips returned to fix his car’s defogger, he noticed something else—the defogger line somewhat resembled a fused-silica column in shape and color. Part of his research at the time involved developing on-column GC sample inlet devices, and he was intrigued that this electrically conductive paint might improve the systems he was using. He took the defogger kit to his laboratory, applied the paint to a section of a GC column, connected the resistive piece to a power supply, and briefly heated it after connecting the head of the column to the headspace of a sample vial containing a volatile organic compound.
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FIGURE 1. Basic 2-D GC systems. (a) Heartcut (GC–GC) vs (b) comprehensive (GC GC). 1, first-dimension inlet; 2, first-dimension column; 3a, diversion valve; 3b, modulator; 4, detector; 5, second-dimension column; 6, second-dimension detector.
The result of all his efforts was a chromatogram of a single, sharp peak that corresponded to the effluent that was trapped and then released by the heat pulse in the painted section of the column. The key advantage of the paint was its low thermal mass on the GC fused-silica capillary column, which, in contrast to other, more sophisticated GC inlet systems, enabled rapid heating and cooling of the sample inlet device. Thus was born a simple, cheap component that his graduate students could easily manufacture for their research projects (1– 4). In the late 1980s, Zaiyou Liu, a fellow graduate student in Phillips’ research group, used the device as an interface between two tandemconnected GC columns, essentially as an on-column sample inlet for the second column (5–7 ). Comprehensive 2-D GC was thus achieved due to the simplicity of the interface and its highspeed operation. In the years since its invention, the instrumentation has evolved from an academic prototype to a fully integrated commercial system. The on-column injector has matured from the original painted resistor to more rugged, mechanically engineered designs that, although sophisticated, retain the operational qualities of the original units. Even the original term, C2DGC, has been replaced by GC GC, which more aptly describes the true multiplicative nature of the technique’s separation power (8). In this article, GC GC will be presented in the context of its potential for a wide array of analytical applications. This emerging technology has great promise for enhancing the resolution of complex volatile organic samples. As it transitions from an instrument development project to a method development tool, the analytical community will continue to debate its true scope in the spectrum of GC techniques (9, 10). 168 A
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The need for multidimensional GC was recognized early in the development of GC itself as a means to increase peak capacity for the resolution of congested areas in the 1-D chromatogram (11, 12). True multidimensional systems require two independent separation mechanisms and the conservation of the primary column separation into the secondary column (13, 14). In selective or heartcut 2-D GC, two columns are typically connected in series through a flow-switching interface that directs specific portions of the primary column effluent into the secondary column (Figure 1a; 15, 16). Two detectors are generally required—one detector monitors the primary column effluent, and the second is located at the end of the secondary column. The goal of heartcut analysis is the targeted resolution of critical regions of the 1-D chromatogram. Methods are developed after 1-D retention information has been obtained, and that information is used to divert the portions of interest into the secondary column. The efficiency of selective multidimensional methods increases as more becomes known about the sample matrix. In GC GC, the entire first-dimension effluent is sampled onto a second column, which is operated at high-speed GC conditions (Figure 1b) that allow rapid sampling of the primary column effluent without compromising the integrity of each second-dimension chromatogram. The interface between the two separation dimensions is a modulator that increases the amplitude of the chemical signal and facilitates its transmission through the second dimension of the system, similar to the way a frequency modulator transmits radio waves. Modulators function like a traffic light as they periodically “trap” and then release smaller, more manageable portions of a continuous stream of traffic. The first-dimension sample effluent is thus continuously transferred in small portions to the second-dimension column throughout the chromatographic run, and each transferred pulse generates a high-speed secondary gas chromatogram. The GC GC moniker is appropriate because the collection of a large number of second-dimension chromatograms effectively results in the multiplication of the peak capacities of the first and second dimensions, as proposed by Giddings (14). In GC GC, second-dimension columns offer lower resolution power than secondary columns in heartcut systems, because shorter columns are used to satisfy the high-speed requirement. However, the cumulative effect of the series of high-speed secondary separations throughout the GC GC run produces chromatograms with a much greater peak capacity (16, 17 ). The construction of a GC GC chromatogram is shown in Figure 2. A single detector records the output of the secondary column (Figure 2a), which is simply a continuous sample stream. The fact that the stream entering the detector consists of many distinct high-speed secondary separations is irrelevant, because the raw trace is subsequently processed on the basis of the modulation frequency (Figure 2b), which is fixed for a par-
ticular run. A 4-s modulation period was used in Figure 2. Samples eluting out of the first dimension are often sampled more than once onto the second-dimension column, depending on the width of the peaks in the first dimension relative to the modulation period. All the second-dimension chromatographic segments or frames are then rearranged in a matrix array (Figure 2c). The resulting 2-D chromatogram can be viewed in several formats, including surface, contour (Figure 2d), and peak apex plots. (In a peak apex plot, the data are reduced to the retention coordinates of
the local peak maximum of each separated compound.) Data reduction options such as peak apex plots are not used as frequently as contour plots, although they are more manageable for advanced data-processing tasks such as pattern recognition (18). The key features of GC GC are its large resolution power and its ability to separate substances into classes. GC GC chromatograms can have peak capacities exceeding 20,000, whereas 1-D gas chromatograms rarely exceed 1000. GC GC separations can usually be done in a time comparable to 1-D GC separations because the higher speed of the second di-
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FIGURE 2. Construction of a GC GC chromatogram. (a) Raw detector signal. (b) Segmentation into second-dimension chromatograms. Tick marks on the x-axis denote each modulation of the sample into the secondary column. (c) 3-D surface plot showing the rearrangement of second-dimension “frames” into a matrix array. The colors are used to visualize the orientation of the secondary chromatograms obtained from Figure 2b. (d) Contour plot of a larger portion of the GC GC chromatogram with the area of interest indicated by the box. Signal intensity: blue (low) to green to yellow to red (high).
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mension allows increased peak capacity without increasing the length of the analysis. The separation of substances into classes through structured chromatograms provides an additional means of identification and reduces the probability of peak overlap between members of different chemical classes. For example, Figure 2d can be viewed as a structured volatility versus polarity plot. The two alkanes nonane and decane are separated by their boiling points but have similar polarity, and therefore similar retention times, in the second dimension. The aldehydes heptanal and octanal are also separated by volatility in the first dimension. In the second dimension, both aldehydes elute at a retention time that indicates they are more polar than the alkanes.
Theoretical considerations The retention of a given substance in the second dimension of a GC GC system depends on the chemistries of its interactions with both first- and second-dimension column stationary phases and can be expressed as µ°D2 µ°D1
[ ( )]
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in which D1 and D2 refer to the first- and second-dimension columns, k is the retention factor, µ° is the chemical potential of a substance interacting with a stationary phase, is the phase ratio in the second column, and C is a constant determined by the gas constant R and a proportionality factor related to the ratio of the temperatures in the two columns (19). Adjusting C by changing the temperature of the second column controls the range of retention and the separating power of the second column. As seen in the equation, second-dimension retention is determined by the ratio of the chemical potentials of a substance interacting with the two stationary phases. The first column’s stationary phase influences second-dimension retention as much as the second column’s stationary phase. The chemical potential on the first column’s stationary phase is a reference point against which the chemical potential on the second column is compared. If the two phases appear to be the same to a given substance, then the ratio is 1. Only deviations from 1 contribute to the spread of substances in the second dimension. Contributions to chemical potential that are shared between the two phases are eliminated from the second dimension. Thus, retention simply due to volatility is minimized and determined by other, more relevant molecular properties. The most common cause of a deviation is molecular polarity. If the second-column stationary phase is more polar than the first, polar substances will have stronger retention in the second column. Subtler retention mechanisms, such as molecular shape interactions, can also contribute to the deviation in chemical potential. The equation also shows that two different classes of substances can be separated in the second dimension. The members of two classes will probably have different kinds of interactions with either the first- or second-column stationary phases, and consequently their chemical potential ratios will vary. The ratios are independent of volatility and much more likely to be consistent 170 A
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for all members of a class than are the chemical potentials themselves. The separation of two classes of substances can be predicted from a set of chemical potentials that can be calculated from 1-D GC data. The ability to make these predictions is helpful for proper selection of stationary-phase combinations for a particular application in a GC GC system (20).
Modulators The primary functions of an efficient modulator are highfrequency operation as well as reproducible trapping and releasing efficiency. A number of different modulator designs developed recently can be classified into two groups: flow-switching modulators that operate as high-frequency diversion valves (0.1–1.0 Hz) and thermal modulators that sample the first dimension more completely (21). Thermal modulators are further divided into three types: heat, cryogenic, and jet-pulsed modulators. In a heat modulator, the sample is trapped by a thick stationary-phase film in the modulator tube to increase the breakthrough time of the analytes beyond the modulation period (6, 22–26). A heat pulse is applied, and the analytes are then released to the secondary column. In a cryogenic modulator, trapping is achieved with a cryogenic zone placed over the modulator tube. The sample portions are released onto the secondary column when the cryogenic zone is removed from the modulator tube, and the analytes resume motion because the oven temperature is reestablished over the modulator tube (27, 28). In a jet-pulsed modulator, trapping and releasing are actively produced by synchronized pulsing of a set of hot and cold jet nozzles that operate on the modulation zone (29, 30). The key parameter of importance in the design of a thermal system is high-frequency operation—rapid trapping and releasing—to fulfill the sampling frequency requirement. Valve modulator GC GC systems function in the same manner as heartcut 2-D GC equipment except that the much higher frequency of the intercolumn sampling valve in GC GC does not require the use of a primary column detector. These modulators are flow-switching devices (31, 32) and, because the amount of diverted material varies between 20% and 90% depending on the design, there has been some discussion as to whether this form of GC GC is truly comprehensive. A critical argument in favor of valve modulators is that as long as they attain or exceed the sampling frequency threshold, the continuity of the signal they generate will be maintained, and no significant information will be lost (33). A high-temperature valve modulator has very recently been reported that extends the temperature range substantially above 200 °C and thus enables the analysis of volatile and semivolatile sample mixtures (34). Valve modulator systems are cheaper, lower-maintenance instruments than thermal modulators. However, thermal modulators are more efficient because the entire primary column effluent is focused and released into the secondary column. The price paid for higher performance modulation is more frequent maintenance (i.e., the use of cryogenic consumables). Nevertheless, modulator technology has evolved to the point where both types of modulators possess the necessary attributes (ruggedness, tem-
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Columns The role of the primary column is to provide the secondary column with smaller subsets of the original sample matrix, and the role of the secondary column is to generate a series of highspeed chromatograms. Independently tunable parameters of the two dimensions include column length and internal diameter, column temperature, and mobile-phase linear velocity. GC GC columns are assembled from conventional GC columns, which can be classified in three major categories based on separation mechanism: volatility, polarity, and shape selectivity (35). Volatility columns offer the most dominant separation mechanism because temperature is considered the most easily varied parameter in GC, and samples are selectively distilled and separated on the basis of their respective boiling points. Nonpolar columns, therefore, are generally selected by users as the primary column in GC GC. In recent work, Haglund et al. have demonstrated the utility of shape-selective columns as primary columns for the enhanced separation of halogenated enantiomers such as polychlorinated biphenyl (PCB) mixtures (36, 37).
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FIGURE 3. GC GC/TOFMS analysis of trace analytes. The high-speed conditions of the secondary (a) Total ion chromatogram contour plot of a congested region of the chromatogram. column result in very sharp peaks (~200 ms (b) Deconvolved chromatographic slice of the area shown in Figure 3a. Each peak marker denotes the presence of a sample component with a unique mass spectrum basewidth on average) that require detectors as identified by the MS deconvolution software. Each colored trace represents a with fast response times (~20 Hz minimum). unique fragment ion. (Adapted with permission from Ref. 44.) Flame ionization detectors (FID) and selective detectors (e.g., electron capture, nitrogen chemiluminescence) that have been develFigure 3 is an example of the resolution of a GC GC oped for high-speed GC (>50 Hz data acquisition rates) have successfully been applied to GC GC (38). Quadrupole and chromatogram further enhanced by TOFMS signal deconvolumagnetic sector high-resolution mass spectrometers also have tion. The contour plot in Figure 3a shows only four visible been interfaced, but the slow nature of their operation (full- peaks, but the individual chromatographic frame or slice indiscan rates are typically 1 Hz) limits their use to the selective cated in Figure 3a and expanded in Figure 3b reveals several mode to speed up detector response time (39 –41). TOFMS additional co-eluters. Peaks 406– 410 are not resolved by GC was recently introduced as a comprehensive detector with an ad- GC, but they possess unique ions that are differentiated by ditional dimension of resolution (42–44). TOFMS spectra are TOFMS. The retention axis shows both the first- and secondnot concentration-dependent because of the pulsed nature of dimension retention times. The 1-D time is the same for all the ionization (45), and thus data-processing methods such as compounds eluting in the same slice. Note that peak 403 is deconvolution can be used to further resolve compounds that composed of three mass fragments, as indicated by the fact that the traces simultaneously reach their peak maxima. are still coeluters after separation (44).
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FIGURE 4. High-resolution GC GC/FID separation of kerosene. (a) 1-D GC separation on a 30 m 250 µm 0.25 µm film nonpolar column. (b) GC GC separation on a 30 m 250 µm 0.25 µm film nonpolar column coupled with a 2 m 100 µm 0.1 µm semipolar 2-D column. Inset is signal amplitude magnified 7.
Data processing performs the tasks outlined in Figure 2 for the visualization of GC GC runs. Qualitative analysis is done on the basis of the retention data in the two dimensions. Quantitative analysis reports a single peak area value for each compound in the chromatogram. Because modulation typically fragments a compound into several peaks in the raw chromatogram, software algorithms are written to recognize the peaks that belong to the same compound and combine the peak areas to provide a total peak area for the compound. Integrated software programs are available on all commercial instruments for modulator control and data processing. The further development and improvement of advanced GC GC data-processing tasks (e.g., chemometrics for sample fingerprinting) is an active area of research and is a critical step in advancing this technology (18, 46, 47 ).
High-resolution GC GC
GC GC is a versatile technique that can be operated in several different modes. The simultaneous optimization of selectivity, sensitivity, speed, and structure of an analytical procedure is the ultimate goal of application development but is difficult to achieve because of conflicting parameters. For example, maximizing selectivity generally comes at the expense of analytical speed because resolving power is improved by increasing column length, but speed is improved by decreasing column length. Most optimization procedures, therefore, focus on one or two parameters and try to find reasonable compromises for 172 A
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the others. The primary operational mode used today is highresolution GC GC. Ordered chromatograms are desirable, especially for complex mixtures such as petroleum (48), tobacco smoke (49), and urban photochemical smog (50) because they reduce the probability of peak overlap and improve the identification process. A typical column set is a 30-m nonpolar first-dimension column coupled to a 2-m second-dimension semipolar column. Figure 4a is a 1-D gas chromatogram that was run under similar temperature programming conditions as the first dimension of the GC GC chromatogram to help match the most salient similarities between the two analyses. The separation in a temperature-programmed, nonpolar 1-D GC column produces a chromatogram with large, regularly spaced n-alkane peaks indicating a carbon range that extends from C8 to C19. The scaling of the retention time on the x-axis is the same in both chromatograms, so the large n-alkane peaks in Figure 4a have corresponding peaks in Figure 4b. Many other, less abundant, petroleum compounds are present as well. Because of their large number, the compounds are poorly resolved and produce an elevated baseline that spans several carbon numbers in the center of the chromatogram. The enhanced separation of these compounds in the second dimension (Figure 4b) allows the resolution of at least an order of magnitude more peaks for this sample. The distribution of compounds in the second dimension on the y-axis is not random; rather, it is based on the relative retention in the more polar (50% phenyl dimethylsiloxane) sec-
ondary column. The least polar compounds, such as the linear and branched alkanes, have the lowest second-dimension retention (e.g., octane and 3-methyloctane at the bottom left region of Figure 4b). One- and two-ring cycloalkanes (e.g., ethylcyclohexane and trans-decahydronaphthalene) have greater polarity and are retained on the second column. One-ring aromatics (e.g., 1,2-dimethylbenzene and 1,2,3-trimethylbenzene) have significant retention due to favorable intermolecular attractions to the secondary-column stationary phase. Fused aromatic, saturated ring compounds have even greater retention than alkylbenzenes (e.g., indan and tetrahydronaphthalene), and multi-ring aromatics have the greatest retention in the secondary column (e.g., naphthalene, 2-methylnaphthalene, and biphenyl). The sorting of these peaks in the 2-D plane produces a structured chromatogram that permits the identification of homologous series. For example, 1,2-dimethylbenzene and 1,2,3-trimethylbenzene are the latest eluting isomers in the C2- and C3-benzene classes, respectively. These compounds are separated by approximately one carbon-number distance. This pattern provides rapid identification for higher substituted benzenes. The C4- through C8-benzenes have observable concentrations in this sample. The inset in Figure 4b highlights an area with numerous lowabundance peaks. Based on the repeating nature of homologous series, peaks in the box most likely belong to the C7- and C8benzenes. Enlarging and rescaling the box show 30 –40 discernible peaks. The dynamic range between the smallest and the largest n-alkane peaks is on the order of 1000:1. Logarithmic visualization of the intensity scale is sometimes advantageous to see both large and small peaks in one image (51).
signal in the 2-D plane is distinguishable from the noise fluctuations. Signal amplitude enhancement in GC GC depends on several factors, such as the extent of the peak broadening in the first dimension and modulation period, and it is not a constant for all compounds in a particular separation. This form of GC GC does not provide a substantial improvement over high-speed 1-D GC methods in which peak broadening is already optimized in the primary dimension, but it significantly enhances sensitivity over more conventional 1-D GC analyses. GC GC can also be operated in a high-throughput mode in which it combines the sample preparation and separation steps of normally time-consuming protocols. Green et al. recently developed several pyrolysis GC GC methods for the analysis of polymer additives (53). For example, determining clarifying agents typically involves >6 h of an elaborate sample extraction followed by a 1-h pyrolysis 1-D GC run. With pyrolysis GC GC, the entire sample preparation is bypassed because the sample is pyrolyzed directly into the instrument and can be analyzed in 1 h. The polar trace additives are easily separated from the rest of the pyrolysis products, and the risk of sample loss due to extraction is eliminated. These types of separations can be further optimized because the goal of the analysis is not the complete resolution of all sample constituents, but rather the separation of specific compounds from a sample matrix. Seeley et al. recently proposed a system in which a firstdimension column is interfaced to two second-dimension columns of dissimilar stationary-phase composition (54). Each modula-
Other operational modes Figure 5 shows the raw chromatographic traces of a high-sensitivity GC GC run. The non-modulated and modulated analyses of a sample of 38 PCBs extracted from human serum are overlaid to compare their signal intensities (52). This chromatogram was acquired with a GC GC/microelectron capture detector system that used a 30-m first-dimension nonpolar column connected to a 20-cm polar second-dimension column. The modulator is intended mainly as a preconcentration device. This system does not target a drastic improvement in resolution power or analysis time; instead, it amplifies the magnitude of the analyte’s signal prior to its detection. In addition, the short second dimension separates the signal from the noise for each modulation. The baseline signal is dispersed in the second dimension, which differentiates this setup from signal amplification devices in 1-D GC, where the amplified signal combines the analytical signal and the noise. The result is a signal amplitude enhancement that can be quite significant (on average, an order of magnitude) when compared with conventional 1-D GC, and it is even more critical at trace levels where the small but localized analyte
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FIGURE 5. Signal amplitude enhancement. Overlay of the modulated (blue) and non-modulated (red) raw traces of a GC GC/ micro-electron capture detector analysis of 38 PCBs most commonly found in human serum. The two trichloro-PCBs (CB-18 and CB-28) are identified with their circled nonmodulated pairs. The deca-PCB (CB-209) is similarly identified.
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tion pulse is fed to both columns, and each column is connected to a separate detector. A library system was created in which compounds are defined by three unique retention time values. This system has potential as an alternative to the use of MS for compound identification. Marriott and Kinghorn have used a “hybrid” GC GC system that can be operated in conventional 1-D GC, selective 2-D GC, or comprehensive 2-D GC mode in a single run (55). Advances in the development of portable GC GC systems have also been reported (56).
2-D or not 2-D, that is the question! GC GC is an exciting technology that offers much promise for enhanced GC separations. It has survived the growing pains of instrument development, and now that the first fully integrated commercial systems are on the market, the number of practitioners is likely to increase. The recent debate over the true merits of GC GC in the literature and at conferences is a typical sign of this stage. It is a necessary phase in the transition from the realm of exotic technology to the arena of public acceptance. Every major analytical instrument has gone through this test, as Ruzicka et al. recently chronicled in the case of flow injection analysis (57 ). The scope of GC GC is not yet defined, but its versatility suggests that it can be applied to a wide variety of analytical problems in a wide variety of fields. The fundamental question when considering GC GC as an option in method development is whether the complexity of the sample warrants an increase in the dimensionality of the analyzer. From this perspective, the use of all GC-based techniques—selective or comprehensive, one- or multi-dimensional—will be considered as an array of complementary tools in solving an analytical problem.
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I dedicate this article to the memory of John B. Phillips (1947–1999). Tincuta Veriotti (Leco Corp.) and Glenn S. Frysinger (U.S. Coast Guard Academy) are gratefully acknowledged for the chromatograms in Figures 3 and 4, respectively. Jean-Marie D. Dimandja is an assistant professor at Spelman College. His research interests cover all aspects of GC GC with particular emphasis on new application areas and method development strategies. Address correspondence about this article to Dimandja at 350 Spelman Lane, SW, Box 279, Atlanta, GA 30314 (
[email protected]).
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