Latex-coated polymeric monolithic ion-exchange stationary phases. 2. Micro-ion chromatography

Latex-coated monolithic polymeric stationary phases are used for micro-ion chromatography (mu-IC) of inorganic anions. Monolithic columns were prepared by the in situ polymerization of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid within fused-silica capillaries of varying internal diameters. Introduction of ion-exchange sites was achieved by coating the anionic polymeric monolith with either Dionex AS10 or Dionex AS18 quaternary ammonium functionalized latex particles to give total ion-exchange capacities in the range 9-24 nequiv for a 30-cm column. The resultant mu-IC columns were used for the separation of anionic analytes using chloride or acetate as the eluent-competing ion and direct UV spectrophotometric detection at 195 nm or using hydroxide as the eluent-competing ion and suppressed or nonsuppressed contactless conductivity detection. Separation efficiencies of 13,000 plates/m were observed (for iodate), and separation efficiency was maintained for large increases in flow rate (up to 42 microL/min, corresponding to a linear flow velocity of 18.5 mm/s), enabling highly reproducible, rapid separations to be achieved (seven analyte anions in less than 2 min). Use of a hollow fiber micromembrane suppressor enabled effective suppression of hydroxide eluents over the range 0.5-5.0 mM, thereby permitting suppressed conductivity detection to be performed. However, the relatively large size of the suppressor resulted in reduced separation efficiencies (e.g., 5400 plates/m for iodate). Detection limits obtained with suppressed conductivity detection were in the range 0.4-1.2 microM.     

Separation of tocopherol isomers using capillary electrochromatography: comparison of monomeric and polymeric C30 stationary phases.

The comparison of tocopherol isomer separation achieved using different stationary-phase alkyl chain lengths (i.e., C18 vs C30) and modes of alkyl phase attachment to the silica-based supports (i.e., polymeric vs monomeric synthesis) using capillary electrochromatography (CEC) has been demonstrated. The smaller alkyl chain does not exhibit the shape selectivity needed to resolve all of the tocopherol isomers. Conversely, both polymeric and monomeric C30 phases show increased tocopherol isomer selectivity. Changing the elutropic strength of the mobile phase had dramatic effects on the selectivity, with small additions of water to a methanol mobile phase yielding the best selectivity. The complete baseline separation of the tocopherol isomers was achieved using the monomerically bound C30 stationary phase and a methanol/ water mobile phase. The differences in stationary-phase selectivity were examined using a NIST standard reference material for determining column selectivity in LC. The results indicated that the monomeric C30 actually had "intermediate" phase characteristics (i.e., high phase loading, end capping, etc). This new CEC-based separation was also used to separate the tocopherols in a vitamin E supplement sample.     

Supercritical fluid generated stationary phases for liquid chromatography and capillary electrochromatography

Chromatographic silica-bonded stationary phases have been prepared using supercritical CO(2) as the reaction medium. (29)Si solid-state NMR spectra of the generated bonded silica phases show unreacted silica species Q(3) and Q(4), alongside important resonances for surface-bound ligands, T(1), T(2), and T(3). Initially, a fluorinated octyl silica (C(8)) phase was produced, by reacting (1)H,(1)H,(2)H,(2)H-perfluorooctyltriethoxysilane with silica particles (3 microm) in sc-CO(2) at 60 degrees C and 450 atm for 3 h. In-house-packed LC columns of this fluorinated sc-C(8) silica phase yielded typical reversed-phase behavior when a standard test mix of benzamide (k' = 1.03), benzophenone (k' = 8.11), and biphenyl (k' = 14.92) was eluted. When packed into fused-silica capillaries for CEC, this fluorinated sc-C(8) silica phase gave linear plots of log k' versus percentage MeOH for benzophenone and biphenyl and, in contrast to octyl or octadecyl silica phases, displayed selectivity for aromatic thioureas when chromatographed among a series of synthetic organic thiourea test solutes. Similarily, an octadecyl silica phase (sc-C(18) silica) was prepared by reaction of n-octadecyltriethoxysilane in sc-CO(2), packed at 9500 psi and examined by LC. The sc-C(18) silica LC column yielded high column efficiency (up to 141 000 N/m (fluorene)) and excellent asymmetry factors (1.06, fluorene) without resource to end-capping. Following a second silylating or end-capping step using hexamethyldisilazane in sc-CO(2), sc-end-capped sc-C(18) silica phases elute N,N-DMA before toluene and the toluidine isomers as a single peak, indicating lowered silanol activity according to the Engelhardt test. A rapid separation of the important pharmaceutical substances, ketoprofen, naproxen, fentoprofen, and ibuprofen, on an sc-end-capped sc-C(18) silica phase is also shown.     

Nanoinjector for capillary electrophoresis and capillary electrochromatography

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.     

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.     

Online sample preconcentration in capillary electrophoresis using analyte focusing by micelle collapse

A dimension for online sample preconcentration in capillary electrophoresis (CE) without modification of current CE commercial instrumentation is introduced. The focusing mechanism is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. More than 2 orders of magnitude improvement in detection sensitivity for model steroidal compounds using sodium dodecyl sulfate micelles as carrier is demonstrated.     

On-line preconcentration in capillary electrochromatography using a porous monolith together with solvent gradient and sample stacking

Preconcentration effects of solvent gradient and sample stacking are investigated on a photopolymerized sol-gel (PSG) in capillary electrochromatography. The porous PSG monolith has a high mass-transfer rate. This characteristic promotes preconcentration of dilute samples. Plugs of samples more than 2 cm in length prepared in the separation solution (nongradient condition) are injected onto the PSG column. The extent of preconcentration is quite significant, showing up to a 100-fold increase in peak heights of the separated analytes. Even larger preconcentrations are achieved under gradient conditions by dissolving the sample in a matrix with a higher concentration of noneluting solvent (water). For eight alkyl phenyl ketones and four polycyclic aromatic hydrocarbons that serve as neutral test analytes, improvements in peak heights obtained under gradient conditions can be more than a 1000-fold. Indeed, injection of a 91.2-cm plug, which is more than 3 times the total length of the capillary, was possible with only a minor loss in resolution. Five peptides serve as charged test analytes. Nongradient conditions in which the sample is hydrodynamically injected onto the PSG column show sizable preconcentration because of sample stacking. The use of a solvent gradient with the same ionic strength, however, does not appear to have practical value because of destacking caused by the changing organic composition that affects the conductivity. As an alternative preconcentration method, we demonstrate that electric field-enhanced sample injection on the PSG yielded up to a 1000-fold improvement in detection sensitivity for the test peptides.     

On-column ion-exchange preconcentration of inorganic anions in open tubular capillary electrochromatography with elution using transient-isotachophoretic gradients. 3. Implementation and method development

A solid-phase extraction method based on an ion-exchange retention mechanism has been used for in-line preconcentration of inorganic anions prior to their separation by capillary electrophoresis (CE). A single capillary containing a preconcentration and a separation zone has been used in a commercial CE instrument without instrumental modification. Analyte anions were retained on a preconcentration zone comprising an adsorbed layer of cationic latex particles, while separation was achieved in a separation zone comprising fused silica modified by adsorption of a cationic polymer. Elution of the adsorbed analytes was achieved using an eluotropic gradient formed by a transient isotachophoretic boundary between a fluoride electrolyte and a naphthalenedisulfonate electrolyte. Optimization of the electrolyte concentrations, sample injection times, and back-flushing times allowed the successful separation of sub-ppb levels of inorganic anions using a 100-min injection at 2 bar pressure, introducing over 40 capillary volumes of sample. A method based on a 10-min injection allowed a 100-fold increase in sensitivity over conventional hydrodynamic injection for Br-, I-, NO3-, CrO4(2-), and MoO4(2-) with a total analysis time of 25 min. Detection limits were dependent on the injection time but were in the range 2.2-11.6 ppb for a 10-min injection time. This approach was used to determine NO3- in Antarctic ice cores where the analysis could be performed using a sample volume 100 times less than that used for ion chromatography.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Chiral separations using polymeric surfactants and polyelectrolyte multilayers in open-tubular capillary electrochromatography

In this study, fused-silica capillaries are modified using a polyelectrolyte multilayer (PEM) coating procedure in open-tubular capillary electrochromatography. The PEM coating was constructed in situ with alternating rinses of positively and negatively charged polymers. The quaternary ammonium salt poly (diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly (sodium N-undecanoyl-l-leucylvalinate) was used as the anionic polymer. Previous studies have shown that the PEM-coated capillaries used for achiral separations have excellent reproducibilities and high stabilities against extreme pH values. In the current study, this PEM coating approach was applied to chiral separations of 1,1'-binaphthyl-2,2'-dihydrogenphosphate (BNP), 1,1'-bi-2-naphthol, secobarbital, pentobarbital, and temazepam. However, the PEM coating procedure used in the achiral studies needed to be significantly modified in order to achieve chiral separations. Optimal conditions were established by varying the additives (sodium chloride, 1-ethyl-3-methyl-1H-imidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate) in the polymer deposition solutions, the salt concentration, the column temperature, and the bilayer number. Reproducibilities were evaluated by use of the relative standard deviation (RSD) values of the electroosmotic flow (EOF) and the first peak ((R)-(+)-BNP). In all cases, the run-to-run and capillary-to-capillary RSD values of EOF were less than 0.5%, and the run-to-run RSD values of the (R)-(+)-BNP peak were less than 1%. In addition, more than 230 runs were performed on a single PEM-coated capillary.     

Enantiomeric separations using bovine serum albumin immobilized on ion-exchange stationary phases.

Bovine serum albumin (BSA) can be readily immobilized on ion-exchange stationary phases by frontal analysis of a proper solution. This provides a simple means of adjusting the amount of BSA contained in the column and of measuring it accurately. Although the immobilization is ionic and not covalent, the columns are stable for extensive periods of time. If needed, they can be easily regenerated by the same frontal analysis procedure.     

Nonaqueous capillary electrochromatographic separation of synthetic neutral polymers by size exclusion chromatography using polymeric stationary phases

In this paper, we report the separations of large, neutral, synthetic polymers using primarily a nonaqueous mobile phase without the use of a supporting electrolyte. The size- exclusion-based mechanism for separation was achieved on sulfonated polystyrene/divinylbenzene stationary phases. The effect of water, voltage, stationary phase exchange capacity, and pore size were investigated. The stationary phase and solvent interactions were studied by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) and a possible mechanism for the generation of EOF in the THF/water system is provided. Linear calibration curves were obtained for polystyrenes ranging in MW from 5K to 2M, for columns made using a combination of high capacity ion exchanger and a neutral polystyrene/divinylbenzene material of varied pore sizes. Analysis of polyurethane, polystyrene, and other polymer samples using CEC correlated well with results obtained by conventional HPLC. The size exclusion CEC separations provide an alternative mode for determining the relative molecular weights of polymers, with reduced solvent consumption.     

Liquid-phase microextraction as an on-line preconcentration method in capillary electrophoresis

A simple and efficient sample preconcentration method for capillary electrophoresis has been developed using liquid-phase microextraction (LPME). A thin layer of an organic liquid was used to separate a drop of the aqueous acceptor phase hanging at the inlet of a capillary from the bulk aqueous donor phase. The donor-phase pH was 1.0, and the acceptor phase pH was 9.5. This pH difference caused the preconcentration of the acidic compounds, fluorescein and fluorescein isothiocyanate, into the acceptor-phase drop. Enrichment factors of 3 orders of magnitude were obtained with 30-min LPME at 35 degrees C.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

Thermal bonding of polymeric capillary electrophoresis microdevices in water

A new method for thermally bonding poly(methyl methacrylate) (PMMA) substrates to form microfluidic systems has been demonstrated. A PMMA substrate is first imprinted with a Si template using applied pressure and elevated temperature to form microchannel structures. This embossing method has been used to successfully pattern over 65 PMMA pieces using a single Si template. Thermal bonding for channel enclosure is accomplished by clamping together an imprinted and a blank substrate and placing the assembly in boiling water for 1 h. The functionality of these water-bonded microfluidic substrates was demonstrated by performing high-resolution electrophoretic separations of fluorescently labeled amino acids. Testing of bond strength in four microdevices showed an average failure pressure of 130 kPa, which was comparable to the bond strength for devices sealed in air. Subsequent profilometry of separated substrates revealed that the dimensions of the channels were well preserved during the bonding process. This new methodology for generation of microfluidic constructs should facilitate the permanent incorporation of hydrated, molecular size-selective membranes in microdevices, thus circumventing problems associated with membrane swelling in microfluidic systems upon exposure to water.     

pH-mediated field amplification on-column preconcentration of anions in physiological samples for capillary electrophoresis.

Two limitations of capillary electrophoresis (CE) are the low sample loadability of the capillary and an incompatibility with high ionic strength samples. Several strategies have been described to preconcentrate and lower the ionic strength of physiological samples prior to CE analysis. These have included both off-capillary and on-capillary approaches. We have previously described a version of on-column field-amplification stacking termed pH-mediated stacking. pH-mediated stacking was initially developed for the separation of cations. In this report, we describe the application of pH-mediated sample stacking to anions. In this method, an electrokinetic injection is used to introduce analyte anions into the CE system and simultaneously replace the sample matrix cations with ammonia from the background electrolyte. Base is then electrokinetically injected to neutralize the sample zone and create a low conductivity region across which the analyte anions will stack. Using this method, a sensitivity enhancement of more than 66-fold was achieved without loss in separation efficiency relative to normal electrokinetic injection. Detection limits of 0.3 microM for four phenolic acids in a physiological sample were achieved using simple UV absorbance detection. The limit to the amount of sample that could be loaded using this technique was the length of the separation capillary. To further increase the amount of sample that could be loaded, a double-capillary system was developed. Using the double-capillary system the sensitivity was increased more than 300-fold and detection limits of 0.06 microM were achieved.     

Preparation and evaluation of a molecularly imprinted polymer derivatized silica monolithic column for capillary electrochromatography and capillary liquid chromatography

A method for preparation of molecularly imprinted polymer (MIP) derivatized onto the surface of a monolithic silica capillary column was successfully developed. The vinyl groups were first introduced onto the silica monolith by immobilization of gamma-methacryloxypropyltrimethoxysilane. Then the MIP coating was copolymerized and anchored onto the surface of the silica monolith. Acetonitrile was selected as porogen (solvent). The other preparation conditions, such as monomer concentration, temperature, and time of polymerization, were systematically studied. The obtained MIP-derivatized silica monolith using l-tetrahydropalmatine (l-THP) and (5S,11S)-(-)-Tr?ger's base (S-TB) as the imprinted template, respectively, was characterized in terms of the retention behavior of thiourea and toluene. Under the optimized CEC conditions, baseline enantioseparations of THP and TB were achieved in 4 min though the effective length of the columns was 8.5 cm. The result indicates that enough recognition sites were on the surface of silica monolith, resulting in strong recognition ability. Compared with a MIP organic monolith, the MIP-derivatized silica monolith exhibits better column efficiency and stability in CEC. Additionally, the comparison of these two kinds of monolithic columns was performed by capillary liquid chromatography. The separation on MIP-derivatized silica monolith was superior to that on the organic monolith.     

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.     

Capillary electrochromatography with novel stationary phases. 3. Retention behavior of small and large nucleic acids on octadecyl-sulfonated-silica

In this investigation, the potentials of porous and nonporous octadecyl-sulfonated-silica (ODSS) microparticles were demonstrated in the capillary electrochromatography (CEC) of small (e.g., nucleotides and dinucleotides) and large (e.g., transfer ribonucleic acids (t-RNAs)) nucleic acids. The ODSS stationary phase comprised two layers: a hydrophilic sulfonated (permanently charged) sublayer and an octadecyl top layer. While the sublayer is to provide a relatively strong electroosmotic flow, the octadecyl top layer is to ensure the retentivity and selectivity required for the separation of the analytes. Mono-, di-, and triphosphate nucleotides were best separated when a small amount of tetrabutylammonium bromide was added to the mobile phase. The tetrabutylammonium bromide functioned as an ion-pairing agent and consequently allowed the rapid separation of 12 different nucleotides. It is believed that the dynamic complex exchange model explains the basis of retention in ion pair reversed-phase CEC. Eight different dinucleotides, which have similar mass-to-charge ratios, separated very well by CEC. These solutes exhibited similar migration times (i.e., little or no separation) in capillary zone electrophoresis (CZE). Similarly, t-RNAs that did not separate by CZE were well resolved in CEC with nonporous ODSS. This demonstrates that CEC is very suitable for the separation of solutes that have similar mass-to-charge ratios but differ in their hydrophobicity.     

Evaluation of a vancomycin chiral stationary phase in capillary electrochromatography using polar organic and reversed-phase modes

A vancomycin chiral stationary phase (CSP) was fully evaluated in capillary electrochromatography (CEC) in reversed-phase and polar organic modes for a number of racemic pharmaceutical compounds. High efficiency and resolution values were obtained for a number of compound classes including thalidomide in both the polar organic mode (190000 plates meter(-1) and Rs = 13.8) and reversed-phase mode (125000 plates meter(-1) and Rs = 13.0). Experimental parameters, including organic modifier, organic solvent ratio, ionic strength, pH, temperature, and voltage, were examined in both the aqueous and nonaqueous modes to deduce their effect on the resultant EOF, retention times, resolution, and efficiency of chiral separations. All results were consistent with and found to be a combination of what is known from existing literature on CEC theory and experience obtained with macrocyclic antibiotic CSPs in LC. Column stability was excellent, and each column packed was found to offer repeatable separations even when switching from the aqueous to the nonaqueous mode.