2D Separation Research Articles

LCxSFC Valve Technologies: Guidelines toward a Successful Online Modulation.

Online two-dimensional liquid chromatography can be used under various separation modes but is sometimes limited in terms of orthogonality, especially for the analysis of neutral compounds. The use of SFC in the second dimension offers a wide choice of mobile and stationary phases, suggesting retention properties complementary to those of the first dimension. Initial works on online LCxSFC coupling published in the literature highlighted the first difficulties of solvent compatibility or potential problems with bubbles created by CO2 in loops. The present work highlights the impact of the interface between the LC and SFC dimensions on the performance of the 2D separation. Six different configurations have been evaluated, differing by the addition of a makeup flow in the first dimension, a division, or a separation of flow paths in the second dimension. Their performances with empty loops eliminated the need for complex trapping columns, regardless of the LC mobile phase nature. Injection in partial fill mode was possible without any problems of CO2 bubble formation, solvent miscibility, pressure, or modulation repeatability in each of the studied configurations. Selected configurations even allowed for the use of a mobile phase split upstream of the valve, so that the online LCxSFC configuration could be as flexible as LCxLC instrumentation in terms of the first dimension flow rate or second dimension injected volume. The obtained results allowed for building guidelines presenting the best interfaces to set up depending on the nature of the mobile phases in both dimensions. Two applications on monomers of lignin and industrial polymers confirmed preliminary results on configuration testing and illustrated that online LCxSFC could now be easily implemented. Precision tests on retention time were conclusive and are promising for the future use of this technique in industrial routines.

13C CSA Tensors and Orientational Order of Model and Dimer Mesogens Comprising of Phenyl Benzoate.

A Model mesogen and its symmetrical Dimer made up of phenyl benzoate core unit are investigated by 13C NMR spectroscopy. The existence of layer order in smectic A and smectic C phases of Dimer mesogen is established by powder X-ray diffraction. The chemical shift anisotropy (CSA) tensors of Model mesogen are determined by 2D separation of undistorted powder patterns by effortless recoupling (SUPER) experiment and are utilized for calculating the order parameters employing the alignment-induced chemical shifts (AIS). Additionally, 2D separated local field (SLF) NMR is availed for extracting 13C-1H dipolar couplings for both mesogens and used for computing the order parameters. A good agreement in the order parameters calculated from 13C-1H dipolar couplings and AIS is observed. Accordingly, the main order parameter (Szz) for the phenyl rings of the Model mesogen is found to be in the range 0.54 - 0.82, and for the Dimer mesogen, the values span 0.64 - 0.82 across mesophases. Since the phenyl benzoate core unit is frequently employed structural moiety for constructing the main chain as well as side chain liquid crystalline polymers and liquid crystalline elastomers, the CSA tensors reported here will be of immense utility for the structural characterization of these materials.

Mean-flow structures of the turbulent boundary layers bounding a two-dimensional separation bubble

Understanding the mean-flow structures of a separated turbulent boundary layer (TBL) is crucial for turbulence modeling. This study investigates the spatial scaling properties of the total shear stress and mixing length in the TBLs bounding a two-dimensional (2D) separation bubble, aiming to derive analytical descriptions for the entire mean-velocity profiles of the TBLs. For the adverse pressure gradient (APG) TBL upstream of the separation bubble, the total shear stress possesses a two-layer structure with an inner layer adhering to a linear law and an outer layer conforming to a defect power law. In contrast, the mixing length profile consists of four layers, namely the viscous sublayer, the buffer layer, the overlap layer, and the wake region. Each of the layers exhibits a power law or a defect power law relationship with the spatial coordinate normal to the wall. In the four-layer structure, three parameters are sensitive to the variation of the APG: the buffer-layer thickness, the relative magnitude of the mixing length at the boundary layer edge, and a defect power law exponent quantifying the extent of the wake region. For the reattached TBL downstream of the separation bubble, the total shear stress consists of two parts. One part is induced by the pressure gradient and retains the two-layer structure, while the other, engendered by the intense turbulence advected from the separated shear layer, exhibits a dual-power-law distribution. The advected turbulence also significantly alters the four-layer structure of the mixing length, resulting in an augmented buffer layer, a diminished overlap layer, and a wake region that mimics a turbulent mixing layer. Via a dilation ansatz to describe the scaling transition between adjacent layers, the study formulates the complete profiles of the total shear stress and mixing length. The formulation leads to the derivation of novel analytical expressions for the entire mean-velocity profiles of the TBLs. The expressions are in precise accord with the direct numerical simulations of an incompressible 2D separation-bubble flow and a 2D impinging shock wave/TBL interaction. The elucidation of the mean-flow structures through this study is anticipated to facilitate the analysis of turbulence models, thereby enhancing their performance in simulating separated TBLs. The construction of the mean-flow descriptions by inspecting the spatial scaling properties of turbulence paves a promising way for theoretical exploration of complex nonequilibrium TBLs.

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis

BackgroundN-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. ResultsIn this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. Significance and noveltyIn conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 μg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

Highly sensitive two-dimensional ion chromatography mass spectrometry method for nitrite determination in hydroxypropyl methylcellulose

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension (1D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension (2D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5–5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5–10.1 % and recovery of 90.5–102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.

Flow-modulation comprehensive two-dimensional enantio-gas chromatography: A valid and flexible alternative to heart-cutting multidimensional enantio-gas chromatography

The present research is focused on the proposal of use of flow-modulation comprehensive two-dimensional enantio-gas chromatography (FM eGC × GC) as a valid, flexible, and possibly superior alternative to heart-cutting multidimensional enantio-GC (eMDGC). The latter, a technique of demonstrated utility, is used specifically for the targeted separation of chiral compounds, whereas FM eGC × GC can produce both targeted and high-resolution untargeted information in a single run. It is clearly possible to use eMDGC for untargeted analysis, often with a flame ionization detector (stand-by analysis), to monitor a first-dimension (1D) separation, of much lower peak capacity compared to FM eGC × GC. If eMDGC is used with mass spectrometry (MS), it is normally exploited to monitor the second-dimension (2D) separation.The analytical instrument consisted of automated solid-phase microextraction (SPME), and a low duty-cycle FM eGC × GC system (with time-of-flight MS), equipped with an enantioselective 1D column (2,3-di-O-methyl-6-t-butyl silyl β-cyclodextrin derivative) and a 2D polyethylene glycol one. Ten Marsala wines were subjected to analysis, for the determination of chiral lactones (many at the low ppb level, due to the high concentration capacity of SPME) and for general analyte profiling. In many instances, highly complex chromatograms were attained, with statistical analysis (ANOVA-simultaneous component analysis and partial least squares discriminant analysis) used for sample differentiation.

High performance mixed-dimensional assembled MXene composite membranes for molecular sieving

Two-dimensional (2D) separation membranes have proved their superiorities in addressing global water scarcity and recycling resources. However, current 2D membranes usually suffer from swelling and irregular stacking issues, which result in poor separation performance. Herein, we developed novel mix-dimensional assembled MXene composite membranes with high permeability and selectivity by intercalating 1D carboxylated cellulose nanofibers (CNFs) into 2D lamellar MXene nanosheets. The effects of chain length of CNFs and MXene/CNFs mass ratios on the morphologies, physicochemical properties, and separation performances of composite membranes were systematically investigated. The optimized MXC-3-L membrane exhibited superior rejection to Congo red (CR, >99%) and remarkable separation efficiency of dye/salt. The separation factors for the CR/NaCl and CR/Na2SO4 mixtures were 512.0 and 517.2, respectively. Moreover, the MXC-3-L membrane showed distinct rejections to perfluoroalkyl substances (PFAS) (>95%) with a nearly 8-fold increase of permeance in comparison with the commercial polyamide membrane. The 2D X-ray diffraction analysis verified that the intercalation of 1D CNFs into 2D MXene nanosheets via strong hydrogen bonding can regulate the nanochannel into more stable and regularly stacking lamellar structures, which plays a key role in high permeance and excellent molecular separation efficiency. In addition, 1D CNFs play an essential role as “threading” and “stitching”, shielding the defects between adjacent MXene nanosheets and improving the separation efficiencies. Our work provides new insights into design of defect-free and highly selective 2D materials-based membranes for enhanced wastewater treatment and molecular sieving.

An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine.

Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6-7.5%), precision (relative standard deviation = 0.8-4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.

Two Multidimensional Chromatography/High-Resolution Mass Spectrometry Approaches Enabling the In-Depth Metabolite Characterization Simultaneously from Three Glycyrrhiza Species: Method Development, Comparison, and Integration.

Two offline multidimensional chromatography/high-resolution mass spectrometry systems (method 1: fractionation and online two-dimensional liquid chromatography, 2D-LC; method 2: fractionation and offline 2D-LC) were established to characterize the metabolites simultaneously from three Glycyrrhiza species. Ion exchange chromatography in the first-dimensional (1D) separation was well fractionated between the acidic (mainly triterpenoids) and weakly acidic components (flavonoids). These obtained subsamples got sophisticated separation by the second (2D) and third dimension (3D) of chromatography either by online reversed-phase chromatography × reversed-phase chromatography (RPC × RPC) or offline hydrophilic interaction chromatography × RPC (HILIC × RPC). Orthogonality for the 2D/3D separations reached 0.73 for method 1 and 0.81 for method 2, respectively. We could characterize 1097 compounds from three Glycyrrhiza species based on an in-house library and 33 reference standards, involving 618 by method 1 and 668 by method 2, respectively. They exhibited a differentiated performance and complementarity in identifying the multiple subclasses of Glycyrrhiza components.

Solid-Phase Extraction at High pH as a Promising Tool for Targeted Isolation of Biologically Active Fractions of Humic Acids.

A search for novel sources of biologically active compounds is at the top of the agenda for biomedical technologies. Natural humic substances (HSs) contain a large variety of different chemotypes, such as condensed tannins, hydrolyzable tannins, terpenoids, lignins, etc. The goal of this work was to develop an efficient separation technique based on solid-phase extraction (SPE) for the isolation of narrow fractions of HS with higher biological activity compared to the initial material. We used lignite humic acid as the parent humic material, which showed moderate inhibition activity toward beta-lactamase TEM 1 and antioxidant activity. We applied two different SPE techniques: the first one was based on a gradient elution with water/methanol mixtures of the humic material sorbed at pH 2, and the second one implied separation by a difference in the pKa value by the use of sequential sorption of HS at pH from 8 to 3. SPE cartridges Bond Elute PPL (Agilent) were used in the fractionation experiments. The first and second techniques yielded 9 and 7 fractions, respectively. All fractions were characterized using high-resolution mass spectrometry and biological assays, including the determination of beta-lactamase (TEM 1) inhibition activity and antioxidant activity. The acidity-based separation technique demonstrated substantial advantages: it enabled the isolation of components, outcompeting the initial material at the first step of separation (sorption at pH 8). It showed moderate orthogonality in separation with regard to the polarity-based technique. Good perspectives are shown for developing a 2D separation scheme using a combination of polarity and acidity-based approaches to reduce structural heterogeneity of the narrow fractions of HS.

Multi-effective components and their target mechanism of Ziziphi Spinosae Semen in the treatment of insomnia

Insomnia is a common and refractory disease. Since more than 2000 years ago, people have been using Ziziphi Spinosae Semen (ZSS). However, there are lack of molecular mechanisms of sleep promotion effects of ZSS. The purpose of this study is to clarify the active ingredients in ZSS that are used to treat insomnia. Using a method called cellular label-free integrative pharmacology (CLIP), we established five insomnia-related target models, including serotonin (5HT2A and 5HT1A), melatonin (MT1), dopamine (D2) and epinephrine (β2) receptors. The one-dimensional (1D) fractions of ZSS extract were prepared on a RZC18 column and assayed on five models. Subsequently, the active fraction was further analyzed, fractionated and quantified using a two-dimensional (2D) liquid phase method coupled with a charged aerosol detector (CAD), This CAD-coupled 2D-LC method requires micro-fractions from the 1D separation and thus it greatly saves sample amounts and corresponding preparation time, and quickly conduct activity screening. The composition of the active 2D fractions was then determined using three-dimensional (3D) HPLC-MS, and molecular docking was separately carried out for the described compounds on the targets for activity prediction. Seven compounds were predicted to be active on 5HT2A, and two compounds on D2. We experimentally verified the prediction and found that vitexin exhibited D2 agonistic activity, and nuciferine exhibited 5HT2A antagonistic activity. This study revealed the effective components and their targets of ZSS in the treatment of insomnia, also highlighted the potential of the CLIP technique and bioactivity guided multi-dimensional HPLC-MS in molecular mechanism elucidation for traditional Chinese medicines.

Development and Application of a Novel Multiloop Splitter-Based Non-cryogenic Artificial Trapping Modulation System in Comprehensive Two-Dimensional Gas Chromatography.

A multiloop splitter-based non-cryogenic artificial trapping (M-SNAT) modulation technique was established, which applied the first (1D) nonpolar and the second (2D) polar columns, deactivated fused silica (DFS) columns, a microfluidic Deans switch (DS), and splitters located between the 1D column outlet and the DS. The splitters were connected into multiple loops with a progressively doubled perimeter of the next loop. This enabled a duplex splitting mechanism within each loop consisting of splitting of analyte pulses, the pulse delay, and their combination which led to equally split peaks of the same analytes with the number of split peaks (nsplit) equal to 2m (m = number of loops). This system resulted in local profiles of artificially split-and-trapped analytes prior to their selective transfers onto the 2D column by means of periodic multiple heart-cuts (H/C). The developed SNAT approach can be successful, providing that the ratio of modulation period to sampling time (PM/tsamp) is equal to nsplit. The approach with nsplit = 16 was further developed into a single device platform and applied for the modulation of a wide range of compounds in waste tire pyrolysis samples with the RSD of ≤0.01 and <10% for the one-dimensional modulated peak times and peak areas, respectively (n = 50). The method enabled an artificial modulation mechanism without cryogen consumption and enhanced the 2D peak capacity (2nc) and 2D separation by use of a longer 2D column.

Enzyme activity- and chemometrics-assisted comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of honeysuckle

A unique and effective comprehensive two-dimensional liquid chromatography system was established and applied for the analysis of bioactive components in honeysuckle. Under the optimal conditions, Eclipse Plus C18 (2.1 × 100 mm, 3.5 μm, Agilent) and SB-C18 (4.6 × 50 mm, 1.8 μm, Agilent) columns were chosen for the first dimension (1D) and the second dimension (2D) separation. The optimal flow rates of 1D and 2D were 0.12 mL/min and 2.0 mL/min, respectively. Additionally, the proportion of organic solution was optimized to enhance orthogonality and integrated shift, and full gradient elution mode was adopted to improve chromatographic resolution. Furthermore, a total of 57 compounds were identified by molecular weight, retention time and collision cross-section value obtained from ion mobility mass spectrometry. Based on the data obtained from the principal component analysis, partial least squares discriminant analysis, and hierarchical cluster analysis, the categories of honeysuckle in different regions were significantly different. Moreover, the half maximal inhibitory concentration values of most samples were between 0.37 and 1.55 mg/mL, and most samples were potent α-glucosidase inhibitors, which is better for the evaluation of the quality of drugs from two aspects of substance content and activity.

A detailed evaluation of the advantages and limitations of online RP-LC×HILIC compared to HILIC×RP-LC for phenolic analysis

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC × LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension (1D) separation mode, and RP-LC in the second dimension (2D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC × HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC × RP-LC-MS configuration.Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC × RP-LC- and RP-LC × HILIC-MS in order to compare the separation performance.Overall, the kinetic performance of HILIC × RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC × HILIC analyses using similar column dimensions. Dilution of the 1D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2D HILIC separation, which resulted in severe 2D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC × HILIC-MS analyses was observed due to higher ESI-MS response for the 2D HILIC mobile phase and greater sample loading capacity of the 1D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC × HILIC-MS separations. These findings point to the potential advantage of RP-LC × HILIC as a complementary configuration to HILIC × RP-LC for phenolic analysis.

Wind-sea and swell separation of 1D wave spectrum by deep learning

In this study, three existing methods to separate wind-sea and swell from 1D wave spectra, namely the Pierson–Moskowitz (PM) method, the steepness method (HP method), and the overshooting method (JP method), were evaluated statistically using data from 38 meteorological buoys from National Data Buoy Center (NDBC) over the period 2010–2020. Among the three methods, the PM method shows the best agreement with the 2D separation method, because it uses the wind speed information as an input term. Using these buoy data, a new 1D wind-sea-swell separation method based on deep learning is proposed. This new method can directly compute the wind-sea or swell significant wave height (SWH) from a 1D wave spectrum with or without wind speed data as input. When the wind speed is used as an input term, the overall root-mean-square error (RMSE) of the method for wind-sea/swell SWH is 0.27/0.36 m compared to the 2D separation method, which outperforms all existing 1D separation methods. When there is no wind speed data, the RMSE of wind-sea/swell SWH can still reach 0.36/0.41 m, which is similar to the accuracy of the PM method uses wind speed as input. Both methods are robust and have no significant geographical dependence.

Valve-based comprehensive two-dimensional gas chromatography with quadrupole mass spectrometry detection using a porous layer open tubular column in the first dimension

A comprehensive two-dimensional gas chromatography instrument with quadrupole mass spectrometry (GC×GC-qMS) is developed and evaluated, by retrofitting a commercial GC-qMS instrument with a high temperature diaphragm valve (HT-DV) modulator. A porous layer open tubular (PLOT) column in the first dimension (1D), and a more polar wall coated open tubular (WCOT) column in the second dimension (2D) were used. The HT-DV can modulate analytes of any volatility and the combination of the PLOT and WCOT columns provides ample separation selectivity for samples containing an extended range of volatility from highly volatile analytes to analytes of more moderate volatility. In addition to instrument development, we describe data visualization considerations when upgrading from GC-qMS to GC×GC-qMS. Unlike time-of-flight mass spectrometry (TOFMS), the mass spectrum cycle time for qMS (defined as the sum of scan time and interscan delay) is a linear function of the range of mass channels (m/z) analyzed. If the cycle time does not divide evenly into the modulation period, PM, visual distortions will appear in the GC×GC chromatogram. We present two approaches to address this GC×GC chromatogram distortion issue. The first method does not require post-run computations; however, care must be taken in selecting an appropriate mass range to minimize the visual distortion issue. A half of the modulation period of total shift in the 2D separations with a PM of 2 s was determined tolerable for a 15 min separation, with 44 mass ranges (and their accompanying scan rate) deemed suitable out of 350 possible mass ranges. Also, a computational “resampling” method, which can be used to correct any qMS scan rate used was implemented. Figures-of-merit for the HT-DV modulated GC×GC-qMS instrument were also examined. Reproducible peak height (average RSDheight = 9.2%, s = 3.5%) and areas (average RSDarea = 9.3%, s = 2.8%) were obtained with low LODs (average LOD = 4.2 mg/L, s = 2.5 mg/L).

Solid Phase Microextraction-Multicapillary Column-Ion Mobility Spectrometry (SPME-MCC-IMS) for Detection of Methyl Salicylate in Tomato Leaves.

Methyl salicylate (MeSA) is a plant-signaling molecule that plays an essential role in the regulation of plant responses to biotic and abiotic pathogens. In this work, solid phase microextraction (SPME) and a multicapillary column (MCC) are coupled to ion mobility spectrometry (IMS) to detect MeSA in tomato leaves. The SPME-MCC-IMS method provides two-dimensional (2D) separation by both MCC and IMS, based on the retention and drift times. The effect of the IMS polarity on the separation efficiency of MCCs was also investigated. In the positive polarity, ionization of MeSA resulted in [MeSA + H]+ formation while, in the negative, deprotonated ions, [MeSA - H]-, and the O2- adduct ion, [MeSA + O2]-, were formed. In the real sample analysis, the negative polarity operation resulted in the suppression of many matrix molecules and thus in the reduction of interferences. Four different SPME fibers were used for head space analysis, and four MCC columns were investigated. In the negative polarity, complete separation was achieved for all of the MCCs columns. The limits of detection (LODs) of 0.1 μg mL-1 and linear range of 0.25-12 μg mL-1 were obtained for the measurement of MeSA in a standard solution (H2O/CH3OH, 50:50) by the SPME-IMS method with a 5 min extraction time using an SPME with a PDMS fiber, in the negative mode of IMS. The MeSA contents of fresh tomato leaves were determined as 1.5-9.8 μg g-1, 24-96 h after inoculation by tomato mosaic ringspot virus (ToRSV).

Portable comprehensive two-dimensional micro-gas chromatography using an integrated flow-restricted pneumatic modulator

Two-dimensional (2D) gas chromatography (GC) provides enhanced vapor separation capabilities in contrast to conventional one-dimensional GC and is useful for the analysis of highly complex chemical samples. We developed a microfabricated flow-restricted pneumatic modulator (FRPM) for portable comprehensive 2D micro-GC (μGC), which enables rapid 2D injection and separation without compromising the 1D separation speed and eluent peak profiles. 2D injection characteristics such as injection peak width and peak height were fully characterized by using flow-through micro-photoionization detectors (μPIDs) at the FRPM inlet and outlet. A 2D injection peak width of ~25 ms could be achieved with a 2D/1D flow rate ratio over 10. The FRPM was further integrated with a 0.5-m long 2D μcolumn on the same chip, and its performance was characterized. Finally, we developed an automated portable comprehensive 2D μGC consisting of a 10 m OV-1 1D μcolumn, an integrated FRPM with a built-in 0.5 m polyethylene glycol 2D μcolumn, and two μPIDs. Rapid separation of 40 volatile organic compounds in ~5 min was demonstrated. A hybrid 2D contour plot was constructed by using both 1D and 2D chromatograms obtained with the two μPIDs at the end of the 1D and 2D μcolumns, which was enabled by the presence of the flow resistor in the FRPM.

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.