Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates

This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GC × GC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC × GC columns sets (twin-GC × GC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GC × GC analysis of both saturated and unsaturated fractions. The benefits of SFC–twin-GC × GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC × GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GC × GC run, and for the first time, an extended PIONA analysis of diesel samples is presented.     

Amino acid analysis by using comprehensive two-dimensional gas chromatography

The separation characteristics of alkylchloroformate-derivatised amino acids (AAs) by using comprehensive two-dimensional gas chromatography (GC×GC) is reported. The use of a low-polarity/polar column set did not provide as good a separation performance as that achieved with a polar/non-polar column set, where the latter appeared to provide less correlation over the separation space. The degree of component correlation in each column set was estimated by using the correlation coefficient (r²; for ¹t_R and ²t_R data) with the low-polarity/polar and polar/low-polarity sets returning correlation coefficients of 0.86, and 0.00 respectively, under the respective conditions employed for the experiments. The 1.5-m non-polar ²D column (0.1-mm ID; 0.1-m film thickness) gave peak halfwidths of the order of 50–80 ms. Linearity of detection was good, over a three order of magnitude concentration range, with typical lower detection limit of ca. 0.01 mg L^–1, compared with 0.5 mg L^–1 for normal GC operation with splitless injection. The method was demonstrated for analysis of AAs in a range of food and beverage products, including wine, beer and honey. The major AA in these samples was proline. The Heineken beer sample had a relatively more complex and more abundant AA content compared with the other beer sample. The wine and honey samples also gave a range of AA compounds. Repetition of the sample preparation/analysis procedure for the honey sample gave acceptable reproducibility for individual AAs.     

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC × GC/TOFMS), respectively. In the GC/MS analysis, serially coupled columns were used. By comparing the GC/MS results with GC × GC/TOFMS results, many more components in the essential oil could be found within the two-dimensional separation space of GC × GC. The quantitative determination of components in the essential oil was performed by GC × GC with flame ionization detection (FID), using a method of multiple internal standards calibration.     

A flexible loop-type flow modulator for comprehensive two-dimensional gas chromatography

The present investigation is focused on a simple flow modulator (FM), for comprehensive two-dimensional gas chromatography (GC×GC). The interface is stable at high temperatures, and consists of a metallic disc (located inside the GC oven) with seven ports, which are connected to an auxiliary pressure source via two branches, to the first and second dimension, to a waste branch (linked to a needle valve) and to an exchangeable modulation loop (2 ports). The ports are connected via micro-channels, etched on one of the inner surfaces of the disc. Modulation is achieved using a two-way electrovalve, connected on one side to the additional pressure source, and to the two metal branches, on the other. An FM enantio-GC×polar-GC method (using a flame ionization detector) was optimized (a 40-μL loop was employed), for the analysis of essential oils. As an example, an application on spearmint oil is shown; the method herein proposed was subjected to validation. Finally, an FM GC×GC diesel experiment was carried out, using an apolar-polar column combination, to demonstrate the effectiveness of the modulator in the analysis of a totally different sample-type.     

Convex hull: A new method to determine the separation space used and to optimize operating conditions for comprehensive two-dimensional gas chromatography

It is important to develop methods of optimizing the selection of column sets and operating conditions for comprehensive two-dimensional gas chromatography. A new method for the calculation of the percentage of separation space used was developed using Delaunay's triangulation algorithms (convex hull). This approach was compared with an existing method and showed better precision and accuracy. It was successfully applied to the selection of the most convenient column set and the geometrical parameters of second column for the analysis of 49 target compounds in wastewater.     

Development of a switchable multidimensional/comprehensive two-dimensional gas chromatographic analytical system

In this study, a new system for analysis using a dual comprehensive two-dimensional gas chromatography/targeted multidimensional gas chromatography (switchable GC × GC/targeted MDGC) analysis was developed. The configuration of this system not only permits the independent operation of GC, GC × GC and targeted MDGC analyses in separate analyses, but also allows the mode to be switched from GC × GC to targeted MDGC any number of times through a single analysis. By incorporating a Deans switch microfluidics transfer module prior to a cryotrapping device, the flow stream from the first dimension column can be directed to either one of two second dimension columns in a classical heart-cutting operation. Both second columns pass through the cryotrap to allow solute bands to be focused and then rapidly remobilized to the respective second columns. A short second column enables GC × GC operation, whilst a longer column is used for targeted MDGC. Validation of the system was performed using a standard mixture of compounds relevant to essential oil analysis, and then using compounds present at different abundances in lavender essential oil. Reproducibility of retention times and peak area responses demonstrated that there was negligible variation in the system over the course of multiple heart-cuts, and proved the reliable operation of the system. An application of the system to lavender oil, as a more complex sample, was carried out to affirm system feasibility, and demonstrate the ability of the system to target multiple components in the oil. The system was proposed to be useful for study of aroma-impact compounds where GC × GC can be incorporated with MDGC to permit precise identification of aroma-active compounds, where heart-cut multidimensional GC-olfactometry detection (MDGC-O) is a more appropriate technology for odour assessment.     

Headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry for the determination of volatile compounds from marine salt

In this work, a methodology to characterise the volatile and semi-volatile compounds from marine salt by headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC/TOFMS) was developed. Samples from two saltpans of Aveiro, in Portugal, with diverse locations, obtained over three years (2004, 2005, and 2007) were analysed. A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane SPME fibre was used. The volatiles present in the headspace of the solid salt samples (crystals) were equilibrated overnight at 60 °C and extracted for 60 min prior to injection in the GC × GC/TOFMS. 157 compounds, distributed over the chemical groups of hydrocarbons, aldehydes, esters, furans, haloalkanes, ketones, ethers, alcohols, terpenoids, C₁₃ norisoprenoids, and lactones were detected across the samples. Furans, haloalkanes and ethers were identified for the first time in marine salt. The large number of co-elutions on the first column that were resolved by the GC × GC system revealed the complexity of marine salt volatile composition. The existence of a structured 2D chromatographic behaviour according to volatility, in the first dimension (¹D), and primarily polarity, in the second dimension (²D), was demonstrated, allowing more reliable identifications. The resolution and sensitivity of GC × GC/TOFMS enabled the separation and identification of a higher number of volatile compounds compared to GC–qMS, allowing a deeper characterisation of this natural product.     

Metabolic profiling of infant urine using comprehensive two-dimensional gas chromatography: Application to the diagnosis of organic acidurias and biomarker discovery

Comprehensive two-dimensional gas chromatography (GC × GC) time-of-flight mass spectrometry (ToFMS) was applied to the analysis of urinary organic acids from patients with inborn errors of metabolism. Abnormal profiles were obtained from all five patients studied. Methylmalonic academia and deficiencies of 3-methylcrotonyl-CoA carboxylase and medium chain acyl-CoA dehydrogenase gave diagnostic profiles while deficiencies of very long chain acyl-CoA dehydrogenase and mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gave profiles with significant increases in dicarboxylic acids suggestive of these disorders. The superior resolving power of GC × GC with ToFMS detection was useful in separating isomeric organic acids that were not resolved using one-dimensional GC. A novel urinary metabolite, crotonyl glycine, was also discovered in the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase sample which may be a useful specific diagnostic marker for this disorder. The quantitative aspects of GC × GC were investigated using stable isotope dilution analyses of glutaric, glyceric, orotic, 4-hydroxybutyric acids and 3-methylcrotonylglycine. Correlation coefficients for linear calibrations of the analytes ranged from 0.9805 to 0.9993 (R²) and analytical recoveries from 77% to 99%. This study illustrates the potential of GC × GC–ToFMS for the diagnosis of organic acidurias and detailed analysis of the complex profiles that are often associated with these disorders.     

Measurement of retention in comprehensive two-dimensional gas chromatography using flow modulation with methane dopant

Flow modulation of methane-doped carrier gas is used to visualize the second dimension hold-up time in GC × GC continuously throughout the run. This provides an internal reference of hold-up time and presents a straightforward means of examining retention in each dimension of GC × GC. Retention factors on similar and dissimilar column pairs are examined. Stationary phase bleed is shown to be retained by the second dimension column.     

High-temperature two-dimensional gas chromatography of hydrocarbons up to nC60 for analysis of vacuum gas oils

In a tense energetic context, the characterization of heavy petroleum fractions becomes essential. Conventional comprehensive two-dimensional gas chromatography (2D-GC or GC × GC) is widely used for middle distillates analysis, but only a few applications are devoted to these heavier fractions. In this paper, it is shown how the optimization of GC × GC separation allowed the determination of suitable high-temperature (HT) conditions, adjusting column properties and operating conditions. 2D separations were evaluated using 2D separation criteria and a new concept of 2D asymmetry (As_2D). New HT conditions allowed the extension of GC × GC range of applications to heavier hydrocarbons, up to nC₆₀. A first application of high-temperature two-dimensional gas chromatography (HT-2D-GC) to a full vacuum gas oil (VGO) feed stock is described. Comparisons with other standardized methods illustrate the high potential of HT-2D-GC for heavy fractions analysis.     

Identification of potent odourants in wine and brewed coffee using gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography

Volatile constituents in wine and brewed coffee were analyzed using a combined system incorporating both GC-olfactometry (GC-O) and comprehensive two-dimensional GC-flame ionization detection (GC×GC-FID). A column set consisting of a 15m first dimension ((1)D; DB-FFAP (free fatty acid phase)), and a 1.0m (2)D column (DB-5 phase) was applied to achieve the GC×GC separation of the volatile extracts isolated by using solid phase extraction (SPE). While 1D GC resulted in many overlapping peaks, GC×GC allowed resolution of co-eluting compounds which coincided with the odour region located using GC-O. Character-impact odourants were tentatively identified through data correlation of GC×GC contour plots across results obtained using either time-of-flight mass spectrometry (TOFMS), or with flame photometric detection (FPD) for sulfur speciation. The odourants 2-methyl-2-butenal, 2-(methoxymethyl)-furan, dimethyl trisulfide, 2-ethyl-5-methyl-pyrazine, 2-octenal, 2-furancarboxaldehyde, 3-mercapto-3-methyl-1-butanol, 2-methoxy-3-(2-methylpropyl)-pyrazine, 2-furanmethanol and isovaleric acid were suspected to be particularly responsible for coffee aroma using this approach. The presented methodology was applied to identify the potent odourants in two different Australian wine varietals. 1-Octen-3-ol, butanoic acid and 2-methylbutanoic acid were detected in both Merlot and a Sauvignon Blanc+Semillon (SV) blend with high aroma potency. Several co-eluting peaks of ethyl 4-oxo-pentanoate, 3,7-dimethyl-1,5,7-octatrien-3-ol, (Z)-2-octen-1-ol, 5-hydroxy-2-methyl-1,3-dioxane were likely contributors to the Merlot wine aroma; while (Z)-3-hexen-1-ol, β-phenylethyl acetate, hexanoic acid and co-eluting peaks of 3-ethoxy-1-propanol and hexyl formate may contribute to SV wine aroma character. The volatile sulfur compound 2-mercapto-ethyl acetate was believed to contribute a fruity, brothy, meaty, sulfur odour to Australian Merlot and SV wines.     

Profiling analysis of volatile compounds from fruits using comprehensive two-dimensional gas chromatography and image processing techniques

An image processing approach originating from the proteomics field has been transferred successfully to the processing of data obtained with comprehensive two-dimensional gas chromatographic separations data. The approach described here has proven to be a useful analytical tool for unbiased pattern comparison or profiling analyses, as demonstrated with the differentiation of volatile patterns (“aroma”) from fruits such as apples, pears, and quince fruit. These volatile patterns were generated by headspace solid phase microextraction coupled to comprehensive two-dimensional gas chromatography (HS-SPME-GC × GC). The data obtained from GC × GC chromatograms were used as contour plots which were then converted to gray-scale images and analyzed utilizing a workflow derived from 2D gel-based proteomics. Run-to-run variations between GC × GC chromatograms, respectively their contour plots, have been compensated by image warping. The GC × GC images were then merged into a fusion image yielding a defined and project-wide spot (peak) consensus pattern. Within detected spot boundaries of this consensus pattern, relative quantities of the volatiles from each GC × GC image have been calculated, resulting in more than 700 gap free volatile profiles over all samples. These profiles have been used for multivariate statistical analysis and allowed clustering of comparable sample origins and prediction of unknown samples. At present state of development, the advantage of using mass spectrometric detection can only be realized by data processing off-line from the identified software packages. However, such information provides a substantial basis for identification of statistically relevant compounds or for a targeted analysis.     

Comprehensive two-dimensional gas chromatography improves separation and identification of anabolic agents in doping control

The application of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS) for the analysis of six anabolic agents (AAs) in doping control is investigated in this work. A non-polar–polar column configuration with 0.2 μm film thickness (d_f) second dimension (²D) column was employed, offering much better spread of the components on ²D when compared to the alternative 0.1 μm d_f²D column. The proposed method was tested on the “key” AA that the World Anti-Doping Agency (WADA) had listed at the low ng mL⁻¹ levels (clenbuterol, 19-norandrosterone, epimethendiol, 17α-methyl-5α-androstane-3α,17β-diol, 17α-methyl-5β-androstane-3α,17β-diol and 3′-OH-stanozolol). The compounds were spiked in a blank urine extract obtained by solid-phase extraction, hydrolysis and liquid–liquid extraction; prior to analysis they were converted to the corresponding trimethylsilyl (TMS) derivatives. The limit of detection (LOD) was below or equal to the minimum required performance limit (MRPL) of 2 ng mL⁻¹ defined by WADA, and the correlation coefficient was in the range from 0.995 to 0.999. The method allows choosing an ion from the full mass spectra which shows the least interference from the matrix and/or the best sensitivity; this can only be done if full scan mass spectral data are available. The advantage of GC × GC over classical one-dimensional GC (1D GC), in terms of separation efficiency and sensitivity, is demonstrated on a positive urine control sample at a concentration of 5 ng mL⁻¹. The obtained similarity to the in-house created TOFMS spectra library at this level of concentration was in the range from 822 to 932 (on the scale from 0 to 999). Since full mass spectral information are recorded, the method allows the retro-search of non-target compounds or new “designer steroids”, which cannot be detected with established GC–MS methods that use selected ion monitoring (SIM) mode.     

Data analysis programs for comprehensive two-dimensional chromatography

User-friendly and easy-to-use laboratory-written programs for visualisation and interpretation of comprehensive two-dimensional chromatographic data were developed. The programs that are not tied to any particular commercial instrument, and data obtained either by comprehensive two-dimensional liquid (LC × LC) or gas (GC × GC) chromatography can be analysed. Operations of the programs allow visualisation of 2D and 3D plots, comparison of two 2D plots at a time, as well as determination of retention times and peak heights and volumes.     

Factors affecting peak shape in comprehensive two-dimensional gas chromatography with non-focusing modulation

In the case of a non-focusing modulator for comprehensive two-dimensional gas chromatography (GC × GC), the systematic distortions introduced when the modulator loads the second-dimension column give rise to a characteristic peak shape. Depending on the operating conditions this systematic distortion can be the dominant component of the second-dimension elution profiles in the GC × GC peak. The present investigation involved a systematic investigation of peak shape in pulsed-flow modulation (PFM)–GC × GC. It is shown that low flow ratio can lead to significant peak skewing and increasing the flow ratio reduces the magnitude of peak skewing. Validation of the peak shape model is made by comparison with experimental data. The residuals from the fitting process (normalised to the maximum detector response) vary between –1.5% and +2.6% for an isothermal model and between –1.0% and +3.0% for a temperature-programmed model.     

Profiling food volatiles by comprehensive two-dimensional ga schromatography coupled with mass spectrometry: Advanced fingerprinting approaches for comparative analysis of the volatile fraction of roasted hazelnuts (Corylus avellana L.) from different origins

This study examined how advanced fingerprinting methods (i.e., non-targeted methods) provide reliable and specific information about groups of samples based on their component distribution on the GC × GC chromatographic plane. The volatile fractions of roasted hazelnuts (Corylus avellana L.) from nine different geographical origins, comparably roasted for desirable flavor and texture, were sampled by headspace-solid phase micro extraction (HS-SPME) and then analyzed by GC × GC-qMS. The resulting patterns were processed by: (a) “chromatographic fingerprinting”, i.e., a pattern recognition procedure based on retention-time criteria, where peaks correspondences were established through a comprehensive peak pattern covering the chromatographic plane; and (b) “comprehensive template matching” with reliable peak matching, where peak correspondences were constrained by retention time and MS fragmentation pattern similarity criteria. Fingerprinting results showed how the discrimination potential of GC × GC can be increased by including in sample comparisons and correlations all the detected components and, in addition, provide reliable results in a comparative analysis by locating compounds with a significant role. Results were completed by a chemical speciation of volatiles and sample profiling was extended to known markers whose distribution can be correlated to sensory properties, geographical origin, or the effect of thermal treatment on different classes of compounds. The comprehensive approach for data interpretation here proposed may be useful to assess product specificity and quality, through measurable parameters strictly and consistently correlated to sensory properties and origin.     

Dynamic interconversion of chiral oxime compounds in gas chromatography

A number of chiral oxime compounds have been synthesised and their gas chromatographic analysis on both a polyethelene glycol phase column and two chiral column phases was investigated. Of particular interest to this work is the observation of dynamic interconversion behaviour, both in a single dimensional analysis, and by using comprehensive two-dimensional gas chromatography (GC × GC). A number of non-chiral compounds were studied as a means to understand the nature of the behaviour observed. As expected, the achiral compound on both the wax column and the chiral column generated two isomeric compounds—the E and Z isomers. On the wax column, a characteristic interconversion zone representing the dynamic process was observed, with extent of interconversion dependent on the conditions used. For the chiral compounds, two isomers and the interconversion zone were exhibited on the wax column, however on the chiral column 4 isomeric peaks were found—the (R) and (S) enantiomers of each of the E and Z isomers. In the case of the chiral column, the extent of interconversion was negligible, and this appears to correlate with the use of low polarity columns. In order to encourage dynamic interconversion, a polyethylene glycol column was coupled to the chiral column, by placing it either before or after the chiral column. In this case a monitor detector was employed between the two columns in order to isolate the effects of the first column from the behaviour on the second. In a further study, the most appropriate column arrangement from the earlier study was placed into a comprehensive two-dimensional gas chromatography instrument, with a wax-phase column in the second dimension. The unique location of peaks for each of the molecules in 2D space and patterns for the interconversion processes is interpreted phenomenologically.     

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GC × GC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GC × GC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5–1000 and rapid spectral acquisition (≤500 spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The ‘unknown’ components were identified by comparison with mass spectra stored in a library database.     

Analysis of special surfactants by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Multidimensional gas-chromatographic analyses of olesochemically based nonionic, anionic and several cationic surfactants in industrial cleaners are demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the simultaneous determination of fatty alcohols, fatty alcohol sulphates and alkyl polyglucosides. In addition, the determination of fatty alcohol ethoxylates up to C₁₀EO₈ (highest degree of ethoxylation) and C₁₈EO₅ (longest C-chain at an ethoxylation degree of five) and the analysis of fatty alcohol alkoxylates that contain ethoxy (EO) and propoxy (PO) groups could be realized. Because of decomposition in the injector and a weak EI-fragmentation, cationic surfactants such as alkyl benzyl dimethyl ammonium chloride could also be identified by their characteristic fragments. Thermogravimetric analyses confirmed that the temperature in a normal GC injector is not high enough to cause thermal decomposition of esterquats. However, we could demonstrate that a modified silylation procedure forms decomposition products of esterquats in the GC injector which are detectable by GC × GC–(TOF)MS and allows the identification of such GC-atypical analytes.     

Application of solid-phase micro-extraction and comprehensive two-dimensional gas chromatography (GC×GC) for flavour analysis

Summary Headspace solid-phase micro-extraction (HS SPME), comprehensive two-dimensional GC (GC×GC), and flame ionization detection (FID) have been examined for their suitability and compatibility for rapid sampling, separation, and detection of garlic flavour volatiles. This approach (HS-SPME-GC×GC-FID) is distinctly superior to use of one-dimensional GC, i. e., HS-SPME-GC-FID. Direct comparison of the experimental results showed that a 10–50-fold increase in sensitivity is obtained, separating power is substantially enhanced, and the peak capacity is up to ten times higher. As a consequence, much more detailed flavour analysis can be performed; this results in better information about the aroma-active compounds.