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.     

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.     

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.     

Fused-core silica column high-performance liquid chromatography/tandem mass spectrometric determination of rimonabant in mouse plasma

A high-performance liquid chromatography (HPLC) method using a fused-core silica particle packing was evaluated to allow fast and efficient separation for the analysis of pharmaceutical compounds. Fused-core particles are produced by "fusing" a porous silica layer onto a solid silica particle. The efficiencies of columns packed with 2.7 microm "fused-core" particles (a 0.5 microm porous shell fused to a solid 1.7 microm silica core particle) and 1.7 microm porous particles were compared in reversed-phase HPLC using rimonabant as an analyte. The fused-core silica materials providing the shorter diffusional mass transfer path for solutes are less affected in resolving power by increases in mobile-phase velocity than the sub-2 microm porous silica packings resulting in faster separations and higher sample throughput. This fast HPLC technology is comparable with ultrahigh-pressure liquid chromatography (UHPLC) in terms of chromatographic performance but demands neither expensive ultra-high-pressure instrumentation nor new laboratory protocols. The column effluent was directly connected to the atmospheric pressure chemical ionization (APCI) source prior to tandem mass spectrometric detection. In this work, the described fast HPLC-MS/MS and UHPLC-MS/MS approaches requiring approximately 1.5 min per sample were applied and compared for the determination of the rimonabant in mouse plasma samples at the low nanograms per milliliter region in support of a pharmacodynamic study.     

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.     

Evaluation of automated isocratic and gradient nano-liquid chromatography and capillary electrochromatography

An automated liquid nano-separation system has been developed for nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) using both isocratic and gradient elution. One fused-silica nanocolumn, typically 75 μm i.d. × 39 cm (25 cm effective packed length), packed with Spherisorb ODS 1, 3 μm particle size, can be used with either technique without having to remove the column upon switching from one mode to the other. The mobile phase is delivered by two reciprocating micro-LC pumps at a flow rate of 30 μL/min to a postinjection splitter that houses the nanocolumn inlet. The splitter is directly connected to a micro-injection valve with a 0.5 μL injection volume. In the CEC mode, pressure is not applied (no restriction on splitter) to the column inlet or outlet and the voltage is continuously applied during sample injection and mobile phase delivery. In the nano-LC mode, the restrictor is coupled to the splitter. Using the same nanocolumn under isocratic conditions, the repeatabilities of retention time and peak area for nano-LC were better than 0.2% and 4%, respectively, and those for CEC were better than 0.6% and 6%, respectively. On average, column efficiency was 57% higher in CEC compared to nano-LC. Gradient elution separations of parabens and polynuclear aromatic hydrocarbons (PAHs) were accomplished by CEC.     

Analysis of corticosteroids in biofluids by capillary electrochromatography with gradient elution

Capillary electrochromatography (CEC) with gradient elution was used to separate mixtures of corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracts of equine urine and plasma. Urine samples were first purified using solid phase extraction. Two purification steps were necessary to prevent contamination of the CEC column. Plasma was purified using automated dialysis. A laboratory-built CEC interface, connected to a gradient HPLC system, delivered samples and mobile phase to the CEC column. CEC was performed in fused-silica capillaries of 50 μm i.d., 24 cm total length, and 16 cm effective length packed with Apex ODS, 3 μm particle size. The mobile phase was ammonium acetate (5 mM) in water/acetonitrile. Acetonitrile in the mobile phase was varied from 9 to 80% (v/v) using the gradient HPLC system. Detection was by UV absorbance at 240 nm. Samples, 10-250 μL, were injected into the mobile phase stream and loaded onto the CEC column under an applied field of 1.04 kV cm(-1) and a CEC column head pressure of 12 bar. Mobile phase flow rate through the sampling interface was 100 μL min(-1). The system was reproducible and could be left in unattended operation for long periods. After injection of 200 urine extracts, a broadening of peaks was observed but the CEC column was still serviceable.     

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.     

Comprehensive two-dimensional gas chromatographic separations with a microfabricated thermal modulator

Rapid, comprehensive two-dimensional gas chromatographic (GC × GC) separations by use of a microfabricated midpoint thermal modulator (μTM) are demonstrated, and the effects of various μTM design and operating parameters on performance are characterized. The two-stage μTM chip consists of two interconnected spiral etched-Si microchannels (4.2 and 2.8 cm long) with a cross section of 250 × 140 μm(2), an anodically bonded Pyrex cap, and a cross-linked wall coating of poly(dimethylsiloxane) (PDMS). Integrated heaters provide rapid, sequential heating of each μTM stage, while a proximate, underlying thermoelectric cooler provides continual cooling. The first-dimension column used for GC × GC separations was a 6 m long, 250 μm i.d. capillary with a PDMS stationary phase, and the second-dimension column was a 0.5 m long, 100 μm i.d. capillary with a poly(ethylene glycol) phase. Using sets of five to seven volatile test compounds (boiling point ≤174 °C), the effects of the minimum (T(min)) and maximum (T(max)) modulation temperature, stage heating lag/offset (O(s)), modulation period (P(M)), and volumetric flow rate (F) on the quality of the separations were evaluated with respect to several performance metrics. Best results were obtained with a T(min) = -20 °C, T(max) = 210 °C, O(s) = 600 ms, P(M) = 6 s, and F = 0.9 mL/min. Replicate modulated peak areas and retention times were reproducible to <5%. A structured nine-component GC × GC chromatogram was produced, and a 21 component separation was achieved in <3 min. The potential for creating portable μGC × μGC systems is discussed.     

High-speed gas chromatography using synchronized dual-valve injection

A novel injection technique for high-speed gas chromatography is demonstrated. Synchronized dual-valve injection is shown to provide peak widths as low as 1.5 ms (width at half-height) for an unretained analyte. This was achieved using a 0.5-m DB-5 column with an internal diameter of 100 microm and a film thickness of 0.4 microm operated at a temperature of 150 degrees C with a column absolute head pressure of 85 psi, resulting in a dead time of only t(o) = 26 ms ( approximately 1900 cm/s, 26 mL/min). Using the DB-5 column in a 1-m length under the same instrumental parameters, with a resulting linear flow velocity of 935 cm/s (12.7 mL/min, t(o) = 117 ms), a minimum peak width of 3.3 ms was obtained. During an isothermal separation, 10 analytes were separated in a time window of 400 ms. A rigorous comparison of experimental and theoretical band-broadening data based on the Golay equation showed that band broadening is limited almost entirely by the chromatographic band broadening terms expressed by the Golay equation and not by extra column band broadening due to the injection process. Synchronized dual-valve injection offers a rugged and inexpensive design, providing extremely reproducible injections with peak height precision of 2.4% (RSD) and low run-to-run variation in retention times, with an average standard deviation less than 0.1 ms. Herein, synchronized dual-valve injection is demonstrated as a proof of principle using high-speed diaphragm valves. It is foreseen that the injection technique could be readily implemented using a combination of thermal modulation and high-speed valve hardware, thus optimizing the mass transfer and not significantly sacrificing the limit of detection performance for high-speed GC. Further implications are that, if properly implemented, high-speed temperature programming coupled with this new technology should lead to very large peak capacities for approximately 1-s separations.     

Capillary Ion Chromatography with On-Line High-Pressure Electrodialytic NaOH Eluent Production and Gradient Generation

A small inexpensive system is described that allows high-performance suppressed anion chromatography on a capillary scale. A fully computer-controlled stepper motor-driven syringe-type dispenser, equipped with a 500 μL-capacity glass syringe is capable of pumping at pressures up to 1000 psi when equipped with an appropriate inlet check valve. Fused-silica capillary columns ～50 cm in length and 180 μm i.d., packed in-house with a commercial packing, provide excellent performance, significantly exceeding the efficiencies observed for the same packing in commercially available 2 mm bore format. The system operates with a pressure drop of <800 psi at a flow rate of 2 μL/min. The system utilizes a novel electrodialytic NaOH eluent generator that is deployed on the high-pressure side of the pump and thus requires no special measures for electrolytic gas removal. This device permits both isocratic and gradient operation with excellent eluent purity; the NaOH concentration is generated linearly with applied current with near-Faradaic efficiency, up to a concentration of at least 100 mM.     

High-temperature ultrafast liquid chromatography

A novel liquid chromatographic system which enables high temperature ultrafast liquid chromatography (HTU-FLC) has been designed through the careful consideration of heat transfer, band broadening, and pressure drop. Studies of the effect of linear velocity on the HETP show that column efficiency at high velocity, especially of well-retained solutes, dramatically improves at higher temperatures. At 150 degrees C, at a flow rate of 15 mL/min with a 5 cm by 4.6 mm (i.d.) column packed with 3 microns polystyrene-coated zirconia porous particles, long chain alkylphenones were completely resolved, and the analysis time could be decreased by a factor of 50 compared to that at room temperature (25 degrees C) at a conventional flow rate (4 mL/min). In addition, using pure water as the mobile phase, five phenols were separated in less than 30 s.     

Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes

The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high-performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600-min gradient, with average chromatographic peak widths of less than 1 min. At room temperature, a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.     

Preparation and characterization of monolithic porous capillary columns loaded with chromatographic particles

Using sol-gel technology, a porous glass matrix (xerogel) is formed in a capillary column and acts as a support for a stationary phase of chromatographic particles used in capillary electrochromatography. Preparation of the sol-gel matrix and immobilization of the octadecylsilica (ODS) stationary phase occur in a single step. The presence of the particles in the column greatly reduces matrix cracking caused by internal pressure differentials within the pores of the sol-gel matrix. Good electroosmotic flow is achieved in part because of the inherent negative charge of both the particles and the sol-gel matrix. The performance of these sol-gel/ODS capillary columns was evaluated with a mixture of aromatic and nonaromatic organic compounds. Efficiencies of up to 80?000 plates/m were observed in columns with immobilized 3-μm ODS particles. The efficiency and resolution are enhanced when 3-μm ODS particles are used in place of the 5-μm particles.     

Use of 1.5-microm porous ethyl-bridged hybrid particles as a stationary-phase support for reversed-phase ultrahigh-pressure liquid chromatography

A new ethyl-bridged hybrid packing material was evaluated in terms of its suitability for ultrahigh-pressure liquid chromatography (UHPLC). The 1.5-microm particles were obtained and packed into 30-microm-i.d. fused-silica capillary columns up to 50 cm in length. The particles were evaluated by isocratic reversed-phase UHPLC at pressures up to 4500 bar (65,000 psi). The chromatographic performance of these particles was found to be similar to the performance of 1.0-microm nonporous silica particles. The mechanical strength of the ethyl-bridged hybrid material was evaluated by running a 15-cm-long column at pressures up to 4500 bar. No breakdown of the particles in the packed bed was observed. The sample loading capacity of the hybrid material was evaluated and compared to 1.0-microm nonporous silica material by observing analyte peak width versus amount injected. The observed improvement in loading capacity for the hybrid material versus nonporous silica was consistent with the improvement predicted by comparing the phase ratios of the two materials.     

In Vitro Release Behavior of Toremifene Citrate from Sol-Gel Processed Sintered Silica Xerogels

Factors affecting the adsorption and desorption of toremifene citrate (TC) on sintered silica xerogels were investigated in vitro. TC was attached onto sol-gel processed sintered silica xerogel grains or disks by adsorption. The adsorption of TC on the surface of silica was pH dependent. The results support the conclusion that large pore size results in highest drug adsorption. Adsorption of TC was most effective in xerogels sintered at 700°C and containing the largest pores and lowest specific surface area of the silica xerogels studied in the adsorption tests. The release of TC from the xerogel matrix was linear with respect to the square root of time. The release of TC from the grains was very rapid for the first 5 hr, followed by a slower release. All drug was released from the grains, and 60% to 80% was released from the disks in 24 hr. All drug-silica xerogel formulations showed sustained in vitro release profiles.     

Solid phase extraction of chromium(VI) from aqueous solutions by adsorption of its ion-association complex with cetyltrimethylammoniumbromide on an alumina column

A simple approach has been developed for the solid phase extraction of chromium(VI) based on the adsorption of its ion-association complex with cetyltrimethylammoniumbromide (CTABr) on an alumina column. The effect of various parameters such as acidity, stability of the column, sample volume, effect of diverse ions, etc. have been studied in detail. The adsorbed complex could be eluted using sodium hydroxide and the concentration of chromium has been established using visible spectrophotometry after complexation with diphenyl carbazide. The calibration graph was linear in the range 0-0.5 microg mL(-1) chromium(VI) with a detection limit of 5 microg L(-1). A highest preconcentration factor of 25 could be obtained for 250 mL sample volume. The data from the column studies were also studied using the Thomas model of adsorption. The experimental results obtained were correlated with the proposed model of adsorption. The Thomas rate constant k was found to be 0.0025 L/min mg and the maximum adsorption capacity q(o) was found to be 0.36 mgCr/g alumina at an initial chromium(VI) concentration of 1 mg L(-1). The validity of the method has been checked by applying it to study the recovery of chromium in spiked water samples and electroplating wastewater.     

Simple and comprehensive two-dimensional reversed-phase HPLC using monolithic silica columns

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 cm long (4.6-mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated (C(18)) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4-0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase (C(18)) or different phases, C(18) and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of approximately 1000 in approximately 60 min in cases 1 and 2 and in approximately 30 min in case 3. The three stationary phases, FR, C(18), and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns.     

Supercritical fluid chromatography and high-performance liquid chromatography/tandem mass spectrometric methods for the determination of cytarabine in mouse plasma

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed-column supercritical fluid chromatography (pSFC) was achieved on bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate. SFC is commonly assumed to be only applicable to nonpolar and relatively low-polarity compounds. In this work, a broader range of compound polarities amenable to pSFC with appropriate mobile-phase modifiers and additives under normal-phase retention mechanism was demonstrated. The pSFC was integrated with an atmospheric pressure chemical ionization source and a tandem mass spectrometer (MS/MS) to enhance the sensitivity, selectivity, and speed of the assay. The influence of mobile-phase components on chromatographic performance and ionization efficiency of the test compounds was investigated for improving the sensitivity and separation for the analyte and the internal standard. The pSFC-MS/MS approach requiring approximately 2.5 min/sample for the determination of ara-C at nanograms per milliliter in mouse plasma was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C obtained by the pSFC-MS/MS method were found to be consistent with those determined by various reversed-phase, high-performance liquid chromatography methods using a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion-pairing agent coupled to a tandem mass spectrometer.