Multidimensional Chromatography

Figure 15.8 Multidimensional GC – MS separation of urinary acids after derivatization with methyl chloroformate: (a) pre-column chromatogram after splitless injection; (b) Main-col- umn selected ion monitoring chromatogram (mass 84) of pyroglutamic acid methyl ester. Adapted from Journal of Chromatography, B 714, M. Heil et al., ‘Enantioselective multidimensional gas chromatography – mass spectrometry in the analysis of urinary organic acids’, pp. 119 – 126, copyright 1998, with permission from Elsevier Science.

separation of unleaded gasoline from their system is shown in Figure 15.10. The chromatographic system consisted of a DB-1701 pre-column, followed by the parallel traps, and then a DB-5 analytical column, mounted inside an HP 5890 gas chromatograph. Customized valves were used to reduce extra-column band broadening that might be caused by the traps and tubing. This technique could be easily applied

to the fingerprinting of gasoline and other flammable materials. The first chromatogram (Figure 15.10(a)) shows the GC-infrared chromatogram from the first dimension. There are approximately 110 peaks, which are significantly overlapped. Four heart-cuts (A – C) were taken. On the second chromatogram (Figure 15.10(b)), the GC/MS analysis of heart-cut C is shown. Twelve overlapping peaks that were initially trapped were resolved into over 30 separated peaks. The combination of non-destructive IR detection following the first stage and MS detection following the second stage presents especially powerful third and fourth dimensions, thus allowing spectral confirmation of peak identities and identification of overlaps.

The fragrance compounds commonly used in cosmetics and household products are some of the most common causes of contact dermatitis. These products often contain complex matrix interferences such as emulsifiers, thickeners, stabilizers, pigments, antioxidants and others, thus making the analytes of interest difficult to analyze. Tomlinson and Wilkins have applied multidimensional GC, coupled to infrared and mass spectrometry, to the analysis of common irritants from these complex mixtures (24). These authors modified a commercially available GC – IR – MS system to accommodate the second analytical column. By using an intermediate polarity (Rtx-1701) initial column and a variety of second columns, they obtained separations of a wide variety of fragrances. In order to evaluate the re-injection efficiency of their system, they used a Grob test mixture (25). This was injected on to the intermediate polarity column, and then the entire chromatogram was re-focused into a trap and re-injected onto an Rtx-5 non-polar column. They estimated a reinjection efficiency of about 85% between the two columns. The Grob test mixture chromatograms are shown in Figure 15.11. The first chromatogram (Figure 15.11(a)) shows a Grob test mix separated on the first-dimension column. The entire chromatogram is cryogenically trapped and then re-injected onto the second column, to give the chromatogram shown in Figure 15.11(b). This provides an excellent means for assessing the efficiency of the interface between the two columns. Thorough reviews of multispectral detection methods, such as GC – IR – MS, have been provided by Ragunathan et al. (26) and Krock et al. (27).

Authenticity evaluation has recently received increased attention in a number of industries. The complex mixtures involved often require very high resolution analyses and, in the case of determining the authenticity of ‘natural’ products, very accurate determination of enantiomeric purity. Juchelka et al. have described a method for the authenticity determination of natural products which uses a combination of enantioselective multidimensional gas chromatography with isotope ratio mass spectrometry (28). In isotope ratio mass spectrometry, combustion analysis is combined with mass spectrometry, and the 13C/12C ratio of the analyte is measured versus a CO2 reference standard. A special interface, employing the necessary oxidation and reduction reaction chambers and a water separator, was used employed. For standards of 5-nonanone, menthol and (R)- -decalactone, they were able to determine the correct 12C/13C ratios, with relatively little sample preparation. The technical details of multidimensional GC – isotope ratio MS have been described fully by Nitz et al. (29). A MDGC – IRMS separation of a natural cis-3-hexen-1-ol fraction is

Figure 15.11 (a) Total ion chromatogram of a Grob test mixture obtained on an Rtx-1701 column, and (b) re-injection of the entire chromatogram on to an Rtx-5 column. Peak identification is as follows: a, 2,3-butanediol; b, decane; c, undecane; d, l-octanol; e, nonanal; f, 2,6- dimethylphenol; g, 2-ethylhexanoic acid; h, 2,6-dimethylaniline; i, decanoic acid; methyl ester; j, dicyclohexylamine; k, undecanoic acid, methyl ester; l, dodecanoic acid, methyl ester. Adapted from Journal of High Resolution Chromatography, 21, M. J. Tomlinson and C. L. Wilkins, ‘Evaluation of a semi-automated multidimensional gas chromatography–infrared– mass spectrometry system for irritant analysis’, pp. 347 – 354, 1998, with permission from Wiley-VCH.

shown in Figure 15.12. In the heart-cut(shown as an inset), taken from 25 – 30 min, several of the components are further resolved. In earlier studies, also related to authenticity determination, Mosandl et al. have described methods for the determination of several chiral natural product (30).

Figure 15.12 GC – GC chromatogram of a natural cis-3-hexen-1-ol fraction. Peak identification is as follows: 1, ethyl-2-methylbutyrate; 2, trans-2-hexenal; 3, 1-hexanol; 4, cis-3-hexen-1- ol; 5, trans-2-hexen-1-ol. Adapted from Journal of High Resolution Chromatography, 15, S. Nitz et al., Multidimensional gas chromatography – isotope ratio mass spectrometry, (MDGC – IRMS). Part A: system description and technical requirements’, pp. 387 – 391, 1992, with permission from Wiley-VCH.

Comprehensive two-dimensional gas chromatography, originally proposed by Schomburg (31) and developed by Phillips and co-workers (32–35), in which the effluent from a traditional analytical column is sampled into a short, narrow-bore, thin-film second column, also shows promise in the analysis of the complex mixtures commonly found in forensic analysis. This technique offers a very rapidly obtained second dimension and high peak capacity which is necessary for complex mixtures, with thousands of peaks being possible in a single chromatogram. The method has been applied mostly in the petroleum and the environmental industries. Several authors have recently reported the use of comprehensive two dimensional gas chromatography on petroleum related samples (36–38).

Although comprehensive two-dimensional gas chromatography has not been applied to any great extent in forensic analysis, the technique shows great promise when samples or sample matrices are complex. For example, when oil is spilled into waterways, assigning responsibility for the economic and environmental damage is often difficult. Gaines et al. employed comprehensive two-dimensional GC in the forensic analysis of samples collected at oil-spill sites and were able to obtain results which were comparable to those obtained by classical methods (39). This article also

17 of the components are the main features here. However, it was noted that one of the internal standards (heptadecanoic acid) co-eluted with major interferences, so even with two-dimensional separation, complete separation of the analytes of interest from a complex matrix is still difficult. The sample preparation used in this case was typical solid phase extraction. These authors also showed reproducibility and linear ranges that were easily competitive with those from traditional GC methods.

Solid phase micro-extraction (SPME) (41, 42) has also been employed by Gaines et al. (43), along with comprehensive two-dimensional GC in the analysis of trace components from aqueous samples. This combination fills the need for a rapid, high sensitivity and high resolution analysis of complex mixtures. These authors examined the analysis of oxygenated and aromatic compounds from water. While these are not strictly forensic analytes, they do provide effective models for other applications. As described above, Gaines and co-workers employed a two-column scheme, with the first analytical column being non-polar and essentially separating compounds by volatility, while the second (fast) column separated analytes of similar volatility by their polarity. In this way, they were able to demonstrate the low ppb analysis of various gasoline components spiked into water. The adaptation of this method to the analysis of volatile and semi-volatile components from water, fire debris, biological material and other forensic matrices would seem to be reasonably straightforward.

15.5 ON-LINE SAMPLE PREPARATION

Although on-line sample preparation cannot be regarded as being traditional multidimensional chromatography, the principles of the latter have been employed in the development of many on-line sample preparation techniques, including supercritical fluid extraction (SFE) – GC, SPME, thermal desorption and other on-line extraction methods. As with multidimensional chromatography, the principle is to obtain a portion of the required selectivity by using an additional separation device prior to the main analytical column.

The coupling of supercritical fluid extraction (SFE) with gas chromatography (SFE – GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SFE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SFE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45).

change is seen for the later-eluting components. As automated systems become more common, on-line sample preparation systems of this type will see tremendous growth as a forensic and toxicological analysis technique in the near future.

15.6 CONCLUSIONS

The applications of multidimensional chromatography presented here show that such techniques provide great promise in the solution of the complex problems involved in forensic and toxicological analysis. These include complex matrices such as urine, blood and natural products, and difficult analytes such as drugs, aromas and natural products. When compared to environmental analysis, forensic and toxicological analysis using multidimensional chromatography has received relatively little attention, although the possibilities are many and the potential is bright. The environmental and pharmaceutical methods described in this present chapter could be readily adapted to forensic problems. In particular, multidimensional chromatography offers the forensic scientist high resolution, high sensitivity and short analysis times.

ACKNOWLEDGEMENT

The author gratefully acknowledges the influential work of Professor John Phillips, which has touched nearly all areas of multidimensional gas chromatography.

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