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.     

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 5 mL/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 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.     

Size separation of colloidally dispersed nanoparticles using a monolithic capillary column

We developed a method to separate colloidally dispersed nanoparticles on monolithic capillary columns. Silica nanoparticles were eluted according to their sizes, and the plots of the logarithm of the size of nanoparticles against their elution volume showed good linearity (r=0.992) over wide range of sizes. Because of the high porosity of the monolithic column (porosity; 88%), the column's length could be increased without clogging of the dispersed samples and the pressure in a long column (500 mm × 0.2 mm i.d.) was low, with a value of 5.8 MPa at a flow rate of 1 μL/min. We demonstrate that this method using monolithic capillary columns could be used as a powerful tool for size separation of nanometer-size materials, which will open a new pathway to quality control of nanomaterials in nanotechnology applications.     

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography

In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Comparison of the migration behavior of nanoparticles based on polyethylene glycol and silica using micellar electrokinetic chromatography

Nanoparticles, spherical particles with diameters less than 100 nm, are promising theranostic devices for noninvasive diagnosis and therapy. In this study, nanoparticles composed of polyethylene glycol and silica were prepared, and their migration behavior was examined using capillary electrophoresis. The effects of the sodium dodecyl sulfate concentration in the electrolyte, the nanoparticle size, and the encapsulated molecule on the migration were examined. The addition of sodium dodecyl sulfate into the electrolyte had a significant effect on the electrophoretic mobility of polyethylene glycol nanoparticles, but a small effect on that of silica nanoparticles. As for the size effect, the mobility became a little faster for smaller nanoparticle sizes for both polyethylene glycol and silica nanoparticles. The encapsulated molecule affected the mobility of the nanoparticles through interactions between the encapsulated molecules and sodium dodecyl sulfate. We propose that the large effect of sodium dodecyl sulfate on the migration of the polyethylene glycol nanoparticles was due to the large spaces within the nanoparticles. These results indicate that nanoparticle migration is mainly determined by the nanoparticle components.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.     

Preparation of particle-fixed silica monoliths used in capillary electrochromatography

Fused-silica capillarieswere packed with porous 1 microm bare silica microspheres and immobilized by potassium silicate-formamide in order to obtain columns with silica-based monolithic packing. After curing, the particle-fixed monolithic columns were octadecylated insitu with dimethyloctadecylchlorosilane. The columns were mechanically strong and permeable. No noticeable loss in efficiency was found after using a column continuously for 1 month. The performances of the particle-fixed silica monolithic columns were evaluated for CEC under RP conditions. High separation efficiency (about125 200 plates/m) was obtained by using these new types of columns.     

Structure and performance of silica-based monolithic HPLC columns

Silica-based monolithic columns were prepared for HPLC with systematic variations of the tetramethoxysilane (TMOS) and polyethylene oxide (PEO) content as reactants in a sol-gel process accompanied by phase separation. The resulting monoliths showed differences in the macropore and silica skeleton diameter as well as in the corresponding domain sizes (the sum of macropore and skeleton diameter). All monoliths were synthesized with a diameter of 4.6 mm and cladded with a suitable polyaryletheretherketone (PEEK) polymer in a standardized and optimized manner for the subsequent chromatographic evaluation of the resulting monolithic HPLC columns. The columns were tested under normal phase conditions using n-heptane/dioxane (95:5 v/v) as a mobile phase and 2-nitroanisole as a test compound for the determination of separation efficiency and permeability. Two different sets of columns were prepared: the first one in which the amount of PEO was stepwise decreased to yield monoliths with identical macropore volumes and variations in the domain sizes. The second group of materials was synthesized adjusting both TMOS and PEO quantities to yield monolithic columns with identical macropore diameters of about 1.80 microm but different skeleton diameters and macropore volumes. The chromatographic results suggest that an increase in the column performance cannot be achieved by just arbitrarily decreasing the domain size of a given column. From a certain point of "downsizing" the monolithic structure a loss of structural homogeneity can be observed, which is apparently responsible for a lower chromatographic performance.     

Electrophoretic separation of nitrogen-containing drugs using silica capillaries modified with citrate-stabilized gold nanoparticles, 6,10-ionene, N-(3-sulfo,3-carboxy)-propionylchitosan and dextran sulfate

Gold nanoparticles have been widely applied in different fields of science over the last decade, e.g., in physicochemical separation methods. Two fused silica capillaries for capillary electrophoresis (CE) modified layer-by-layer with citrate-stabilized gold nanoparticles, 6,10-ionene, N-(3-sulfo,3-carboxy)-propionylchitosan (SCPC), and dextran sulfate (DS) were obtained and investigated. It has been found that the separation of the mixture of tetrahydrozoline, pindolol, terbutaline, nadolol, and hydroxyzine is fastest and most effective when the capillaries are modified with gold nanoparticles than with polysaccharide and polymers only. The most successful system for CE was obtained using DS as the polysaccharide. The enantiomers of tetrahydrozoline were separated using the capillaries modified with gold nanoparticles, 6,10-ionene, and dextran sulfate.     

Thermo-responsive monolithic materials

One of the recent major improvements of HPLC was the introduction of monolithic silica columns, which have the advantage of faster separation and lower back pressure as compared to common silica beads. Here, we present an interesting alternative to such reversed-phase monolithic columns by a convenient coupling route of a thermo-responsive polymer to hydrophilic silica monoliths. Poly(N-isopropylacrylamide) (PNIPAM) was polymerized in solution via a reversible addition fragmentation chain transfer (RAFT) polymerization technique and coupled then in situ onto an amino-modified silica monolithic column. These columns were compared with RP-18 monolithic columns in the separation of steroids under isocratic condition in aqueous mobile phase. Separation is optimized just by changing the temperature, instead of changing the mobile phase composition.     

Fast high performance liquid chromatography analysis in lipidomics: Separation of radiolabelled fatty acids and phosphatidylcholine molecular species using a monolithic C18 silica column

HPLC procedures using conventional C₁₈ columns are usually used to separate simple and complex lipid mixtures but these methods of separation remain often laborious and very slow. Here, monolithic columns were successfully applied to separate lipids - radiolabelled fatty acid mixtures and individual phosphatidylcholine (PC) molecular species. For that, isocratic elution was performed using two Chromolith™ Performance RP-18e columns connected in series. Detection was achieved by online measurement of radioactivity for radiolabelled fatty acids and by UV absorbance at 205 nm for PC molecular species. The performances of such silica rods were compared to conventional reverse-phase silica columns. Monolithic stationary phase separated radiolabelled fatty acids and PC molecular species two times and four times faster, respectively. In each analysis, monolithic columns allowed better separation efficiency per unit of time, with lower inlet pressure. The main advantages of this method for lipid separation are that, under isocratic conditions, it is simpler and much faster, while remaining accurate and selective when compared to conventional methods. Therefore, monolithic columns may represent a powerful tool for the near future in the field of lipidomics.     

Titania-coated monolithic silica as separation medium for high performance liquid chromatography of phosphorus-containing compounds

A method of preparing titania-coated monolithic silica stationary phase has been developed to achieve liquid chromatographic separation of phosphorus-containing compounds, which have recently been attracting increasing attention in biochemical research. The titania-coated silica columns exhibited efficient separation with low pressure drop, which is a typical feature of monolithic structures, and also possessed phospho-selectivity, which is a unique property of the titania surface. The material characteristics of titania-coated monolithic silica were examined, and then resin-clad columns were applied to the HPLC analysis of phosphorylated compounds. Highly efficient separation of phosphorylated substances indicated that the novel titania-coated monolithic silica column will find applications as a useful tool in the field of biochemistry, especially in post-genomic analyses.     

Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography

This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.     

Single-step approach to beta-cyclodextrin-bonded silica as monolithic stationary phases for CEC

A novel single-step sol-gel approach for the preparation of beta-CD-bonded silica monolithic electrochromatographic columns is established. The porous silica networks were fabricated inside fused-silica capillaries using sol-gel processing of tetramethoxysilane and an organfunctional silicon alkoxide that contains beta-CD. Scanning electron micrographs and nitrogen adsorption-desorption data showed that these functional monolithic columns have double pores structures with micrometer-size co-continuous through-pores and silica skeletons with open mesopores. The beta-CD monolithic columns have successfully been applied to the separation of several neutral and negatively charged isomers by CEC. The column performance was evaluated by using positional isomers of naphthalenedisulfonic acid as model compounds. A plate height of less than 10 mum for the first eluted isomer of naphthalenedisulfonic acid was obtained at an optimal flow rate (0.47 mm/s) of the mobile phase. Moreover, the columns have been proved to be stable for more than 100 runs during 3 months period and show reasonable column reproducibility.     

Rapid and high-resolution analysis of geometric polyprenol homologues by connected octadecylsilylated monolithic silica columns in high-performance liquid chromatography

A Chromolith Performance octadecylsilyl (ODS) monolithic silica column (Merck) was compared with a conventional microparticulate ODS-bonded silica column in the high-performance liquid chromatography separation of natural polyprenols. A system comprising two connected monolithic columns afforded an equivalent separation at half the analysis time of the conventional method. Furthermore, ten connected columns achieved a tremendously high-resolution separation, in which the complicated series of homologous polyprenols with geometric isomerism were fully separated.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

金纳米粒子修饰毛细管硅胶整体柱的制备及其电色谱性能研究

通过毛细管硅胶整体柱表面修饰十八烷基硫醇金纳米粒子,制备了一种新型毛细管电色谱固定相.制备金纳米粒子修饰整体柱时,采用溶胶-凝胶法制备毛细管硅胶整体柱,并在其表面化学修饰3-巯基丙基三甲氧基硅烷;通过巯基基团固载金纳米粒子于整体柱上,再共价键合十八烷基硫醇于金纳米粒子表面.以甲苯为探针,对理论塔板高度与流动相线速度之间...