Secondary Column Research Articles

Microfluidic Deans Switch for Comprehensive Two-Dimensional Gas Chromatography

A microfluidic Deans switch was used as a comprehensive two-dimensional gas chromatography (GCxGC) modulator. The simplicity and wide temperature range of the Deans switch make it a promising alternative to existing modulation techniques. However, the Deans switch is a low duty cycle modulator; that is, it samples only a small portion of the primary column effluent. Like all low duty cycle modulators, the Deans switch produces inconsistent transfer of components from the primary to the secondary column if the primary peaks are undersampled. Theoretical simulations and experimental studies show that the relative standard deviation (RSD) of the fraction of material transferred from the primary column to the secondary column is less than 1% if the modulation ratio is greater than 2.5. But the RSDs increase rapidly as the modulation ratio is decreased below 2.5. Deans switch GCxGC was validated by analyzing the aromatic content of gasoline. A fast analysis (<10 min) produced narrow primary peaks and a modulation ratio of 1.7. The quantitative results were in good agreement with results obtained with differential flow modulation GCxGC and GC/MS, but the RSDs of single-component levels were approximately three times greater. The Deans switch modulator was also used for a slower gasoline analysis (33 min run time) that produced modulation ratios near 5. In this case, the quantitative results and RSDs were in excellent agreement with the differential flow GCxGC and GC/MS results. These studies demonstrate that a Deans switch can be an effective modulator provided that modulation ratios greater than approximately 2.5 are employed.

Multidimensional liquid chromatography system with an innovative solvent evaporation interface

An orthogonal two-dimensional liquid chromatographic (2D-LC) system was developed by using a vacuum-evaporation loop-type valve interface. Normal-phase liquid chromatography (NPLC) with a bonded CN phase column was used as the first dimension, and reversed-phase liquid chromatography (RPLC) with a C 18 column was used as the second dimension. All the solvents in the loop of the interface were evaporated at 90 °C under vacuum conditions, leaving the analytes on the inner wall of the loop. The mobile phase of the second dimension dissolved the analytes in the loop and injected them onto the secondary column, allowing an on-line solvent exchange of a selected fraction from the first dimension to the second dimension. The chromatographic resolution of analytes on the two dimensions was maintained at their optimal condition. Sample loss due to evaporation in the interface was observed that depended on the boiling point of the compound. Separation of sixteen polycyclic aromatic hydrocarbon mixtures and a traditional Chinese medicine Angelica dahurica was demonstrated.

Generating Multiple Independent Retention Index Data in Dual-secondary Column Comprehensive Two-dimensional Gas Chromatography

A method producing simultaneously three retention indexes for compounds has been developed for comprehensive two-dimensional gas chromatography by using a dual secondary column approach (GC x 2GC). For this purpose, the primary flow of the first dimension column was equally diverted into two secondary microbore columns of identical geometry by means of a three-way flow splitter positioned after the longitudinally modulated cryogenic system. This configuration produced a pair of comprehensive two-dimensional chromatograms and generated retention data on three different stationary phases in a single run. First dimension retention indexes were determined on a polar SolGel-Wax column under linear programmed-temperature conditions according to the van den Dool approach using primary alcohol homologues as the reference scale. Calculation of pseudoisothermal retention indexes in both second dimensions was performed on low-polarity 5% phenyl equivalent polysilphenylene/siloxane (BPX5) and 14% cyanopropylphenyl/86% dimethylpolysiloxane (BP10) columns. To construct a retention correlation map in the second dimension separation space upon which KovAts indexes can be derived, two methods exploiting "isovolatility" relationships of alkanes were developed. The first involved 15 sequential headspace samplings of selected n-alkanes by solid-phase microextraction (SPME), with each sampling followed by their injection into the GC at predetermined times during the chromatographic run. The second method extended the second dimension retention map and consisted of repetitive introduction of SPME-sampled alkane mixtures at various isothermal conditions incremented over the temperature program range. Calculated second dimension retention indexes were compared with experimental values obtained in conventional one-dimensional GC. A case study mixture including 24 suspected allergens (i.e., fragrance ingredients) was used to demonstrate the feasibility and potential of retention index information in comprehensive 2D-GC.

Implementing a SIMS ion source on the NRL trace element accelerator mass spectrometer

The NRL trace element accelerator mass spectrometer facility has recently added a modified Cameca IMS 6F SIMS instrument as a high-performance ion source, providing the advantages of accelerator mass spectrometry to SIMS analysis. These advantages include removal of molecular interferences, a greatly reduced background, and composition analysis using molecular ions (after breaking them into atomic constituents before detection). This last feature compensates for a requirement to use only negative secondary ions, as it enables measurement of electropositive elements. In addition, the NRL facility provides for parallel mass analysis, thus improving isotope ratio precision and practical sensitivity. Spatial analysis of the sample is still provided by raster of the primary Cs beam. The modified Cameca includes the Cs primary column, sample chamber, and the secondary ion column up to the standard electrostatic analyzer. The NRL accelerator facility supplants the normal mass spectrometer portion of the IMS 6F instrument. Additional recent facility upgrades provide improved mass spectrometry performance, both for a prior radiocarbon ion source and the now installed SIMS. An example measurement, using silver ions, demonstrates the system's capabilities.

Towards a comprehensive 2‐D‐LC–MS separation

A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column. This cycle is repeated throughout the chromatogram. The use of dual secondary columns provides the secondary columns with additional time to generate high-speed chromatograms. Each secondary column generates alternate chromatograms which when combined generate the entire chromatogram. The primary column separation is comparable to conventional HPLC, whereas the secondary column separation is fast. With both the columns operating in reverse phase mode, one would expect strong correlation in the two-dimensional retention and hence inefficiency in separation. However, differences in column operation modes, interaction mechanisms, and vendor silica result in a complementary separation. The system was evaluated by comparing it to one-dimensional counterparts and coupled column chromatography. Although some correlations were observed in 2-D-LC-MS, peaks do show two-dimensional distribution with superior UV and MS data as co-elution is minimized. Also, the ease of converting conventional systems to 2-D-LC-MS is discussed.

Determination of beef tallow in lard through a multidimensional off‐line non‐aqueous reversed phase–argentation LC method coupled to mass spectrometry

The presence of tallow in lard is not easy to determine, due to the similarity of the composition of these two animal fats, which differ mainly in the distribution of fatty acids (FA) in the three positions of the glycerol molecule. The determination of the composition of the triacylglycerol (TAG) fraction of lard, tallow, and their mixtures was investigated by HPLC in combination with atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The presence of tallow in lard was determined through the study of the sn-POP/sn-PPO ratio by multidimensional HPLC. The off-line bidimensional system was attained through the coupling of non-aqueous reversed phase (NARP)-HPLC and silver ion (Ag)-HPLC. The primary column eluate was fractionated and the fraction containing POP/PPO isomers was injected onto the secondary column, allowing the separation of positional isomers, unresolved in the first dimension. Peak assignment was carried out by combining retention data with APCI-MS spectral information. The fatty acid distribution along the glycerol backbone, determined by Ag-HPLC, was confirmed through diglyceride ion ratios derived from APCI-MS analysis. Method validation was carried out in preliminary applications on standard TAGs. The analytical results obtained show that even a 5% addition of tallow to lard modifies the distribution of positional isomers.

Effect of first-dimension column film thickness on comprehensive two-dimensional gas chromatographic separation

In comprehensive two-dimensional gas chromatography (GC × GC), samples experience two-dimensional separation implemented by a modulator which helps preserve the first-dimension separation and facilitates the second-dimension separation by periodically collecting, focusing and launching the material from the primary column onto the secondary column with a different stationary phase. Column overloading in GC × GC is a considerable problem, aggravated by the fact that two columns are involved. Broad first-dimension peaks of an analyte help produce smaller fractions of the analyte in the second-dimension, reducing the chance of secondary column overloading. One of the means to generate broad peaks in the first-dimension is to use thick film primary columns. A series of primary columns of various film thickness were tested in the study, and the results indicate that when other conditions are kept constant, 1 μm film columns often provide better resolution in both first and second-dimension but at the expense of a much longer separation time; 0.1 μm is clearly inadequate for GC × GC separation; 0.5 and 0.25 μm film columns seem to be the best compromises.

Two-dimensional liquid chromatography with mixed mode stationary phases

Mixed mode stationary phases with ion-pairing reagent (acidic or basic) as integral part of hydrophobic chain offers unique selectivity, and hence, are ideal for multidimensional separations. The retention of hydrophobic components is a function of organic content, whereas that of charged species is a function of organic content, ionogenic modifier and its level in the mobile phase. Hence, by controlling the parameters influencing component retention (stationary phase and mobile phase), the selectivity of chemical components in the two-dimensional plane can be manipulated to improve the separation. A two-dimensional liquid chromatograph has been developed by coupling similar and dissimilar mixed mode stationary phases in the two dimensions. This technique has immense potential in resolving co-eluting components as the retention mechanism in the two-dimensions are complementary. However, with only part of the primary column eluent sampled into the secondary column, the technique is limited to qualitative analysis.

Partial modulation method via pulsed flow modulator for comprehensive two-dimensional gas chromatography.

A partial modulation method by using a pulsed-flow modulator for comprehensive two-dimensional gas chromatography is proposed. The method is based on the fact that when a pulsed flow of inert gas is introduced into the conjunction between a primary and a secondary column, the concentration of analyte is disturbed, and a plug of higher or lower concentration is created. The plug, which forms a spike signal coupled to the primary GC signal, is then separated in a secondary column, creating a new dimension of GC information. The modulation is partial because only a fraction of the primary signal is modulated and converted into the secondary signal; the remaining primary signal stays unchanged. Therefore, this method yields a comprehensive two-dimensional chromatogram and a primary one-dimensional chromatogram in a single GC run. In this study, the modulation mode, modulation index, and modulation percentage are discussed and the reproducibility of peak areas and retention time are investigated. With a 5.8% modulation percentage and a primary peak half-width 1.7 times wider than the modulation time, the standard deviation for the peak areas are 0.15% for the primary and 0.78% for the secondary chromatograms. Chromatograms of laboratory-mixed hydrocarbons and of high-temperature fuel oil no. 6 standard are demonstrated.

Simulation and optimisation of extractive distillation with water as solvent

Acetone–methanol, methyl acetate–methanol and methanol–chloroform binary extractive single column distillation systems were simulated with the HYSYS software platform, to investigate the effects of solvent feed entry stages, solvent split stream feed and solvent condition on the separation. Water was used in all of the simulations as the solvent. The simulations supported data and findings from experimental column studies of the same systems. A rigorous simulation of the acetone–methanol system including a secondary stripping column and recycle loop was established to simulate an industrially relevant situation. This simulation enabled an economic evaluation of the process to be made. It was found for feed mixtures containing 25, 50 and 75 mol% methanol, the optimum reflux ratios were found to be 3.5, 3.5 and 4.2, respectively. As a consequence one column design could separate binary feed of varying composition between 25 and 75% methanol. The optimum number of ideal stages for the primary column for an equimolar binary feed was determined to be 73. When maintaining a constant solvent flow, the distance between the split feed entry stages had no effect on the economic potential (EP) of the system.

Off-line coupling of non-aqueous reversed-phase and silver ion high-performance liquid chromatography–mass spectrometry for the characterization of rice oil triacylglycerol positional isomers

The determination of the triacylglycerol (TAG) profile in real world matrices is rather difficult as these compounds present a complex composition and are characterized by similar physico-chemical properties. This investigation is based on the high-performance liquid chromatography (HPLC) multidimensional determination of the TAG profile in terms of TAG species and positional isomers in a rice oil sample. The off-line bi-dimensional system was attained through the coupling of non-aqueous reversed-phase HPLC and silver ion (Ag)-HPLC. The primary column eluate was fractionated and the fractions of interest were then injected onto the secondary column, allowing the separation of several TAG positional isomers, unresolved in the first dimension. Peak assignment was carried out by combining retention data with atmospheric pressure chemical ionisation (APCI) MS spectra information. The fatty acid distribution along the glycerol backbone, determined by Ag-HPLC, was confirmed through diglyceride ion ratios derived from APCI-MS analysis. Method validation, where both precision and accuracy were measured, was carried out in preliminary applications on standard compounds. The analytical results obtained show that rice oil TAGs follow a distribution which can be considered typical for vegetable oils.

Comprehensive two-dimensional normal-phase (adsorption)-reversed-phase liquid chromatography.

A comprehensive two-dimensional HPLC system has been developed. It is based on the use of a microbore silica column operated in normal-phase (adsorption) mode (NP) in the first dimension and a monolithic type C18 column operated in reversed-phase (RP) mode in the second dimension. The interface was a 10-port, 2-position valve equipped with two storage loops. The first column was operated at a flow rate of 20 microL/min in isocratic mode, while the monolithic column flow rate was 4 mL/min and was operated in gradient mode. The sample loops had a volume of 20 microL each, and the analysis time in the second dimension was 1 min. In this way, every fraction from the first dimension was transferred on-line to the second dimension switching the automated valve every minute. A photodiode array detector has been used after the secondary column. The use of normal- and reversed-phase mode in the two dimensions can be helpful in the separation of complex mixtures of a natural origin that contain uncharged molecules of comparable dimension, different in polarity and hydrophobicity. The use of a microbore column in the first dimension permits the injection of a small volume in the secondary column, making the transfer of incompatible solvents from the first to the second dimension possible. Since the mobile phase in the NP separation is always stronger than the mobile phase at the head of the secondary column operated in RP mode, the initial eluent strength is important in order to obtain an effective focusing of the sample. The use of a monolithic type column in the second dimension permits the performance of very fast analysis operating at higher flow rates without loss of resolution, due to a higher permeability and increased mass-transfer properties in comparison to conventional particulate columns. Due to the brief reconditioning time necessary for monolithic columns, repetitive gradients can be carried out, extending the field of application to mixtures that contain components with different polarities. The utility of the system has been demonstrated in the analysis of the oxygen heterocyclic fraction of cold-pressed lemon oil, made up of coumarins and psoralens. These components may contain hydroxyl, methoxyl, isopentenyl, isopentenyloxyl, and geranyloxyl groups and oxygen-containing modification of the terpenoid side-chain groups, such as epoxides or vicinal diol groups. The relative location of the components in the 2D plane varied in relation to their chemical structure and allowed positive peak identification. The UV spectra recorded with the photodiode array detector supplied additional information that was used for the characterization of the studied sample.

Chromatographic Evaluation of Resolution and Secondary Mechanisms of Pure and Mixed Sets of SEC Columns: TSK-Gel HHR and TSK-Gel HXL

Size exclusion chromatographic (SEC) evaluation of secondary mechanisms and column specific resolution, of five solvent/polymer systems in four sets of pure and mixed organic columns packings based on polystyrene/divinylbenzene copolymer, TSK-Gel HHR and TSK-Gel HXL, has been carried out. The combination of columns employed has been: (i) three HHR columns (set A); (ii) two HHR and one HXL column (set B); (iii) one HHR and two HXL columns (set C) and (iv) three HXL columns (set D). Both packings offer similar characteristics (pore size, particle size, efficiency) but some differences have been found when eluting the same systems in different combination of both of them. Values of the chromatographic partition coefficient, Kp, of the volume fraction of the network in the swollen state, φ3, and the concentration effect on the retention volume have been related. It can be concluded that the higher the φ3, the higher the crosslinking degree and Kp, and the higher the concentration effect on the retention volume for a given solvent/polymer system. We also observe that, in general, a decrease of Kp comes along with an increase of specific resolution, RS, and that the sets of mixed columns show lower secondary effects different from pure size exclusion (lower Kp) and higher RS than the two sets of pure HHR or HXL columns.

Flow-switching device for comprehensive two-dimensional gas chromatography

A simple flow-switching device has been developed as a differential flow modulator for comprehensive two-dimensional gas chromatography (GC×GC). The device is assembled from tubing, four tee unions, and a solenoid valve. The solenoid valve is located outside the oven of the gas chromatograph and is not in the sample path. The modulation technique has no inherent temperature restrictions and passes 100% of the primary column effluent to the secondary column(s). Secondary peaks are produced with widths at half maximum less than 100 ms when operating in GC×2GC mode with a 2.0 s modulation period. The efficacy of this approach is demonstrated through the analysis of a standard mixture of volatile organic compounds (VOCs) and diesel fuel.

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).

An automated orthogonal two-dimensional liquid chromatograph.

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.

An automatic method for the determination of carcinogenic 1-nitropyrene in extracts from automobile exhaust particulate matter.

An automatic method for the determination of carcinogenic 1-nitropyrene in extracts from automobile exhaust particulate matter has been developed. Organic matter was extracted from particulate matter and the extract was concentrated. Then the extract was injected into a two-dimensional (2D) high-performance liquid chromatograph (HPLC) with a fluorescence detection system. To improve the sensitivity a large sample volume (300 microl) was applied to the HPLC system using the double on-column focusing technique. To achieve full automation and high selectivity a reduction column packed with platinum black was introduced after the 2D HPLC system using a C18 column as the primary column and a pentabromobenzyl column as the secondary column. This HPLC system could determine 0.01-3,000 ng ml(-1) of 1-nitropyrene in an extract from diesel and gasoline exhaust particulate matter at 40 min intervals.

Theoretical study of incomplete sampling of the first dimension in comprehensive two-dimensional chromatography

The process of regularly transferring material from the primary column to the secondary column is critical in producing comprehensive two-dimensional separations. A series of calculations have been performed to determine how sampling period, duty cycle, and sampling phase affect (1) the fraction of material transferred from the primary column to the secondary column, (2) the accuracy of primary retention time determination, and (3) the effective peak width along the primary retention axis. The results demonstrate that comprehensive two-dimensional separations can be produced without a substantial loss in quantitative precision and with only a moderate loss in primary column resolution if the sampling period is less than 1.5 times the primary peak standard deviation. The quantitative precision of total peak areas (for duty cycles less than 1.0) and primary retention time determination are rapidly reduced as the sampling period is increased above 1.5 times the primary peak standard deviation.

Application of comprehensive two-dimensional gas chromatography (GC×GC) to the enantioselective analysis of essential oils

A new method for the enantioselective analysis of essential oils is described, using comprehensive two-dimensional gas chromatography (GC×GC). The column set comprised a primary column containing a cyclodextrin derivative as a chiral selector, and a secondary column containing a polyethylene glycol stationary phase. A cryogenic modulation system was used to achieve the GC×GC experiment. The enantiomeric compositions of a number of monoterpene hydrocarbons and oxygenated monoterpenes in Australian tea tree (Melaleuca alternifolia), including α-thujene, sabinene, β-pinene, α-phellandrene, limonene, trans-sabinene hydrate, cis-sabinene hydrate, linalool, terpinen-4-ol, and α-terpineol are reported. The GC×GC resolution advantage is shown to improve the efficiency of enantioselective essential oil analyses. In a single temperature programmed analysis, the individual antipodes of optically active components can be separated, and are effectively free from matrix interferences.

Application of comprehensive two-dimensional gas chromatography (GC×GC) to the enantioselective analysis of essential oils

A new method for the enantioselective analysis of essential oils is described, using comprehensive two-dimensional gas chromatography (GC×GC). The column set comprised a primary column containing a cyclodextrin derivative as a chiral selector, and a secondary column containing a polyethylene glycol stationary phase. A cryogenic modulation system was used to achieve the GC×GC experiment. The enantiomeric compositions of a number of monoterpene hydrocarbons and oxygenated monoterpenes in Australian tea tree (Melaleuca alternifolia), including α-thujene, sabinene, β-pinene, α-phellandrene, limonene, trans-sabinene hydrate, cis-sabinene hydrate, linalool, terpinen-4-ol, and α-terpineol are reported. The GC×GC resolution advantage is shown to improve the efficiency of enantioselective essential oil analyses. In a single temperature programmed analysis, the individual antipodes of optically active components can be separated, and are effectively free from matrix interferences.