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.     

Modulation-induced error in comprehensive two-dimensional gas chromatographic separations

There is a fundamental difference between data collected in comprehensive two-dimensional gas chromatographic (GCxGC) separations and data collected by one-dimensional GC techniques (or heart-cut GC techniques). This difference can be ascribed to the fact that GCxGC generates multiple sub-peaks for each analyte, as opposed to other GC techniques that generate only a single chromatographic peak for each analyte. In order to calculate the total signal for the analyte, the most commonly used approach is to consider the cumulative area that results from the integration of each sub-peak. Alternately, the data may be considered using higher order techniques such as the generalized rank annihilation method (GRAM). Regardless of the approach, the potential errors are expected to be greater for trace analytes where the sub-peaks are close to the limit of detection (LOD). This error is also expected to be compounded with phase-induced error, a phenomenon foreign to the measurement of single peaks. Here these sources of error are investigated for the first time using both the traditional integration-based approach and GRAM analysis. The use of simulated data permits the sources of error to be controlled and independently evaluated in a manner not possible with real data. The results of this study show that the error introduced by the modulation process is at worst 1% for analyte signals with a base peak height of 10xLOD and either approach to quantitation is used. Errors due to phase shifting are shown to be of greater concern, especially for trace analytes with only one or two visible sub-peaks. In this case, the error could be as great as 6.4% for symmetrical peaks when a conventional integration approach is used. This is contrasted by GRAM which provides a much more precise result, at worst 1.8% and 0.6% when the modulation ratio (MR) is 1.5 or 3.0, respectively for symmetrical peaks. The data show that for analyses demanding high precision, a MR of 3 should be targeted as a minimum, especially if multivariate techniques are to be used so as to maintain data density in the primary dimension. For rapid screening techniques where precision is not as critical lower MR values can be tolerated. When integration is used, if there are 4-5 visible sub-peaks included for a symmetrical peak at MR=3.0, the data will be reasonably free from phase-shift-induced errors or a negative bias. At MR=1.5, at least 3 sub-peaks must be included for a symmetrical peak. The proposed guidelines should be equally relevant to LCxLC and other similar techniques.     

Programmed automation of modulator cold jet flow for comprehensive two-dimensional gas chromatographic analysis of vacuum gas oils

A method is described for automating the regulation of cold jet flow of a comprehensive two-dimensional gas chromatograph (GCxGC) configured with flame ionization detection. This new capability enables the routine automated separation, identification, and quantitation of hydrocarbon types in petroleum fractions extending into the vacuum gas oil (VGO) range (IBP-540 degrees C). Chromatographic data acquisition software is programmed to precisely change the rate of flow from the cold jet of a nitrogen cooled loop modulator of a GCxGC instrument during sample analysis. This provides for the proper modulation of sample compounds across a wider boiling range. The boiling point distribution of the GCxGC separation is shown to be consistent with high temperature simulated distillation results indicating recovery of higher boiling semi-volatile VGO sample components. GCxGC configured with time-of-flight mass spectrometry is used to determine the molecular identity of individual sample components and boundaries of different molecular types.     

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).     

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.     

Image background removal in comprehensive two-dimensional gas chromatography

This paper describes a new technique for removing the background level from digital images produced in comprehensive two-dimensional gas chromatography (GCxGC). Background removal is an important first step in the larger problem of quantitative analysis. The approach estimates the background level across the chromatographic image based on structural and statistical properties of GCxGC data. Then, the background level is subtracted from the image, producing a chromatogram in which the peaks rise above a near-zero mean background. After the background level is removed, further analysis is required to determine the quantitative relationship between the peaks and chemicals in the sample. The algorithm is demonstrated experimentally to be effective at determining and removing the background level from GCxGC images. The algorithm has several parametric controls and is incorporated into an interactive program with graphical interface for rapid and accurate detection of GCxGC peaks.     

A comprehensive two-dimensional gas chromatography method for analyzing extractable petroleum hydrocarbons in water and soil

A flow-switching two-dimensional gas chromatography (GCxGC) apparatus has been constructed that can operate at temperatures as high as 340 degrees C. This system is employed to analyze complex hydrocarbon mixtures such as diesel fuel, gas-oil, motor oil, and petroleum contaminated environmental samples. The GCxGC system generates two-dimensional chromatograms with minimal overlap between the aliphatic and aromatic regions This allows these compound classes to be independently quantitated without prior fractionation. The GCxGC system is used to analyze extracts of spiked water samples, wastewater, and soil. The accuracy of the method is compared to that of the Massachusetts Extractable Petroleum Hydrocarbons (MA EPH) method. The GCxGC system generates a quantitative accuracy similar to the MA EPH method for the analysis of spiked water samples. The GCxGC method and the MA EPH method generate comparable levels of total hydrocarbons when wastewater is analyzed, but the GCxGC method detects a significantly higher aromatic content and lower aliphatic content. Both the GCxGC method and MA EPH method measure comparable levels of aromatics in the soil samples.     

Development of an algorithm for peak detection in comprehensive two-dimensional chromatography

A method for peak detection in two-dimensional chromatography is presented. The algorithm applies first the methods developed for peak detection in one-dimensional chromatography to detect peaks in one dimension. In a second step, a decision tree is applied to decide which one-dimensional peaks are originated from the same compound and have to be 'merged' into one two-dimensional peak. To this end, different features of the peaks (second-dimension peak regions and second-dimension retention times) are compared and different criteria (common peak regions, retention time differences, unimodality in the first dimension) are applied. Different options can be used, depending on the nature of the data. The user controls this decision tree by establishing several options and "switches". The algorithm was tested with GCxGC chromatograms obtained for a commercial air-freshener sample, detecting and merging the modulated peaks belonging to the same compound. Recommendations for the set of options and switches are given. A utility that calculates and sums peak areas from merged peaks is added to facilitate automated quantification. Although the algorithm was developed for GCxGC, its application to comprehensive two-dimensional liquid chromatography (LCxLC) data should at most require minor modifications.     

A linear modulation-based stochastic resonance algorithm applied to the detection of weak chromatographic peaks

A simple stochastic resonance algorithm based on linear modulation was developed to amplify and detect weak chromatographic peaks. The output chromatographic peak is often distorted when using the traditional stochastic resonance algorithm due to the presence of high levels of noise. In the new algorithm, a linear modulated double-well potential is introduced to correct for the distortion of the output peak. Method parameter selection is convenient and intuitive for linear modulation. In order to achieve a better signal-to-noise ratio for the output signal, the performance of two-layer stochastic resonance was evaluated by comparing it with wavelet-based stochastic resonance. The proposed algorithm was applied to the quantitative analysis of dimethyl sulfide and the determination of chloramphenicol residues in milk, and the good linearity of the method demonstrated that it is an effective tool for detecting weak chromatographic peaks. Figure The linear modulation-based stochastic resonance algorithm (LSRA) improved the output chromatographic peak of chloramphenicol     

Comprehensive two-dimensional gas chromatography of volatile and semi-volatile components using a diaphragm valve-based instrument

A high-temperature configuration for a diaphragm valve-based gas chromatography (GCXGC) instrument is demonstrated. GCxGC is a powerful instrumental tool often used to analyze complex mixtures. Previously, the temperature limitations of valve-based GCxGC instruments were set by the maximum operating temperature of the valve, typically 175 degrees C. Thus, valve-based GCxGC was constrained to the analysis of mainly volatile components; however, many complex mixtures contain semi-volatile components as well. A new configuration is described that extends the working temperature range of diaphragm valve-based GCxGC instruments to significantly higher temperatures, so both volatile and semi-volatile compounds can be readily separated. In the current investigation, separations at temperatures up to 250 degrees C are demonstrated. This new design features both chromatographic columns in the same oven with the valve interfacing the two columns mounted in the side of the oven wall so the valve is both partially inside as well as outside the oven. The diaphragm and the sample ports in the valve are located inside the oven while the temperature-restrictive portion of the valve (containing the O-rings) is outside the oven. Temperature measurements on the surface of the valve indicate that even after a sustained oven temperature of 240 degrees C, the portions of the valve directly involved with the sampling from the first column to the second column track the oven temperature to within 1.2% while the portions of the valve that are temperature-restrictive remain well below the maximum temperature of 175 degrees C. A 26-component mixture of alkanes, ketones, and alcohols whose boiling points range from 65 degrees C (n-hexane) to 270 degrees C (n-pentadecane) is used to test the new design. Peak shapes along the first column axis suggest that sample condensation or carry-over in the valve is not a problem. Chemometric data analysis is performed to demonstrate that the resulting data have a bilinear structure. After over 6 months of use and temperature conditions up to 265 degrees C, no deterioration of the valve or its performance has been observed.     

Comparison of comprehensive two-dimensional gas chromatography and gas chromatography--mass spectrometry for the characterization of complex hydrocarbon mixtures

In this paper, we compare the current separation power of comprehensive two-dimensional gas chromatography (GCxGC) with the potential separation power of GC-mass spectrometry (GC-MS) systems. Using simulated data, we may envisage a GC-MS contour plot, that can be compared with a GCxGC chromatogram. Real examples are used to demonstrate the current potential of the two techniques in the field of hydrocarbon analysis. As a separation technique for complex hydrocarbon mixtures, GCxGC is currently about as powerful as GC-MS is potentially powerful. GC-MS has not reached its potential separation power in this area, because a universal, soft ionization method does not exist. The greatest advantage of GCxGC is, however, its potential for quantitative analysis. Because flame-ionisation detection can be used, quantitative analysis by GCxGC is much more robust, reliable and reproducible.     

Towards comprehensive hydrocarbons analysis of middle distillates by LC-GCxGC

The detailed characterization of middle distillates is essential for a better understanding of reactions involved in refining processes. Owing to a higher resolution power and an enhanced sensitivity, but especially to a group-type ordering in the chromatographic plane, comprehensive two-dimensional gas chromatography (GCxGC) offers unsurpassed characterization possibilities for petroleum samples. However, GCxGC fails to totally discriminate naphthenes from unsaturates occurring in hydrotreated diesel samples. This article aims at promoting the implementation of LC-GCxGC for the quantitative determination of hydrocarbon distribution in middle distillates, including naphthenes. In this configuration, liquid chromatography (LC) enables the separation of hydrocarbons into two fractions (viz., saturated and unsaturated) before the subsequent analysis of each fraction by GCxGC. In this paper, the choice of GCxGC conditions in order to achieve the separation and identification of hydrocarbons by chemical class is discussed; under these conditions, naphthenes are separated according to the number of saturated rings. For the first time, the presence of di-, tri-, and tetra-naphthenes resulting from the hydroconversion of aromatics can clearly be evidenced. A quantitative procedure for the determination of the distribution of hydrocarbons, including the distribution of naphthenes according to the number of saturated rings, is also proposed and discussed in detail. LC-GCxGC is found to provide an unequalled degree of information that will widely contribute to a better understanding of hydroconversion processes.     

Targeted multidimensional gas chromatography for the quantitative analysis of suspected allergens in fragrance products

Two approaches are described and compared for the analysis of suspected allergens (SAs) in fragrance products, which are defined by the Scientific Committee of Cosmetics and Non-Food Products (SCCNFP). The first consists of a comprehensive two-dimensional gas chromatography (GCxGC) experiment using both a "conventional" non-polar/polar column combination and an "inverse" polar/non-polar column set. The second approach uses a targeted multidimensional gas chromatography (MDGC) system employing a Deans type pneumatic switch and a longitudinally modulated cryogenic system (LMCS). It was found that the conventional and inverse column sets complement each other well, providing identification of SAs present. Compounds well retained on the second dimension of one column set were the first to be eluted from the other. In some instances SAs co-eluting with matrix components on the second dimension for a given column set were clearly resolved on the other, although this has the disadvantage of requiring two analytical runs. Targeted MDGC with a non-polar/polar column set, successfully separated all SAs identified within a fragrance product. The instrument is set up in a similar fashion to a GCxGC system though with longer second dimension ((2)D) column, a cryogenic trap at the beginning of the second column, and a pneumatic switch coupling both columns. The data are easier to process than for a GCxGC experiment. The targeted MDGC method has the capacity to deliver far greater efficiency to targeted regions of a primary separation than a GCxGC experiment, whilst still maintaining overall run times similar to those of a conventional one-dimensional (1D) GC experiment. Cryogenic focussing at the beginning of the (2)D column delivers enhanced sensitivity, accurate (2)D retention times and narrow peak widths; these are responsible for an increased resolution obtained from the fast, relatively short ( approximately 5m) (2)D column. The two column set GCxGC analysis provided a quick and effective means to qualitatively determine the presence of six SAs in a commercially available air freshener, however all were not adequately resolved from matrix components. In contrast, quantitation was straightforward using the targeted MDGC method.     

Analysis of organic compounds of water-in-crude oil emulsions separated by microwave heating using comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry

In this work the higher peak capacity and resolution of comprehensive two-dimensional gas chromatography (GCxGC) has been successfully applied, for the first time, to tentatively identify several polar organic compounds of organic extracts of aqueous phases resulting from microwave demulsification process of water-in-crude oil emulsions. Results have shown that higher temperatures and longer exposure time to microwave irradiation produced water phases with a wider variety of polar organic compounds. The microwave process showed to be suitable for the extraction of several polar compounds classes of petroleum. The proposed microwave extraction method and GCxGC identification of polar compounds of petroleum samples are of practical interest for the petrochemical industry due to corrosion and related problems associated with these polar compounds in refinery equipments. The GCxGC/time-of-flight MS technique shows to be very important in the total separation of different classes of compounds and allows the identification of many compounds in these classes.     

Compehensive two-dimensional gas chromatography (GC×GC) and its applicability to the characterization of complex (petrochemical) mixtures

In general, petrochemical products contain only a limited number of chemical classes of compounds (sample dimensionality). The enormous number of individual components within these classes, however, soon puts limitations upon a single chromatographic technique when it comes to adequate characterization of these products. Comprehensive two-dimensional gas chromatography (GC×GC) clearly opens the possibility of estimating the composition of hydrocarbon mixtures in a far more detailed fashion than hitherto possible. Although the emphasis of papers of GCxGC thus far almost exclusively applies to the unsurpassed peak-capacity, in the oil industry there is a need for characterization, rather than for analyzing all the individual compounds. In principle a GCxGC system can provide an almost perfect match between its intrinsic properties and the dimensionality of oil samples. To establish the applicability of GCxGC towards petrochemical analytical challenges, a commercially aavailable prototype instrument was subjected to an exhaustive characterization of a typical hydrocarbon precess stream and a fast characterization of a light gas oil. Although there are no fundamental limitations towards the quantitative aspects of a GCxGC system, this paper confines itself to qualitative results only. Quantitative aspects of GCxGC will be published in a forthcoming paper.     

Time-resolved cryogenic modulation reveals isomer interconversion profiles in dynamic chromatography.

The dynamic chromatographic study of interconversion of E and Z forms of oximes has been investigated by using a novel cryogenic modulation method in a two-dimensional gas chromatographic array. The primary column is a conventional capillary GC column on which the molecular interconversion proceeds. In this case, the molecular dynamical process leads to a peak profile describing the kinetics and thermodynamics of the interconverting molecules during its chromatographic elution. Thus an interconversion region intercedes the elution of the individual stereoisomers of the reaction. Since the molecules are isomers, classical molecular identification methods such as gas chromatography-mass spectrometry are unable to study the individual instantaneous amounts of each of the compounds. Hence the infinitesimal profiles of interconversion along the entire column have never been experimentally observed; rather the total profile is normally subjected to mathematical modelling studies in order to match experiment with theory, and to gain the kinetic parameters of the process. In the present study, an instantaneous ratio of the individual isomers can be found during the chromatographic elution by direct measurement. This is achieved by using a cryogenic zone focussing process, with rapid longitudinal modulation of a cold trap and continual pulsing of collected zones into a fast-analysis high-resolution capillary column on which isomer interconversion is minimized. The data can be displayed as a two-dimensional contour plot to demonstrate the individual isomer profiles. The two-dimensional analysis also allows easy measurement of the peak ratios of the two isomers which is an indicator of the extent of interconversion that has taken place. Two model systems, acetaldoxime and butyraldoxime, were chosen to illustrate the use of the cryogenic modulation procedure. It is anticipated that the procedure could be applied to other molecules which exhibit gas-phase isomerizations or reactions.     

Estimation of individual ultraviolet spectra in incomplete two-component separations by high-performance liquid chromatography

A method is described for the estimation of spectral features in a two-component chromatographic peak by means of a u.v. diode-array detector. The calculation relies on the assumption that the front of a fused chromatographic peak contains a single pure component. The spectrum of this component is used in calculating the concentration profile of the other component, thus allowing the determination of a solution band for the spectrum of the second component. The boundaries of the solution band are based on non-negativity restrictions of chromatographic and spectral features. The method does not require the use of principal components analysis.     

Application of comprehensive two-dimensional gas chromatography with nitrogen-selective detection for the analysis of fungicide residues in vegetable samples

Comprehensive two-dimensional gas chromatography (GCxGC) with nitrogen-phosphorus detection (NPD) has been investigated for the separation and quantitation of fungicides in vegetable samples. The detector gas flows (H(2), N(2) and air) were adjusted to achieve maximum response of signal whilst minimizing peak width. The comparison of different column sets and selection of the temperature program were carried out with a mixture of nine N-containing standard fungicides, eight of which were chlorinated. The results from GCxGC-NPD and GCxGC with micro electron-capture detection (muECD) were compared. External calibrations of fungicides were performed over a concentration range from 1 to 1,000 microgL(-1). The peak area calibration curves generally had regression coefficients of R(2)>0.9980, however for iprodione which was observed to undergo on-column degradation, an R(2) of 0.990 was found. The limit of detection (LOD) and limit of quantitation (LOQ) were less than about 74 and 246 ng L(-1), respectively. The intra-day and inter-day RSD values were measured for solutions of concentration 0.100, 0.500 and 1.50 mg L(-1). For the 0.500 mg L(-1) solution, intra- and inter-day precision of peak area and peak height for most of the pesticides were about 2% and 8%, respectively. Excellent linearity was observed for these standards, from 0.001 to 25.00 mg L(-1). The standard mixture peak positions were identified by using GCxGC with quadrupole mass spectrometry (qMS). To illustrate the potential and the versatility of both GCxGC-NPD and GCxGC-muECD, the method was applied to determination of fungicides in a vegetable extract. Decomposition of one fungicide standard (iprodione) during chromatography elution was readily observed in the two-dimensional (2D) GCxGC plot as a diagonal ridge response in the 2D chromatogram between the degrading compound and the decomposition product.     

Characterization of polymer-based monolithic capillary columns by inverse size-exclusion chromatography and mercury-intrusion porosimetry

Comprehensive two-dimensional gas chromatography (GCxGC) offers favourable resolution and sensitivity compared with conventional one-dimensional gas chromatography (1D-GC), as reported in many studies. These characteristics are of major interest when analytes are in trace concentration, and are present in complex mixtures, as is the case of polycyclic aromatic hydrocarbons (PAHs) in atmospheric particulate samples. Whilst GCxGC has been widely applied to identification of different types of analytes in several matrices, less seldom has it been used for quantification of these analytes. Although several quantitative methods have been proposed, they may be tedious and/or require considerable user development. Whereas quantification in 1D-GC is a routine and well-established procedure, in GCxGC, it is not so straightforward, especially where novel or untested procedures have yet to be incorporated into software packages. In the present study, it is proposed that a subset of the modulated peaks generated for each solute may be summed, based on the specific target ion mass of each compound present in a certified standard reference material (SRM) 1649a (urban dust). The ratio between a PAH and its corresponding deuterated (PAH-d) form showed that there is no statistical loss of sensitivity when this ratio is calculated based on whether the total sum of modulated peaks, or if only the two or the three most intense modulated peaks, are employed. Manual integration may be required, and here was found to give more acceptable values than automatic integration. Automated integration has been shown here to underestimate the modulated peak responses when low concentrations of PAHs were analyzed. Although for most PAHs good agreement with the certified values were observed, the analytical method needs to be further optimized for some of the other PAH, as can be see with those PAH with high variability in the range of urban dust analyzed.     

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).