Semi-micro-monolithic columns using macroporous silica rods with improved performance

Monolithic silica columns in semi-micro-format have been synthesized using poly(acrylic acid) as a phase-separation inducer via a sol–gel route. The absence of a thick skin layer accompanied by deformation of the micrometer-sized gelling skeletons on the outermost part of the macroporous silica rod contributed to improve the efficiency of monolithic silica columns as thick as 2.4 mm in diameter. The kinetic plot analysis revealed that monolithic silica columns with macropore diameter of 1 μm and skeleton thickness of 1 μm with decreased macroporosity behave similarly to columns packed with 3 μm particles with slightly lower back pressure.     

Double Pore Silica Gel Monolith Applied to Liquid Chromatography

Silica gels retaining double pore structure in the size ranges of micrometer and nanometer have been applied to the rod-shaped monolithic column for liquid chromatography. The macropore structure was designed by controlling the phase separation process induced by the hydrolysis and polycondensation of alkoxysilane, whereas the mesopore structure was tailored by the solvent exchange treatments on wet gels. The size exclusion chromatograms on polystyrene standards exhibited almost similar features for octadecyl-modified rod and conventional packed beads columns. The dependence of plate height on the velocity of mobile phase determined for amylbenzene was by far weaker in the rod column than in the packed beads column, suggesting that additional geometrical factors should be considered in describing the separation mechanism in the rod column.     

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile-water (80/20, v/v) mobile phase at 40°C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K(F) about 2.4×10(-14) m(2) (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000-120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates (N) is greater than 50,000.     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.     

Formation of Hierarchical Pore Structure in Silica Gel

By inducing a phase separation parallel to the sol-gel transition of alkoxy-derived silicate systems, gels with well-defined macroporous structure can be prepared. Depending on the post-gelation treatment such as aging and solvent exchange, the final pore structure in the nanometer range of dried and heat-treated gels exhibits a considerable variation. With an aim of completely controlling the hierarchical pore structure in the discrete size ranges of nanometers and micrometers, systematic experimental studies have been performed. The macroporous nature of the wet gels allows an efficient solvent exchange process compared with conventional gels only with mesopores. In addition, the surface chemistry of the wet gel skeleton affects the mesopore formation process by the solvent exchange to a great extent. The median size of mesopores larger than 5 nm can be controlled by adjusting the basic solvent exchange conditions such as pH value, temperature and bath ratio for any kind of macroporous silica gel. On the other hand, the control of pore volume independent of the mesopore size is possible only in the system incorporated with the micelle-forming surfactant. Some examples of the effects of controlled mesopores on the analytical performance of monolithic-type chromatographic columns are also presented.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Sol–gel synthesis of macro–mesoporous titania monoliths and their applications to chromatographic separation media for organophosphate compounds

We have developed a method of independently tailoring the macro- and mesoporous structures in titania (TiO₂) monoliths in order to achieve liquid chromatographic separations of phosphorous-containing compounds. Anatase TiO₂ monolithic gels with well-defined bicontinuous macropores and microstructured skeletons are obtained via the sol–gel process in strongly acidic conditions using poly(ethylene oxide) as a phase separator and N-methylformamide as a proton scavenger. Aging treatment of the wet gels in the mother liquor at temperatures of 100–200 °C and subsequent heat treatment at 400 °C allow the formation and control of mesoporous structures with uniform pore size distributions in the gel skeletons, without disturbing the preformed macroporous morphology. The monolithic TiO₂ rod columns with bimodal macro–mesoporous structures possess the phospho-sensitivity and exhibit excellent chromatographic separations of phosphorus-containing compounds.     

Monolithic bed structure for capillary liquid chromatography

Monolithic stationary phases show promise for LC as a result of their good permeability, ease of preparation and broad selectivity. Inorganic silica monoliths have been extensively studied and applied for separation of small molecules. The presence of a large number of through pores and small skeletal structure allows the chromatographic efficiencies of silica monoliths to be comparable to columns packed with 5 μm silica particles, at much lower back pressure. In comparison, organic polymeric monoliths have been mostly used for separation of bio-molecules; however, recently, applications are expanding to small molecules as well. Organic monoliths with high surface areas and fused morphology rather than conventional globular morphology have shown good performance for small molecule separations. Factors such as domain size, through-pore size and mesopore size of the monolithic structures have been found to govern the efficiency of monolithic columns. The structure and performance of monolithic columns are reviewed in comparison to particle packed columns. Studying and characterizing the bed structures of organic monolithic columns can provide great insights into their performance, and aid in structure-directed synthesis of new and improved monoliths.     

Molded separation media: An inexpensive,efficient, and versatile alternative to packed columns for the fast HPLC separation of peptides,proteins, and synthetic oligomers and polymers

A simple molding process carried out within the confines of a chromatographic column has been used for the preparation of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) rods. The novel monolithic separation media that are obtained are useful for the HPLC separation of biological and synthetic polymers. The presence of large pores with a diameter of about 1 μm makes the molded rod columns easily permeable to eluents. Therefore, the back pressure of these columns is modest even at high flow rates. In contrast to the conventional HPLC columns packed with beads, all of the mobile phase flows through the continuous monolithic medium. As a result of this total convection, the efficiency of the molded media is almost independent of the flow rate. This improves significantly the separation ability of the rod columns and very fast separations of macromolecules such as peptides, proteins, and synthetic polymers have been demonstrated.     

Rapid reversed-phase separation of proteins and peptides using optimized 'moulded' monolithic poly(styrene-co-divinylbenzene) columns

Monolithic macroporous poly(styrene-co-divinylbenzene) stationary phases have been prepared by free radical polymerization within the confines of 4.6-mm I.D. chromatographic columns. The optimized porous properties allow the mobile phase to flow through these columns at flow-rates of up to 10 ml/min. As opposed to the simultaneously tested columns packed with either silica or synthetic polymer beads, the monoliths exhibit only modest back pressure. The monolithic columns were able to separate mixtures of peptides and proteins in a very short time. Under the optimized conditions, the separation of five proteins can be easily achieved in less than 20 s.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Permeability, porosity, and structure of monolithic capillary columns in gas chromatography

The permeability of monolithic silica gel capillary columns with respect to the helium carrier gas was studied using gas chromatography. The results obtained by gas chromatography and liquid chromatography were found to be in close agreement. The permeability of monolithic capillary columns was compared to that of hollow capillary columns and columns packed with finely dispersed sorbents. It was demonstrated that the permeability of the monolithic capillary columns studied is almost three orders of magnitude lower than that of hollow capillary columns of the same diameter but two orders of magnitude higher than that of columns packed with micron-scale particles. The interstitial fraction of the monolithic columns was found to be very high, 0.95.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

Tailoring Mesopores in Monolithic Macroporous Silica for HPLC

Mesopore formation in silica gels having continuous macropores has been investigated. The macroporous wet silica gel prepared by the sol‐gel process including phase separation was aged in a basic solvent making use of hydrolysis of urea in a closed condition. The mesopore structure was finally obtained by subsequent evaporation drying of solvent and heat‐treatment at 600°C for 2 h. The dissolution‐reprecipitation kinetics at the interfaces between wet gel skeletons and an external solvent affected the size and volume of pores formed within the skeletons. Below 120°C, mesopores suitable for various chromatographic applications have been formed typically within 24 h. On the other hand, at 200°C, the pore size attained the macropore dimensions (>50 nm), and the whole macroporous morphology was significantly modified.     

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

Design and evaluation of synthetic silica-based monolithic materials in shrinkable tube for efficient protein extraction

Sample pretreatment is a required step in proteomics in order to remove interferences and preconcentrate the samples. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transport compared with more common packed beds. These features are due to the micro-structure within the monolithic silica column which contains both macropores that reduce the back pressure, and mesopores that give good interaction with analytes. The aim of this work was to fabricate a continuous porous silica monolithic rod inside a heat shrinkable tube and to compare this with the same material whose surface has been modified with a C(18) phase, in order to use them for preconcentration/extraction of proteins. The performance of the silica-based monolithic rod was evaluated using eight proteins; insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, ovalbumin, hemoglobin, and bovine serum albumin at a concentration of 60 μM. The results show that recovery of the proteins was achieved by both columns with variable yields; however, the C(18) modified silica monolith gave higher recoveries (92.7 to 109.7%) than the non-modified silica monolith (25.5 to 97.9%). Both silica monoliths can be used with very low back pressure indicating a promising approach for future fabrication of the silica monolith inside a microfluidic device for the extraction of proteins from biological media.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

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Monolithic Silica Columns for HPLC,Micro‐HPLC,and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR‐FS) and the other prepared in a mold and clad with an engineering plastic (poly‐ether‐ether‐ketone) (SR‐PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5‐μm ODS‐silica particles and of an ODS‐silica monolith prepared in a mold and wrapped with PTFE tubing (SR‐PTFE). SR‐FS gave a lower pressure drop than a column packed with 5‐μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR‐PEEK showed higher flow‐resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8–10 μm and a lower pressure drop than popular commercial columns packed with 5‐μm particles. The evaluation of SR‐FS columns in a CEC mode showed much higher efficiency than in a pressure‐driven mode.