Porous Layer Open Tubular Research Articles

Multiplex immunoassays using surface modification-mediated porous layer open tubular capillary

We proposed an innovative surface modification-mediated porous layer open tubular (PLOT) capillary, which was modified via an in situ biphasic reaction. This capillary comprised three-dimensional homogeneous and porous structures, which could increase the surface-area-to-volume ratio for antibody immobilization. The PLOT capillary was shown as an ideal immunoreaction base to enhance the sensitivity of immunoassays and shorten analysis time. By connecting two separate PLOT capillaries using a suitable sleeve tube, we can perform multiplex targets detection in the same sample. We developed a sensitive, rapid, and multiplex PLOT capillary-mediated immunosensor for the simultaneous identification of alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) in clinical serum samples with good accuracy. The detection sensitivity of the PLOT immunosensor improved by 10-fold compared with that of bare-capillary sensor, and the whole analysis could be completed within 1 h. This work suggest that suitable surface modification strategy is an effective tool to improve the analytical performance of conventional immunoassay and our study provided a feasible, sensitive, and multi-target assay for the detection of cancer biomarkers, which would be of valuable application in clinical diagnosis.

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.

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.

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.

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.

Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.

Preparation and electrochromatographic characterization of new chiral β-cyclodextrin poly(acrylamidopropyl) porous layer open tubular capillary columns

A novel chiral stationary phase consisting of an amidopropyltrimethylammonium chloride divinylbenzene (APAT–DVB) polymer containing the chiral selector heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin (HSβCD) has been employed in porous layer open tubular (PLOT) capillary column format with various conditions evaluated to optimize the polymerization and chiral selector immobilization. Scanning electron microscopy demonstrated a near homogenous longitudinal open path in the column with a polymer film of uniform thickness. IR spectroscopy characterized the functional groups of the polymer and energy-dispersive X-ray spectroscopy provided further evidence of the successful polymer modification with HSβCD. Optimum electrochromatographic separation conditions were elaborated with respect to organic solvent content and pH of the background electrolyte. Colum-to-column and long-term reproducibility was excellent. The effectiveness of the new capillary column was demonstrated with the successful separation of d-and l-aspartic acid, d- and l-tyrosine and d-and l-lysine.

A porous layer open tubular monolith on microstructured optical fibre for microextraction and online GC-MS applications

A microextraction device comprising a porous layer open tubular (PLOT) polymer housed in a microstructured optical fiber has been shown to be an attractive tool for analyte extraction and coupling to GC-MS as a cost-effective alternative to traditional SPME. This paper details the fabrication of a poly(styrene-co-divinylbenzene) PLOT optical fibre microextraction device and its use in the extraction of polyaromatic hydrocarbons (PAHs) in aqueous solution. Good linear calibrations were obtained with R2 values above 0.970 for five PAHs analyzed, with percent relative standard deviation values of 22.2 to 43.6% for PAH standards with concentrations ranging from 0.1 to 100 ppm. In addition, the robust poly(styrene-co-DVB) PLOT optical fibre porous polymer microextraction (PPME) device appears to retain its effectiveness with repeated use over an extended period of time.

Infrared photo-initiated fabrication of monolithic porous layer open tubular (monoPLOT) capillary columns for chromatographic applications

Investigation into the development of a fabrication approach for capillary porous layer open tubular (PLOT) chromatographic columns via infrared (IR) photo-initiated polymerisation and the optimisation of the technique is presented in this work.

Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.

Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using “sub-chip” columns (10–20 μm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 μm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 μm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3–5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.

The Application of TD/GC/NICI–MS with an Al2O3-PLOT-S Column for the Determination of Perfluoroalkylcycloalkanes in the Atmosphere

A modified method for the quantitative determination of atmospheric perfluoroalkylcycloalkanes (PFCs) using thermal desorption coupled with gas chromatography and detection by negative ion chemical ionization–mass spectrometry was developed. Using an optimized analytical system, a commercially available Al2O3 porous layer open tubular (PLOT) capillary column (30 m × 0.25 mm) deactivated with Na2SO4 was used for separation of PFCs. Improvements in the separation of PFCs, the corresponding identification and the limit of detection of PFCs using this method and column are presented. The method was successfully applied to determine the atmospheric background concentrations of a range of PFCs from a number of samples collected at a rural site in Germany. The results of this study suggest that the method outlined using the Al2O3-PLOT-S capillary column has good sensitivity and selectivity, and that it can be deployed in a routine laboratory process for the analysis of PFCs in the future research work. In addition, the ability of this column to separate the isomers of one of the lower boiling PFCs (perfluorodimethylcyclobutane) and its ability to resolve perfluoroethylcyclohexane offer the opportunity for single-column analysis for multiple PFCs.

Separation optimization of long porous‐layer open‐tubular columns for nano‐LC–MS of limited proteomic samples

The single-run resolving power of current 10 μm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 μm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.

Method and apparatus for pyrolysis—Porous layer open tubular column—Cryoadsorption headspace sampling and analysis

In previous work, dynamic headspace vapor collection on short, porous layer open tubular (PLOT) capillary columns maintained at low temperature was introduced. In this paper, that metrology is extended with the introduction of a small in situ pyrolysis platform that provides for rapid heating and rapid vapor capture for a wide variety of samples. The new approach is referred to as pyro-PLOT-cryo. The pyrolysis platform is made from two small copper lead wires that hold a basket formed from small diameter, high resistance stainless steel or NiCr wire. The basket is formed to accept a small sample, the mass of which can typically range from 0.2 to 0.05mg. The pyrolysis is performed by use of a resistor capacitor circuit of the type used in spot welders. We have provided examples of the application of this technique with the analysis of facial cosmetics, plastic explosives, organometallic gasoline additives, polymers, and in micro scale chemical reactions. Additional modifications and future work are also discussed.

Synthesis and crystal chemistry of the STA-12 family of metal N,N′-piperazinebis(methylenephosphonate)s and applications of STA-12(Ni) in the separation of gases

The crystal chemistry of divalent metal N,N′-piperazinebis(methylenephosphonates) of the STA-12 family, (M2(H2O)2(O3PCH2NC4H8NCH2PO3)·xH2O, M=Mg, Mn, Fe, Co, Ni) is compared. The different metal analogues are isostructural in the hydrated forms, possessing R3¯ symmetry, but their reversible dehydration behaviour and resultant porosity are strongly dependent on the metal cation. Whereas the Co and Ni forms change symmetry to triclinic upon dehydration, giving permanent porosity to N2 of 0.14cm3g−1 and 0.27cm3g−1, respectively, the Mn and Fe forms remain rhombohedral but exhibit a strong decrease in unit cell volume (e.g. 23% for STA-12(Mn)). Structure determination of dehydrated STA-12(Mn) indicates a topotactic transformation with a change in coordination of the phosphonate O atoms. Negligible permanent porosity is observed in either dehydrated STA-12(Mn) or (Fe), suggesting the presence of non-crystallographic pore blocking. Dehydration of STA-12(Mg) results in loss of some long range order, preventing structural determination of the fully dehydrated form, but does give appreciable permanent porosity for N2 of 0.20cm3g−1. Infrared spectroscopy (and for STA-12(Mg) solid-state NMR spectroscopy) have been used to follow the changes upon dehydration. Applications of the most porous and stable STA-12 structure, the Ni form, have also been investigated. CO2 adsorption selectivity over CH4 and CO has been measured via analysis of breakthrough curves, and a Porous Layer Open Tubular (PLOT) gas chromatographic column has been prepared and used to separate a mixture of low molecular weight alkanes.

Microproteomic analysis of 10,000 laser captured microdissected breast tumor cells using short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis and porous layer open tubular liquid chromatography tandem mass spectrometry

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC–MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC–MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

High‐Separation Performance of Chromatographic Capillaries Coated with MOF‐5 by the Controlled SBU Approach

Recently developed MOF surface-coating techniques, the controlled SBU approach (CSA) for the generation of MOF-5, and the use of self-assembled monolayers have been combined to generate a wall-bonded, crosslinked stationary phase for gas chromatographic capillary columns displaying excellent performance in the separation of natural gas components. The chromatographic performance of this new type of column has been compared to the state-of-the-art solution for this separation problem, namely a coated silica column of the porous layer open tubular (PLOT) type. Chromatographic parameters such as separation, resolution, and tailing factors, as well as plate numbers and heights in the case of isothermal operation, have been determined. Kinetic and thermodynamic parameters characterizing the analyte-stationary phase interaction have been determined for various C1-C4 analytes.