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Principles and applications of comprehensive two-dimensional gas chromatography

Philip Marriott*, Robert Shellie Australian Centre for Research on Separation Science, Department of Applied Chemistry, RMIT University, GPO Box 2476V, Melbourne, Victoria 3001, Australia

This issue of Trends in Analytical Chemistry celebrates 50 years of gas chromatography (GC) the greatest enabling technology for chemical analysis of volatile compounds. However, what may be considered the most powerful separation tool in GC comprehensive two-dimensional gas chromatography (GCGC) is a development born of the 1990s. It was rst described and almost fully established in the last decade of the twentieth century. The coming decades can be expected to see it ourish into a major operating mode of GC, when applications and fundamental principles will be further expanded, and, most importantly, its universal acceptance will be unquestioned. This article describes why the pioneers of GCGC have so much faith in the new opportunities afforded by this exciting technology. # 2002 Published by Elsevier Science B.V. All rights reserved.
Keywords: Comprehensive two-dimensional gas chromatography; GCGC; High resolution; Modulation; Multi-dimensional gas chromatography; Orthogonal separation

The columns and injector are not particularly specic to GCGC methodology, and presently available injectors and columns are satisfactory. Detectors must be capable of performance that matches GCGC requirements (see below). So GCGC is essentially dependent upon the modulation process or the interface between the two columns for its development. In essence, GCGC may be considered a special case of multi-dimensional GC (MDGC), which has a long history in GC, and generally has made a valuable contribution to enhanced resolution in GC. We will therefore introduce MDGC briey as a prelude to the discussion of GCGC below.

2. Multi-dimensional concepts leading to GCGC

Multi-dimensional analysis in chromatography may be considered to be any technique that combines two or more distinct separation/analysis steps, where at least one of the steps or dimensions involves a chromatographic separation. Thus LC-GC, GC-GC and GC-spectroscopic detection are typical multi-dimensional methods. Focusing on methods involving two separation dimensions, LC-GC is of interest. The aim is to simplify the mixture presented to the GC step, by reducing the overlap of different classes of compounds. Of course, this is achieved at the expense of increasing the total analysis time, since each broad fraction delivered to the GC must be individually analysed in a conventional GC procedure. Multi-dimensional separation techniques are not conned just to analyses
# 2002 Published by Elsevier Science B.V. All rights reserved.

1. Introductory comments on GCGC

Phillips described in 1991 [1] an experiment that produced the GCGC result, which was the practical demonstration of the concepts outlined by Giddings [2]. The key to GCGC resides in the manner in which sequential segments of a rst column (dimension 1; D1) separation are continually passed to the second column (D2).
*Corresponding author. Tel.: +61-3-99252632; Fax: +61-396391321. E-mail: philip.marriott@rmit.edu.au

0165-9936/02/$ - see front matter PII: S0165-9936(02)00814-2

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where there is an overwhelming number of peaks. They are of use whenever a critical separation of compounds cannot be achieved on one column or phase type, but requires the use of two sequential separations on two different phase columns. Thus MDGC, using a heart-cut process, is capable of isolating small regions of a primary column separation and transferring them to a second column where column selectivity now gives enhanced resolution of the heart-cut zone(s) (refer to [3] for further details). Clearly neither of these options is able to give the best possible resolution for all sample components. Contrasted with these is the GCGC experiment, where a single run gives expanded separation capacity, and the aim of complete resolution may indeed be achieved, depending upon experimental considerations. The discussion below will describe how GCGC is achieved.

3. Implementation of GCGC
Fig. 1 illustrates the generic GCGC set-up with two columns (D1 and D2) and the option for either one-column or two-column oven operation.

3.1. Modulators
The modulator either discretely samples, or samples in an accumulative manner, the efuent

from D1, and then pulses the segment of analyte to D2. Modulators may be classied into those that conserve mass or those that sample only parts of the D1 efuent. The latter are generally based on valves. The former use directly coupled columns. To accumulate analyte into narrow bands in the modulator, essentially it can use only thermal means: elevated temperature to accelerate solute into a narrow band (the thermal sweeper approach); or, cryogenic means to retard analyte and cause on-column trapping or focusing of bands. The latter was rst described by the present research group, and is called the longitudinally modulated cryogenic system (LMCS). A later variant of this uses two cryogenic jets to focus compounds at two close, but distinct, regions of the capillary column to decouple the sample-trapping and sample-remobilisation events. Conceptually, this works according to the same requirements as the LMCS. Fig. 2 presents a comparison of the different modulators. In Fig. 2A, an electrically-heated sleeve rapidly ushes sample out of the modulated column zone and thereby compresses that region of column contents into a narrower zone [1]. The need to heat and cool this region cyclically led to a lack of robustness. It was essentially replaced by the rotating, heated-sweeper modulator (Fig. 2B). Apparently, this was difcult to set up experimentally, but the experienced

Fig. 1. Generic GCGC instrument showing the modulator located between D1 and D2 columns. It is possible to have both columns in one oven or to use different ovens for each column.

Fig. 2. Different modulators may be classied as mass conservation types (A.D.) or sampling (valve) types (E. and F.) The modulator is located between D1 and D2.

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user was able to generate reliable results [4]. The LMCS unit (Fig. 2C) uses a cryogenically-cooled trap located about a column segment of 23 cm [5]. By oscillating back and forth, solute may be sequentially trapped then remobilised. The dual cryogenic jet system (Fig. 2D) [6,7] serves the same purpose as the LMCS. It focuses solute in two regions. The downstream region expels solute into D2. Solute remobilisation is achieved by either turning off the jet, blowing hot gas over the cooled column or diverting the jet. The second class of modulator, which relies on ow switching, is represented in Figs. 2E and 2F. The arrangement in Fig. 2F samples only a small segment of the chromatographic band [8]. Fig. 2E incorporates a large volume collecting tube that is lled with (part of) the migrating peak, and, upon switching the valve, the sampled segment is pushed with high gas ow into D2 in a differential ow arrangement [9]. Whilst there are fundamental differences between the two classes of modulators, for the purposes of the present review, their performances and relative merits will not be contrasted and most of the discussion will pertain to the mass-conservation systems.

The reason for requiring multiple peak pulses on D2 is to allow pulse proles to be compared back to the D1 peak shape or envelope, and, importantly, to ensure that peak resolution in D1 is not degraded excessively by the pulsing process. Murphy, et al. discussed aspects of this in LCLC [10]. Loss of D1 resolution will occur if two neighbouring (resolved) peaks on D1 are collected in the same accumulation step. However, these may still show resolution if the D2 phase can separate the components. If solutes are well resolved in D2, then the requirement for the number of pulses across a D1 peak may be reduced or relaxed. The phase of modulation also plays a role in the degree of resolution and apparent peak width in D1, since it alters the peak-pulse presentation [11]. Here, phase refers to the timing of the modulation process with the peak prole entering the modulator. Pulses may be symmetric about a single maximum (in-phase), or two maxima (180 out-of-phase), as shown by the two modulated peaks in Fig. 4. Since the time at which a peak enters the modulator is not

3.2. Generation of peak pulses

Modulation of the D1 peak using the mechanisms described above generates a series of peak pulses. Based on this, Fig. 3 demonstrates how two overlapping peaks are effectively deconvoluted into two interleaved series of pulses. The data stream from the detector is converted into a 2D array, based on the modulation period and detector data-acquisition rate. Conventional wisdom states that, for a peak eluted from D1, at least four peak pulses should be generated on D2 (see Fig. 3). Whilst this may be subject to challenge or re-consideration, it suggests that modulation periods of about 1wb/ 2 or faster will be recommended (where 1wb is peak base-width on D1). Since 1 w b 4s, then Pm < 2s should be used (where Pm is the period of modulation). This further implies that the effective solute-elution time on D2 should be about 2s.

Fig. 3. Illustration of how two overlapping peaks emerging from D1 (A) are resolved in GCGC after passage to D2 (B). The two components are labelled d and f and they map out a very good correspondence with the non-modulated peak distribution. Thus pulse P1 contains only the d component, whereas pulse P2 has a larger amount of d and a small amount of f. Provided D2 can resolve the d and f components, as shown, then the desired resolution of peaks is achieved.

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a controlled variable, any pattern between these two distributions may be obtained experimentally.

3.4. Relative peak widths

Peak widths generated by D2 will depend upon the efciency of band remobilisation in the modulation step, and the extent of band spreading on D2. Peak widths in D2 will depend on carrier ow-rate, liquid-phase loading, capillary length and solute-retention factor. For a 1 m column, of 0.1 mm ID and a solute of k=4 with tM=0.5 s, it can be estimated that a peak base-width of about 0.1 s can be expected. However, the high carrier ow-velocity suggests that the column is not operated at optimum, and a broader peak than indicated here will result, so 0.2 s may be a better estimate. This means that in a D2 elution window (determined by the modulation period) of 4 s, a capacity of about 20 peaks is expected. Thus, for unresolved peaks on D1, there is adequate opportunity for separation on D2, subject to suitable distribution-coefcient differences on the D2 phase for the components to be resolved.

3.3. Column dimensions

Since GCGC relies upon fast analysis of accumulated bands, it is usual for D1 to give a normal GC elution, and D2 to be a fast-elution column. The dimensions of D1 are not critical. Rather, it is more important to consider the peak widths generated by D1. D2 must be able to achieve complete elution of pulsed bands within the modulation period of the system. This is most effectively achieved by use of short, narrow-bore, thin-lm columns. Thus a column set might comprise of a 25 m0.25 mm ID0.25 mm lm thickness (df) column as D1, with a 1 m0.1 mm ID0.1 mm df column as D2. Note that the differential ow-valve procedure is able to use reduced-length capillary columns of normal dimensions at high ow rates for fast analysis on D2 [9]. Complete elution within a time frame of 28 s is usual on D2. It is also possible to use a short D1 column (e.g. 5 m), which will be of thick-lm dimensions, combined with a fast D2 column [12].

3.5. Detection
With a 2wb of 0.2 s and classical acceptance that at least 10 points per peak half-width are required across a peak for it to be suitably measured by a chromatographic detector, a detector data-acquisition rate of 50200 Hz will be necessary. At present, this limits detection technologies to ame-ionisation detection (FID) and electron-capture detection (ECD), for nonspectroscopic detection, and time-of-ight mass spectrometry (TOF-MS). It is expected that other detection principles will soon be available for GCGC. Whilst faster detector data acquisition reduces signal-to-noise ratio by (dataacquisition rate)1/2, so a 100 Hz detector dataacquisition rate gives a signal-to-noise ratio about three times lower than a 10 Hz rate, the zone-compression effect in GCGC leads to greater mass ux in the detector. If the peakresponse increase is proportional to the peakwidth reduction, we expect a sensitivity increase of about 30-fold in GCGC. The net effect is a gain in sensitivity. This benets analytical

Fig. 4. The phase of the modulator may be dened as the difference between the mean of the peak region sampled by the modulator and the centre of the whole peak. An inphase process is shown for the rst peak, with a single, central peak pulse and a symmetric pulse distribution, and 180 out-of-phase pulsing by the second peak where two peak maxima are obtained. Any phase between these two limiting phases can be obtained, and this is entirely at random for a given sample analysis, since the time of emergence of a peak into the modulator is not controlled.

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methodology with respect to sample handling and other aspects, but will not be described here. Mass spectrometric detection is an important issue. Scanning MS systems generally cannot be used for fast-GC peak proles because of mass spectral skewing (where peak-abundance changes in the ion source occur so fast that spectra exhibit mass spectral bias). It is possible that a quadrupole instrument under regular analysis conditions can acquire not even one scan per GCGC peak. Rapid spectral acquisition by TOF-MS technology, with presentation of 100 or more spectra per second and instantaneous measurement of all masses for each TOF pulse, should ideally give unbiased spectra. This has implications for peak deconvolution, which has been widely promoted in fast-GC-TOF-MS methodology [13]. The TOF-MS opportunity in GCGC requires further discussion, albeit briey here. First, quantitation by MS depends on correct response factors, and these are both compound dependent and highly dependent on the m/z value chosen. Further, in single-column GC, where overlapping peaks occur, it is impossible to quantify individual compounds accurately without MS detection. Hence, the conventional approach requires that every sample must be analysed by GC-MS. In GCGC, the greater separation power means that many compounds are now completely separated, so there is less need for, or reliance on, GC-MS identication/ quantitation for routine samples. The 2Dseparation space reproducibility and pattern or structured retention are important factors to consider in qualitative studies. Notwithstanding this, the correct MS identication of components separated in 2D is even more demanding in GCGC, simply because there are so many more components that can now be detected.
3.5.1. Consideration of retention in GCGC

compounds according to dispersion forces (boiling points (BPs)) and specic phasesolute interactions. It is not possible to isolate the BP elution property from the overall GC retention, but for a non-polar column, elution according to BP will be a major determinator of peak retention. In this case, peak co-elution will imply that they have the same or similar BP. If these components are then passed to a second, polar column, at the time they co-elute from the rst column, they will be separated on D2 almost entirely because of their specic polar interactions with the D2 phase. We can then say that the two columns provide orthogonal separation. The chemical nature of the sample components will determine the column combination required for the separation task. For essential oils, a non-polar/polar (polyethylene glycol) column set has been found to be useful. For petroleum samples, a non-polar/polar (high phenyl content phase) column set is reported to be excellent. The availability of a broader selection of columns (such as liquid crystalline columns) will be important in the future, enabling sophisticated and highly specic GCGC separations. The 2D space gives a clue to component chemistry in a manner not possible in a single-column GC analysis. For petroleum oils, this is a powerful tool, since saturated, cyclic saturated, olenic, heteroatomic and aromatic elution regions can be dened within the 2D space. And, for example, in the aromatic region, relationships or patterns of elution of different alkylated species can be found. It can be appreciated that this is a more efcient means of high-resolution sample analysis and the power of such a sample representation cannot be overestimated. Combined with 2D-separation space reproducibility, this should prove to be a completely new analytical tool for sample ngerprinting.

The 2D-separation space is a chemical map of compound properties, which derive from the ability of the two columns to impose their individual separation mechanisms on the sample components. A single column will elute

4. Applications of GCGC
In addition to the general benets of GCGC (sensitivity and separation enhance-

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ment), various studies have revealed additional specic advantages of GCGC for particular sample types. Thus, for petrochemicals, the ability to separate a sample into classes within the 2D space is a distinct advantage that immediately gives the petroleum chemist valuable fundamental information about the chemical nature of the sample; e.g. its aromatic content. For essential oils, the direct comparison or ngerprinting of different samples is useful. In environmental analysis, the ability to use the GCGC system to provide an extra dimension of interference removal from target analytes can potentially simplify sample work-up. Table 1 summarises a reasonably comprehensive range of GCGC applications (excluding review papers and papers describing technology) reported over the last four years or so, a period of much activity in exploring the scope of GCGC. A selection of these chosen for further comment follows.

the relevance of GCGC technology in providing value-added data in such an industrial application. One can only ask why this has not been picked up much more widely in the petrochemical industry. The fact that the above analysis can be achieved using just a FID, rather than having to rely on GC-MS with the problems of divergent component-response factors, is another factor weighing heavily in favour of GCGC. Xu, et al. showed that new information can be derived for geochemical sample analysis, when they discovered a hitherto unknown compound in an organic extract of a Black Sea sediment sample [15]. Again, the possibilities that await exponents of GCGC are amply illustrated by this study. This discovery was not serendipitous, but merely the result of having a more powerful tool that can reveal such chemical nuances.

4.2. Essential oils

Like petrochemicals, essential oils offer similarly complex sample types, comprising a range of chemical-compound classes, so they provide an equally valuable study area for GCGC. Dimandja, et al. from the Centers for Disease Control and Prevention (CDCP, Atlanta, USA), amongst their other work, reported the study of comparison of various oilspeppermint and spearmintby GCGC [16]. They showed that equality of retention times in the 2D plane was sufcient for component identication. The present group reported the analysis of essential oils from a range of samples, with the highly productive vetiver oil rst shown to reveal its full compositional complexity [17]. Monoterpene and sesquiterpene hydrocarbons, and their oxygenated analogues, can be located readily in the 2D plot. Fig. 6 is a typical essential oil GCGC trace showing the above features. The role of GCGC-TOF-MS in identifying components of lavender was subsequently reported. The ability to resolve otherwise overlapping components was said to improve library matching. This will undoubtedly lead to more extensive studies as TOF-MS becomes more widely available for GCGC research in the

4.1. Petrochemical and geochemical analysis

This was the rst major study area of GCGC. The work of the groups at Shell Amsterdam (Blomberg, Beens, Schoenmakers) and the US Coast Guard Academy (Frysinger, Gaines) is noteworthy. The former group can be considered to have pioneered and strongly promoted this eld of GCGC, and quickly demonstrated the structural features that can be drawn out of a carefully tuned analysis. The pictorial interpretive possibilities that arise from structured 2D chromatograms were apparent in papers by both groups where groupings of different chemical classes are readily recognised. Fig. 5 shows clusters of alkanes, and mononaphthenes and di-naphthenes for a non-aromatic, middle-distillate sample. Similar studies enabled such facile determinations as total aromatic content of a sample based on the clustering of aromatics in the 2D plane [14]. The correlation of such data with chemical measures on similar samples using industry standard methods was very good, with acceptable precision, but GCGC was less technically demanding and faster. This is a clear demonstration of

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Table 1 References listed here represent the work reported in various publications. Grouped according to application, they serve to illustrate the variety of studies in which the GCGC technique has become a valuable analytical tool. In some cases, a reference may be listed more than once where its subject material is appropriate to more than one application Reference Organic chemicals/Petrochemicals [1] [20] [21] [8] [22] [14] [23] [24] [25] [26] [27] [28] [12] [29] [6] [30] [18] [31] [32] [15] Essential oils [16] [17] [33] [34] [19] Fatty acids [35] [36] Environmental/Pollution [37] [38] [39] [40] [41] [24] [42] [43] [44] [45] [46] [47] [48] Toxicology/Forensic [49] [50] [51] Year 1991 1993 1997 1998 1998 1999 1999 1999 1999 1999 2000 2000 2000 2000 2000 2000 2000 2001 2001 2001 2000 2000 2000 2001 2001 2001 2001 1996 1997 1997 1998 1998 1999 2000 2000 2001 2001 2001 2001 2001 1994 2000 2000 Sample First demonstration of GCGC; analysis of sample of coal liquids Kerosene Hydrocarbon process stream; light gas oil White gas Kerosene Determination of total aromatics in gasoline GCGC-MS of petroleum Oil-spill source identication Petroleum White gas Non-aromatic middle-distillate solvent Jet fuel Determination of oxygenates in gasoline Diesel Kerosene Numerous complex hydrocarbon mixtures Group-type identication of oil (GCGC-TOF-MS) Separation and identication of petroleum biomarkers Aromatic and naphthene content in naphtha Black Sea sediment; novel alkanone identied Comparison of peppermint oil and spearmint oils Vetiver oil Comparison of lavender oil and tea tree oil GCGC-TOF-MS, lavender oil Enantioselective analysis, tea tree oil Fatty acids in biological oil samples separation of FAME (fatty acid methyl ester) isomers Environmental toxicants Chlorinated biphenyls PCBs Trace-level oxygenates & aromatic compounds in water Environmental toxicants Oil-spill source identication VOCs in air VOCs in air Surface-water contaminants PCBs PCBs VOCs in air Faecal sterols in natural waters Pesticides extracted from human serum Environmental toxicants in serum and urine Technical & biota samples containing chlorinated biphenyls

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future. Cramers, et al. at Eindhoven University of Technology have presented various studies on GCGC-TOF-MS [18]. Successful enantioselective analysis using a chiral/achiral column combination was recently reported [19]. The use of a chiral rst column is counterintuitive for conventional MDGC, since the role of MDGC is to isolate the enantiomers for particular compounds, then transfer them to the subsequent column for enantiomeric ratio measurement. This can only be done with a chiral analytical (D2) column. In GCGC, a chiral rst column can be used. Since all sample is transferred from D1 to D2, there is mass conservation. The use of a polar second column is able to provide resolution of enantiomers from potentially-interfering components that may otherwise co-elute on the rst column (this is the most troublesome problem in single chiral column analysis).

4.3. Environmental analysis

The major benets of GCGC in environmental analysis are improvements in trace analysis by increasing sensitivity and the resolution of target compounds from co-extracted impurities and interferences. A further advantage, and one that is still to be fully investigated, is the opportunity for trace multi-residue analysis. The 2D space has capacity available for many thousands of individual components, and so its ability to locate many different volatile/semivolatile components of different chemical nature suggests that this study should be tried. Leading groups in GCGC in this area are: CDCP, USA; Free University of Amsterdam, The Netherlands; The Netherlands Institute for Fisheries Research, which has reported applications in the pesticide area and work on polychlorinated biphenyls (PCBs); and our own collaboration with the group at Umea University, Sweden. Thus organochlorine compound analysis has been a major focus, as well as polyaromatic hydrocarbons (PAHs) and various standards of environmental signicance. Fig. 7 demonstrates the opportunity that GCGC holds for advanced separation studies with such samples. The structure of the 2D separation for PAH and PCB samples is valuable, although it is not entirely clear what the structure-retention relationship is that leads to the observed separations in all cases. The most urgent question will be to validate the use of GCGC as an alternative tool to high-resolution MS, which is the only current method available for speciation of individual co-eluting congeners of different chlorine number for PCBs and dioxins.

5. Future perspectives
Fig. 5. Example of a non-aromatic, middle-distillate, petroleum sample analysis by GCGC. The column set was D1=10m, DB-1 phase, with D2=2.5 m, BPX50 phase, with oven temperature program of 2  C/min. from 30  C. Distinct bands of related compounds are readily delineated. (Reproduced with permission from [27]).

Where will GCGC be in 10 years time? What major developments might be introduced into GCGC methodologies and interpretations over this period? Trying to gaze into a crystal ball is fraught with danger. Either it is

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Fig. 6. Example of essential oil analysis using GCGC. Kanuka essential oil. The column set was D1=30 m, BPX5 phase, with D2=1.0 m, BP20 phase, with oven temperature program of 2  C/min. from 60  C.

Fig. 7. PCB sample (Clophen A50) analysed using (A) 1D-GC and (B) GCGC. The modulation period was 3 s with an oven temperature program of 60  C for 2 min., to 140  C at 20  C/min., then to 275  C at 10  C/min. (Reproduced with permission from [45]).

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completely off the mark, or the eld advances far beyond what one may imagine. First, there will be more sophisticated datahandling packages available. This must come eventually because of the widely expressed desire to have such data-processing facilities. Second, GCGC-TOF-MS will have been established as the most powerful separation/ identication tool in the hands of the volatile organic compound (VOC) analyst. Third, chemometrics will have been applied to a wide range of analytical studies for sample comparison, and for contrasting samples to identify major differences. This will lead to sophisticated ngerprinting of samples based on the 2Dseparation space. The concept of targeted analysis will be much more readily accepted, where selected components in the 2D space will be easily identied and compared from sample to sample, and will be chosen for their representative character for the whole sample; the analyst will be much more involved in the selection of target components that will simplify the analytical measurement of these components. Finally, the use of complex elution patterns will aid researchers to recognise much more easily the chemical construction of samples to discover new compounds, and to match patterns to assist in identication of components. Acknowledgements The authors wish to express their thanks to Peter Dawes, SGE International, for continuing support for our research work on GCGC. Agilent Technologies has supported facility development and is similarly acknowledged. Paul Morrison has been pivotal in managing and developing our research projects.

[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]

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