Porous Layer Open Tubular Columns Research Articles

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography.

Porous monoliths are well-known stationary phases in high-performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica-based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene-co-divinylbenzene), polymethacrylate, and silica-based monolithic columns while expanding to polymer-based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low-pressure gas chromatography will be addressed.

The fabrication of poly (polyethylene glycol diacrylate) monolithic porous layer open tubular (mono-PLOT) columns and applications in hydrophilic interaction chromatography and capillary gas chromatography for small molecules.

The easy shrinkage and swelling of polymer monolithic column when exposed to mobile phase with different polarity is a problem that cannot be ignored. To overcome this drawback, a convenient aqueous two-phase polymerization approach was used to prepare poly (polyethylene glycol diacrylate, PEGDA) monolithic porous layer open tubular (mono-PLOT) columns (150μm). The poly(PEGDA) mono-PLOT column with homogeneous polymer porous layer was synthesized successfully. A maximum plate number of 41,500 plates per meter for allyl thiourea was obtained under a velocity of 1.8mm/s. Several kinds of polar molecule were separated on the proposed mono-PLOT column and a typical hydrophilic interaction retention mechanism was observed. High speed separation of benzoic acids was also carried out, baseline separation of five benzoic acids was successfully achievedwithin 5min with a 70cm mono-PLOT column at 50°C. Furthermore, the resulting PLOT column was also successfully applied to separate standard analytes of three DNA oxidative damage products and RNA-modified nucleosides and four chlorophenols. At last, the column could separate alcohols, alkanes, and aromatic isomers via GC. It had more than 20,000 plates per meter for butanol - higher than commercial coatings open tubular columns.

Surface modification with highly-homogeneous porous silica layer for enzyme immobilization in capillary enzyme microreactors

Immobilized enzyme micro-reactors (IMERs) are of vital importance in developing miniaturized bioanalytical systems and have promising applications in various biomanufacturing. An inherent limitation in designing IMERs is the one-dimensional cylindrical geometry of micro-channels that offers limited exposed surface area for molecular reorganization and enzyme immobilization. In this study, we report a robust capillary-IMER based on a three dimensional porous layer open tubular (3D-PLOT) column which is prepared by an easy-to-control surface modification strategy via single-step in situ biphasic reaction. The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels, showing the beneficial effects for enzyme immobilization to enhance reaction efficiency and shorten analysis time. Taking trypsin as a model enzyme, enzymatic activities of immobilized enzyme are analyzed. We compare enzyme assays using the proposed 3D-PLOT-IMER with those using normal capillary-IEMR without surface modification as well as free trypsin. The 3D-PLOT-IMER exhibits excellent stability and inter/intra-day reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate enzyme assay. The feasibility of the proposed method for potential application in biological analysis is demonstrated by coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins, cell extraction and living Hela cells. Our study show that the surface modification with the proposed 3D-porous layer is a simple and efficient approach for enzyme immobilization, and could be widely suitable for different kinds of IMERs.

Reversed-Phase Functionalised Multi-lumen Capillary as Combined Concentrator, Separation Column, and ESI Emitter in Capillary-LC–MS

For the first time, a multi-lumen capillary (MLC) (126 parallel channels of 4.2 µm i.d) has been modified to produce a C18-functionalised silica porous layer open tubular (PLOT) capillary column for both on-capillary preconcentration and separation. The modified multi-lumen capillary used in this dual mode provided significant advantages over typical nano/capillary-LC–MS systems, in that it facilitated both higher sample loading capacity, the use of elevated flow rates, and simplified equipment requirements. Following modification, 100% of the channels displayed a homogenous porous silica layer, 257 ± 36 nm thick. The PLOT-MLC was first evaluated for on-capillary solid-phase extraction. Extraction of caffeine, ofloxacin, atrazine, and diuron was carried out offline using an 8-cm-long PLOT-MLC, with quantification achieved using HPLC coupled to a quadrupole-time of flight (QTOF) mass spectrometer. The results confirmed reversed-phase selectivity and average recoveries obtained were around 70%. Subsequently, a 65-cm-long PLOT-MLC was evaluated as a separator column using a capillary liquid chromatography (Cap-LC) system equipped with a nano-injector and coupled to the mass spectrometer. The short PLOT-MLC provided a baseline separation in isocratic mode [water:acetonitrile (each with 0.1% formic acid) = 70:30, v/v] of ofloxacin, atrazine, and diuron. Finally, direct coupling of the PLOT-MLC with the QTOF via a capillary electrosprayer facilitated the simultaneous use of the modified capillary as a solid-phase concentrator, separator column (carrying out concentration-focusing-separation on the PLOT-MLC) and electrospray emitter. This configuration greatly simplifies the traditional capillary-LC–MS equipment requirements, via the removal of all connectors and additional capillary between injector and MS inlet, and is demonstrated herein with large volume sample loading and step-gradient elution/separation with sensitive MS detection.

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) was prepared in two steps. First, a partially filled porous layer open tubular capillary column containing active epoxy groups was synthesized by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N′-methylenebisacrylamide. Then, an aqueous solution of commercial carrier ampholytes (CAs, Pharmalyte) was focused in the prepared polymer column. With the column immobilized by Pharmalyte 4.5–6.0 (narrow pH range) or Pharmalyte 3.0–10.0 (wide pH range), proteins with known pIs were separated and a good linear correlation between pI values and migration times was obtained. Meanwhile, resolution was improved compared to the traditional gradient with a wide pH range. A mixture of low molecular weight amino acids was separated with the narrow pH range PLOT-IPG column; from the obtained results, the resolution of our new column was calculated to be at most 0.09 pH unit. In addition, human serum proteins were analyzed with the newly developed wide and narrow pH range PLOT-IPG columns; its sensitivity and resolution in the narrow pH range one were better than capillary electrophoresis.

Selection of the porous layer open tubular columns for separation of light components in comprehensive two-dimensional gas chromatography

Up to the present, comprehensive two-dimensional gas chromatography was commonly performed using combinations of columns with stationary liquid phases. There are singular examples with a porous layer open tubular (PLOT) column used only in the second dimension. However, GC × GC systems with two PLOT columns are not reported in the literature. Our work describes the application of two PLOT columns in the GC × GC mode. In the first and second dimensions we used columns with the sorbents based on organic porous polymers with different selectivity: Rt-Q-BOND (nonpolar column consist of 100% divinylbenzene-styrene), Rt-S-BOND (intermediate polar column, which contain 4-vinylpyridine), Rt-U-BOND (polar column based on divinylbenzene - ethylene glycol dimethylacrylate copolymer), DVB-VIm (divinylbenzene-vinylimidazole copolymer), a column PTMSP with poly-(1-trimethylsilyl-1-propyne) sorbent, and a GASPRO column with porous silica as a stationary phase.The degree of orthogonality was calculated for five different combinations of columns, where a column with the porous polymer was used in the first dimension, and GASPRO column – in the second dimension. Orthogonality was estimated from the correlation coefficient of retention times in the first and second dimensions.Examples of the separation of C1-C5 hydrocarbons on a combination of PTMSP - GASPRO and Rt-Q-BOND - GASPRO columns are reported. It is shown that not only light hydrocarbons but also compounds belonging to different chemical classes can be separated with the use of PLOT columns.

Determination of hydrogen isotopes and isomers by different deactivated alumina PLOT columns

To determine residual H2 and D2 concentrations in high purity HD, gas chromatography was used to separate the hydrogen isotopes in a 5 A molecular sieve porous layer open tubular (PLOT) column cooled in a slowly warmed dewar. It took more than 15 min to produce one chromatogram in which a platform existed between o-H2 and p-H2, and resolution between o-D2 and p-D2 was hardly investigated. In this study, to improve thermal stability of the column oven, the column is designed to be immersed in pure liquid nitrogen instead of a mixture of liquid nitrogen and isopentane. Several alumina PLOT columns under different deactivation conditions are used to separate the hydrogen isotopes at 78 K. Results show that the sulfate deactivated column performs higher elution resolution than the chloride deactivated column. The platform was decreased in a 15 m sodium sulfate deactivated column; o-H2 and p-H2 was baseline separated within 1 min; o-H2: p-H2 = 72:28 according to peak areas. o-D2 and p-D2 was separated in less than 2 min in the same column, the ratio o-D2:p-D2 = 67:33. Relative short analysis time would help reduce the platform. Residual concentrations of high purity HD (HD = 96%) were determined within 3 min in a 15 m sulfate deactivated column modified by magnesium sulfate.

Highly uniform porous silica layer open-tubular capillary columns produced via in-situ biphasic sol–Gel processing for open-tubular capillary electrochromatography

We report a highly uniform porous layer open tubular (PLOT) column for capillary electrochromatography (CEC) analysis. The PLOT column is easily fabricated using a single-step in-situ biphasic reaction, producing homogeneous porous-layer modified surface with ∼240 nm thickness in a 50 μm-id capillary. CEC performance of the PLOT column has been investigated and optimized under various experimental parameters. Using a mixture of naphthalene and biphenyl as the test sample, we show that the PLOT column exhibits good separation efficiency with resolution >3.0 and theoretical plate numbers over 6 × 104, as well as good intra-/inter-day repeatability and column-to-column repeatability. The column has been successfully applied for CEC analysis of three different types of samples without any further modification of the columns, including complicated peptide products from tryptic-digestion of proteins (lysozyme and BSA), β-blockers (basic samples) and polycyclic aromatic hydrocarbons (neutral samples). Efficient separation has been achieved, which could be attributed to the enhanced surface-to-volume ratio of the PLOT column that will increase the interaction between solid phase and mobile phase in CEC. In addition, base-line separation of neutral samples indicates the reversed phase chromatographic property of the PLOT column, which could be induced by the residue of hexadecyltrimethylammonium bromide used in the fabrication process. Our study show that the present PLOT column is a promising approach that can significantly enhance CEC separation efficiency and could be of potential value in analysis of various different samples.

Preparation and Application of Porous Layer Open Tubular Capillary Columns with Narrow Bore in Liquid Chromatography

In recent years, micro/nano-scaled separation technique has attracted increasing attention due to its inherent advantages. The porous layer open tubular (PLOT) capillary column is an important micro-column form. Compared to wide-bore PLOT capillary columns (inner diameter > 25 μm), the PLOT capillary columns with narrow inner diameters yield higher separation efficiencies and lower reagent consumption. In this paper, the preparation methods for porous layer open tubular capillary columns with narrow inner diameters, less than or equal to 25 μm, are reviewed. Detection techniques combined with mass spectrometry and their applications in liquid chromatography are also discussed.

Extension of Golay plate height equation for open-tubular columns

Golay plate height equation with its B-term equal to 2Dm/u (Dm is molecular diffusivity of a solute in the carrier gas and u is the gas velocity) has been developed for WCOT (wall-coated open-tubular) GC columns with liquid stationary phases. In a more general case of OTC (open tubular columns) such as the PLOT (porous layer open-tubular) columns and others, the B-term is 2(Dm+kDS)/u (DS and k are the solute net diffusivity in the stationary zone and the retention factor, respectively) which can be substantially larger than 2Dm/u. A simple proof is provided together with other adjustments in Golay equation, and its interpretation.

Experimental and Computational Investigation of Gas Separation in Adsorbent-coated Microchannels

An experimental and computational investigation of gas separation in adsorbent-coated microchannels is performed. Experiments on porous-layer-open-tubular (PLOT) columns containing zeolite 5A using a ternary mixture of helium (He), nitrogen (N2) and carbon dioxide (CO2) show sequential breakthrough of N2 and CO2 and gradual saturation of adsorption sites with trace water. Multiple tests conducted for the adsorbent breakthrough investigation result in determination of adsorbent layer properties. The heat and mass transfer model results for a pressure drop range of 5–55kPa and channel lengths from 1 to 4m are found to be in reasonable agreement with the data with an average absolute deviation (AAD) of 14% for adsorption time and 41% for temperature rise (ΔT). The larger error in ΔT is thought to be due to uneven adsorbent loading of the PLOT columns. Therefore, the adsorption experiments are also conducted with custom-made adsorbent-coated microchannels with known adsorbent mass and layer thickness. The AADs for adsorption time and ΔT then decrease to 4% and 25%, respectively, indicating that the overall gas separation process is described well by the adsorption stage models, with local variation due to fabrication variability.

High-throughput gas chromatography for volatile compounds analysis by fast temperature programming and adsorption chromatography.

The synergy of combining fast temperature programming capability and adsorption chromatography using fused silica based porous layer open tubular columns to achieve high throughput chromatography for the separation of volatile compounds is presented. A gas chromatograph with built-in fast temperature programming capability and having a fast cool down rate was used as a platform. When these performance features were combined with the high degree of selectivity and strong retention characteristic of porous layer open tubular column technology, volatile compounds such as light hydrocarbons of up to C7 , primary alcohols, and mercaptans can be well separated and analyzed in a matter of minutes. This analytical approach substantially improves sample throughput by at least a factor of ten times when compared to published methodologies. In addition, the use of porous layer open tubular columns advantageously eliminates the need for costly and time-consuming cryogenic gas chromatography required for the separation of highly volatile compounds by partition chromatography with wall coated open tubular column technology. Relative standard deviations of retention time for model compounds such as alkanes from methane to hexane were found to be less than 0.3% (n = 10) and less than 0.5% for area counts for the compounds tested at two levels of concentration by manual injection, namely, 10 and 1000ppm v/v (n = 10). Difficult separations were accomplished in one single analysis in less than 2min such as the characterization of 17 components in cracked gas containing alkanes, alkenes, dienes, branched hydrocarbons, and cyclic hydrocarbons.

Pico-HPLC system integrating an equal inner diameter femtopipette into a 900 nm I.D. porous layer open tubular column.

A picoflow high performance liquid chromatography (pico-HPLC) system was developed, which could directly pipette femtoliter samples using a separation column tip driven by an electroosmotic pump. Amino acid enantiomers were separated in the 900 nm I.D. porous layer open tubular column at a flow rate of 13.50 pL min-1.

Isotopic separation of acetaldehyde and methanol from their deuterated isotopologues on a porous layer open tubular column allows quantification by stable isotope dilution without mass spectrometric detection

An isotopic separation of acetaldehyde and acetaldehyde-2,2,2-d3 was achieved in a temperature programmed run on a porous layer open tubular (PLOT) capillary column coated with particles of divinylbenzene ethylene glycol/dimethylacrylate (Rt®-U-BOND). This is the prerequisite for the development of quantitative analytical methods based on a stable isotope dilution assay (SIDA) without a mass spectrometric detection (non-MS SIDA). For routine analysis a flame ionization detector (FID) can thus be applied as a robust and low-cost alternative. In a preliminary study, static headspace extraction and gas chromatographic separation (HS-GC-FID) of acetaldehyde in aqueous solutions was shown as an application. Good linearity was obtained in a calibration range from 0.4 to 40mgL−1, with peak integration benefitting from the inverse isotope effect encountered on the specific porous polymer. Furthermore, separation of methanol and deuterated methanol (d3) could also be achieved under the same chromatographic conditions. The achieved isotopic separation of these important volatile compounds now allows non-MS SIDA-based methods that are still to be developed.

In Situ Measurement of Polymer Layer Thickness in Porous Layer Open Tubular (PLOT) Columns Using Optical Absorbance in the Near-IR Range

Highly reproducible fabrication of porous layer open tubular (PLOT) structures in fused silica capillaries is often challenging; thus, methods to measure layer thickness growth in real time represent a powerful tool for the production of such columns. The work presented herein demonstrates the application of optical absorbance in the near-infrared (near IR) range for the in-process measurement of polymer layer growth inside fused silica capillaries during the fabrication of PLOT columns. The proposed technique can be used for both on- and off-line measurements of layer thickness for thermal- and photo- initiated polymerisation methods, performed in either polytetrafluoroethylene (PTFE)- or polyimide-coated capillaries. Measurements of layer thickness were carried out at λ 700 nm, using 100 μm and 8 μm optical fibres, yielding relative standard deviation (%RSD) values of 27% and 22%, respectively.

Preparation and evaluation of 3 m open tubular capillary columns with a zwitterionic polymeric porous layer for liquid chromatography.

A 3 m zwitterionic polymeric porous layer open tubular column (3 m × 25 μm id × 375 μm od) with a polymeric porous layer thickness of 4 μm was fabricated by the copolymerization of [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide and N,N'-methylenebis(acrylamide). The effects of the diameter of the capillary, reaction temperature, and polymerization time on the preparation of the open tubular column were investigated. Characterized by scanning electron microscopy, the zwitterionic layer was observed to be rough and throughout the fused-silica capillary homogenously, which increased the phase ratio. The separation of neutral, basic, and acidic compounds demonstrates the strong hydrophilicity of the poly[2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide coating. In addition, the poly[2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide porous layer open tubular column was applied for the analysis of flavonoids from the rootstalk of licorice, revealing the potential in separating complex samples. The relative standard deviation of retention time for run-to-run (n = 5), day-to-day (n = 3), and column-to-column (n = 3) of toluene, N,N-dimethylformamide, formamide, and thiourea were below 1.2%, exhibiting good repeatability.

Preparation and characterization of micro-cell membrane chromatographic column with silica-based porous layer open tubular capillary as cellular membrane carrier.

Cell membrane chromatography (CMC) is a powerful tool to study membrane protein interactions and to screen active compounds extracted from natural products. Unfortunately, a large amount of cells are typically required for column preparation in order to carry out analyses in an efficient manner. Micro-CMC (mCMC) has recently been developed by using a silica capillary as a membrane carrier. However, a reduced retention of analytes is generally associated with mCMC mostly due to a low ligand (cellular membrane) capacity. To solve this common problem, in this work a silica-based porous layer open tubular (PLOT) capillary was fabricated and, to the best of our knowledge, for the first time applied to mCMC. The mCMC column was prepared by physical adsorption of rabbit red blood cell (rRBC) membranes onto the inner surface of the PLOT capillary. The effects of the PLOT capillaries fabricated by different feed compositions, on the immobilization amount of cellular membranes (represented by the fluorescence intensity of the capillary immobilized with fluorescein isothiocyanate isomer-labeled cellular membranes) and on the dynamic binding capacity (DBC) of verapamil (VP, a widely used calcium antagonist which specific interacts with L-type calcium channel proteins located on cellular membrane of rRBC) have been systematically investigated. The fluorescence intensity of the mCMC column when combined with the PLOT capillary was found to be more than five times higher than the intensity using a bare capillary. This intriguing result indicates that the PLOT capillary exhibits a higher cellular membrane capacity. The DBC of VP in the PLOT column was found to be more than nine times higher than that in the bare capillary. An rRBC/CMC column was also prepared for comparative studies. As a result, mCMC provides similar chromatographic retention factors and stability with common CMC; however, the cellular membrane consumption for mCMC was found to be more than 460 times lower than that for CMC. Graphical Abstract Comparision of mCMC chromatograms and SEM images between bare capillary and PLOT capillary.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Direct Measurement of Trace Elemental Mercury in Hydrocarbon Matrices by Gas Chromatography with Ultraviolet Photometric Detection.

We introduce a technique for the direct measurement of elemental mercury in light hydrocarbons such as natural gas. We determined elemental mercury at the parts-per-trillion level with high precision [<3% RSD (n = 20 manual injection)] using gas chromatography with ultraviolet photometric detection (GC-UV) at 254 nm. Our approach requires a small sample volume (1 mL) and does not rely on any form of sample preconcentration. The GC-UV separation employs an inert divinylbenzene porous layer open tubular column set to separate mercury from other components in the sample matrix. We incorporated a 10-port gas-sampling valve in the GC-UV system, which enables automated sampling, as well as back flushing capability to enhance system cleanliness and sample throughput. Total analysis time is <2 min, and the procedure is linear over a range of 2-83 μg/m(3) [correlation coefficient of R(2) = 0.998] with a measured recovery of >98% over this range.

Wall modified photonic crystal fibre capillaries as porous layer open tubular columns for in-capillary micro-extraction and capillary chromatography

Wall modified photonic crystal fibre capillary columns for in-capillary micro-extraction and liquid chromatographic separations is presented. Columns contained 126 internal parallel 4 μm channels, each containing a wall bonded porous monolithic type polystyrene-divinylbenzene layer in open tubular column format (PLOT). Modification longitudinal homogeneity was monitored using scanning contactless conductivity detection and scanning electron microscopy. The multichannel open tubular capillary column showed channel diameter and polymer layer consistency of 4.2 ± 0.1 μm and 0.26 ± 0.02 μm respectively, and modification of 100% of the parallel channels with the monolithic polymer. The modified multi-channel capillaries were applied to the in-capillary micro-extraction of water samples. 500 μL of water samples containing single μg L−1 levels of polyaromatic hydrocarbons were extracted at a flow rate of 10 μL min−1, and eluted in 50 μL of acetonitrile for analysis using HPLC with fluorescence detection. HPLC LODs were 0.08, 0.02 and 0.05 μg L−1 for acenaphthene, anthracene and pyrene, respectively, with extraction recoveries of between 77 and 103%. The modified capillaries were also investigated briefly for direct application to liquid chromatographic separations, with the retention and elution of a standard protein (cytochrome c) under isocratic conditions demonstrated, proving chromatographic potential of the new column format, with run-to-run retention time reproducibility of below 1%.