Comprehensive two-dimensional gas chromatography in metabolomics

One of the major objectives in metabolomics is the identification of subtle changes in metabolite profiles as affected by genetic or environmental factors. Comprehensive two-dimensional gas chromatography (GC × GC) hyphenated to a fast-acquisition mass spectrometer is a well-established analytical technique to study the composition of complex samples due to its enhanced separation capacity, sensitivity, peak resolution, and reproducibility. This review reports applications of GC × GC to metabolomics studies of sample of different types (biofluid, cells, tissue, bacteria, yeast, plants), and discusses its advantages and limitations.     

Comprehensive two-dimensional gas chromatography analysis of high-ethanol containing motor fuels

A comprehensive 2-D GC (GC x GC) instrument equipped with a flow-switching modulator was used to determine the concentration of ethanol and eight other alcohols in a retail pump sample of E85 fuel. E85 is a mixture of ethanol and gasoline where the ethanol concentration can range from 70 to 85 vol%. The increased peak capacity and selectivity generated by GC x GC analysis allowed the alcohols to be fully resolved from the gasoline hydrocarbons. GC x GC analysis was compared to the performance obtained with the standard analytical method for determining ethanol in fuel ethanol (ASTM D5501) and the standard method for determining oxygenate concentrations in gasoline (ASTM D4815). The GC x GC analysis required 14 min while the combined ASTM D5501 and ASTM D4815 analyses required more than 60 min. The ethanol concentration obtained by GC x GC was in excellent agreement with the value obtained by the D5501 method. Poorer agreement was observed between the GC x GC and D4815 concentrations for the other alcohols present in E85. In all cases, the differences could be attributed to deficiencies in the D4815 method that led to coelutions between the alcohols and gasoline hydrocarbons.     

Comprehensive two-dimensional gas chromatography in stop-flow mode

The theory and proof of concept of a new mode of operation for comprehensive two-dimensional gas chromatography (GC x GC) is presented. In current GC x GC interface designs, the modulation period defines the separation time allowed in the second dimension. In the stop-flow GC x GC mode, flow in the primary column is periodically stopped for brief periods of time. Consequently, the modulation period for the primary column and the amount of time available for second dimension separation become independent variables. This allows the separations in both the primary and secondary dimensions of the GC x GC system to be carried out under more optimised conditions, allowing extended periods of time for second dimension separations without sacrificing the separation in the primary dimension. This new technique has the potential to offer increased separation power and overall resolution as it is further developed.     

Enhanced resolution comprehensive two-dimensional gas chromatography applied to the analysis of roasted coffee volatiles

The present research is based on the full exploitation of the separation power of a 0.05 mm internal diameter (ID) capillary, as a comprehensive two-dimensional (2D) GC (GC × GC) secondary column, with the objective of attaining very high-resolution second dimension separations. The aim was achieved by using a split-flow system developed in previous research [P.Q. Tranchida, A. Casilli, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 79 (2007) 2266], and a dual-oven GC × GC instrument. The column combination employed consisted of a polar 30 m × 0.25 mm ID column connected, by means of a T union, to a detector-linked high-resolution 1.1 m × 0.05 mm ID apolar analytical column and to a 0.33 m × 0.05 mm ID retention gap; the latter was connected to a manually operated split valve. As previously demonstrated, the use of a split valve enables the regulation of gas flows through both analytical columns, generating the most appropriate gas linear velocities. Comprehensive 2D GC experiments were carried out on Arabica roasted coffee volatiles (previously extracted by means of solid-phase microextraction) with the split-valve closed (equal to what can be defined as conventional GC × GC) and with the split-valve opened at various degrees. The reasons why it is absolutely not effective to use a 0.05 mm ID column as second dimension in a conventional GC × GC instrument will be discussed and demonstrated. On the contrary, the use of a 0.05 mm ID column as second dimension, under ideal conditions in a split-flow, twin-oven system, will also be illustrated and discussed.     

Comprehensive two-dimensional gas chromatography for the analysis of Fischer-Tropsch oil products

The Fischer-Tropsch (FT) process involves a series of catalysed reactions of carbon monoxide and hydrogen, originating from coal, natural gas or biomass, leading to a variety of synthetic chemicals and fuels. The benefits of comprehensive two-dimensional gas chromatography (GC×GC) compared to one-dimensional GC (1D-GC) for the detailed investigation of the oil products of low and high temperature FT processes are presented. GC×GC provides more accurate quantitative data to construct Anderson-Schultz-Flory (ASF) selectivity models that correlate the FT product distribution with reaction variables. On the other hand, the high peak capacity and sensitivity of GC×GC allow the detailed study of components present at trace level. Analyses of the aromatic and oxygenated fractions of a high temperature FT (HT-FT) process are presented. GC×GC data have been used to optimise or tune the HT-FT process by using a lab-scale micro-FT-reactor.     

Comprehensive two-dimensional gas chromatography in the analysis of dietary fatty acids

Comprehensive two-dimensional gas chromatography utilising a semi-rotating cryogenic modulator was applied to the analysis of dietary milk derived fatty acids. Four column combinations were tested: two polar-nonpolar and two different nonpolarpolar column combinations. Best results were obtained with a nonpolar-polar column set, with narrow-bore (50 microm ID) Carbowax column as the second column. The GC x GC method clearly improved the separation efficiency of the fatty acids in comparison with one-dimensional GC analysis and proved to be suitable for the quantification of the analytes. The relative standard deviations (RSD) of the retention times in the first and second dimensions were on average 0.06% and 4.9%, respectively. The relative RSD for peak volumes was on average 2.9%.     

Comprehensive two-dimensional gas chromatography/mass spectrometric analysis of pepper volatiles

The headspace compositions of 13 pepper and peppercorn samples of different species, colloquially also referred to as pepper, were analyzed, and more than 300 compounds were tentatively characterized by means of comprehensive two-dimensional gas chromatography in tandem with flame ionization detection, quadrupole mass spectrometric detection and time-of-flight mass spectrometric detection (GC x GC-FID, GC x GC/qMS and GC x GC/TOFMS, respectively). The analysis of volatile organic compounds (VOCs) was performed after solid-phase microextraction (SPME) using a 75-microm PDMS/DVB fibre. Fingerprint comparison between the three techniques permitted peaks to be assigned in the GC x GC-FID experiment based on the analogous MS analysis, taking into account retention shifts arising from method variations. When using GC x GC/TOFMS, about five times more peaks were identified than in GC x GC/qMS. Retention indices for all peaks were calculated in the bi-dimensional column set comprising of a 5% phenyl polysilphenylene-siloxane primary column and a polyethylene glycol second column. The spectra obtained by both mass detection techniques (qMS and TOFMS) give very similar results when spectral library searching was performed. The majority of the identified compounds eluted as pure components as a result of high-resolution GC x GC separations, which significantly reduces co-elution, and therefore increases the likelihood that pure spectra can be obtained. The differences between TOFMS and qMS (in fast scanning mode) spectra were generally small. Whilst spectral quality and relative ion ratios across a narrow peak (e.g. w(b) approximately 100-150 ms) do vary more for the fast peaks obtained in GC x GC/qMS operation, than with TOFMS, in general adequate spectral matching with the library can be achieved.     

Comprehensive two-dimensional chromatography in food analysis

Comprehensive two-dimensional (2D) chromatographic techniques can be considered innovative methods, only quite recently developed. Since their introduction to the chromatographic community, these techniques have been used in several fields and have gained an excellent reputation as valuable and powerful analytical tools. The revolutionary aspect of comprehensive multidimensional (MD) techniques, in respect to classical MD chromatography, is that the entire sample is subjected to the 2D advantage. The resulting unprecedented separating capacity makes these approaches prime choices when analysts are challenged with highly complex mixtures. Furthermore, in the case of automated systems, instrumental analysis times are roughly the same as in monodimensional applications. The present review reports various comprehensive chromatographic applications on different food matrices. The GC x GC section highlights two fundamental aspects for component separation/identification: the exceptional peak capacity and the formation of group types on the 2D space plane. The LC x LC section reports the employment in food analysis of a recently developed multidimensional normal-phase (NP)-reversed-phase (RP) high performance liquid chromatography (HPLC) system. Also reported are comprehensive LC x GC and packed column supercritical fluid chromatography (pSFC x pSFC) applications in this field.     

Comprehensive two-dimensional gas chromatography of polybrominated diphenyl ethers

Comprehensive two-dimensional gas chromatography with micro electron-capture detection (GCxGC-muECD) was evaluated for the separation of 125 polybrominated diphenyl ethers (PBDEs). From among the six column combinations that were evaluated, DB-1x007-65HT was found to be the most suitable because of: (i) the highest number of BDE congeners separated; (ii) the least decomposition of higher brominated congeners; and (iii) the most suitable maximum operating temperature. The separation of the 125 BDE congeners from five hydroxy- and two methoxy-BDEs and nine other brominated flame retardants (polybrominated biphenyls, tetrabromobisphenol-A, methyl-tetrabromobisphenol-A and hexabromocyclododecane) was also studied. Fluorinated BDEs were found to be valuable internal standards for the determination of BDEs because of their very similar physico-chemical properties and excellent separation from the parent BDEs, mainly in the second dimension. GCxGC-time-of-flight MS and GCxGC-muECD were shown to be useful tools to identify decomposition products of nona- and deca-substituted BDEs, which are formed during the GC run. Three nona-BDEs were shown to be the major decomposition products of BDE 209.     

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection applied to the determination of pesticides in food extracts

The separation provided by conventional gas chromatography (1D-GC) can be significantly enhanced by using comprehensive two-dimensional GC (GC X GC) instead. Combination with mass spectrometric detection is desirable for unambiguous confirmation of target compounds and the provisional identification of unknowns. A GC X GC system using a cryogenic modulator was coupled to a time-of-flight mass spectrometric (TOF MS) detector. With the determination of pesticides in vegetable extracts as an example, it was demonstrated that GC X GC improves the separation dramatically. All 58 pesticides of interest could be identified using their full-scan mass spectra, which was not possible when using ID-GC-TOF MS. In addition, the high scan speed of the TOF MS allowed the deconvolution of compounds partly co-eluting in GC X GC.     

Application of two-dimensional gas chromatography combined with pixel-based chemometric processing for the chemical profiling of illicit drug samples

The chemical profiling of illicit drugs is a complex process. The results are affected by many different factors such as sample size, the sample processing conditions, the used analytical technique as well as the statistics that are applied. Within this proof-of-concept study, which was done in cooperation with the German Federal Criminal Police Office (Bundeskriminalamt, BKA), the adaptability of comprehensive two-dimensional (2D) gas chromatography (GCxGC) combined with a pixel-based chemometric data processing method is demonstrated. Samples of heroin and cannabis are extracted and analyzed with GCxGC-TOF-MS (time-of-flight mass spectrometry) and GCxGC-FID (flame ionization detection). The obtained second-order data are then used to identify possible marker compounds for the discrimination of the samples according to their chemical profile. The pixel-based chemometric process includes preprocessing steps (background correction, alignment of chromatograms and normalization) followed by an adaptation of hierarchical clustering to identify chemically similar samples, and finally a subsequent calculation of Fisher criterion based on the found clustering in order to identify promising marker compounds. The results of the pixel-based data analysis are compared to a limited peak-based study for cannabis and to a well-established standard method for the chemical profiling of heroin.     

Comprehensive two-dimensional gas chromatography to characterize hydrocarbon mixtures in lithic materials

The analysis of organic biomarkers in chert samples offers key information about the environmental conditions in which these samples were formed, and this information can be used to track the lithic materials of many archaeological artifacts. Since the content of the organic fraction is very low and the complexity of the organic extracts is quite high, we have optimized the GC×GC separation of these mixtures. Making use of mixture of C(16)H(34)-C(44)H(90)n-alkane standards, a central composite design was carried out taking into account the carrier flow in the first and second columns, the modulation period and the discharge time. Regarding the measured responses, though the initially considered one was the peak volume, we have also evaluated the effects on the number of modulated peaks per analyte, the symmetry of the modulated peaks and the number of detected compounds. The final optimum conditions were defined as follows: a hydrogen flow of 1.2 mL/min in the first column and 18 mL/min in the second one, a modulation period of 1.4 s and a discharge time of 0.1 s and under these conditions all the response variables showed optimum values. Based on this optimized method several chert samples obtained from different stratigraphic levels in an ancient quarry were studied and we were able to distinguish them on the basis of the different constituents of organic biomarkers, such as mono-methylated alkanes, cyclic n-alkanes, branched alkanes, steranes and hopanes.     

Application of comprehensive two-dimensional gas chromatography to sterols analysis

The applicability of comprehensive two-dimensional gas chromatography (GCxGC) for sterol analysis was investigated by separation and identification of endogenous sterols in standards, and spiked in human urine. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of sterols was compared on two complementary column sets. Whilst the BPX5/BPX50 column set offers better overall separation, BPX50/BPX5 provides better peak shape and sensitivity. Comparison of the identification power of GCxGC-TOFMS against both the NIST05 MS library and a laboratory created (in-house) TOFMS library was carried out on a free sterols extract of urine, derivatised and spiked at the World Anti-Doping Agency (WADA) limit of 2 ng mL(-1). The average match quality for 19 analysed sterols on the BPX50/BPX5 column set was 950/1000 when searched against the in-house library; only four were identified against the NIST05 library, at a match threshold of 800. The match quality of GCxGC-TOFMS spectra was superior to that for analysis using 1D GC-TOFMS for sterols spiked in urine at 10 ng mL(-1). An r(2)>0.997 was obtained for the concentration range between 0.25 ng mL(-1) and 10 ng mL(-1) for three selected sterols. The lowest limit of detection (LOD) was obtained for estrone (0.1 ng mL(-1)) and the highest LOD was for 5alpha-androstan-3alpha,11beta-diol-17-one, epitestosterone and cholesteryl butyrate (1 ng mL(-1)), using a match threshold of at least 800 and signal-to-noise ratio of at least 10. TOFMS coupled to GCxGC enabled satisfactory identification of sterols in urine at their LOD. A minimum acceptable match (MAM) criterion for urinary sterols using 2D retention times and TOF mass spectra is introduced. This study shows that GCxGC-TOFMS yields high specificity for steroids derived from urine, with detection limits appropriate for use in doping control.     

Comprehensive two-dimensional gas chromatography of the 209 polychlorinated biphenyls

Comprehensive two-dimensional gas chromatography (GC x GC) of the 209 polychlorinated biphenyls (CBs) was carried out using a longitudinally modulated cryogenic system (LMCS) and liquid carbon dioxide as cryogen. The effluent from a non-polar column was modulated and further separated on either a polar or a shape-selective second-dimension column. Five GC x GC column combinations were evaluated, with DB-XLB as the first column in each case. DB-XLB separates more congeners than any other GC column currently available. When combined with a biscyanopropyl siloxane (SP-2340 or BPX70) or smectic liquid crystal (LC-50) second-dimension column in a GC x GC system many additional CBs can be separated. In total, 176 and 181 of the 209 congeners were separated (Rs = 0.5) on the column combinations DB-XLB/SP-2340 and DB-XLB/LC-50, respectively. Of the 136 CBs present in any Aroclor mixture at concentrations greater than 0.05% (w/w), 126 were resolved using either of these two column combinations. The seven frequently measured CBs 28, 52, 101, 118, 138, 153, 180, and the WHO-PCBs 77, 81, 105, 114, 118, 123, 126, 156. 157, 167, 169 and 189 were all separated from Aroclor CBs on the DB-XLB/LC-50 column set, whereas CBs 118 and 131 coeluted on the DB-XLB/SP-2340 column set. In addition, three technical CB formulations (Aroclors 1232, 1248 and 1260) and a seal blubber sample (Halichoerus grypus) from the Baltic Sea were analyzed. Similar peak patterns were found for Aroclor 1260 and the CBs in the seal blubber, facilitating use of this technical CB formulation to identify the CBs in the blubber by GC x GC. Individual CBs in environmental samples, such as seal blubber, may be identified semi-automatically by matching the samples GC x GC profiles to a template generated using a standard containing all 209 CBs. Using such a template, 64 CBs were identified in the grey seal blubber sample.     

Comprehensive two-dimensional gas chromatography for the analysis of synthetic and crude-derived jet fuels

Fully synthetic jet fuel (FSJF) produced via Fischer-Tropsch (FT) technology was recently approved by the international aviation fuel authorities. To receive approval, comparison of FSJF and crude-derived fuel and blends on their qualitative and quantitative hydrocarbon composition was of utmost importance. This was performed by comprehensive two-dimensional gas chromatography (GC×GC) in the reversed phase mode. The hydrocarbon composition of synthetic and crude-derived jet fuels is very similar and all compounds detected in the synthetic product are also present in crude-derived fuels. Quantitatively, the synthetic fuel consists of a higher degree of aliphatic branching with less than half the aromatic content of the crude-derived fuel. GC×GC analyses also indicated the presence of trace levels of hetero-atomic impurities in the crude-derived product that were absent in the synthetic product. While clay-treatment removed some of the impurities and improved the fuel stability, the crude-derived product still contained traces of cyclic and aromatic S-containing compounds afterwards. Lower level of aromatics and the absence of sulphur are some of the factors that contribute to the better fuel stability and environmental properties of the synthetic fuel. GC×GC was further applied for the analysis of products during Jet Fuel Thermal Oxidation Testing (JFTOT), which measures deposit formation of a fuel under simulated engine conditions. JFTOT showed the synthetic fuel to be much more stable than the crude-derived fuel.     

Comprehensive two-dimensional gas chromatography with capillary flow modulation to separate FAME isomers

A method to separate FAME and the linoleic and linolenic acids isomers by GCxGC using an apparatus equipped with a capillary flow technology (CFT) based modulator and a FID detector has been developed. Four different column combinations (one conventional and three inverted phase sets) were used in these experiments. The conventional set first involved a DB5-MS non-polar column followed by a highly polar HP-INNOWax column in the second dimension. The inverted phase set comprised of a highly polar BPX-70 column in the first dimension and a non-polar ZB5-MS column for the second dimension. Furthermore, the influence of the length of the second dimension column on FAME isomer separation was studied in the inverted phase sets, along with other parameters like the modulation time and column flow. The best results in terms of the time required for the analysis and number of FAME identified with the inverted set were achieved with the shorter second dimension column. After supercritical fluid extraction, the method was applied to identify FAMEs in broccoli leaves from three different cultivars (Naxos, Nubia and Viola).     

Portable capillary liquid chromatography for pharmaceutical and illicit drug analysis

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high‐pressure syringe pumps capable of delivering capillary‐scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub‐2 μm particles are housed in cartridges that can be inserted into the system and easily connected through high‐pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over‐the‐counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug‐related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.     

Application of micellar electrokinetic capillary chromatography to the analysis of illicit drug seizures

The application of micellar electrokinetic capillary chromatography (MECC) to the analysis of illicit drug seizures is presented. Areas investigated include general screening and qualitative and, in some instances, quantitative analysis of various drugs, including heroin, opium, cocaine, amphetamines, LSD and anabolic steroids. Due to its high efficiency, high selectivity and general applicability, MECC is well suited for forensic drug analyses.