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).     

Multidimensional gas chromatography using a double cool-strand interface

Commercial interfaces for multidimensional gas chromatography (MDGC) are based either on a valve or a pneumatic switching between columns. Both exhibit significant drawbacks and only few suppliers exist. An extremely simple interface has been set up to overcome these limitations without requiring any pneumatic control or valves switching. This new MDGC design is based on the cryo-control of the analyte transfer from the first to the second column through two cool strands of a capillary. This technique is simple to implement and does not require any special column connections. Applications involve non-polar/polar phase combinations, as well as chiral analysis, hyphenation to a conventional mass spectrometer, and olfactometric detection. In contrast to conventional MDGC configuration, the present configuration allows the use of a single oven to operate both columns at different temperatures.     

Feasibility of two multidimensional techniques, heart-cut MDGC and GCxGC, for the separation of PCBs and PBDEs

The feasibility of heart-cut MDGC-ECD and GCxGC-muECD for the separation of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) has been evaluated. Among the different column combinations tested, the best results were obtained when using either a DB-5 type or an HT-8 column as the first dimension combined with a mid-polar stationary phase (50% phenyl) as the second dimension. The co-elutions between the target PCBs and PBDEs in the first dimension were satisfactorily resolved once transferred to the second column. Repeatability and intermediate precision were satisfactory in both systems, considering retention times in both dimensions. Nevertheless, peak area/volume precision was better for heart-cut MDGC since software for the data analysis in the case of GCxGC was still under development. The need for multiple injections of the same extract on the heart-cut MDGC system was the main drawback since the analysis time it increased. GCxGC becomes then a valuable alternative able to achieve the same performance in one injection. However, the GCxGC data analysis is still very time-consuming and needs further development. The applicability of both separation techniques was shown using a human breast milk sample with low concentrations of PCBs (0.2-227 pg/g fresh weight) and PBDEs (1.2-41 pg/g fresh weight).     

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.     

Semi-rotating cryogenic modulator for comprehensive two-dimensional gas chromatography

A laboratory-made cryogenic modulator for comprehensive two-dimensional gas chromatography (GCxGC) was constructed and evaluated. Analytes were trapped with carbon dioxide and desorbed with heated GC oven air. The GCxGC system included a non-polar first-dimension column and a semi-polar second-dimension column connected to a flame ionisation detector. A laboratory-written Matlab-based program was used for the data analysis. Peak widths at half height for n-alkanes, obtained with use of new modulator, were at narrowest 60 ms.     

Time-resolved cryogenic modulation for targeted multidimensional capillary gas chromatography analysis

Multidimensional gas chromatography (MDGC) is performed in a new manner, described in this paper. The method incorporates two directly coupled columns and employs a longitudinally modulated cryogenic trap located between the columns. No heartcutting process is used, but rather a method better termed selected zone compression pulsing is used. Compared with normal MDGC, where primary column effluent has to be temporarily diverted either to a monitor detector or to the second dimension column, the new procedure in its simplest mode passes all of the first column effluent to the second column. It is simply the times at which the modulation of the trap is performed that determines which target solutes will be selected for enhanced separation. This approach allows almost instantaneous separation of selected zones on the second column, and has the potential to significantly simplify the MDGC method. Since data are presented in a time-response format, and do not require transformation as previously described for comprehensive GC when using the longitudinal modulator, quantitation and report generation are essentially the same as in any GC method and data system. Advantages also include significant sensitivity improvement. By using cryofocussing, and benefiting from the zone compression effects along with fast GC conditions on the second dimension, new possibilities for MDGC can be realised. The method is demonstrated by using a mixture of semi-volatile aromatic hydrocarbons.     

Comprehensive two-dimensional gas chromatography: metrics,potentials, limits

Metrics for evaluation of separation performance of comprehensive two-dimensional gas chromatography (GCxGC) and for quantitative comparison of that performance with similar performance of its 1D (one-dimensional) counterparts are described. The performance improvement can be expressed via reduction in the saturation of a chromatogram or-in the case of the uniform distribution of peaks along the second dimension--via the peak capacity gain due to GCxGC. An order of magnitude peak capacity gain due to the comprehensive GCxGC is possible under optimal conditions. Optimal parameters of the second dimension column as well as the optimal operational conditions for that column and for the modulator in a comprehensive GCxGC are also presented.     

On retentivity tuning by flow in the second column of different comprehensive two dimensional gas chromatographic configurations

Retentivity tuning in comprehensive two dimensional GC separations of aliphatics (linear and cyclic hydrocarbons) and aromatics in gasoline by changing the carrier gas flows in the column series at constant working temperature parameters of both columns is discussed. Comprehensive 2D techniques studied include GC×GC with cryogenic and differential flow modulation and non-modulated transfer (NMT). In all configurations, the first dimension was a non-polar column and the second dimension a polar column. Using three different flows (0.6, 1.0 and 1.4mL/min) of helium carrier gas in cryogenic modulated GC×GC illustrated that, as expected, retention of the solutes on the (1)D and (2)D columns increased but the separation quality was nearly constant. A change of carrier gas pressure (p(m)=175-125kPa) on the (1)D and (2)D columns joint point at constant inlet pressure (p(i)=525kPa) in NMT, induces an increase of the carrier gas flow rate on the (1)D and a decrease on the (2)D column, respectively. The higher retentivity of the (2)D column improved the group type separation of aliphatic/cyclic hydrocarbons and aromatics and a higher distribution of aromatics on the 2D retention plane was noted. Retentivity tuning was also performed in flow modulated GC×GC by operating the (1)D column at 0.8mL/min and the (2)D column at 20 and 26mL/min. The higher retentivity at 20mL/min improved the group type separation of aliphatic/cyclic hydrocarbons and aromatics in the 2D retention plane.     

Comprehensive two-dimensional gas chromatography for fingerprint pattern recognition in cachaça production

Cacha?a samples were studied by means of comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GCxGC/TOFMS) during the fermentation process and after ageing in different wood materials. The analyses of the aroma compounds were performed after headspace-solid phase microextraction method (HS-SPME) using an 85microm polyacrylate (PA) fibre. Fingerprint monitoring of the distillation process allowed the easy determination of the turning points of the process and high-resolution comparison of cabe?a (head), cora??o (core) and cauda (tail) fractions. The ageing process in different wood materials was well characterised through fingerprint similarity observations; in the absence of a suitable metric for expressing the overall similarity, here we use a visual and retention time comparison to identify co-incident peaks and those that differ between samples. For quality control purposes, a simple observation of the contour plots obtained can thus allow the identification of the type of wood used in the ageing process, and the process of ageing, without further statistical treatment or peak identifications. In this manner, peaks, which discriminated most between the different mixtures studied were readily found, i.e. unique compounds were identified in each stage of the distillation process. Approximate first dimension linear retention indices (LRI) for these identified compounds were calculated in a bi-dimensional polar/non-polar column set in the GCxGC experiment and were used in conjunction with mass spectral library searching for tentative identification. Along the progression of the distillation process, 70 compounds appear to visually discriminate between samples and their retention indices are indicated, presenting good correlation with literature data.     

Optimization in multidimensional gas chromatography applying quantitative analysis via a stable isotope dilution assay

Trace level analyses in complex matrices benefit from heart-cut multidimensional gas chromatographic (MDGC) separations and quantification via a stable isotope dilution assay. Minimization of the potential transfer of co-eluting matrix compounds from the first dimension (¹D) separation into the second dimension separation requests narrow cut-windows. Knowledge about the nature of the isotope effect in the separation of labeled and unlabeled compounds allows choosing conditions resulting in at best a co-elution situation in the ¹D separation. Since the isotope effect strongly depends on the interactions of the analytes with the stationary phase, an appropriate separation column polarity is mandatory for an isotopic co-elution. With 3-alkyl-2-methoxypyrazines and an ionic liquid stationary phase as an example, optimization of the MDGC method is demonstrated and critical aspects of narrow cut-window definition are discussed.

Higher mass loadability in comprehensive two-dimensional gas chromatography-mass spectrometry for improved analytical performance in metabolomics analysis

A major challenge in metabolomics analysis is the accurate quantification of metabolites in the presence of (extremely) high abundant metabolites. Quantification of metabolites at low concentrations can be complicated by co-elution and/or peak distortion when these metabolites elute close to high abundant metabolites. To increase the separation efficiency a comprehensive two-dimensional gas chromatographic-mass spectrometric method (GC x GC-MS) was set up, in which a polar first dimension column and an apolar second dimension column were used to maximize the peak capacity. The feasibility of using wider bore, thicker film columns in the second dimension to improve the mass loadability and inertness of the analytical system was investigated. Several column combinations with varying second dimension column dimensions were compared with a setup with a narrow bore column (0.1mm I.D.) in the second dimension. With a wider bore column (0.32 mm I.D.) in the second dimension the mass loadability was improved 10-fold, and the more inert column surface of the thicker film second dimension column resulted in a more accurate (automated) quantification and improved linearity in the presence of high concentrations of matrix compounds or metabolites. These benefits amply compensated the observed decrease in peak capacity of 40% compared to the narrow bore (0.1mm I.D.) thin film second dimension column. Compared to GC-MS and conventional GC x GC-MS, better performance for quantification of metabolites for typical metabolomics samples was achieved.     

Comprehensive two-dimensional gas chromatography for fast screening of wash oils

Fast screening of wash oils is demonstrated using comprehensive two-dimensional gas chromatography (GCxGC). Wash oils are used in ethylene production plants to minimize compressor fouling. The composition of a wash oil determines its effectiveness in solubilizing heavy hydrocarbons. In particular, the relative amount of 1- and 2-ring aromatics is important. The presence of oxygenates is undesirable because of adverse effects to the process. It is shown that GCxGC is well suited for this application. Species in wash oils are separated and grouped into three bands: a nonpolar aliphatics band, 1- and 2-ring aromatics band, and polyaromatics band. For a given polar secondary column, the spacing between bands in the second dimension can be adjusted in a broad range by selecting a primary column and an oven-temperature-programming rate. Integration of GCxGC peaks is evaluated using a standard GC integration program and a new GCxGC integration program. Consistent results are obtained using both programs for well-separated GCxGC peaks with relative differences for individual peak ranging from 0.04% to 1.6%. Peak responses are integrated by the GCxGC software, and the relative amounts of aromatics content and aliphatics content are estimated by peak response percent with relative standard deviations ranging from 0.15% to 2.8% (n = 3).     

Fast enantiomeric analysis of a complex essential oil with an innovative multidimensional gas chromatographic system

The present research is focussed on the evaluation of a recently developed high performance multidimensional gas chromatographic (MDGC) system employed in the fast analysis of a series of chiral compounds contained in rosemary essential oil. The heart of the MDGC system consists in a simple transfer device for the rapid sequential re-injection of analyte "heart-cuts" from the first to the second dimension. The transfer system has no temperature restrictions, presents very low dead volumes and achieves multidimensional analysis through a pressure-balance mechanism. The MDGC set-up is characterized by two GC ovens (enabling independent temperature programming) and the possibility of mass spectrometric (MS) and/or flame ionization detection (FID). Multiple-cut conventional and fast MDGC-FID methods were developed and the results obtained compared, in order to evaluate the effectiveness of the system. In this respect, the rapid method provided the same analytical result in a greatly reduced time (approximately five times less). Furthermore, quali/quantitative data reproducibilty was very good. Fast MDGC was achieved by using micro-bore (0.1mm I.D.) columns in both dimensions.     

Retention time reproducibility in comprehensive two-dimensional gas chromatography using cryogenic modulation an intralaboratory study

A survey was conducted to determine the reproducibility of retention times in both the first (D1) and second dimension (D2) axes of the two-dimensional separation space, in the comprehensive two-dimensional gas chromatographic analysis of an essential oil sample using cryogenic modulation. The retention times in the two dimensions for a number of individual components comprising hydrocarbon, alcohol, ester and ketone chemical classes in a Melaleuca alternifolia essential oil were recorded from replicate analyses using four separate column sets and two identical gas chromatographs. Run-to-run, day-to-day, instrument-to-instrument, and column set-to-column set reproducibility were demonstrated from the experimental design. A total of 60 GC x GC analyses were conducted. The longitudinally modulated cryogenic system produced reproducible modulation start times and consistent modulation phase profiles for individual components in all experiments, and retention time variations in both dimensions were negligible. The average run-to-run reproducibility of 43 components for six replicate injections was found to be 0.12% RSD in the first dimension, and 0.74% RSD in the second dimension. Day-to-day reproducibility showed statistically "significant" difference (F-test), but this was partly ascribable to the excellent within-day reproducibility that led to apparent day-to-day differences. Confidence in absolute retention times (hence component positions) in the two-dimensional separation space is critical to component identification.     

Enantiomeric separation of chiral polychlorinated biphenyls on beta-cyclodextrin capillary columns by means of heart-cut multidimensional gas chromatography and comprehensive two-dimensional gas chromatography. Application to food samples

Three commercially available chiral capillary columns, Chirasil-Dex, BGB-176SE, and BGB-172, have been evaluated for the separation into enantiomers of the 19 chiral polychlorinated biphenyls (PCB) congeners stable at room temperature. The enantiomers of 15 chiral PCBs were, at least to some extent, separated using these beta-cyclodextrin based columns. Multidimensional techniques, such as heart-cut multi-dimensional gas chromatography (heart-cut MDGC) and comprehensive two-dimensional gas chromatography (GC x GC), were investigated for their ability to solve coelution problems with other PCBs present in commercial mixtures and real-life samples. Heart-cut MDGC improved the separation as compared to one-dimensional GC, and enantiomeric fractions of the investigated chiral PCBs could be determined free from interferences. However, limitations on the number of target compounds that can be transferred to the second column in a single run and, therefore, the time consumption, have led to the evaluation of GC x GC as an alternative for this type of analysis. With GC x GC, two column set-ups were tested, both having a chiral column as first-dimension column, and two different polar stationary phase columns in the second dimension. On using both column combinations, congeners 84, 91, 95, 132, 135, 136, 149, 174, and 176 could be determined free from coelutions with other PCBs. Results on the application of heart-cut MDGC to food samples such as milk and cheese are given, as well as the first results on the application of GC x GC to this type of samples.     

Optimization of separation and detection conditions for the multiresidue analysis of pesticides in grapes by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry

A comprehensive GCxGC-TOFMS method was optimized for multiresidue analysis of pesticides using a combination of a non-polar (RTX-5MS, 10 m x 0.18 mm x 0.2 microm) and a polar capillary column (TR-50MS, 1 m x 0.1 mm x 0.1 microm), connected in series through a dual stage thermal modulator. The method resolved the co-elution problems as observed in full scan one-dimensional GC-MS analysis and allowed chromatographic separation of 51 pesticides within 24 min run time with library-searchable mass spectrometric confirmation. Four pesticides, viz. chlorpyrifos-methyl, vinclozoline, parathion-methyl and heptachlor could be baseline separated on GCxGC, which were otherwise closely eluting and interfering each other's detection in 1D GC-MS run. Similarly, it could be possible to separate myclobutanil, buprofezin, flusilazole and oxyfluorfen on GCxGC. Although in 1D GC-MS, these closely eluting compounds could be identified through deconvolution algorithm and 'peak-find' option of the Chromatof software but the spectral purity significantly improved on GCxGC analysis. Thorough optimization was accomplished for the oven temperature programming, ion source temperature and GCxGC parameters like modulation period, duration of hot pulses, modulation-offset temperature, acquisition rate, etc. to achieve best possible separation of the test compounds. The limit of detection significantly improved by 2-12 times on GCxGC-TOFMS against GC-TOFMS because of sharper and narrower peak shapes. The method was tested for grape matrix after preparing the samples using previously described method and recoveries of the entire test pesticides were within 70-110% at 10 ng/g level of fortification. GCxGC-TOFMS was found to be an excellent technique for library-based screening of pesticides with high accuracy and sensitivity.     

Detection of volatile organic compounds by temperature-programmed desorption combined with mass spectrometry and Fourier transform infrared spectroscopy

A temperature-programmed desorption (TPD) device connected to a mass spectrometer was used to detect volatile organic compounds from air samples. The main aim was to develop an analytical method, by which both non-polar and polar organic components can be detected in the same run. In TPD, the adsorbed compounds are desorbed from the resin more slowly than in the conventional trapping techniques, such as purge-and-trap technique, in which the resin is flash-heated and the compounds are desorbed at the same time to a cryogenic trap or an analytical column. In TPD, the adsorbent resin acts also as an analytical column. In this way it is possible to obtain more rapid analysis, and also a more simple instrumentation, which can be used on-line and on-site. In this work, a new version of TPD device, which uses a resistor for heating and a Peltier element for rapid cooling, was designed and constructed. Various adsorbent resins were tested for their adsorption and desorption properties of both polar and non-polar compounds. When using a mixture of adsorbent resins, Tenax TA and HayeSep D, it was possible to analyze both polar, low-molecular weight compounds, such as methanol and ethanol, and non-polar volatile organic compounds, such as benzene and toluene, in the same run within 15 min including sampling. The same TPD principle was also tested using a Fourier transform infrared spectrometer as an analytical instrument, and the results showed that it was possible to obtain a separation of similar compounds, such as hexane and heptane, and still retaining the same sensitivity as the original on-line FTIR instrument.     

Determination of aromatic hydrocarbons in gasolines by flow modulated comprehensive two-dimensional gas chromatography

Valve based/flow modulated comprehensive two-dimensional gas chromatography-flame ionization detection (GC x GC-FID) was used for quantification of C6 through C12 aromatic hydrocarbons by carbon number in gasolines. A 0.53 mm i.d. non-polar first dimension column was coupled to a 0.53 mm i.d. polar second dimension column through a double loop eight port valve modulator. Depending on the sample type, normalized percent and internal standard (I.S.) quantification was performed. For normalized percent quantification, a one-point calibration performed with one aromatic compound per carbon number/class provided an average % accuracy of 2.1% and a short-term n--1 relative standard deviation of 1.0%. For total aromatic compounds good agreement with the more complex conventional multidimensional GC technique was obtained. However, GC x GC has certain advantages over most other methods, mainly increased selectivity for total and carbon number aromatic content. The identification of the aromatic hydrocarbons was confirmed by GC x GC-MS.     

Low cost, robust, in-house hardware for heart cutting two-dimensional gas chromatography

Natural materials are so complex that no single column can separate all of the components. Heart cutting 2-D GC (GC-GC) using a Deans switch provides maximum separation, but the requisite gas flow configurations have earned a reputation for being fiddly and time consuming to set up and tune, unless electronic gas controls and costly software are employed. A design for a vented Deans switch is presented that can be assembled in-house from standard commercial components. A vent to atmosphere replaces the balancing resistor, which solves the problem of balancing the pressures and flows, and requires no compensation for changes in gas viscosity and back-pressure during temperature programming. First and second dimension columns can both be run at optimum flow rates, even if they are of different diameters. Analyte detectability is preserved by cutting only the target fraction from the first column and transferring the whole of it to the second column. Cryotrapping the selected fraction allows cuts of any width, and transfer of analytes to the second column as sharp bands. I have applied the vented Deans switch-cold trap to the identification of flavour and fragrance compounds from South African plants and foods; cuts are very repeatable, and the detectability of trace components is enhanced. Its capabilities are demonstrated by examples from analyses of essential oils and flavour extracts.