Second Dimension Separation Research Articles

Development of a Two-Dimensional Supercritical Fluid Chromatography System in Multiple Heart-Cutting Modes.

In this study, we present the development of a loop-based two-dimensional supercritical fluid system in multiple heart-cutting modes (mSFC-SFC), with diode-array and mass spectrometric detection. The instrument design was developed to be as simple as possible, based on a single SFC instrument, with the sole addition of three external 2-port 6-position valves. The objective was to achieve the most complete transfer of a peak from the first to the second dimension, whatever the composition of the mobile phase, i.e., whatever the proportion of carbon dioxide and methanol cosolvent along a wide gradient elution. Thanks to fine adjustment of the valve switching times, the first-dimension peaks were parked in 50 μL or 100 μL loops and later discharged to the second dimension. The interest of this instrument was then demonstrated with a sample application on a natural product: an extract of Citrus aurantium L. bark was analyzed, with a particular focus on chiral flavonoids, neohesperidin, and naringin. In this system, the first dimension was an achiral separation of the flavonoids, based on a polar diethylamine-bonded silica stationary phase (ACQUITY Torus DEA), while the second dimension used a stereoselective polysaccharide stationary phase (CHIRALPAK IB-3) to resolve flavonoid diastereomers. Excellent repeatability was demonstrated, with relative standard deviation values on retention times and peak areas all below 2%, together with excellent peak capacity and peak shapes (no distortion observed), making it possible to quantify diastereomers in the second-dimension separation. This good repeatability was also shown for the transfer rate between the two dimensions, which reached a value of 83%. Finally, transferring a compressible sample from the first to the second dimension is demonstrated to yield excellent performance, despite the large loop volume.

Coupling multidimensional chromatography with plasmonic sensing: an exploration of electrochemical SERS as a detection modality for 2D-LC

Complex mixtures that may contain hundreds, if not thousands, of unique compounds pose an analytical challenge for complete and detailed analyses. For example, green tea is a well-known complex substance requiring advanced separation techniques and ultrasensitive detection methods for full characterization. Separation and identification of components in green tea help gauge tea quality, as green tea is one of the most consumed beverages in the world. In this work, the coupling of two-dimensional liquid chromatography (2D-LC) and electrochemical surface-enhanced Raman spectroscopy (EC-SERS) was explored for the first time to analyze compounds in green tea. 2D-LC offers enhanced separation compared to conventional HPLC systems due to the second dimension of separation. EC-SERS was used as an offline detection modality and qualitative tool to help identify the compounds in green tea fractions collected from the second dimension separation. 2D-LC and EC-SERS are coupled in this work for the first time and offer a pathway forward for sensitive and selective separation and identification of components in complex mixtures.

Three-dimensional characteristic chromatogram by online comprehensive two-dimensional liquid chromatography: Application to the identification and differentiation of ginseng from herbal medicines to various Chinese patent medicines

One bottleneck problem in the quality control of traditional Chinese medicine (TCM) is the accurate identification of easily confused herbal medicines from Chinese patent medicine (CPM). Ginseng products derived from the multiple parts (e.g., root/rhizome, leaf, and flower bud) of multiple Panax species (P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. japonicus var. major) are globally popular; however, their authentication is very challenging. Using online comprehensive two-dimensional liquid chromatography (LC × LC), we propose the concept of a three-dimensional characteristic chromatogram (3D CC) by integrating enhanced LC × LC separation and a contour plot that visualizes the stereoscopic chromatographic peaks and examine its performance in authenticating various ginseng products. Targeted at the resolution of 17 ginsenoside markers, an online LC × LC/UV system with a 56 min analysis time was constructed: a CORTECS UPLC Shield RP 18 column running at 0.1 mL/min for the first-dimensional chromatography and a Poroshell SB-Aq column at 2.0 mL/min in shift gradient mode in the second dimension of separation. In particular, ginsenosides Rg1/Re and Rc/Ra1 were well resolved. According to the presence/absence of stereo peaks consistent with the main ginsenoside markers in the 3D CC and the depth of shade (depending on peak volume), it was feasible to use a single method to identify and distinguish among 12 different ginseng species as the drug materials and the use of ginseng simultaneously from 21 CPMs. Conclusively, a practical solution enabling the accurate identification of easily confused TCMs was provided, covering both the drug materials and the compound preparations.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa.Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape.The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.

Packed modulation loops to reduce band broadening in two-dimensional liquid chromatography

Modulation interfaces employing sample loops are applied in many hyphenated separations such as two-dimensional liquid chromatography (2D-LC). When the first-dimension effluent in 2D-LC is eluted from the modulation loop, dispersion effects occur due to differences in the laminar flow velocity of the filling and emptying flow. These effects were recently studied by Moussa et al. whom recommended the use of coiled loops to promote radial diffusion and reduce this effect. In the 1980s, Coq et al. investigated the use of packed loops, which also promote radial diffusion, in large volume injection 1D-LC. Unfortunately, this concept was never investigated in the context of 2D-LC modulation. Our work evaluates use of packed loops in 2D-LC modulation and compares them to unpacked coiled and uncoiled modulation loops. The effect of the solvents, loop volume, differences in filling and emptying rates, and loop elution direction on the elution profile was investigated. Statistical moments were used as a pragmatic tool to quantify elution profile characteristics. Decreased dispersion was observed in all cases for the packed loops compared to unpacked loops and unpacked coiled loops. In particular for larger loop volumes the dispersion was reduced significantly. Furthermore, countercurrent elution resulted in narrower elution profiles in all cases compared to concurrent elution. We found that packed modulation loops are of high interested when analytes are not refocussed in the second-dimension separation (e.g. for size-exclusion chromatography). Moreover, our work suggests that the use of packed loops may aid in prevention of loop overfilling.

Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns

Comprehensive characterization of the lipidome remains a challenge requiring development of new analytical approaches to expand lipid coverage in complex samples. In this work, offline two-dimensional liquid chromatography-mass spectrometry was investigated for lipidomics from human plasma. Hydrophilic interaction liquid chromatography was implemented in the first dimension to fractionate lipid classes. Nine fractions were collected and subjected to a second-dimension separation utilizing 50 cm capillary columns packed with 1.7 µm C18 particles operated on custom-built instrumentation at 35 kpsi. Online coupling with time-of-flight mass spectrometry allowed putative lipid identification from precursor-mass based library searching. The method had good orthogonality (fractional coverage of ∼40%), achieved a peak capacity of approximately 1900 in 600 min, and detected over 1000 lipids from a 5 µL injection of a human plasma extract while consuming less than 3 mL of solvent. The results demonstrate the expected gains in peak capacity when employing long columns and two-dimensional separations and illustrate practical approaches for improving lipidome coverage from complex biological samples.

Flow Dynamics and Analyte Transfer in a Microfluidic Device for Spatial Two-Dimensional Separations

In the last decade, chip-based separations have become a major area of interest in the field of separation science, especially for the development of “spatial” two-dimensional liquid chromatography (xLC × xLC). In xLC × xLC, the analytes are first separated by migration to different positions in a first-dimension (1D) channel and subsequently transferred with the aid of a flow distributor in a perpendicular direction to undergo a second-dimension (2D) separation. In this study, several designs for 2D separations are explored with the aid of computational fluid dynamics simulations. There were several aims of this work, viz. (1) to investigate the possible anomalies arising from the location of analyte bands in the first-dimension channel before transfer to the second dimension induced by the flow distributor, (2) to study the distribution ratio of the analytes across the different outlets of the 1D channel, and (3) to study the flow behaviour confinement in the flow distributor. In all designs, the simulated absolute flow velocity was not equal in all regions of the 1D channel. The extreme segments showed higher velocities compared to the central zones. This will eventually influence the migration times (first moments) and the variances (second moments), as confirmed by CFD results. The study has contributed to the understanding of the effects of the peak locations and, ultimately, to progress in spatial 2D-LC separations.

Evaluation of Gas Chromatography Columns with Radially Elongated Pillars as Second-Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

The present study investigated the use of a dedicated gas chromatography (GC) column (L = 70 cm, 75 μm deep, and 6.195 mm wide) with radially elongated pillars (REPs) as the second column in a comprehensive two-dimensional gas chromatography (GC × μGC) system. Three stationary phases [apolar polydimethylsiloxane (PDMS), medium polar room-temperature ionic liquid (RTIL) based on monocationic phosphonium, and polar polyethylene glycol (PEG-1000)] have been coated using the static method at constant pressure or using an original vacuum pressure program (VPP) from 400 to 4 mbar. The best efficiency reached up to N = 62,000 theoretical plates for a film thickness of 47 nm at 100 °C for an iso-octane peak (k = 0.16) at an optimal flow rate of 4.8 mL/min. The use of the VPP improved the efficiency by approximately 15%. Efficiencies up to 28,000 and 47,000 were obtained for PEG-1000 and RTIL, respectively. A temperature-programmed separation of a mixture of 11 volatile compounds on a PDMS-coated chip was obtained in less than 36 s. The PDMS-, PEG-1000-, and RTIL-coated chips were tested as the second column using a microfluidic reverse fill/flush flow modulator in a GC × μGC system. The REP columns were highly compatible with the operating conditions in terms of flow rate and with more than 30,000 plates for the iso-octane peak. Moreover, a commercial solvent called white spirit containing alkanes and aromatic compounds was injected in three sets of columns in normal and reverse modes, demonstrating the great potential of the chip as a second-dimension separation column.

Combining Photodegradation in a Liquid-Core-Waveguide Cell with Multiple-Heart-Cut Two-Dimensional Liquid Chromatography

Photodegradation greatly affects everyday life. It poses challenges when food deteriorates or when objects of cultural heritage fade, but it can also create opportunities applied in advanced oxidation processes in water purification. Studying photodegradation, however, can be difficult because of the time needed for degradation, the inaccessibility of pure compounds, and the need to handle samples manually. A novel light-exposure cell, based on liquid-core-waveguide (LCW) technology, was embedded in a multiple-heart-cut two-dimensional liquid chromatography system by coupling the LCW cell to the multiple-heart-cut valve. The sample was flushed from the heart-cut loops into the cell by an isocratic pump. Samples were then irradiated using different time intervals and subsequently transferred by the same isocratic pump to a second-dimension sample loop. The mixture containing the transformation products was then subjected to the second-dimension separation. In the current setup, about 30–40% of the selected fraction was transferred. Multiple degradation products could be monitored. Degradation was found to be faster when a smaller sample amount was introduced (0.3 μg as compared to 1.5 μg). The system was tested with three applications, that is, fuchsin, a 19th-century synthetic organic colorant, annatto, a lipophilic food dye, and vitamin B complex.

Characterization and comparison of smokeless powders by on-line two-dimensional liquid chromatography

Smokeless powders (SPs) are one of the most commonly used propellants for ammunition but can also be abused as energetic material in improvised explosive devices (IEDs) such as pipe bombs. After a shooting or explosion, unburnt or partially burnt particulates may be observed which can be used for forensic investigation. SPs comprise mainly nitrocellulose (NC) and additives. Therefore, the characterization of both NC and the additives is of significant forensic importance. Typically, the identification, classification, and chemical profiling of smokeless powders are based exclusively on the analysis of the additives. In this study, information regarding the NC base component was combined with the chemical analysis of the additives using two-dimensional liquid chromatography (2D-LC). The system combines size-exclusion chromatography (SEC) and reversed-phase liquid chromatography (RPLC) in an on-line heart-cut 2D-LC configuration. In the first dimension, the NC is characterized by its molecular-weight distribution (MWD) while being separated from the additives. The additives are then transferred to the second-dimension separation using a novel analyte-transfer system. In the second dimension, the additives are separated to obtain a detailed profile of the low-molecular-mass compounds in the SP. With this approach, the MWD of the NC and the composition of the additives in SP have been obtained within an hour. A discrimination power of 90.53% was obtained when studying exclusively the NC MWD, and 99.47% for the additive profile. This novel combination enables detailed forensic comparison of intact SPs. Additionally, no extensive sample preparation is required, making the developed method less labor intensive.

Strategies to circumvent the solvent strength mismatch problem in online comprehensive two-dimensional liquid chromatography.

On-line comprehensive two-dimensional liquid chromatography is a powerful technique for the separation of highly complex samples. Due to the addition of the second dimension of separation, impressive peak capacities can be obtained within a reasonable analysis time compared to one-dimensional liquid chromatography. In online comprehensive two-dimensional liquid chromatography, the separation power is maximized by selecting two separation dimensions as orthogonal as possible, which most often requires the combination of different mobile phases and stationary phases. The online transfer of a given solvent from the first dimension to the second dimension may cause severe injection effects in the second dimension, mostly due to solvent strength mismatch. Those injection effects may include peak broadening, peak distortion, peak splitting or breakthrough phenomenon. They are often found to reduce significantly the peak capacity and the peak intensity. To overcome such effects, arising specifically in online comprehensive two-dimensional liquid chromatography, different methods have been developed over the years. In this review, we focused on the most recently reported ones. A critical discussion, supported by a theoretical approach, gives an overview of their advantages and drawbacks.

An enhanced strategy integrating offline two-dimensional separation with data independent acquisition mode and deconvolution: Characterization of metabolites of Uncaria rhynchophylla in rat plasma as a case

The importance to clarify the drug metabolites is beyond doubt in view of their potential efficacy and safety. However, due to the complex matrix interference, relatively low content and the co-eluting effect, it is of a great challenge to comprehensively and systematically characterize the metabolites in vivo, especially for the traditional Chinese medicines (TCMs) due to the numerous types of components. In the present study, a comprehensive off-line two-dimensional separation system combining with data independent acquisition (DIA) mode and multi-dimensional data deconvolution method was established for chromatographic separation, data acquisition and data procession of indole alkaloids in rat plasma after intragastrically administrated with the extract of Uncaria rhynchophylla at the dose of 1 g/kg. The orthogonality of the off-line 2D separation system consisting of HILIC for first-dimensional separation and the PRLC for second-dimensional separation was valuated with the “asterisk” equations, and the results showed that off-line 2D separation system had passable orthogonality (A0 = 53.3%). Furthermore, the DIA mode was applied to capture MS/MS spectra in view of its advantage in acquiring MS data, and an effective multi-dimensional deconvolution method integrating the calculation of chemical formula, the extraction of diagnostic ion, the filter of ring double bond (RDB) and the judgement of neutral loss was established to parse the spectra for the complicated DIA data for comprehensive analysis of metabolites in rat plasma. Ultimately, a total of 127 indole alkaloids were tentatively characterized, and the main metabolic pathways were inferred as demethylation, dehydrogenation, hydroxylation and deglycosylation. The off-line two-dimensional separation system was applied for the comprehensive characterization of metabolites in vivo for the first time. This study suggested a new approach to enable the enrichment, separation and analysis of the low content components in vivo.

Polypropylene capillary-channeled polymer fiber column as the second dimension in a comprehensive two-dimensional RP × RP analysis of a mixture of intact proteins.

Online, comprehensive two-dimensional liquid chromatography (2D-LC) has become an attractive option for the analysis of complex samples of relevance in various fields (e.g., environmental, food, biology, and polymer sciences). In general, the second-dimension (2D) separation plays a more crucial role than the first (1D) in the total performance of LC × LC systems. Speed and efficiency of the 2D separation are of primary importance. These challenges are amplified in the case of protein separations where mass transfer limitations effect elution kinetics. The research presented here leverages the developments of capillary-channeled polymer (C-CP) fiber stationary phases directed at high-throughput and rapid protein separations, evaluating them as 2D material in comprehensive reversed-phase × reversed-phase (RP × RP) 2D-LC versus conventional, packed-bed columns as 2D. The ability to operate fiber columns at high linear velocities (> 75mms-1 at < 1000psi) without sacrifice in resolution is the first step towards potential 2D implementation, alleviating the need for ultrahigh-pressure pumping (i.e., UHPLC) in that stage. What must be realized are fast elution gradients and rapid re-equilibration to minimize cycling times (modulation periods). Different flow rates and gradient programs were evaluated. Rapid elution allows the use of shorter shift-gradient windows. At the optimized gradient recycling time on this instrument, 36s, there is no significant degradation in peak capacity. Finally, a graphic comparison of the separation efficiency between seven commercial reversed-phase columns and the polypropylene C-CP fiber column is presented for a synthetic, eleven-protein mixture using the recommended operating conditions for each of the 2D columns. The kinetic advantages of the fiber columns are clearly demonstrated.

Separation and characterization of products from acidic methanolysis of plasmalogenic lipids by two-dimensional gas chromatography with online reduction

The complexity of determining the composition of animal tissue lipids is greatly increased by the presence of plasmalogens in which the alkyl chain is linked to glycerol by an enol ether bond instead of being esterified. Acidic methanolysis of animal tissue lipids provides the simultaneous scission of acyl and alkenyl ether moieties, but the complexity of the products of reaction poses a great challenge in their gas chromatographic analysis. Two-dimensional gas chromatography with online reduction (GC-OR × GC) provided the resolution of all components contained in acid methanolyzed animal lipids, taking advantage of the selective hydrogenation of alkenyl ether methanolysis products prior to the second-dimension separation (2D). In this study, we also studied the chemical transformations occurring during the acidic methanolysis of animal lipids and the subsequent gas chromatographic analysis. In particular, we observed that using methanolysis reagents contaminated with water resulted in the undesired formation of fatty aldehydes, and we made recommendations on how to avoid these side reactions using proper methanolysis conditions. Products of acidic methanolysis were studied by GC-OR × GC, GC-MS, NMR spectroscopy, and GC with flame ionization detection (GC-FID). We defined the GC-FID elution order of animal lipid acidic methanolysis products using 100 m × 0.25 mm 100% bis(cyanopropyl)siloxane columns and two different set of elution conditions: isothermal elution at 180°C, and a temperature program optimized for dairy fats. A simple procedure for isolating dimethyl acetals (DMA) prior to GC analysis is also described.

Development of a rapid and improved two‐dimensional electrophoresis method for the separation of protein lysate

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.

Experimental and numerical study of band-broadening effects associated with analyte transfer in microfluidic devices for spatial two-dimensional liquid chromatography created by additive manufacturing

Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (2D) separation, flow confinement during the first-dimension (1D) separation, and band-broadening effects during analyte transfer from the 1D separation channel to the 2D separation area.In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the 2D development and analyte transfer from 1D to 2D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the 2D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the 1D channel had a great effect on the analyte transfer from the 1D to the 2D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed.

Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses.

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Two-dimensional insertable separation tool (TWIST) for flow confinement in spatial separations

Spatial comprehensive two-dimensional liquid chromatography (xLC×xLC) may be an efficient approach to achieve high peak capacities in relatively short analysis times, thanks to parallel second-dimension separations [1,2]. A key issue to reach the potential of xLC×xLC is to achieve adequate flow control and confinement of the analytes to the desired regions, i.e. confinement in the first-dimension direction and subsequently homogeneous flow in the second dimension. To achieve these goals we propose the TWIST concept (TWo-dimensional Insertable Separation Tool), a modular device that includes an internal first-dimension (1D) part that is cylindrical and rotatable. This internal part features a series of through-holes, each of which is perpendicular to the direction of the 1D flow. The internal part is inserted in the cylindrical casing of the external part. The internal diameter of the casing is marginally larger than the external diameter of the internal part. The external part also comprises a flow distributor and second-dimension (2D) channels. During the 1D injection and development, the channel is placed in a position where the through-holes are facing the wall of the external part, such that the liquid remains confined within the 1D channel. Thereafter, to realize the transfer to the second dimension (2D injection), the 1D channel is rotated, so that the holes of the internal part are aligned with the holes on the external part, allowing a transversal flow of the 2D mobile phase from the distributor through the 1D channel and eventually into the 2D area.

Separation of lignans from Schisandra chinensis by two-dimensional reversed-phase liquid chromatography

A two-dimensional reversed-phase liquid chromatography (2D-RPLC) method was established to separate lignan components and pure compounds from Schisandra chinensis. This method was based on the modules of separation-enrichment and self-developed with independent intellectual property rights. First, a C18 column (250 mm×4.6 mm, 5 μm) was used as the analytical column. A water-methanol mixture was selected as the mobile phase. Separation resolution and retention time were selected as indexes. Gradient chromatography of the lignan extracts was repeated four times and the information was input into XTool, a chromatographic separation software. The conditions for the first-and second-dimensional chromatographic separation were optimized by XTool. Then, linear amplification was used for preparative chromatography. C18 column (250 mm×30 mm, 10 μm) was selected as the first-and second-dimensional separation column. The C18 columns (80 mm×30 mm, 10 μm) were selected as enriching columns, and water was chosen as the diluent solution. Lignans from Schisandra chinensis were separated and purified by 2D-RPLC. Finally, nine lignan components were obtained by the first-dimensional separation. Totally, 20 high-purity compounds, containing six monomers, were obtained by the second-dimensional separation. Experimental results showed that this method has good reproducibility. This method thus allows for the systematic separation of lignans from Schisandra chinensis.

Centrifugal partition chromatography a first dimension for biomass fast pyrolysis oil analysis

Biomass fast pyrolysis oils contain molecules having a large variety of chemical functions and a wide range of molecular weights (from several tens to several thousand grams per mole). The good knowledge of their complex composition is essential for optimizing the conversion of bio-oils to biofuels, thereby requiring powerful separation techniques. In this work, we investigate the interest of centrifugal partition chromatography (CPC) as a first dimension for the analysis of a bio-oil. A CPC method is proposed to separate oxygen containing compounds according to their partition coefficients in the solvent system. This approach is a powerful and easy-to-use technique that enables fractionation of a bio-oil at a semi-preparative scale, without any sample loss related to adsorption on the stationary phase. Collected fractions are then injected in liquid chromatography as a second dimension of separation. Contour plot representations of the CPC × LC separation are established to discuss the potential of this approach. These representations can be used as a veritable fingerprint in the comparison of different samples or samples at different steps of a conversion process but also as a powerful tool to identify new compounds and describe the entire composition of the bio-oil.