Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.     

Performance of monolithic silica capillary columns with increased phase ratios and small-sized domains

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of approximately 1.3 microm and silica skeletons of approximately 0.9 microm) showed a plate height of less than 5 microm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of approximately 1, and approximately 7 microm at u = 8 mm/s. With a permeability similar to that of a column packed with 5-microm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-microm particles, and showed performance in the "forbidden region" for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15,000-30,000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.     

Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography

Polymer-based strong cation-exchange monolithic capillary columns with different capacities were constructed for ion chromatography by radical polymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate in a 250-microm-i.d. fused-silica capillary and its subsequent sulfonation based on ring opening of epoxides with 1 M Na(2)SO(3). The cation-exchange capacities can easily and reproducibly be controlled in the range of up to 300 microequiv/mL by changing the immersion time of the epoxy-containing polymer in the Na(2)SO(3) solution. The chromatographic performance of the produced monolithic capillary columns was evaluated through the separation of a model mixture of common cations such as Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+). As an example, these cations could be well separated from one another on a 15-cm-long cation-exchange monolithic column (column volume, 7.4 microL) with a capacity of 150 microequiv/mL by elution with 10 mM CuSO(4). The pressure drop of this 15-cm column was approximately 1 MPa at a normal linear velocity of 1 mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for the cations were above 3000 plates/15 cm. This GMA-based cation-exchange monolithic column could withstand high linear velocities of at least 10 mm/s. Over a period of at least two weeks of continuous use, no significant changes in the selectivity and resolution were observed. The applicability of a flow rate gradient elution and the feasibility of direct injection determination of major cations in human saliva sample were also presented.     

Porous polymer monoliths functionalized through copolymerization of a C60 fullerene-containing methacrylate monomer for highly efficient separations of small molecules

Monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns, which incorporate the new monomer [6,6]-phenyl-C(61)-butyric acid 2-hydroxyethyl methacrylate ester, have been prepared and their chromatographic performance have been tested for the separation of small molecules in the reversed phase. While addition of the C60-fullerene monomer to the glycidyl methacrylate-based monolith enhanced column efficiency 18-fold, to 85,000 plates/m at a linear velocity of 0.46 mm/s and a retention factor of 2.6, when compared to the parent monolith, the use of butyl methacrylate together with the carbon nanostructured monomer afforded monolithic columns with an efficiency for benzene exceeding 110,000 plates/m at a linear velocity of 0.32 mm/s and a retention factor of 4.2. This high efficiency is unprecedented for separations using porous polymer monoliths operating in an isocratic mode. Optimization of the chromatographic parameters affords near baseline separation of 6 alkylbenzenes in 3 min with an efficiency of 64,000 plates/m. The presence of 1 wt % or more of water in the polymerization mixture has a large effect on both the formation and reproducibility of the monoliths. Other factors such as nitrogen exposure, polymerization conditions, capillary filling method, and sonication parameters were all found to be important in producing highly efficient and reproducible monoliths.     

Chemically L-phenylalaninamide-modified monolithic silica column prepared by a sol-gel process for enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography

A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LE-CEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 x 10(4) plates m(-1) for the D-enantiomer and 6.6 x 10(4) plates m(-1) for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

High-efficiency liquid chromatographic separation utilizing long monolithic silica capillary columns

Long monolithic silica-C18 capillary columns of 100 microm i.d. were prepared, and the efficiency was examined using reversed-phase HPLC under a pressure of up to 47 MPa. At linear velocities of 1-2 mm/s, 100,000-500,000 theoretical plates could be generated with a single column (90-440 cm in length) using an acetonitrile-water (80/20) mobile phase with a column dead time (t0) of 5-40 min. It was possible to prepare columns with a minimum plate height of 8.5 +/- 0.5 microm and permeability of (1.45 +/- 0.09) x 10(-13) m(2). The chromatographic performance of a long octadecylsilylated monolithic silica capillary column was demonstrated by the high-efficiency separations of aromatic hydrocarbons, benzene derivatives, and a protein digest. The efficiency for a peptide was maintained for an injection of up to 0.5-2 ng. When three 100 microm i.d. columns were connected to form a 1130-1240 cm column system, 1,000,000 theoretical plates were generated for aromatic hydrocarbons with retention factors of up to 2.4 with a t0 of 150 min. The fact that very high efficiencies were obtained for the retained solutes suggests the practical utility of these long monolithic silica capillary columns.     

Monolithic column with zwitterionic stationary phase for capillary electrochromatography

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycyclic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 1. Optimization of polymerization conditions, porous properties, and chemistry of the stationary phase

Monolithic columns for chiral capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine , ethylene dimethacrylate, and glycidyl methacrylate or 2-hydroxyethyl methacrylate in the presence of mixture of cyclohexanol and 1-dodecanol as a porogenic solvent. The porous properties of the monolithic columns can easily be controlled through changes in the composition of the binary porogenic solvent. Although both thermal- and UV light-initiated polymerizations afford useful capillary columns, monoliths prepared using the former approach exhibit better chromatographic properties. The ability to control pore size independently of the polymerization mixture composition enables the preparation of monoliths with varying percentages of the chiral monomer and cross-linker, as well as the optimization of their separation properties. Very good separations of model racemate (R,S)-N-3,5-dinitrobenzoylleucine were achieved using an optimized monolithic CEC column, with high efficiencies of up to 74000 plates/m for the retained peaks.     

Separation of peptides from myoglobin enzymatic digests by RPLC. Influence of the mobile-phase composition and the pressure on the retention and separation

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long C18-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 2. Effect of chromatographic conditions on the chiral separations

The effect of chromatographic conditions on the performance of chiral monolithic poly(O-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroqui nidine-co-ethylene dimethacrylate-co-2-hydroxyethyl methacrylate) columns in the capillary electrochromatography of enantiomers has been studied. The flow velocity was found to be proportional to the pore size of the monolith and both the pH and the composition of the mobile phase. The length of both open and monolithic segments of the capillary column was found to exert a substantial effect on the run times. The use of monoliths as short as 8.5 cm and the "short-end" injection technique enabled the separations to be achieved in approximately 5 min despite the high retentitivity of the quinidine selector. Very high column efficiencies of close to 250000 plates/m and good selectivities were achieved for the separations of numerous enantiomers using the chiral monolithic capillaries with the optimized chromatographic conditions.     

Preparation of perphenylcarbamoylated β-cyclodextrin-silica hybrid monolithic column with "one-pot" approach for enantioseparation by capillary liquid chromatography

Perphenylcarbamoylated β-cyclodextrin-silica (Ph-β-CD-silica) hybrid monolithic columns for enantioseparation in capillary liquid chromatography (cLC) have been prepared by a "one-pot" approach via the polycondensation of alkoxysilanes and in situ copolymerization of mono (6(A)-N-allylamino-6(A)-deoxy)-Ph-β-CD and vinyl group on the precondensed siloxanes. The morphologies of the Ph-β-CD-silica hybrid monolithic columns were characterized by optical microscopy and scanning electron microscopy (SEM), showing the uniform monolithic matrixes tightly bonded onto the capillary wall. The content of Ph-β-CD incorporated in monolithic matrix by the "one-pot" approach was ca. 2.9 times higher than that by postmodification method. The permeability of the Ph-β-CD-silica chiral hybrid monolithic column was 3.63 × 10(-14) m(2), and the minimum plate height was 12 μm corresponding to 83,300 theoretical plates/meter. Enantioseparations of 13 racemates were achieved by the Ph-β-CD-silica hybrid monolithic column. In this work, since the prepolymerization system mainly consisted of organic solvent (methanol (MeOH), N,N-dimethylformamide (DMF)), the limitation and difficulty of the use of water insoluble organic monomers in the previously reported "one-pot" method was circumvented. Therefore, various β-CD derivatives as well as other hydrophobic monomers could thus be used to prepare organic-silica hybrid monolithic columns with the "one-pot" process.     

Analysis of bile acids and their conjugates by capillary electrochromatography/electrospray ion trap mass spectrometry

Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.     

Sol-gel monolithic columns with reversed electroosmotic flow for capillary electrochromatography

Sol-gel chemistry was used to prepare porous monolithic columns for capillary electrochromatography. The developed sol-gel approach proved invaluable and generates monolithic columns in a simple and rapid manner. Practically any desired column length ranging from a few tens of centimeters to a few meters may be readily obtained. The incorporation of the sol-gel precursor, N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, into the sol solution proved to be critical as this reagent possesses an octadecyl moiety that allows for chromatographic interactions of analytes with the monolithic stationary phase. Additionally, this reagent served to yield a positively charged surface, thereby providing the relatively strong reversed electroosmotic flow (EOF) in capillary electrochromatography. The enhanced permeability of the monolithic capillaries allowed for the use of such columns without the need for modifications to the commercial CE instrument. There was no need to pressurize both capillary ends during operation or to use high pressures for column rinsing. With the developed procedure, no bubble formation was detected during analysis with the monolithic capillaries when using electric field strengths of up to 300 V cm(-1). The EOF in the monolith columns was found to be dependent on the percentage of organic modifier present in the mobile phase. Separation efficiencies of up to 1.75 x 10(5) plates/m (87,300 plates/column) were achieved on a 50 cm x 50 microm i.d. column using polycyclic aromatic hydrocarbons and aromatic aldehydes and ketones as test solutes.     

A protein-encapsulation technique by the sol-gel method for the preparation of monolithic columns for capillary electrochromatography

A novel protein-encapsulation technique using sol-gels was developed for the preparation of monolithic capillary columns for capillary electrochromatography. Two chiral compounds, bovine serum albumin (BSA) and ovomucoid (OVM) from chicken egg white, were encapsulated in tetramethoxysilane-based hydrogel and their chiral selectivity was evaluated for the separation of some selected enantiomers (tryptophan, benzoin, eperisone, chlorpheniramine). The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. Two different proteins, BSA and OVM, were successfully encapsulated into the gel matrixes by changing the alkoxysilane compositions of the gel. Run-to-run repeatability was quite satisfactory. The consecutive analysis of the neutral compound, benzoin, by the OVM-encapsulated column showed good repeatability in the retention time (RSD = 1.23% for the first peak, N = 10). Under optimized conditions, the theoretical plate number for the first peak of benzoin reached 72,000 plates/m.     

Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples

Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-high-pressure multidimensional liquid chromatography system that operates up to approximately 20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constant-flow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved approximately 30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversed-phase column (approximately 50 cm) for MudPIT-type experiments are also discussed.     

On-line preconcentration prior to on-column derivatization monolith octadecasiloxane capillary electrochromatography for the determination of biogenic amines

To expand the applications of the on-line preconcentration technique with capillary electrochromatography (CEC) to biogenic amines that have no specific chromophore or fluorophore in their molecules, a method of on-line preconcentration prior to on-column derivatization CEC is presented. A monolithic ODS capillary column (20 cm effective length x 75 microm i.d.) for CEC was fabricated using a thermal sol-gel reaction of tetraethyl orthosilicate to capture ODS particles (5-microm particle diameter) in a capillary tube. A standard model biogenic amine solution consisting of histamine, methylhistamine, and serotonin was electrokinetically injected from the anodic site of the capillary column with 5 kV, and these amines were effectively concentrated at the inlet site of the capillary column by a field-amplified sample stacking, a gradient effect mode, or both. This preconcentration occurred whenever the several types of solvent for reconstitution of the amines, e.g., water (noneluting solvent or low-conductivity solvent), 0.9% sodium chloride (noneluting solvent or high-conductivity solvent), or 60% acetonitrile in 10 mM borate buffer (pH 10) (eluting solvent) were employed. After concentration, the amines were subsequently derivatized, separated, and detected during CEC with an optimum CEC run buffer solution containing 60% acetonitrile in 5 mM o-phthalaldehyde/2-mercaptoethanol-10 mM borate buffer (pH 10) when 5 kV was continuously applied. Using the present system, equipped with a fluorescence detector instead of a UV/visible detector, the detection sensitivity for amines reached a 0.1 microM level, which increased sensitivity by a factor of 10(3) times greater than that of normal on-column derivatization CEC.     

High-efficiency solvating gas chromatography using packed capillaries

In this study, column efficiency in packed capillary column solvating gas chromatography (SGC) was investigated. Long (>3 m) fused silica capillaries with an inner diameter of 250 μm were packed with 10 and 15 μm spherical porous (300 ?) octadecyl bonded silica particles using a CO(2) slurry packing method. A 336 cm × 250 μm i.d. fused silica capillary containing 10 μm particles provided a total column efficiency of 264?000 plates (k = 0.41), corresponding to a reduced plate height of 1.27, using CO(2) as the mobile phase at a column inlet pressure of 260 atm. A minimum plate height of 12.7 μm and a maximum plate number per unit time of 813 plates/s were obtained using packed capillary SGC. Retention factors were dependent on the column inlet pressure but independent of the pressure gradient along the column. Gasoline and diesel samples were separated under SGC conditions, and the results were comparable to those obtained using typical open tubular column gas chromatography.     

Separation of neutral saccharide mixtures with capillary electrochromatography using hydrophilic monolithic columns

While developing a combination of capillary electrochromatography (CEC) with tandem mass spectrometry (MS) for the benefit of characterizing complex oligosaccharide mixtures, we needed highly efficient CEC columns operating in an "MS-friendly" mode. We demonstrate here novel types of polar, monolithic CEC columns that separate effectively complex mixtures of saccharides with the use of mobile phases containing acetonitrile/dilute ammonium formate buffers. Using the positive-ion mode of detection for neutral saccharides, the detection conditions were optimized down to the low-femtomole sensitivities with the use of an ion trap mass spectrometer. This column technology provides a nearly universal system that can separate a wide range of carbohydrates: mono- and oligosaccharides with the intact reducing end, as well as saccharide alditols. Even the anomers formed due to mutarotation could be resolved with a high content of organic phase.     

A graphitized-carbon monolithic column

The preparation of a novel carbon monolithic column for high performance liquid chromatography is described. A phenolic resin rod with embedded 10-microm silica beads was prepared by acid-catalyzed polymerization of a resorcinol/iron(III) complex and formaldehyde. This rod was carbonized and graphitized under inert atmosphere with a programmed temperature cycle from room temperature to 1250 degrees C. Subsequently, the silica beads along with iron catalysts were removed, leaving a porous carbon rod. Imaging of this monolithic rod by scanning and transmission electron microscopies revealed a highly interconnected bimodal porous structure. The porosity and pore size distribution of the mesopores were characterized by N2 absorption/desorption. Graphene sheets were found in the TEM images of the carbon rod, and the graphite index was characterized by Raman spectrum and X-ray diffraction. A monolithic column prepared with the aforementioned carbon rod was evaluated using a mixture of alkylbenzenes. It exhibited an excellent separation power and a low hydraulic resistance. The bundle-of-capillaries model was used to characterize the hydrodynamics of this monolith. Its permeability was found to agree well with the theoretical one.