Silica-based Monolithic Capillary Columns Research Articles

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.

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.

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.

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.

Photopolymerization of acrylamide as a new functionalization way of silica monoliths for hydrophilic interaction chromatography and coated silica capillaries for capillary electrophoresis

A simple, rapid and localizable photochemical process for the preparation of hydrophilic coated capillary and silica-based monolithic capillary columns is described. The process involves the free radical polymerization of acrylamide monomers onto acrylate pre-activated silica surface triggered by UV photoinitiation. The experimental conditions (monomer content, time of irradiation) were optimized on silica monolithic columns by monitoring the evolution of the chromatographic properties (retention, permeability, efficiency) in HILIC mode using a set of nucleosides as test solutes. Compared to thermal polymerization process, the photoinitiation allows the preparation of highly retentive and efficient HILIC monolithic columns in less than 10min of irradiation. This process was then successfully applied to the surface coating of fused silica capillary walls. In addition to its relative high stability and ability to reduce the electroosmotic flow, this polyacrylamide coating is localizable. Benefits of this localizable photochemical process are highlighted through the conception of an in-line integrated bimodal microseparation tool combining a SPE preconcentration step on a photografted silica monolith and an electrokinetic separation step in a polyacrylamide photopolymerized capillary section. Two neuropeptides are used as model solutes to illustrate the suitability of this approach.

Silica-based monolithic capillary columns modified by liposomes for characterization of analyte–liposome interactions by capillary liquid chromatography

This study introduces a silica-based monolith in a capillary format (0.1mm×100mm) as a support for immobilization of liposomes and its characterization in immobilized liposome chromatography. Silica-based monolithic capillary columns prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea were modified by (3-aminopropyl)trimethoxysilane, whereby amino groups were introduced to the monolithic surface. These groups undergo reaction with glutaraldehyde to form an iminoaldehyde, allowing covalent binding of pre-formed liposomes containing primary amino groups. Two types of phospholipid vesicles were used for column modification; these were 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidyl choline with and without 1,2-diacyl-sn-glycero-3-phospho-l-serine. The prepared columns were evaluated under isocratic separation conditions employing 20mM phosphate buffer at pH 7.4 as a mobile phase and a set of unrelated drugs as model analytes. The liposome layer on the synthesized columns significantly changed the column selectivity compared to the aminopropylsilylated monolithic stationary phase. Monolithic columns modified by liposomes were stable under the separation conditions, which proved the applicability of the suggested preparation procedure for the synthesis of capillary columns dedicated to study analyte–liposome interactions. The column efficiency originating from the silica monolith was preserved and reached, e.g., more than 120,000 theoretical plates/m for caffeine as a solute.

Zwitterionic silica-based monolithic capillary columns for isocratic and gradient hydrophilic interaction liquid chromatography

This study introduces zwitterionic monolithic capillary columns intended for isocratic and gradient HILIC separations. Silica-based monolithic capillary columns (100μm×150mm) prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea were modified by a sulfoalkylbetaine zwitterion ([2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide) to HILIC stationary phase. The prepared columns were evaluated under the isocratic and gradient separation conditions using a standard mixture containing nucleic acid bases, nucleosides, and 2-deoxynucleosides. Mobile phases contained high concentration of acetonitrile (95–85%, v/v) and 5–50mM ammonium acetate or ammonium formate in the pH range of 3–6. The synthesized columns showed a long-term stability under the separation conditions while the high permeability and efficiency originating from dual structure of the silica monolith were preserved. The relative standard deviations (RSDs) for the retention times of tested solutes were lower than 2% under the isocratic conditions and lower than 3.5% under the gradient conditions.

Silica-based monolithic capillary columns—Effect of preparation temperature on separation efficiency

The temperature effects during the sol–gel process and ageing of the silica-based monolith on the structure and separation efficiency of the capillary columns (100 μm i.d., 150 mm) for HPLC separations were studied. The tested columns were synthesized from a mixture of tetramethoxysilane, polyethylene glycol and urea under the acidic conditions. The temperature was varied from 40 °C to 44 °C and formation of bypass channels between the silica mold and the capillary wall was examined. The temperature of 43 °C was estimated as optimal for preparation of efficient silica capillary columns which were subsequently modified by octadecyldimethyl- N, N-diethylaminosilane or covered by poly(octadecyl methacrylate) and tested using standard mixture of alkylbenzenes under the isocratic conditions.

MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics

Silica-based monolithic capillary columns (25 cm x 10 microm i.d.) with integrated nanoESI emitters have been developed to provide high-quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray operation at flow rates of approximately 10 nL/min. In an initial application with a linear ion trap MS, we identified 5510 unique peptides that covered 1443 distinct Shewanella oneidensis proteins from a 300-ng tryptic digest sample in a single 4-h LC-MS/MS analysis. The use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and provided good run-to-run reproducibility.

Capillary electrochromatography with monolithic silica columns. IV. Electrochromatographic characterization of polar bonded monolithic stationary phases having surface‐bound cyano functionalities

Two polar ligands, namely 3-hydroxypropionitrile and 1H-imidazole-4,5-dicarbonitrile (IDCN) were covalently attached to epoxy-activated silica-based monolithic capillary columns via an epoxide ring-opening reaction to yield CN-OH-Monolith and 2CN-OH-Monolith, respectively. The silica monolith was prepared by a sol-gel process, and the resulting "rod-like" stationary phase was subjected to pore tailoring with an alkaline solution to convert small pore domains to mesopore domains, thus yielding a monolith with bimodal pore structure consisting of flow through pores (i.e., flow channels for mobile-phase flow) and mesopores that provide most of the adsorption capacity of the monolith toward the separated solutes. The two polar monoliths, CN-OH-Monolith and 2CN-OH-Monolith, were evaluated in normal-phase CEC with organic-rich mobile phases less polar than the stationary phase. The 2CN-OH-Monolith bearing more polar functions than the CN-OH-Monolith exhibited more retention and improved selectivity toward model polar solutes.

Preparation of 20-μm-i.d. Silica-Based Monolithic Columns and Their Performance for Proteomics Analyses

We describe the preparation and performance of high-efficiency 70 cm x 20 microm i.d. silica-based monolithic capillary LC columns. The monolithic columns at a mobile-phase pressure of 5000 psi provide flow rates of approximately 40 nL/min at a linear velocity of approximately 0.24 cm/s. The columns provide a separation peak capacity of approximately 420 in conjunction with both on-line coupling with microsolid-phase extraction and nanoelectrospray ionization-mass spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and approximately 15-amol detection limits for peptides were obtained using a conventional ion trap and MS/MS for peptide identification. The sensitivity and separation efficiency enabled the identification of 2367 different peptides covering 855 distinct S. oneidensis proteins from a 2.5-microg tryptic digest sample in a single 10-h analysis. The number of identified peptides and proteins approximately doubled when the effective separation time was extended from 200 to 600 min. The number of identified peptides increased from 32 to 390 as the injection amount was increased from 0.5 to 100 ng. Both the run-to-run and column-to-column reproducibility for proteomic analyses were also evaluated.

Silica-based monoliths for capillary electrochromatography: methods of fabrication and their applications in analytical separations.

This review article is intended to provide the reader with the recent advances made in the fabrication of silica-based monolithic capillary columns for use in capillary electrochromatography (CEC). The silica-based monoliths can be produced by three different approaches, namely (i) fusion of silica particles by thermal sintering, (ii) cross-linking/entrapping silica particles in a packed bed using the sol-gel process, and (iii) polymerization of silicon alkoxide precursors using the sol-gel process. Thus far, approach (iii) is the most widely used for fabricating silica monoliths. After providing a thorough description of each of the three approaches used for the production of silica-based monolithic capillary columns, the analytical separations performed by CEC on each kind of monolith are discussed.

Silica-based monoliths for rapid peptide screening by capillary liquid chromatography hyphenated with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Capillary liquid chromatography based on particulate and monolithic stationary phases was used to screen complex peptide libraries by fast gradient elution coupled on-line to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). A slightly modified commercial electrospray interface consisting of a fused-silica transfer capillary and low dead volume stainless steel union at which the electrospray voltage was grounded enabled the effluent of all the capillary columns to be directly sprayed into the mass spectrometer. Stable electrospray conditions were generated over a wide range of mobile phase compositions, alleviating the need for a tapered end of the spray capillary, pneumatic assistance or preheated nebulizer gas. Since the identification of complex samples containing numerous isobaric substances is facilitated by chromatographic separation prior to mass spectrometry, stationary phase materials have been employed which offer a fast, efficient elution and, due to the complexity of samples, a high loading capacity. Silica-based monolithic capillary columns combine these three characteristics in a unique manner due to a tailored adjustment of both macro- and mesopore sizes in the highly porous silica structure. As we demonstrate by a comparative study of the silica-based monolithic and packed capillaries for LC/MS analysis of complex peptide libraries, silica monoliths show superior performance over packed beds of small-diameter particles with respect to analysis time and separation efficiency. Libraries with more than 1000 different peptides could be screened in less than 20 min.