Silica-based Monolithic Column Research Articles

An all-glass based micro gas chromatographic column for light hydrocarbon separation with HKUST-1 as stationary phase

BackgroundThe gas chromatography column is one of the key components of the gas chromatograph and typically be miniaturized using micro-electro-mechanical system (MEMS) technology. Due to the limited area of the Si wafer, the column length of micro gas chromatographic column (μGCC) is usually much smaller than that of commercial chromatographic columns. Therefore, it is always difficult to use μGCCs to separate small molecule gas components such as light hydrocarbons. More importantly, the heterogeneous microchannel surface formed by silicon glass bonding causes uneven stationary phase coating, further preventing the improvement of separation performance. ResultsIn this paper, a novel all-glass based μGCC with 2 m length for the separation of light hydrocarbons is proposed. The microchannels of the μGCC were directly prepared in the glass substrate by ultrafast laser assisted chemical etching (ULAE). The all-glass microchannels make the coating of the hydrophilic metal-organic frameworks (MOFs) stationary phase continuously because of the homogeneous material composition. Therefore, a widely used copper based hydrophilic MOFs HKUST-1 was used as stationary phase for coating and testing. The test results show that the μGCC which is an open tubular column can realize the baseline separation of light hydrocarbons at 100 °C. And the resolution of difficult separated compounds, methane and ethane, can reach 12.98, which is 201.86 % higher than the silica-based monolithic capillary column in the relevant research. The resolution of ethane and ethylene reaches 6.81 at 120 °C. SignificanceThe μGCC fabricated by ULAE method is composed of all-glass and has the uniform stationary phase coating because of the homogeneous microchannel surface which greatly improve the separation performance, resulting in a large resolution for methane and ethane. The all-glass μGCC has broad application prospects in light hydrocarbon separation.

Silica-Based Monolithic Columns as a Tool in HPLC-An Overview of Application in Analysis of Active Compounds in Biological Samples.

Monolithic fillings used in chromatography are of great interest among scientists since the first reports of their synthesis and use were published. In the 20 years since silica-based monolithic columns were introduced into the commercial market, numerous papers describing their chromatographical properties and utility in various branches of industry and scientific investigations were presented. This review is focused on possible applications of commercially available silica-based HPLC monolithic columns in the analysis of biological samples.

Applicability of a Monolithic Column for Separation of Isoquinoline Alkalodis from Chelidonium majus Extract.

Isoquinoline alkaloids are the main group of secondary metabolites present in Chelidonium majus extracts, and they are still the object of interest of many researchers. Therefore, the development of methods for the investigation and separation of the alkaloids is still an important task. In this work, the application potential of a silica-based monolithic column for the separation of alkaloids was assessed. The influence of the organic modifier, temperature, salt concentration, and pH of the eluent on basic chromatographic parameters such as retention, resolution between neighboring peaks, chromatographic plate numbers, and peak asymmetry were investigated. Based on the obtained results, a gradient elution program was developed and used to separate and quantitatively determine the main alkaloids in a Chelidonium majus root extract.

Monolithic Column Ion-Pair Chromatography with Indirect Ultraviolet Detection for the Determination of Three Acid Radical Anions Containing Fluorine in Ionic Liquids

A rapid ion-pair chromatographic method was developed on a monolithic column for the determination of trifluoroacetate, trifluoromethanesulfonate, and tetrafluoroborate. The three anions were successfully detected using indirect ultraviolet detection. These analyte anions were separated using a tetrabutylammonium hydroxide - p-toluenesulfonic acid – acetonitrile mobile phase on a silica-based monolithic column with an octadecyl functional group. The effects of detection wavelength, mobile phase, column temperature and flow rate on the retention of the anions were investigated. The chromatographic conditions were optimized and the retention properties were evaluated. Under the selected optimal conditions, the successful separation of the anions was achieved within 3.5 min. The detection limits were less than 1.0 mg/L for trifluoroacetate, trifluoromethanesulfonate, and tetrafluoroborate. The relative standard deviations for five repeated measurement of retention time and peak area were no more than 0.2%. The linear ranges extended across two orders of magnitude. The three anions in synthetic ionic liquid samples were successfully separated and detected by this method. The results show that the recoveries of this method were between 94.0% and 99.0%. The method was rapid, simple, and easy to use.

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.

Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50×4.6mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10mM phosphate buffer (pH7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Comprehensive two-dimensional monolithic liquid chromatography of polar compounds.

Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.

Monolithic Silica Capillary Columns with Improved Retention and Selectivity for Amino Acids

A strategy for the preparation of silica-based monolithic capillary columns (150 × 0.1 mm) with high selectivity to amino acids is presented. The zwitterionic columns were prepared by coating the silica monolith with [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. The columns were evaluated under isocratic conditions in hydrophilic interaction liquid chromatography. The best separation of amino acids was obtained on the monolithic column prepared by a stepwise modification procedure where the modification step was repeated four times. The mixture of fifteen amino acids was separated within 13 min using the mobile phase consisting of 75% acetonitrile and 25% 5 mmol/L ammonium acetate at pH 4.5.

Immobilization of a phosphonium ionic liquid on a silica monolith for hydrophilic interaction chromatography

A methodology for preparing phosphonium-based ionic liquid modified silica-based monolithic capillary columns is presented. The silica monolithic columns with dimensions of 150 × 0.1 mm were modified by a phosphonium-based ionic liquid (trioctyl(3/4-vinylbenzyl)phosphonium chloride) via 3-(trimethoxysilyl)propyl methacrylate. The prepared columns were evaluated under hydrophilic interaction liquid chromatography separation conditions, employing a sample mixture containing purine and pyrimidine bases and nucleosides. Detection was made by UV. The high efficiency of the original silica monolith was preserved even after modification, and it reached values in the range of 98,000–174,000 theoretical plates/m. The effects of the concentration of acetonitrile in the mobile phase, the presence of additives in the mobile phase, such as, acetic acid or ammonium acetate, and the pH of the mobile phase on the separation of some selected analytes were investigated. The prepared columns showed different separation selectivity compared to silica, phenyl and sulfobetaine stationary phases.

Rapid Determination of Pyrrolidinium Cations by Ion-Pair Chromatography With Imidazolium Ionic Liquids.

This article describes a pioneering ion-pair chromatography with indirect ultraviolet detection with imidazolium ionic liquids (ILs) as ultraviolet absorption reagent and eluting agent for determination of pyrrolidinium cations. Three pyrrolidinium cations were analyzed on a reversed-phase silica-based monolithic column by isocratic elution with imidazolium ILs-ion-pair reagents-methanol mixtures. The effects of the ultraviolet absorption reagents, detection wavelength, ion-pair reagents, imidazolium ILs, organic solvents, column temperature and flow rate on the separation and determination of pyrrolidinium cations were investigated. Under the selected chromatographic conditions, the separation time was reduced to <2.5 min while maintaining excellent peak shapes and sufficient resolution. The detection limits for N-methyl-N-ethylpyrrolidinium cation, N-methyl-N-propylpyrrolidinium cation and N-methyl-N-butylpyrrolidinium cation were 0.12, 0.08 and 0.19 mg/L, respectively. Relative standard deviations for peak area were <0.59%. This simple and practical method has been successfully applied to the determination of three IL samples synthesized by the chemistry laboratory. The method is accurate, reliable and meets the requirements for quantitative analysis. In general, the analytical system and its associated methods appear to be widely applicable to determination of pyrrolidinium cations.

Self-packed core shell nano liquid chromatography columns and silica-based monolithic trap columns for targeted proteomics

Self-preparation of nano liquid chromatography (nLC) columns has advantages regarding cost and flexibility. For targeted proteomics, we evaluated several approaches for particle-packing nLC columns and manufacturing fritless silica-based monolithic trap columns (50μm inner diameter). Our preferred approach for nLC column preparation was to magnetically stir Accucore core shell particles (C18 stationary phase) in ACN/water (80/20, v/v) suspensions during pressure-driven filling of polymer-fritted standard fused silica capillaries. The columns were ready for use about one hour after preparation had begun. They had comparable peak capacities (peptides) to commercial columns, and satisfactory within/between-column retention time repeatability, suited for targeted proteomics. Packing with commercial capillary housings/nanospray emitters did not improve performance compared to packing with in-house fritted stock fused silica capillary tubing.For trap columns, several recipes for narrow bore silica-based monolithic columns were evaluated, and we found the recipe by Zou et al. (2005) to be reproducible. Compared to the standard C18 trap column for Accucore nLC columns, monolith trap columns (C8 stationary phase) significantly reduced peak widths. The readily prepared in-house columns were used for targeted detection of the enzyme CYP27A1 in cancer cells, which is associated with proliferation and metastasis of breast cancer.

Bridged polysilsesquioxane-based wide-bore monolithic capillary columns for hydrophilic interaction chromatography

The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10−14m2 and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5μm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.

Rapid and simultaneous determination of piperidinium and pyrrolidinium ionic liquid cations by ion pair chromatography coupled with direct conductivity detection

A method of ion-pair chromatography with direct conductivity detection was developed on a silica-based monolithic column for the fast and simultaneous determination of piperidinium and pyrrolidinium ionic liquid cations. The effects of the mobile phase, column temperature and flow rate on the retention of the cations were investigated. The retention rules were discussed. As an ion-pair reagent, sodium heptanesulfonate is more suitable than sodium pentanesulfonate for the separation and determination of piperidinium and pyrrolidinium cations. The increase of ion-pair reagent concentration led to the increased retention time of the cations. When acetonitrile content and mobile phase flow were increased, the retention time of the cations became shorter. The retention of piperidinium and pyrrolidinium cations is an exothermic process, and the retention of the cations conforms to the carbon number rule. The chromatographic analysis was performed using the Chromolith Speed ROD RP-18e column, 0.5mmol/L sodium heptanesulfonate-5% acetonitrile as the mobile phase at a flow rate of 3.0mL/min and column temperature of 30°C. Separation of N-methyl-N-ethyl piperidinium, N-methyl-N-propyl piperidinium, N-methyl-N-butyl piperidinium and N-methyl-N-ethyl pyrrolidinium, N-methyl-N-propyl pyrrolidinium, N-methyl-N-butyl pyrrolidinium cations were achieved within 10min. The detection limits (S/N=3) were between 0.19 and 3.08mg/L. Relative standard deviations (n=5) for peak areas were less than 1.2%. The method has been applied to the determination of piperidinium and pyrrolidinium cations in ionic liquid samples. The spiked recoveries of ionic liquid cations were between 96% and 111%. The method is accurate, reliable, rapid, and has a better practicability.

Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns

A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.

Effect of polyethylene glycol on pore structure and separation efficiency of silica-based monolithic capillary columns

Monolithic silica materials (first unclad monolith rods, then monolithic capillary columns) were prepared using various amounts of polyethylene glycols (PEGs) with different molecular weight (MW). The monolith rods were used to examine the mesoporosity by argon physisorption technique, and the macroporosity by mercury intrusion porosimetry. Subsequently, silica-based monolithic capillary columns with an inner diameter of 100μm were produced using the same preparation conditions as used for the rods. The results obtained with the monolith rods showed the following important findings: (1) it is feasible to fabricate monolithic silica rods possessing macropore size of 0.5–1.4μm by tuning the amount of PEGs (independently of the MW), whereas the macropore volume and the mesoporosity remain similar. (2) the smallest macropore size (0.5μm) rod prepared with PEG having a MW=20,000g/mol provided a narrower macropore size distribution than with PEG with MW=10,000g/mol. The monolithic capillary columns produced with the different PEG type showed similar retention factors for hexylbenzene (k=2.3–2.4) and similar t0-based column permeability (Kv0=2.3–2.4×10−14m2) in 20:80% (v/v) water:methanol, as expected from the results obtained with the monolith rods. The column prepared with PEG of MW=20,000g/mol gave a plate height of H=4.0μm for hexylbenzene at an optimal linear velocity of u0=2.6mm/s in 20:80% (v/v) water containing 0.1% formic acid:acetonitrile. To the best of our knowledge, this is the lowest plate height ever recorded for a monolithic column. Comparing the kinetic performance at 30MPa shows that the best monolithic silica column obtained in the present study performs better than the second-generation monolithic silica columns developed up till now in the practically most relevant range of plate numbers (N≤40,000). In this range, the performance is now similar to that of 2.7μm core-shell particle columns.

Separation Behavior of Short Oligonucleotides by Ion-Pair Reversed-Phase Capillary Liquid Chromatography Using a Silica-Based Monolithic Column Applied to Simple Detection of SNPs

The separation behaviors of short oligonucleotides using capillary liquid chromatography have been investigated comparatively on an octadecylsilane (ODS) packed column (0.3 mm inner diameter, particle size 3 μm) and a monolithic ODS column (0.5 mm inner diameter, through pore size 2 μm). The present paper clearly demonstrates that the resolution obtained using the monolithic ODS packed column is much higher than that using a semi-μ-high-performance liquid chromatography (HPLC) column on a semi-μ-HPLC system. Furthermore, it was surprising that complete baseline resolution of oligonucleotides, typically shorter than 30-mer, with single nucleotide units was achieved using the monolithic column, although this was not possible using the ODS packed column. Good separation of short oligonucleotides was achieved at 40–60 °C with a linear 40–60 % gradient of methanol content. Separation conditions on the monolithic column were optimized regarding the type of counterion, methanol content, temperature, and flow rate. Using the present method, detection of single-nucleotide polymorphisms was achieved without using a mass-spectrometry (MS) detector.

Silica-Based Monolithic Coupled Column for the Simultaneous Determination of Echitamine, Nb-Demethylalstogustine, and Loganetin in Alstonia scholaris by RP-HPLC and Optimization of Extraction Method

A simple, rapid, and sensitive high performance liquid chromatography (HPLC) method for the simultaneous quantification of echitamine (1), Nb-demethylalstogustine (2), and loganetin (3) in Alstonia scholaris was developed using silica-based monolithic coupled column. The samples were analyzed on Chromolith RP-18 coupled column (100 + 50 = 150 mm × 4.6 mm) using isocratic elution with acetonitrile: 0.01 M buffer (KH2PO4) containing 0.1% trifluoroacetic acid (20:80, v/v) at 0.5 mL/min flow rate and detection at 254 nm. The limits of detection and quantitation were 0.64 and 2.15 µg/mL for echitamine, 0.72 and 2.43 µg/mL for Nb-demethylalstogustine, and 0.78 and 2.62 µg/mL for loganetin, respectively. Standard curves were linear in the range of 3–15 µg/mL (r2 = 0.999). The method showed good repeatability (%RSD < 1.14), reproducibility (%RSD < 1.16), and recovery (mean% > 99.48). This is the first report on loganetin isolation from A. scholaris. Furthermore, various extraction methods and parameters were investigated to optimize markers extraction from the sample matrix. Finally, the optimized extraction method was used in the extraction of markers from different parts of the plant. The validated method was successfully used in the quantification of markers in various parts of the plant, which can be applied in the overall quality assessment of A. scholaris.

Feasibility of the preparation of silica monoliths for gas chromatography: Fast separation of light hydrocarbons

The preparation conditions of silica monoliths for gas chromatography were investigated. Silica-based monolithic capillary columns based on sol–gel process were tested in the course of high-speed gas chromatographic separations of light hydrocarbons mixture (C1–C4). The impact of modifying the amount of porogen and/or catalyst on the monolith properties were studied. At the best precursor/catalyst/porogen ratio evaluated, a column efficiency of about 6500 theoretical plates per meter was reached with a very good resolution (4.3) for very light compounds (C1–C2). The test mixture was baseline separated on a 70cm column. To our knowledge for the first time a silica-based monolithic capillary column was able to separate light hydrocarbons from methane to n-butane at room temperature with a back pressure in the range of gas chromatography facilities (under 4.1bar).

Separation of nucleobases, nucleosides, and nucleotides using two zwitterionic silica-based monolithic capillary columns coupled with tandem mass spectrometry

The capability of employing synthesized zwitterionic silica-based monolithic capillary columns (140mm×0.1mm) for separation of highly polar and hydrophilic nucleobases, nucleosides, and nucleotides in hydrophilic interaction chromatography is reported. The suitability of the columns for on-line conjunction with electrospray tandem mass spectrometry was explored. Our results show that the grafted layer of zwitterionic monomer ([2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide or 2-methacryloyloxyethyl phosphorylcholine) on the silica monolithic surface significantly improved the separation selectivity and reproducibility, as compared to the bare silica monolith. The stepwise elution from 90% to 70% of acetonitrile enabled separation of a complex sample mixture containing 21 compounds with a total analysis time less than 40min.

The pore size distribution of the first and the second generation of silica monolithic stationary phases

The mesopore structure (pore size and its distribution) for the first and second generations of silica-based monolithic columns was determined by inverse size-exclusion chromatography. The effect of pore size distribution was considered via the molecular theory of size-exclusion chromatography. The molecular theory of chromatography allows taking into account the kinetics of the pore ingress and egress processes, the heterogeneity of the pore sizes and polymer polydispersity. Besides, the mesopore structure, the characteristic domain sizes of the macropores present in the first and second generations of silica-based monolithic columns were also characterized.