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.     

Comprehensive two‐dimensional gas chromatography coupled with static headspace sampling to analyze volatile compounds: Application to almonds

The hyphenation of static headspace sampling with comprehensive 2D GC equipped with a modulator based on capillary flow technology and a flame ionization detector was used to separate and identify 43 representative target volatile compounds (light hydrocarbons, carbonyls, pyrazines, alcohols, furans, and benzenes) frequently detected in the roasting process of nuts. Five column combinations with differing degrees of orthogonality (one conventional and four inverted phase sets) were tested in order to obtain the best conditions for analyzing these volatile compounds. Optimization of the working conditions for each of the different column combinations was performed by means of a central composite design. The best results in terms of separation and differentiation among the different chemical groups were achieved with a combination of inverted phase columns (first dimension: highly polar, INNOWax; second dimension: mid‐polar, ZB‐35). Additionally, a reference template was developed to provide an effective and rapid analysis of the target compounds. Finally, the proposed method was successfully employed to identify volatile compounds in raw and roasted almond samples from the Spanish cultivar Largueta.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

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.     

Comparison of GC stationary phases for the separation of fatty acid methyl esters in biodiesel fuels

The fatty acid methyl ester (FAME) content of biodiesel fuels has traditionally been determined using gas chromatography with a polar stationary phase. In this study, a direct comparison of the separation of FAMEs present in various biodiesel samples on three polar stationary phases and one moderately polar stationary phase (with comparable column dimensions) was performed. Retention on each column was based on solubility in and polarity of the phase. Quantitative metrics describing the resolution of important FAME pairs indicate high resolution on all polar columns, yet the best resolution, particularly of geometric isomers, is achieved on the cyanopropyl column. In addition, the separation of four C18 monounsaturated isomers was optimized and the elution order determined on each column. FAME composition of various biodiesel fuel types was determined on each column to illustrate (1) chemical differences in biodiesels produced from different feedstocks and (2) chemical similarities in biodiesels of the same feedstock type produced in different locations and harvest seasons.     

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.     

Covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography and hydrophilic interaction chromatography

The mixed sulfated/methacryloyl polysaccharide derivative was prepared and successfully immobilized onto the surface of porous silica particles by polymerization. Polysaccharide derivative was calculated as 10.33% in the stationary phase prepared. The new stationary phase (PMSP) showed both hydrophilic interaction (HILIC) and per aqueous liquid chromatography (PALC) characteristics. The effects of column temperature, the water content, pH and ion strength of mobile phase on the retention time of test compounds in highly aqueous eluents were investigated to evaluate the PALC features of PMSP. The column efficiency is about 31,000 plates/m for benzoic acid in water/ACN (97/3, v/v) mobile phase at a flow rate of 1.0 mL/min. Compared with C18 column, the PMSP had shorter retention time for weak polar and non-polar compounds, but also showed stronger retention for strong polar compounds. It indicated that PALC was a suitable mode of chromatography as replacement of HILIC and complementarity of reversed-phase liquid chromatography (RPLC).     

Characterization of biodiesel and biodiesel blends using comprehensive two-dimensional gas chromatography

In this work the development of a comprehensive 2-D GC flame ionization detection (GC x GC FID) method for biodiesel fuels is reported. This method is used for the analysis of fatty acid methyl esters (FAMEs) in both biodiesel (B100) and biodiesel blend (B5) samples. The separation of FAME was based on component boiling point in the first dimension and polarity in the second dimension by using a BPX5/BP20 column set to provide a measure of 'orthogonality' in the 2-D space. Here the columns are coupled with a cryogenic modulator operating in a novel temperature programmed mode (T(M)) whereby the cryotrap is progressively incremented in temperature as the oven temperature is increased. The final method employs eight cryotrap temperature settings. The developed GC x GC method is able to successfully characterize and identify both B100 and B5 FAME components, which are produced from a variety of vegetable oils, animal fats and waste cooking oils, with high precision. The method is capable of analysing FAME with carbon numbers C4-C24, and is particularly suitable to characterize various types of biodiesel, making it possible to differentiate the origin and type of FAME used in the biodiesel samples.     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Comprehensive two-dimensional gas chromatography in the analysis of volatile samples of natural origin: a multidisciplinary approach to evaluate the influence of second dimension column coated with mixed stationary phases on system orthogonality

The study evaluates the influence of selectivity tuning of the stationary phase of the second dimension on the orthogonality of a comprehensive two-dimensional gas chromatography (GC x GC) system. Two different sets of columns, providing independent and semi-independent separation mechanisms were used. The first consisted of a first dimension separating analytes on a volatility basis (i.e. a non-polar polydimethylsiloxane (OV1) column) combined with a second dimension separating by polarity, using columns coated with 100% polyethylene glycol (CW20M), CW20M/OV1 mixtures in ratios of 25-75%, and polydimethylsiloxane, 7% phenyl, 7% cyanopropyl (OV1701). The second set consisted of a first dimension separating analytes on a polarity basis (100% CW20M column) combined with a second dimension separating by volatility, consisting of columns coated with 100% OV1, OV1/CW20M mixtures in ratios of 25-75%, and 100% OV1701. Medium-complexity mixtures of natural origin (i.e. peppermint essential oil and a standard mixture of suspected allergens) consisting of components in a relatively limited range of molecular weights (MW) and volatilities, but belonging to different classes of compounds in a wide range of polarity (mono- and sesquiterpenoids, hydrocarbons and oxygenated compounds) were analysed with the above sets of columns. Different approaches were used to evaluate peak spreading on the GC x GC separation plane and degree of orthogonality of the column sets, namely: (1) a Factor Analysis (FA) approach, estimating the correlation coefficients and spreading angles of the sample components in the two-dimensional chromatographic plane; (2) an Informational Theory (IT) approach, based on determining a group of parameters including: informational entropy, % synentropy and similarity (H); and (3) an approach based on estimating the amount of separation space used, i.e. a practical parameter that directly refers to the experimental separation plane of the GC x GC chromatogram. Results showed that peak spreading in the chromatographic plane, when CW20M and OV1 are combined in different ratios, can be predicted from retention mechanisms, and that the degree of orthogonality measured with different approaches, is consistent with the divergent nature, in terms of polarity of the stationary phases combined in the GC x GC system.     

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.     

Twin comprehensive two-dimensional gas chromatographic system: concept and applications

A twin GC x GC system has been designed which enables the analysis of a sample by means of two different and independent column combinations simultaneously. Both combinations are incorporated in the same oven, using the same temperature programme, and are fed using a 50:50 column-entrance-split. It is demonstrated that, employing combinations of a conventional non-polar x polar and a reversed-type polar x non-polar column set, the information content is as high, and the analytical performance is as good as when using two separate GC x GC systems. That is, there is an appreciable gain of time and a reduction of costs without any loss of quality. The general usefulness of performing, and comparing two mutually different GC x GC runs is further illustrated with FAMEs in olive oil, and pollutants in a sediment sample.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.     

Separation of phytosterol oxidation products by combination of different polarity gas chromatography capillary columns

The number of characterized phytosterol oxidation products (POPs) from both ring- and side-chain structures has increased during recent decades, resulting in difficulties in the separation of POPs on different gas chromatography (GC) capillary columns. The main objective of this study was to separate a mixture of 29 purified and characterized oxidation products from sito-, campe- and stigmasterol using GC capillary columns with different polarity. For the first time in the area of POPs analysis, the separation efficiency of the combination of two capillary GC columns with different polarities was investigated. A non-polar 5% phenyl coated (DB5-MS) and a mid-polar 35% phenyl coated (DB35-MS) column was combined with a pressfit connector. The main improvement was enhanced base line separation for many of the analyzed POPs, compared with the separations achieved using the individual columns. However, three pairs of POPs co-eluted: 24-hydroxysitosterol/campesterol-5beta,6beta-epoxide, stigmasterol-5beta,6beta-epoxide/campesterol-5alpha,6alpha-epoxide and stigmasterol-5alpha,6alpha-epoxide/campestanetriol.     

Evaluation of New Stationary Phases for the Separation of Fatty Acid Methyl Esters

In the analysis of fatty acids, one of the most commonly used tools is a GC separation of the fatty acid methyl esters (FAME). Many researchers perform this separation using a non-polar phase such the ubiquitous 5% phenyl / 95% methyl capillary columns found in most every chromatography laboratory. Numerous laboratories have also turned recently to polar phases such as 70% cyanopropyl columns, as this type of chemistry provides increased selectivity for unsaturated compounds, and thus improved separation of cis/trans and ω³/ω⁶ FAME isomers. Here, a series of columns nominally having 60, 70, 80, and 90% bis-cyanopropyl content have been tested for the separation of FAME isomers. Trends in retention and the influence of increasing phase polarity on effective and fractional chain lengths are highlighted to provide the FAME chromatographer with insight into which of these novel stationary phases might be best suited to their particular application. In addition, the elution temperatures (T_e) of the FAME and linear alkane standards are presented, as this information will be of value to comprehensive two-dimensional multidimensional GC (GC × GC) users who wish to use these columns in the primary dimension separation.     

Projection of multidimensional GC data into alternative dimensions—exploiting sample dimensionality and structured retention patterns

Comprehensive multidimensional gas chromatography (GC×GC) is a powerful separation technique. One of the features of this technique is that it offers separations with more apparent structure than that offered by conventional one-dimensional GC (1-D GC). While some previous studies have alluded to this structure, and used structured retention patterns for some simple classifications, the topic of structured retention in GC×GC has not been studied in any great detail. Using the separation of fatty acid methyl esters (FAME) on both nonpolar/polar and polar/nonpolar column sets, the interaction between the separation dimensions and the sample dimensions is explored here. The GC×GC separation of a series of compounds is presented as a projection of the sample from sample space, a p-dimensional space with dimensions defined by the dimensionality of the sample, into separation space: for GC×GC, a two-dimensional plane passing through the sample space in an orientation defined by the separation conditions. Using this conceptual model and some a priori knowledge of the sample, it is shown how the image of the sample in the separation space can be used to construct an image of the sample in alternate dimensions, such as second dimension retention factor (²k) vs. chain length in the case of FAME. These projections into alternate dimensions should facilitate the interpretation of the complex patterns found within the GC×GC chromatogram for the identification and classification of compounds.     

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in "trapping" and separating neurotransmitters

Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough.     

New benchmark for basic and neutral nitrogen compounds speciation in middle distillates using comprehensive two-dimensional gas chromatography

This paper reports an analytical method for the comprehensive two-dimensional gas chromatography (GC x GC) separation and identification of nitrogen compounds (N-compounds) in middle distillates according to their types (basicity). For the evaluation of the best chromatographic conditions, a non-polar x polar approach was chosen. The impact of the second dimension (stationary phase and column length) on the separation of basic and neutral N-compounds was evaluated by mean of two-dimensional resolution. This study revealed that the implementation of polar secondary column having free electron pairs improves drastically the separation of N-compounds. Indeed, the presence of permanent dipole-permanent dipole interactions between neutral N-compounds and the stationary phase was enlightened. The comparison of two different nitrogen chemiluminescence detectors (NCD) was also evaluated for GC x GC selective monitoring of N-compounds. Owing to higher resolution power and enhanced sensitivity achieved using developed chromatographic and detection conditions, it was possible to identify univocally and to quantitate N-compounds (i) by class of compounds and (ii), within a class, by carbon number. Finally, quantitative comparison of GC x GC-NCD with conventional gas chromatography illustrates the benefits of GC x GC leading to an excellent correlation with results obtained by American Society for Testing Materials (ASTM) methods for the determination of basic/neutral nitrogen ratio in diesel samples.     

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 enhanced analysis of naphthas: new column combination involving permethylated cyclodextrin in the second dimension

A new column association using comprehensive two-dimensional gas chromatography for the detailed molecular analysis of hydrocarbon mixtures is reported in this paper. In order to compare the impact of two different secondary columns, a novel column combination relying on a GC x 2GC system was used. This system is based on a non-polar first column (PONA) combined with both a permethylated beta-cyclodextrin (beta-Dex 120) stationary phase and a polysilphenylensiloxane (BPX 50) in the second dimension. Compared to BPX 50 stationary phase, the implementation of beta-cyclodextrin columns as the second dimension was found to improve the resolution between paraffins and naphthenes in the naphtha range but not in the middle distillate range. Attempts to improve the results and to understand the interaction mechanism remained unsuccessful. Therefore, the benefits of the beta-Dex 120-column are only demonstrated on heavy naphtha cut for the quantitation of hydrocarbons.