Evaluation of a retention model in comprehensive two-dimensional gas chromatography

Modeling the retention in comprehensive two-dimensional gas chromatography (GC x GC) was achieved using retention indices obtained in conventional GC. Predicted results were compared with experimental data obtained in the two-dimensional separation of a synthetic hydrocarbon mixture. This proved to be helpful in optimizing the operating conditions of GC x GC separation of a complex petroleum sample and in identifying chemical families.     

The Need for Two‐Dimensional Gas Chromatography: Extent of Overlap in One‐Dimensional Gas Chromatograms

The need for two‐dimensional gas chromatography is justified by the extent of peak overlap in one‐dimensional gas chromatograms (GCs) of complex mixtures. Such overlap was predicted long ago by statistical‐overlap theory (SOT). In this paper, SOT is conceptually reviewed and its predictions are shown to be quantitatively accurate. GCs of complex mixtures of polychlorinated biphenyls, pyridine‐ and nitrogen‐containing polynuclear aromatic hydrocarbons, tetrachlorodibenzo‐p‐dioxins and dibenzofurans, fatty acid methyl esters, flavors and fragrances, and naphtha were simulated by commercial GC software on DB‐1, DB‐5, and Stabilwax stationary phases. The numbers of peak maxima in the GCs agreed with predictions of SOT, when the interval of time between successive peaks of pure compounds was described by Poisson statistics. This agreement was realized even though the time intervals actually are deterministic, not statistical. In addition, the numbers of mixture components were predicted with accuracy by regression of peak numbers against SOT. Similar regressions have been reported before, but the theory used here is more sophisticated and its predictions consequently are more accurate. Future directions for finalizing SOT are suggested.     

Validation of theory of n-column separations with gas chromatograms predicted by commercial software

A probability theory for the average number of compounds resolved by the partial separation of complex mixtures on n columns was tested using commercial-software predictions of gas chromatograms. Such n-column separations are traditional means for addressing peak overlap, in which one chooses additional columns of different selectivity to separate compounds that cannot be separated by a single column. Gas chromatograms of five types of complex mixtures containing from 99 to 283 compounds were predicted for eight stationary phases using both optimized and other temperature programs. The number n of columns for different mixtures varied from 2 to 5. The numbers of compounds separated as singlet peaks at different resolution thresholds were compared to predictions, as evaluated with point-process statistical-overlap theory based on a Poisson distribution. A good agreement between theory and results was found in all cases corresponding to low saturation. Both good and poor agreements were found for cases corresponding to high saturation. A good agreement also was found for results based on resolving complex mixtures by a single column subject to two temperature programs. The moments and distribution of the number of resolved compounds were computed by Monte Carlo simulation, thus gauging the significance of departures between results and theory. The potential of such simulations to explore the limitations of theory was briefly investigated.     

Columns and optimum gradient conditions for fast second-dimension separations in comprehensive two-dimensional liquid chromatography

Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two-dimensional liquid chromatography (LC). We compared suitability of five commercial core-shell columns and one monolithic column for fast gradients in the second LC dimension, where the time of separation is strictly limited by the fraction cycle time. In two-dimensional reversed-phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second-dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second-dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second-dimension gradients with single-step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first-dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two-dimensional retention plane. The approach was applied to the two-dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.     

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.     

Relationship between selectivity and average resolution in comprehensive two-dimensional separations with spectroscopic detection

The average value of the multivariate selectivity (SEL) of randomly positioned peaks in a multi-component separation is shown to equal the average fraction of peaks that are singlets, as predicted by statistical-overlap theory (SOT). This equality is the basis for proposing a useful metric, specifically the average minimum resolution of nearest-neighbor peaks, for the performance of comprehensive two-dimensional (2D) separations. Furthermore this metric was computed both without ancillary spectroscopic information and with the assistance of such help, specifically multi-wavelength UV-vis spectra, acquired during the separation. Separations are simulated with randomly positioned peaks over wide ranges of total number of peaks, first- and second-dimension peak capacity, dimensionless first-dimension sampling time, and spectral diversity. The specific version of the general multivariate selectivity concept that is used here--identified as SEL--gives the relative precision of quantification when using the PARAFAC (parallel factor analysis) method, a popular curve resolution algorithm. The SEL values of all peaks were calculated, averaged, and compared to the predictions of SOT. In the absence of auxiliary spectral data, the SEL-based average minimum resolution required to separate two peaks in a 2D separation is 0.256, compared to resolution of 0.5 if no chemometric assistance is available. This was found to be valid over a wide range of conditions and is essentially independent of peak crowding. With the assistance of the spectral data, the requisite minimum resolution substantially improves, that is, it decreases, especially when peak crowding is severe. The requisite minimum resolution decreases even further, up to a limit, as the spectral diversity is increased. In contrast, the SEL-based average under-sampling correction factor is virtually independent of the presence of the additional spectral data, and additionally is about the same as calculated with SOT from the average number of maxima in closely analogous simulations. The use of selectivity greatly increases the fraction of peaks that are singlets, relative to the number of singlet maxima, especially when spectral assistance is added. The insensitivity of the under-sampling correction factor to either the use of selectivity or added spectral data simplifies optimization of the corrected peak capacity in on-line comprehensive 2D separations.     

The dimensionality of chromatographic separations

The box-counting or capacity dimension algorithm, known from the fractal mathematics literature, is used to measure the dimensionality D of chromatographic separation techniques for any number of dimensions. It is shown that D has limit properties that match Giddings' sample dimensionality s. D values are shown to be sensitive to the uniformity of peak spacing. A number of examples are given where D is calculated for various limits in one- and two-dimensional separations and for heart-cutting separations. The use of D as a quantitative measure of multidimensional orthogonality is suggested as D, due to the scale-free nature, is not dependent on the effective separation area. The connection to statistical peak overlap theory is discussed.     

Enantioselective comprehensive two‐dimensional gas chromatography. A route to elucidate the authenticity and origin of Rosa damascena Miller essential oils

The analysis of Bulgarian and Turkish Rosa damascena Miller essential oils was performed by flow‐modulated comprehensive two‐dimensional gas chromatography using simultaneous detection of the second column effluent by flame ionization and quadrupole mass spectrometric detection. Enantioselective separations were obtained by running the samples on 2,3‐di‐O‐ethyl‐6‐O‐tert‐butyldimethylsilyl‐β‐cyclodextrin column as the first column and on polyethylene glycol as the second column. The determination of enantiomeric or diastereomeric excess of some terpenoic solutes is a possible route for quality or authenticity control as well as for the elucidation of the country of origin.     

Elucidation of fatty acid profiles in vegetable oils exploiting group-type patterning and enhanced sensitivity of comprehensive two-dimensional gas chromatography

The present research is focused on the use of comprehensive 2-D GC (GCxGC) for the thorough elucidation of fatty acid (FA) profiles contained in vegetable oils; the samples analysed consisted of extra-virgin olive oil and refined hazelnut oil. The enhanced sensitivity and the formation of group-type patterns provided by GCxGC enabled the identification and quantification of both well-known and rather unexpected FAs contained in the lipid matrices. Peak assignment was, in most cases, supported by using pure standard compounds. Of particular interest was the identification of a series of odd-numbered FAs in both samples. The results attained to demonstrate the usefulness of GCxGC also for the analysis of supposedly low-complex samples.     

Studies on thermionic ionisation detection in comprehensive two-dimensional gas chromatography

This study explores the application of specific thermionic ionisation detection in comprehensive 2-D GC (GC x GC) and represents the first report of GC x GC with nitrogen phosphorus detection (GC x GC-NPD). Of particular interest is the performance of the NPD with respect to peak parameters of asymmetry and sensitivity. Since GC x GC produces much narrower peaks than obtained with fast GC (e.g. 100 ms vs. <1 s) the effect of detector response time and any lack of symmetry arising from the detection step is important if peak separation (resolution) is to be maintained. It was observed that detector gas flows had a significant impact on peak asymmetry and peak magnitude, and that optimisation of the detector was critical, particularly for complex sample analysis by GC x GC-NPD. Peak asymmetries ranging from As = 1.8 to 8.0 were observed under different conditions of detector gas flows. Comparison of GC x GC-NPD with GC x GC-flame ionisation detection (FID) showed the former to be approximately 20 times more sensitive for the detection of nitrogen-containing methoxypyrazines analytes, and GC x GC-NPD had a larger linear detection range compared to GC x GC-FID. Furthermore, comparison of GC x GC-NPD and GC x GC-TOFMS chromatograms for the analysis of coffee head-space demonstrated the benefits of selective detection, ultimately realised in a comparatively simplified contour plot.     

Screening for pesticide residues in oil seeds using solid-phase dispersion extraction and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

In this paper, we describe the development of an oil-absorbing matrix solid-phase dispersion extraction with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry suitable for screening of 68 pesticide residues (PRs) in peanut, soybean, rape seed, sesame, and sunflower seed. The 68 PRs include 27 kinds of organophosphorus, 23 organic chlorines, 11 synthetic pyrethroids, and 7 carbamates. Heptachlor epoxide was used as the internal standard. Aminopropyl silica was chosen as the dispersion sorbent of the oil-absorbing matrix solid-phase dispersion extraction and was applied to capture hydrophobic components from high oil samples. A 35-min orthogonal separation was performed by using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with a nonpolar-polar column set. Identification of 68 PRs in the extract was finished by using the time-of-flight mass spectrometry in the assistance of an automated peak-find and spectral deconvolution software. A screening based on control design was introduced and explained. This screening method considerably reduced the cost for the quantitative and confirmatory analyses. The quality of present screening method was evaluated by the Document No. SANCO/10684/2009. The false positive rate and false negative rate provide a useful tool for the evaluation of screening performance.     

Resolving overlap in two-dimensional NMR spectra: nuclear Overhauser effects in a polysaccharide

Overlapping resonances in the two-dimensional nuclear Overhauser (NOESY) spectrum of the O-antigenic polysaccharide from Escherichia coli O147 have been resolved by recording a tilted projection of a three-dimensional NOESY-HSQC spectrum, where the carbon-13 and proton evolutions are linked together. Through the introduction of small contributions from the appropriate carbon-13 shifts, a cluster of five unresolved proton peaks is separated into its components without the need to perform the full three-dimensional measurement, a time saving of at least an order of magnitude.     

Comprehensive two-dimensional gas chromatographic analysis of commercial lemon-flavored beverages

Fresh lemon juice and lemon-flavored beverages were analyzed by using comprehensive 2-D GC (GC x GC) with flame-ionization detection, with a nonpolar-polar column combination. A low-alcohol, ready-to-drink (RTD) beverage was also analyzed as fresh, and after deterioration for 12 days at 50 degrees C. Identification of some of the components in the 2-D plots was performed by comparison of peak positions of authentic standards and comparison with 1-D GC. However, without the aid of GC x GC-mass spectral data, only 24 components were identified; a large number of components remained unassigned. In some soft drinks obtained in the market, components indicative of deterioration, such as p-methylacetophenone and p-cymen-8-ol were already present in the products. In contrast, even upon heat challenge, a low-alcohol RTD beverage did not generate deterioration products of citral, such as p-methylacetophenone and the intermediates, p-menth-2-ene-1,8-diols. This was apparently related to the fact that the original formulation contained only a minute amount of the citral ingredient.     

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.     

Characterization of crude oil biomarkers using comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry

Oil samples from Recôncavo basin (NE Brazil), previously analyzed by traditional techniques such as gas chromatography coupled to tandem mass spectrometry, were evaluated using comprehensive two‐dimensional gas chromatography coupled to quadrupole mass spectrometry and comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry along with simplified methods of samples preparation to evaluate the differences and advantages of these analytical techniques to better understand the development of the organic matter in this basin without altering the normal distribution of the compounds in the samples. As a result, the geochemical parameters calculated by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry described better the origin, maturity, and biodegradation of both samples probably by increased selectivity, resolution, and sensitivity inherent of the multidimensional technique. Additionally, the detection of the compounds such as, the C(14α‐) homo‐26‐nor‐17α‐hopane series, diamoretanes, nor‐spergulanes, C₁₉–C₂₆ A‐nor‐steranes and 4α‐methylsteranes resolved and detected by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry were key to classify and differentiate these lacustrine samples according to their maturity and deposition conditions.     

Separation of phenol‐containing pyrolysis products using comprehensive two‐dimensional chromatography with columns based on pyridinium ionic liquids

We describe the application of columns with highly polar stationary liquid phases based on pyridinium ionic liquids for the two‐dimensional chromatography separation of bio‐oil and product of coal pyrolysis. By using inverse combination columns—a first ionic liquid column and a second nonpolar column—good separation results have been obtained. In the analysis of coal pyrolysis products, the suggested approach provides a much better resolution between components in comparison with a less polar first‐dimension column (based on polyethylene glycol). A good selectivity for the peaks of phenols is observed, and the group of phenols is well detached and separated from the group of diaromatics. A good separation picture was obtained also for bio‐oil, the groups of phenols and guaiacol derivatives are distinguished with good resolution of substances within each group.     

Theories to support method development in comprehensive two-dimensional liquid chromatography--a review

On-line comprehensive two-dimensional liquid chromatography techniques promise to resolve samples that current one-dimensional liquid chromatography methods cannot adequately deal with. To make full use of the potential of two-dimensional liquid chromatography, optimization is required. Optimization of two-dimensional liquid chromatography is a relatively new yet important research topic the aim of which is to predict combinations of stationary and mobile phases, column formats, and chromatographic conditions that maximize resolving power and minimize analysis time. In on-line two-dimensional liquid chromatography, dilution-related issues play also an important role and these should be taken into account when developing optimization strategies. In this work, state-of-the-art strategies that support method development for on-line two-dimensional liquid chromatography through a rigorous choice of chromatographic parameters are critically reviewed. The final aim is to provide practitioners with a clear understanding of which aspects can be optimized using current on-line two-dimensional liquid chromatography strategies (and which ones cannot). In two-dimensional liquid chromatography, maximizing resolving power for a given analysis time and dilution requires optimizing efficiency, selectivity and retention. While great strides forward have been made in the optimization of efficiency-related issues, considerable effort needs still to be made in terms of (1) developing models that can predict the retention factors that given stationary/mobile phase systems can provide and (2) using this information for choosing the two ones that maximize two-dimensional liquid chromatography orthogonality. Because of this limitation, in two-dimensional liquid chromatography, this aspect is typically dealt with a posteriori through examining chromatograms. This review clearly shows that important progress in the optimization of on-line two-dimensional liquid chromatography has recently been made.     

In vivo determination of the volatile metabolites of saprotroph fungi by comprehensive two‐dimensional gas chromatography

In this work, we discuss the use of multiway principal component analysis combined with comprehensive two‐dimensional gas chromatography to study the volatile metabolites of the saprophytic fungus Memnoniella sp. isolated in vivo by headspace solid‐phase microextraction. This fungus has been identified as having the ability to induce plant resistance against pathogens, possibly through its volatile metabolites. Adequate culture media were inoculated, and its headspace was then sampled with a solid‐phase microextraction fiber and chromatographed every 24 h over seven days. The raw chromatogram processing using multiway principal component analysis allowed the determination of the inoculation period, during which the concentration of volatile metabolites was maximized, as well as the discrimination of the appropriate peaks from the complex culture media background. Several volatile metabolites not previously described in the literature on biocontrol fungi were observed, as well as sesquiterpenes and aliphatic alcohols. These results stress that, due to the complexity of multidimensional chromatographic data, multivariate tools might be mandatory even for apparently trivial tasks, such as the determination of the temporal profile of metabolite production and extinction. However, when compared with conventional gas chromatography, the complex data processing yields a considerable improvement in the information obtained from the samples.