Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation,dual detectors,and multivariate curve resolution

Comprehensive two-dimensional liquid chromatography (LC × LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC × LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC × LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension (¹D) column to the second dimension (²D) column, and the larger relative background signals. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate ¹D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the ²D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution–alternating least squares (MCR–ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the ²D detector data is used to provide relatively pure analyte spectra to the MCR–ALS algorithm, and the final quantitative results are obtained from the resolved ¹D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC × LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC × LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the ¹D chromatogram – this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC × LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the ²D column.     

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.     

Multiplexed dual second-dimension column comprehensive two-dimensional gas chromatography (GC × 2GC) using thermal modulation and contra-directional second-dimension columns

A multiplexed dual-secondary column comprehensive two-dimensional gas chromatography approach (GC × 2GC) designed for complex sample analysis is introduced. The approach splits the first-dimension column effluent into two second-dimension columns with different stationary phases, and recombines the two streams into one detector post-separation. The approach produces two single two-dimensional chromatograms for each injection. Careful manipulation of thermal modulator timing parameters combined with a novel contra-directional modulation regime facilitates this approach. A selection of 34 laboratory reference compounds containing n-alkanes, alcohols, aromatic hydrocarbons, ketones, esters and halogenated hydrocarbons were analysed to demonstrate the approach. The dual two-dimensional chromatogram from this single detector system provides complementary information due to the unique selectivity of the three separation columns. The results of this proof-of-principle investigation provide significant impetus for further development of GC × 2GC–MS methodology.     

A simple approach to reduce dimensionality from comprehensive two-dimensional liquid chromatography coupled with a multichannel detector

Comprehensive two-dimensional liquid chromatographic (LC × LC) systems play an ever increasing role in separation and characterization of complex samples. When coupled with multichannel detectors, such as the diode array detector, these LC × LC systems become especially useful for non-target analysis and identification of patterns based on the information extracted from those complex samples. Nevertheless, due to the large amount of data generated by these systems, the extraction of useful information for the identification of patterns still is one of the major drawbacks for a wider application of this technique. As a preliminary step in data treatment, we have developed a simple and fast way to deal with this large amount of multi-dimensional data by identifying the three-dimensional (3D) regional maxima of each chromatographic peak generated in a LC × LC–DAD system: retention times at the peak maximum in the first- and second-dimensions and the wavelength of the maximum UV absorption. This dataset is then used to build a 3D fingerprinting of the given sample, which alongside the 3D fingerprinting of other samples, can be used to identify different patterns associated with the specific properties of every sample under study. The applicability of the developed methodology was further assessed by performing a non-target LC × LC–DAD analysis of four Portuguese red wine samples.     

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.     

Application of parallel computing to speed up chemometrics for GC×GC-TOFMS based metabolic fingerprinting

Parallel computing was tested regarding its ability to speed up chemometric operations for data analysis. A set of metabolic samples from a second hand smoke (SHS) experiment was analyzed with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). Data was further preprocessed and analyzed. The preprocessing step comprises background correction, smoothing and alignment of the chromatographic signal. Data analysis was performed by applying t-test and partial least squares projection to latent structures discriminant analysis (PLS-DA). The optimization of the algorithm for parallel computing led to a substantial increase in performance. Metabolic fingerprinting showed a discrimination of the samples and indicates a metabolic effect of SHS.     

Multivariate curve resolution combined with gas chromatography to enhance analytical separation in complex samples: A review

This review describes the major advantages and pitfalls of iterative and non-iterative multivariate curve resolution (MCR) methods combined with gas chromatography (GC) data using literature published since 2000 and highlighting the most important combinations of GC coupled to mass spectrometry (GC–MS) and comprehensive two-dimensional gas chromatography with flame ionization detection (GC × GC-FID) and coupled to mass spectrometry (GC × GC–MS). In addition, a brief summary of some pre-processing strategies will be discussed to correct common issues in GC, such as retention time shifts and baseline/background contributions. Additionally, algorithms such as evolving factor analysis (EFA), heuristic evolving latent projection (HELP), subwindow factor analysis (SFA), multivariate curve resolution-alternating least squares (MCR-ALS), positive matrix factorization (PMF), iterative target transformation factor analysis (ITTFA) and orthogonal projection resolution (OPR) will be described in this paper. Even more, examples of applications to food chemistry, lipidomics and medicinal chemistry, as well as in essential oil research, will be shown. Lastly, a brief illustration of the MCR method hierarchy will also be presented.     

Analytical challenges in doping control: Comprehensive two-dimensional gas chromatography with time of flight mass spectrometry,a promising option

Doping control screening based on the enhanced resolution of comprehensive two-dimensional (2D) gas chromatography hyphenated to time of flight mass spectrometer was investigated. The identification of anabolic agents (clenbuterol, norandrosterone, epimetendiol, two methyltestosterone metabolites and 3′-hydroxystanozolol) contained in a spiked urine sample (2 ng/ml) was demonstrated. Special emphasis was given to 3′-hydroxystanozolol, mainly considering the difficulty in its detection. In contrast to conventional GC–MS approaches that must use single-ion monitoring, the GC × GC–TOFMS method enabled the identification of that metabolite through the deconvolution of the full mass spectrum and also resolved the co-eluted peaks of 3′-hydroxystanozolol and an endogenous component.     

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

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

Quantitative analysis by comprehensive two-dimensional gas chromatography using interval Multi-way Partial Least Squares calibration

A new approach for target quantitative analysis for comprehensive two-dimensional gas chromatography (GC × GC), interval Multi-way Partial Least Square (iNPLS) is presented and evaluated in this paper. In iNPLS, the two-dimensional chromatogram is split in small sections; each of these pieces is treated as an independent new chromatogram. Separated conventional NPLS calibration models for the concentration of the target analyte are built for each of the pieces of the whole chromatogram, and the best model is selected for quantitative analysis. An algorithm for iNPLS running on MatLab platform was written, preliminarily evaluated with using solutions of model compounds with different chemical properties and subsequently applied to quantify some allergens in perfume samples. The results were found to be adequate, and good precision and accuracy was obtained even for poorly resolved peaks.     

Effect of column combinations on two-dimensional separation in comprehensive two-dimensional gas chromatography: Estimation of orthogonality and exploring of mechanism

With the analysis of Chinese liquor Moutai as an example, the effect of different column combinations was studied on two-dimensional separation in comprehensive two-dimensional gas chromatography (GC × GC). A method to optimize column combinations was developed for achieving maximum orthogonality. Using a geometric approach to factor analysis, the degree of separation orthogonality was quantitatively estimated. The parameters evaluated include peak spreading angle, retention correlation, and practical peak capacity. When using the “reversed-type” column combinations (a polar column as the first dimension and a non- or less polar one as the second dimension), correlation coefficient was lower than or equal to 0.221, the spreading angle was higher than or equal to 77°, and more than 92% of the theoretical peak capacity was reasonably used. For Moutai liquor mainly consisting of some polar compounds, the HP-Innowax + DB1701 column combination was optimal. In addition, through the test of Grob mixture and McReynolds constant, the mechanism of solute-stationary phase interactions was disclosed in details, which validated the estimation of GC × GC orthogonality in a molecular level.     

Traceability of honey origin based on volatiles pattern processing by artificial neural networks

Head-space solid-phase microextraction (HS-SPME)-based procedure, coupled to comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC × GC–TOF-MS), was employed for fast characterisation of honey volatiles. In total, 374 samples were collected over two production seasons in Corsica (n = 219) and other European countries (n = 155) with the emphasis to confirm the authenticity of the honeys labelled as “Corsica” (protected denomination of origin region). For the chemometric analysis, artificial neural networks with multilayer perceptrons (ANN-MLP) were tested. The best prediction (94.5%) and classification (96.5%) abilities of the ANN-MLP model were obtained when the data from two honey harvests were aggregated in order to improve the model performance compared to separate year harvests.     

Optimized use of a 50 μm ID secondary column in comprehensive two-dimensional gas chromatography–mass spectrometry

The objective of the present research is directed towards the optimized use of a 50 μm ID secondary column, in a comprehensive two-dimensional gas chromatography–quadrupole mass spectrometry (GC × GC–qMS) system. The analytical aim was achieved by exploiting a split-flow GC × GC approach, and a rapid-scanning qMS instrument. The stationary phase combination consisted of an apolar (silphenylene polymer) 30 m × 0.25 mm ID column, linked by means of a Y-union, to an MS-connected 1 m × 0.05 mm ID polar one [poly(ethyleneglycol)], and to a 0.20 m × 0.05 mm ID uncoated capillary segment; the latter was connected to a manually operated split-valve. It will be herein demonstrated that the split-flow GC × GC approach, successfully employed in previous H₂-based, flame ionization detection experiments, provides equally satisfactory results using mass spectrometric detection and helium as carrier gas. An optimized split-flow GC × GC–qMS method was developed and exploited for the analysis of a perfume sample. The results attained were compared with those observed using the same analytical column combination, but with no flow-splitting. It was found that it is not convenient to employ a 50 μm ID secondary column in a conventional GC × GC–MS instrument. On the contrary, the use a 50 μm ID secondary column, in a split-flow, twin-oven system, provided a good performance. A recently developed comprehensive chromatography software was used for data processing.     

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 critical look at the definition of multidimensional separations

Multidimensional (MD) separations, especially comprehensive two-dimensional (2D) separations such as comprehensive 2D LC (LC × LC), and comprehensive 2D GC (GC × GC), are potentially powerful separation techniques. It is important to have a clear definition of MD techniques to better understand the scope and boundaries of the subject. Widely accepted definitions of MD Separations have their roots in the definition proposed by Giddings. Giddings also added several comments that clarified the scope of his definition. However, some researchers extend Giddings’ definitions beyond their intended scope. Doing so disqualifies such comprehensive 2D techniques as LC × LC, GC × GC and 2D TLC from being considered as 2D techniques. In other instances, extended treatment of Giddings’ definition is used as a basis to justify design-parameters of comprehensive 2D separations despite the fact that these parameters lead to sub-optimal implementations. We believe that the shortcomings in the definition and its popular interpretations are serious enough to warrant attention, especially by those interested in designing optimal instrumentation for MD separations like comprehensive 2D GC. After discussion of the weaknesses in the currently used definitions, we propose to define n-dimensional analysis as one that generates n-dimensional displacement information. We believe that this definition captures the spirit of Giddings’ definition while avoiding the problems associated with its popular interpretations.     

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.     

Design of a compressed air modulator to be used in comprehensive multidimensional gas chromatography and its application in the determination of pesticide residues in grapes

In this study, a new modulator that is simple, robust and presents low operation costs, was developed. This modulator uses compressed air to cool two small portions in the first centimeters of the second chromatographic column of a comprehensive multidimensional gas chromatography (GC × GC) system. The results show a variation in the peak area less than 3 and 5% to alkanes and pesticides, respectively. The standard deviations for the retention times in the first and second dimension are around 0.05 min and 0.05 s for all the compounds. The system was optimized with n-alkanes. The GC × GC system proposed was applied in the determination of pyrethroid pesticides (bifenthrin, cypermethrin, deltamethrin, fenvalerate, esfenvalerate, cis- and trans-permethrin) in grape samples. Samples were extracted by the mini-Luke modified method and pesticides were quantified by comprehensive multidimensional gas chromatography with micro electron-capture detection (μECD). The values of method limit of quantification (LOQ) were 0.01–0.02 mg kg⁻¹ for all studied pyrethroid and the values of recovery were between 94.3 and 115.2%, with good precision (RSD < 18.4%), demonstrating that the performance of the total method consisting of a modified Luke extraction method and determination by GC × GC-μECD are satisfactory. This study also showed that the system using a modulator with a double jet of compressed air has the potential for application in the analysis of a wider range of pesticide residues in other commodities since it provides low values of LOQ with acceptable accuracy and precision.     

Quantitative analysis of biodiesel in blends of biodiesel and conventional diesel by comprehensive two-dimensional gas chromatography and multivariate curve resolution

In this paper, a method to determine the composition of blends of biodiesel with mineral diesel (BXX) by multivariate curve resolution with Alternating Least Squares (MRC-ALS) combined to comprehensive two-dimensional gas chromatography with Flame Ionization Detection (GC × GC-FID) is presented. Chromatographic profiles of BXX blends produced with biodiesels from different sources were used as input data. An initial evaluation carried out after multiway principal component analysis (MPCA) was used to reveal regions of the chromatograms were the signal was likely to be dependent on the concentration of biodiesel, regardless its vegetable source. After this preliminary step MCR-ALS modeling was carried out only using relevant parts of the chromatograms. The resulting procedure was able to predict accurately the concentration of biodiesel in the BXX samples regardless of its origin.     

A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time

Simulated chromatographic data were used to determine the precision and accuracy in the estimation of peak volumes (i.e., peak sizes) in comprehensive two-dimensional liquid chromatography in time (LC × LC). Peak volumes were determined both by summing the areas in the second dimension chromatograms and by fitting the second dimension areas to a Gaussian peak. The Gaussian method is better at predicting the peak volume than the moments method provided there are at least three second dimension injections above the limit of detection (LOD). However, when only two of the second dimension signals are substantially above baseline, the accuracy and precision of the Gaussian fit method become quite poor because the results from the fitting algorithm become indeterminate. Based on simulations in which the modulation ratio (M_R = 4¹σ/t_s) and sampling phase (?) were varied, we conclude for well-resolved peaks that the optimum precision in peak volumes in 2D separations will be obtained when the M_R is between two and five, such that there are typically four to ten second dimension peaks recorded over the eight σ width of the first dimension peak. This sampling rate is similar to that suggested by the Murphy–Schure–Foley criterion. This provides an RSD of approximately 2% for the signal-to-noise ratio used in the present simulations. The precision of the peak volume of experimental data was also assessed, and RSD values were in the range of 4–5%. We conclude that the poorer precision found in the LC × LC experimental data as compared to LC may be due to experimental imprecision in sampling the effluent from the first dimension column.     

Application of head-space solid-phase microextraction coupled to comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the determination of multiple pesticide residues in tea samples

A new method has been developed to detect 36 pesticides that may contaminate tea samples (green, black and fruit tea). The hyphenation of solid-phase microextraction in head-space mode with a comprehensive two-dimensional gas chromatography coupled with high-speed time-of-flight mass spectrometry (HS-SPME-GC × GC/TOF MS) proved to be a quick alternative to conventional GC/MS methodology which employs solvent-based extraction. The key parameters for controlling HS-SPME performance were optimized, including fiber coating type, temperature and absorption time settings and tea matrix modification by adding water. Quantification was carried out using matrix-matched calibration. The repeatability of measurements, expressed as relative standard deviation (R.S.D.), was less than 24% for all analytes. The limits of quantification ranged from 1 to 28 μg kg⁻¹. The optimized method was applied to analyze real life samples obtained from a retail market. Results generated by the new SPME procedure and those obtained by using a conventional one involving ethyl acetate extraction and high-performance gel permeation chromatography (HPGPC) clean up agreed with each other for positive (containing residue) samples.