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The concept of biocompatibility with reference to chromatographic stationary phases for separation of biomolecules (including proteins and peptides) is introduced. Biocompatible is a characteristic that indicates resistance to nonspecific adsorption of biomolecules and preservation of their structures and biochemical functions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide- and poly(meth)acrylate-based monoliths] used for protein and peptide separations are reviewed in detail, with emphasis on size exclusion, ion exchange, and hydrophobic interaction chromatographic modes. Biocompatible monoliths for enzyme reactors are also included. The two main synthetic approaches to produce biocompatible monoliths are summarized, i. e., surface modification of a monolith that is not inherently biocompatible and direct copolymerization of hydrophilic monomers to form a biocompatible monolith directly. Integration of polyethylene glycol into the poly(meth)acrylate monolith network is becoming popular for reduction of non-specific protein interactions.

In spite of an earlier report to the contrary and a different attempt that appears to have been unsuccessful, here we show an improved method that allows functionalization of hydrogen- and deuterium-terminated diamond with a dialkyl peroxide. In particular, hydrogen-/deuterium-terminated diamond particles were treated with neat di-tert-amyl peroxide (DTAP, C2H5C(CH3)2OOC(CH3)2C2H5) at elevated temperature. Surface changes were followed by X-ray photoelectron spectroscopy (XPS), diffuse reflectance Fourier transform infrared spectroscopy (DRIFT), and time-of-flight secondary ion mass spectroscopy (ToF-SIMS). After these reactions, the oxygen signal in the XPS spectra increased, the deuterium peak in the negative ToF-SIMS spectra decreased, and DRIFT showed C−H stretches, which were not previously present and which were similar to those of the precursor. In the C−H stretching region, the IR spectrum of adsorbed di-tert-amyl peroxide shows features that are red-shifted with respect to the IR spectrum of the ...

A strong cation-exchange (SCX) monolithic stationary phase was prepared in 75 microm id capillaries by direct in situ polymerization of sulfopropyl methacrylate and polyethylene glycol diacrylate in a ternary porogen system consisting of methanol, cyclohexanol, and water. The resulting monolith exhibited good dynamic binding capacity, fast kinetic adsorption of proteins, and high permeability. The monolith had a dynamic binding capacity of approximately 52 mg/mL of column volume for lysozyme and cytochrome C. The monolith was evaluated for SCX capillary LC of synthetic peptides, natural peptides, and protein standards. Fast separation of proteins was achieved in less than 4 min. The average peak capacity for peptides was 28 using a relatively steep gradient when hydrophobic interactions were suppressed with 40% acetonitrile.

Recently, we reported the synthesis, fabrication, and preliminary evaluation of poly(ethylene glycol) (PEG)-functionalized polymeric microchips that are inherently resistant to protein adsorption without surface modification in capillary electrophoresis (CE). In this study, we investigated the impact of cross-linker purity and addition of methyl methacrylate (MMA) as a comonomer on CE performance. Impure poly(ethylene glycol) diacrylate (PEGDA) induced electroosmotic flow (EOF) and increased the separation time, while the addition of MMA decreased the separation efficiency to approximately 25% of that obtained using microchips fabricated without MMA. Resultant improved microchips were evaluated for the separation of fluorescent dyes, amino acids, peptides, and proteins. A CE efficiency of 4.2 x 10(4) plates for aspartic acid in a 3.5 cm long microchannel was obtained. Chiral separation of 10 different D,L-amino acid pairs was obtained with addition of a chiral selector (i.e., beta-cyclodextrin) in the running buffer. Selectivity (alpha) and resolution (R(s)) for D,L-leucine were 1.16 and 1.64, respectively. Good reproducibility was an added advantage of these PEG-functionalized microchips.

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.

We report the design and performance of a novel radio-frequency (RF) ion-trap mass analyzer, the planar Paul trap, in which a quadrupolar potential distribution is made between two electrode plates. Each plate consists of a series of concentric, lithographically deposited 100-microm-wide metal rings, overlaid with a thin resistive layer. A different RF amplitude is applied to each ring, such that the trapping field produced is similar to that of the conventional Paul trap. The accuracy and shape of the electric fields in this trap are not limited by electrode geometry nor machining precision, as is the case in traps made with metal electrodes. The use of two microfabricated plates for ion trap construction presents a lower-cost alternative to conventional ion traps, with additional advantages in electrode alignment, electric field optimization, and ion-trap miniaturization. Experiments demonstrate the effects of ion ejection mode and scan rate on mass resolution for several small organic compounds. The current instrument has a mass range up to approximately 180 Thomsons (Th), with better than unit mass resolution over the entire range.

The synthesis of 2-naphthyl 2′-dibenzothienyl ether, 1-(1′-naphthyl)-2-(4″-acridinyl)ethane and a series of naphthylethyldibenzothiophenes is reported.

A novel gas chromatograph-mass spectrometer (GC-MS) based on a miniature toroidal ion trap mass analyzer (TMS) and a low thermal mass GC is described. The TMS system has an effective mass/charge (m/z) range of 50–442 with mass resolution at full-width half-maximum (FWHM) of 0.55 at m/z 91 and 0.80 at m/z 222. A solid-phase microextraction (SPME) fiber mounted in a simple syringe-style holder is used for sample collection and introduction into a specially designed low thermal mass GC injection port. This portable GC-TMS system weighs

We report the formation of a highly stable amino stationary phase on diamond and demonstrate its use in solid-phase extraction (SPE). This process consists of spontaneous and self-limiting adsorption of polyallylamine (PAAm) from aqueous solution onto oxidized diamond. Thermal curing under reduced pressure or chemical cross-linking with a diepoxide was shown to fix the polymer to the particles. The resulting adsorbents are stable under even extreme pH conditions (from at least pH 0-14) and significantly more stable than a commercially available amino SPE adsorbent. Coated diamond particles were characterized by X-ray photoelectron spectroscopy (XPS) and diffuse reflectance Fourier transform-infrared spectroscopy (DRIFT). Model silicon surfaces were characterized by spectroscopic ellipsometry and wetting. Solid-phase extraction was demonstrated using cholesterol, hexadecanedioic acid, and palmitoyloleoylphosphatidylcholine as analytes, and these results were compared to those obtained with commercially available materials. Breakthrough curves indicate that, as expected, porous diamond particles have higher analyte capacity than nonporous solid particles.

Electric field gradient focusing (EFGF) is an equilibrium gradient focusing technique that depends on an electric field gradient and a hydrodynamic counterflow to focus, concentrate, and separate charged analytes. In this work, EFGF devices were fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic plastic. The separation channel was formed in an ionically conductive and protein-resistant PEG-functionalized hydrogel, which was cast in a changing cross-sectional cavity in the plastic device. A linear electric field gradient was obtained by applying a voltage lengthwise across the shaped hydrogel. Standard proteins were used as analytes to demonstrate the performance of these EFGF devices. With an increase in counterflow rate or decrease in applied voltage, analyte bands broadened, but resolution increased in agreement with theory. To reduce analyte band dispersion and improve focusing performance, a protein-compatible PEG-functionalized monolith was incorporated in the EFGF channel. Compared with focusing in an open channel, protein bands in the monolith-filled EFGF channel were significantly narrower.

Miniaturized devices for electric field gradient focusing (EFGF) were developed that consist of a cylindrical separation channel surrounded by an acrylic-based polymer hydrogel. The ionic transport properties of the hydrogel enable the manipulation of the electric field inside the separation channel. A changing cross-section design was used in which the hydrogel is shaped such that an electric field gradient is established in the separation channel. One of the challenges with this type of EFGF device has been that experimental resolution between protein analytes is lower than theoretically predicted. In order to investigate this phenomenon, a mathematical transport model was developed using FEMLAB. Model results and experimental observations showed that the reduced performance was caused by concentration gradients formed in the EFGF channel, and that these concentration gradients were the result of an imbalance in cation transport between the open separation channel and the hydrogel. Removing acidic impurities from the monomers that form the hydrogel reduced this tendency and improved the resolution. These transport-induced concentration gradients can be used to establish electric field gradients that may be useful for sample pre-concentration. Both the results of simulation and experiments demonstrate how transport-induced concentration gradients lead to the establishment of electric field gradients.

A novel ion gate for electrospray-ionization atmospheric-pressure ion-mobility spectrometry (ESI-IMS) has been constructed and evaluated. The ion gate consisted of a chopper wheel with two windows--one for periodic ion passage from the ESI source into the drift region and the other for timing and synchronization purposes. The instrument contained a 45.0 cm long drift tube comprising 78 stainless steel rings (0.12 cm thick, 4.90 cm o.d., 2.55 cm i.d.). The rings were connected together in series with 3.34-MOmega resistors. The interface plate and the back plate were also connected with the first and the last rings, respectively, of the drift tube with 3.34-MOmega resistors. A potential of -20.0 kV was applied to the back plate and the interface plate was grounded. The drift tube was maintained at an electric field strength of approximately 400 V cm-1. An aperture grid was attached to the last ring in front of a Faraday plate detector, center-to-center. Several sample solutions were electrosprayed at +5.0 kV with +500 V applied to the ion gate. Baseline separations of selected benzodiazepines, antidepressants, and antibiotics were observed with moderate experimental resolution of approximately 70.

In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 μL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 μL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system.

Over the last four decades, liquid chromatography (LC) has experienced an evolution to smaller columns and particles, new stationary phases and low flow rate instrumentation. However, the development of person-portable LC has not followed, mainly due to difficulties encountered in miniaturizing pumps and detectors, and in reducing solvent consumption. The recent introduction of small, non-splitting pumping systems and UV-absorption detectors for use with capillary columns has finally provided miniaturized instrumentation suitable for high-performance hand-portable LC. Fully integrated microfabricated LC still remains a significant challenge. Ion chromatography (IC) has been successfully miniaturized and applied for field analysis; however, applications are mostly limited to inorganic and small organic ions. This review covers advancements that make possible more rapid expansion of portable forms of LC and IC.

One approach to achieve fast and efficient separations in packed column liquid chromatography (LC) is to reduce eddy diffusion and mass transfer resistance in the mobile phase using short columns packed with small particles. In this study, efficiencies of columns packed with 1.5 and 3.0 microm nonporous and porous particles were compared in reversed-phase LC using nitromethane and a protein as analytes. Nonporous particles provided overall higher efficiency at high linear velocities when nitromethane was used as solute; however, the efficiency difference diminished significantly when the particle size was reduced from 3 to 1.5 microm. Efficiencies for 1.5 microm nonporous particles were considerably higher than those for 1.5 microm porous particles at high linear velocities when a protein, alpha-chymotrypsinogen A (MW 25,000), was used as solute. In addition, the average retention factor for amylbenzene in a column packed with ACQUITY C(18) porous particles was approximately 16 fold higher than for Micra C(18) nonporous particles for aqueous mobile phase compositions containing from 40 to 75% acetonitrile. Pressure drop, sample loading capacity, and separation power were also evaluated and compared for porous and nonporous particles under practical conditions.

A stable poly(2-carboxyethyl acrylate-co-poly(ethylene glycol) diacrylate) monolith was synthesized inside a 75-μm id capillary by direct in situ photo-initiated polymerization in a binary porogenic solvent consisting of methanol and ethyl ether. The resulting monolith was evaluated for weak cation-exchange capillary liquid chromatography of peptides and proteins. A high dynamic binding capacity of 72.7 mg lysozyme per cm3 column volume was measured. Fast mass transfer was demonstrated by steep breakthrough curves. The resulting monolith exhibited negligible hydrophobicity, leading to good separation of peptides and proteins. Peak capacities of 11 for peptides with a 10-min salt gradient and 39 for proteins with a 20-min salt gradient were measured. An efficiency of 37,000 plates/m for proteins was obtained under isocratic conditions. The effects of functional group concentration, porogenic solvent composition, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run relative standard deviation (RSD)1.93% and column-to-column RSD4.63% were achieved.

A 260 nm deep UV LED-based absorption detector with low detection limits was developed and integrated with a small nanoflow pumping system. The detector is small in size (5.2 × 3.0 cm) and weighs only 85 g (without electronics). This detector was specifically designed and optimized for on-column detection to minimize extra-column band broadening. No optical reference was included due to the low drift in the signal. Two ball lenses, one of which was integrated with the LED, were used to increase light throughput through the capillary column. Stray light was minimized by the use of a band-pass filter and an adjustable slit. Signals down to the parts per billion level (nanomolar) were easily detected with a short-term noise level of 4.4 μAU, confirming a low limit of detection and low noise. The detection limit for adenosine-5'-monophosphate was 230 times lower than any previously reported values. Good linearities (3 orders of magnitude) were obtained using sodium anthraquinone-2-sulfonate, adenosine-5'-monophosphate, dl-tryptophan, and phenol. The LC system was demonstrated by performing isocratic separation of phenolic compounds using a monolithic capillary column (16.5 cm × 150 μm i.d.) synthesized from poly(ethylene glycol) diacrylate.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively pre-concentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, the development of an on-line protein G monolithic pre-concentrator-CE device is proposed. To generate active groups for protein immobilization, glycidyl methacrylate (GMA) was used to prepare polymer monoliths. A 1.5-2 cm monolith was cast inside a 75 microm I.D. fused silica capillary that had previously been coated with alternating layers of negatively (dextran) and positively (polybrene) charged polymers. Protein G was covalently bound to GMA. Monoliths from different formulations were prepared and evaluated for binding capacity to optimize the monolith formulation for protein preconcentration. The physical properties of the column considered best for preconcentration were determined by mercury intrusion porosimetry. The total pore area was 4.8m(2)/g, the average pore diameter was 3.3 microm and the porosity was 82%. The monolith had a low flow resistance and was macroscopically homogeneous. The effectiveness of the monolith to rapidly pre-concentrate proteins at flow rates as high as 10 microL/min was demonstrated using a 1.8 microM IgG solution. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available.

Electric field gradient focusing (EFGF) uses a hydrodynamic flow and an electric field gradient to focus proteins in order of electrophoretic mobility. In this paper, we describe several bioanalytical applications using voltage-controlled hollow dialysis fiber-based EFGF with online UV detection. Using bovine serum albumin (BSA) as a model protein, a concentration factor as high as 15,000 and a concentration limit of detection as low as 30 pM were achieved. We also demonstrate the potential of using fiber-based EFGF for protein quantitative analysis. Simultaneous desalting and protein concentration were performed by mixing BSA with 2 M NaCl in a cell culture medium. Online concentration of ferritin and simultaneous removal of albumin from a sample matrix were performed using this EFGF system.

A poly(polyethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monolith was prepared by UV-initiated polymerization. Methanol and ethyl ether were selected as porogens from a variety of organic solvents to achieve the desirable characteristics of the monolith. The preparation of the monolith could be achieved within 10 min. The monolith was macroscopically homogeneous, had low flow resistance, and did not swell or shrink significantly in tetrahydrofuran. Inverse size-exclusion data indicate that the monolith had a total porosity of 75.4% and an internal porosity of 9.1%. The monolith could be used for size-exclusion separation of peptides, although it could not separate proteins with molecular masses between 10 and 100 K due to its unique pore size distribution. It was found to resist adsorption of proteins in capillary liquid chromatography when using 100 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. Complete recovery of both acidic and basic proteins was achieved. The monolith can be used for applications in which inert materials are required for protein analysis.

A new electron ionization source was developed for orthogonal acceleration time-of-flight mass spectrometry (TOFMS) based on the superimposition of a magnetic field around a radio frequency-only (rf-only) ion guide. The cylindrically symmetric magnetic field compresses the electron beam from the electron source into a long narrow volume along the ion guide axis. The magnetic field also helps to maintain a narrow energy distribution of electrons that penetrate the full length of the ion guide despite the influence of the radial rf field. Ionization occurs inside the ion guide with improved efficiency resulting from efficient use of electrons, prolonged interaction time, and nontraditionally large ionization volume. At the same time, the rf field effectively focuses ions radially and confines them to the axis of the ion guide by collisional focusing, leading to high ion transmission efficiency. Furthermore, the source can also be operated in a trap-and-pulse mode to improve the ion sampling duty cycle of orthogonal acceleration TOFMS. To validate the design concept of this new ion source, a simple prototype using a single set of cylindrical rods was constructed and retrofitted to an orthogonal acceleration TOFMS. A significant increase in ion signal intensity was observed by operating the source in a pulsed ion extraction mode. Low detection limits (for example, 12 fg for toluene) were determined at 12.5 spectra s(-1) in the full spectrum mode.

An ODS packing material was specially designed for CEC to achieve high efficiency. The size of the particles was approximately 3.2 μm based on scanning electron microscopy, and the average pore size of the particles was 1500 A by mercury intrusion porosimetry measurement. The packing materials were used in sol-gel bonded continuous-bed columns. The performance of columns containing these 3 μm, 1500 A ODS particles was evaluated for CEC and compared to 3 μm, 80 A ODS particles. The electroosmotic velocity (EOF) for the 1500 A ODS material was slightly greater than that for the 80 A ODS material. An efficiency of 410,000 plates m−1 was obtained for unretained thiourea with a 30 cm × 75 μm i.d. 1500 A ODS column, which was more than 2 times higher than the efficiency obtained from the 80 A ODS column.

The class of equilibrium gradient methods utilizes the opposition of two forces, at least one of which changes in magnitude with position, to separate and concentrate analytes. The drawback of many methods of this type is that the production of two opposing forces requires in comparison to standard methods, such as capillary electrophoresis, a relatively complex apparatus. In addition, for techniques such as electric field gradient focusing, hydrodynamic flow leads to Taylor dispersion, which limits the attainable concentration factor. We propose a new method, gradient field electrophoresis, which achieves analyte separation and focusing with only one spatially varying force, an electric field gradient. A model for the method is developed and used to analyze peak capacity. Experimental results for a protein (R-phycoerythrin) are given and compared to the model.

Electric field gradient focusing (EFGF) is a separation technique that uses an electric field gradient and an opposing hydrodynamic flow to separate and concentrate charged analytes. This work describes miniaturized EFGF devices that are used for protein analysis. These devices employ a unique ionically conductive polymer that enables the required electric field gradient to be established. This polymer has good protein compatibility and allows the transport of small buffer ions while retaining large analytes such as proteins. With the use of an EFGF device, green fluorescent protein was concentrated 10 000-fold and the separation of a protein mixture was demonstrated. The development of these ionically conductive polymer-based devices represents a step toward making EFGF a useful analytical tool for proteomics investigations.

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60–200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. β-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, β-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.

Electrospray ionization has grown to be one of the most commonly used ionization techniques for mass spectrometry, and efforts continue to improve its performance. Typically, the sprayer tip must be very close to the entrance orifice of the mass spectrometer in order to maximize the conduction of ions from the sprayer into the mass spectrometer. However, because of space-charge repulsion, most ions never reach the sampling orifice. In this work, an industrial air amplifier, for which the working mechanism is based on venturi and coanda effects, was added between an electrospray ionization source and a time-of-flight mass spectrometer. When a series of reserpine solutions (0.5, 1.0, 5.0, and 10.0 microM) were monitored using mass spectrometry, an over 5-fold increase in m/z 609.3 ion intensity was measured for a separation distance of 14 mm between the electrospray tip and interface capillary inlet, as compared to when the electrospray tip was in its normal position 1 mm in front of the inlet without the amplifier. When a voltage was applied to the air amplifier to further assist in focusing the electrosprayed ions, an approximately 18-fold increase in m/z 609.3 ion intensity was obtained. In addition, a 34-fold reduction in method detection limit was observed.

Ultrahigh pressure liquid chromatography (UHPLC) is an emerging technique which utilizes pressures higher than 10,000 p.s.i. to overcome the flow resistance imposed when using very small particles as packing materials in fused-silica capillary columns (1 p.s.i.=6894.76 Pa). This technique has demonstrated exceptionally high separation speeds and chromatographic efficiencies. However, safety is a concern when extremely high pressures are used. In this study, the safety aspects of capillary column rupture during operation were identified and carefully evaluated. First, liquid jets may be formed as a result of blow-out of the on-column frits or from rupture of the capillary at or near the column inlet. Second, incorrect installation of the capillary at the injector, failure of the ferrule used in the capillary connection, or rupture of the capillary can produce high speed projectiles of silica particles or column fragments. Experiments were carried out in the laboratory to produce liquid (water) jets and capillary projectiles using a UHPLC system, and the power density, an important parameter describing water jets in industrial practice, was calculated. Experimental results were in accordance with theoretical calculations. Both indicated that water jets and capillary projectiles under ultrahigh pressures might lead to skin penetration under limited conditions. The use of a plexiglass shroud to cover an initial length of the installed capillary column can eliminate any safety-related concerns about liquid jets or capillary projectiles.

Capillary columns packed with small diameter particles typically lead to low permeability and long separation times in high-performance liquid chromatography. Ultrahigh pressures (10,000 p.s.i.; 1 p.s.i. is identical with 6,894.76 Pa) can be used to overcome the limitations that small particles impose. Ultrahigh-pressure liquid chromatography (UHPLC) has demonstrated great potential for high-speed and high-efficiency separations. Decreasing the viscosity of the mobile phase by elevating the temperature could additionally reduce the pressure drop and facilitate the use of longer columns or smaller particles to achieve even higher total plate numbers. For this reason, we investigated the use of elevated temperatures in UHPLC. Water-resistant, flexible heater tape covered with insulation was used to provide the desired heat to the column. Polybutadiene-coated 1 microm nonporous zirconia particles were used because of their chemical stability at elevated temperature. A column efficiency as high as 420,000 plates m(-1) was obtained. The effects of temperature and pressure on the separation of parabens were investigated. Separation of five herbicides was completed in 60 s using 26,000 p.s.i. and 90 degrees C.

A copolymeric stationary phase, consisting of a chiral selective part, i.e. (1R-trans)-N,N'-1,2-cyclohexylenebisbenzamide, and an efficient siloxane oligomeric part, was successfully applied to open tubular column GC analysis. The efficiency and the chiral selectivity of this stationary phase were studied in detail, and high capacity and efficiency at elevated GC temperatures were especially noted. Several drugs and other enantiomeric pairs were separated. The shown examples demonstrate a broad application range for this type of chiral stationary phase in GC analysis.

Two novel polysiloxanes containing the metal complex, Co(TACN)(3+)2 (TACN= 1,4,7-triazacyclononane) were used as coatings for capillary electrophoresis (CE) and capillary electrochromatography (CEC). Through crosslinking and covalent bonding, the positively charged polymers were bonded to silica supports. In both CE and CEC, these coatings exhibited strong, pH-independent, and anodic electroosmotic flow (EOF), and had excellent long-term stability. Successful separations of aromatic acids were achieved in CE. In CEC, separation of alkylbenzenes (7 min) and basic compounds (20 min) was achieved with higher resolving power than conventional octadecyl silica packings. These polymers represent a new class of coatings for CE and CEC that generate pH-independent EOF.

Equilibrium gradient methods belong to a family of separation techniques in which analytes are forced to unique equilibrium points by a force gradient and a counter force along the separation pathway. The basic theory for equilibrium gradient methods where the force gradient is induced by a field gradient is developed in this paper. The results indicate that peak capacity can be dynamically improved by using a nonlinear field-intensity gradient in which the first section is steep, and the following section is shallow. Using electromobility focusing (EMF) as an example, a separation model is established. EMF is an equilibrium gradient method that uses an electric field intensity gradient to induce a force gradient on charged analytes, such as proteins, and a constant hydrodynamic flow as an opposing force. Equations relating operating parameters with separation performance are given. Although simulation results show that a peak capacity of over 10,000 is theoretically possible using a single channel in a separation time just under 2 months, if 100 parallel separation units are utilized in an array format under the same operating conditions, the same peak capacity can be obtained in just over 12 h.

Analytical equilibrium gradient methods are non-linear separation methods in which the separation mechanism involves a force gradient along the separation channel. These methods can be classified into two categories: those in which the gradient is a field gradient applied along the separation channel (i.e., field gradient), and those in which the channel is subjected to a constant field with a gradient formed in some other property (i.e., constant field). Standard deviation of peak width, resolution and peak capacity are important parameters in characterizing equilibrium gradient methods, and general expressions can be obtained from considering both the point of force acting on the analyte and the basic flux equation. Several successful examples, such as density gradient sedimentation, isoelectric focusing and electromobility focusing are discussed. Based on equilibrium gradient methods in the field gradient category, a method to dynamically improve peak capacity is described. An example of such an approach is given using electromobility focusing.

Two novel polysiloxane-based polymers, which contain metal complexes, have been prepared. To prepare the Co(TACN)3+(2) (TACN= 1,4,7-triazacyclononane) based polymers, an orthoamide derivative of TACN was added to bromobutane-substituted methylpolysiloxane and hydrolyzed with base. Co(II) was then coordinated to the TACN, followed by cobalt oxidation to make polymer A or followed by N-octyl TACN coordination and cobalt oxidation to make polymer B. In both materials, TACN forms thermodynamically and kinetically stable Co(TACN)3+(2) complexes in which the six coordination sites of the Co(III) are occupied by nitrogens from the TACN. The polymers were coated on fused-silica capillary columns and spherical silica particles, which were used for capillary electrophoresis and capillary electrochromatography, respectively. The open and packed columns showed strong and pH-independent reversed electroosmotic flow.

Ultrahigh pressure liquid chromatography was demonstrated for fast and efficient chiral separations. Capillary columns approximately 13–24 cm in length packed with nonporous 1.0μm C6-modified silical particles were used. β-Cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were added to the mobile phase as modifiers to produce transient diastereomeric complexes with the analytes. Pressures up to ≈42,000 psi were applied, and efficiencies in excess of 200,000 plates m−1 were obtained for separations that were accomplished in less than 2 minutes.

An ultra-high pressure liquid chromatograph was interfaced to a moderately high-resolution nanoelectrospray ionization ion mobility spectrometer operating at ambient temperature and pressure to achieve fast multidimensional separations. The potential of using ion mobility spectrometry as the second dimension in a comprehensive arrangement with liquid chromatography to offer improved qualitative information for compounds under specific operating conditions, is discussed. Separation and detection of selected benzodiazepines and triazine herbicides are demonstrated. Composite peak capacities of 39 and 33 for benzodiazepine and triazine herbicide mixtures, respectively, were achieved in less than 75 s using a 16.5 cm×50 μm internal diameter fused silica capillary liquid chromatographic column packed with 1.5 μm diameter ODS-bonded silica particles.

Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23 nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.

New monolithic reversed-phase liquid chromatography (RPLC) stationary phases based on single multi-acrylate/methacrylate-containing monomers [i.e., 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), trimethylolpropane trimethacrylate (TRIM) and pentaerythritol tetraacrylate (PETA)] were synthesized using organotellurium-mediated living radical polymerization (TERP), which was expected to produce more efficient monolithic columns than conventional free-radical polymerization. The rationale behind selection of porogens, relative concentrations of reagents and polymerization conditions are described. The new monolithic columns were applied to the separation of small molecules (i.e., alkylbenzenes) under isocratic conditions. Chromatographic efficiencies as high as 60,200 plates/m (71,300 plates/m when corrected for extra-column variance) were obtained, showing a general improvement over previous RPLC monoliths.

A previous investigation from our laboratory showed that the odor of live green peach aphids, Myzus persicae (Sulzer), highly attracts the convergent lady beetle, Hippodamia convergens Guérin-Méneville. In this study, we isolated the odor and identified it as (E)-β-farnesene (an aphid alarm pheromone) using gas chromatography – mass spectrometry. The olfactory response of the convergent lady beetle to (E)-β-farnesene was investigated using an eight-arm air-flow olfactometer and tracing the pathways of the lady beetles. The results clearly indicate that H. convergens can perceive and orient to (E)-β-farnesene released by green peach aphids. These findings suggest that this species of lady beetle has evolved a sensory system for detecting the green peach aphid alarm pheromone as a means of finding its prey.

Experimental peak capacities for capillary gas chromatography (GC), capillary liquid chromatography (CLC), and capillary electrochromatography (CEC) were compared. To obtain a meaningful comparison, the following constraints were applied. First, the same sample (mixture of alkylbenzenes) was used as a test mixture for all three techniques; second, the same packing material and column diameter were used in CLC and CEC; and third, isothermal conditions were used in GC, while isocratic conditions were used both in CLC and in CEC. Comparison of peak capacities for the same total column efficiency (approximately 36000 plates) showed that the peak capacity of GC is greater than those of the liquid-phase separation techniques. Comparison of CEC and CLC for constant retention factor was also carried out. For this condition, the results depend on the particle size used; for 3-microm porous particles, CEC had a peak capacity larger than CLC due to higher efficiency from the flow profile generated by electroosmotic flow. However, when 1.5-microm nonporous particles were used, the peak capacities were approximately the same for both techniques. The effect of linear velocity on peak capacity was also studied for all three techniques. Practical conditions aimed at increasing peak capacities of liquid-phase separation techniques are discussed.

Neat water at temperatures above its normal boiling point was used as a mobile phase for packed capillary column chromatography. The “universal” flame ionization detector (FID) was used for detection. No restrictor or back pressure was imposed at the column outlet. The effects of temperature on column efficiency and retention were investigated using particle-bonded continuous bed columns packed and crosslinked with 3 μm polybutadiene-encapsulated zirconia particles. It was found that when the column inlet pressure was held constant and the temperature was increased to values that were higher than 100°C, the pressure drop decreased rapidly, allowing the use of long columns or small particles. Solute retention factors decreased with increasing temperature; however, column inlet pressure had little effect on retention factor. Mass transfer resistance also decreased with an increase in temperature. Mixtures of alcohols and phenols could be separated within several minutes after the interface between the column and the FID was optimized. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 41–47, 2001

A new gradient system for CEC was designed and adapted to a home-built CEC system. Chromatographic separations are initiated with a predetermined mobile phase composition and, after a desired time period, a syringe pump delivers pure organic solvent at a preset flow rate into a 2 mL vial used as the inlet buffer reservoir. Mixing is achieved by the use of a microstir bar in the buffer reservoir, which in turn is driven by an air-actuated stir plate. In this way, a convex-shaped gradient is generated, and the mobile phase is introduced into the separation column by electro-osmotic flow. Use of this system significantly reduced analysis time while still retaining acceptable reproducibility. Nonaqueous CEC was used for isocratic and gradient separation (

Addition of Gd to Co/SiO2 Fischer–Tropsch synthesis catalysts (1) increases reducibility to Co metal, (2) decreases CH4 selectivity, (3) increases activity for CO hydrogenation based on the mass of Co, and (4) causes carbon deposition at lower temperatures when compared to the unpromoted catalyst. The intrinsic activity, based on H2 chemisorption surface area, is the same for both unpromoted and promoted catalysts.

Isotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient quantities without heart-cutting or splitting. Examples of comprehensive ITP-CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of individual ITP zones. Direct on-line coupling of ITP to CE in this comprehensive arrangement involved the use of columns having different diameters with one directly inserted inside of the other. A computer-controlled counterflow was applied when the isotachophoretic sample stack reached the bifurcation point. Large volume (10 μL) injections were made using an electrically insulated commercial polymeric rotary valve injector for increased reproducibility compared to previous comprehensive ITP-CE studies, with ITP and CE retention time RSD's ranging from 2 to 5%. An ultraviolet (UV) detector positioned at the bifurcation point was used to determine the beginning of CE injection. Application of a splitting voltage at the bifurcation point showed no affect on analyte transfer into the CE column. In using multiple injections of the ITP band(s), CE column overloading was not observed. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 361–370, 2001

Column technologies for capillary electrochromatography (CEC) are reviewed. To achieve high efficiency, the inner diameters of open-tubular and packed columns should be less than 25 and 200 μm, respectively. To obtain acceptable separation speed under typical CEC conditions (e.g. 30 kV, 1 mm s−1 electroosmotic flow velocity, and 2–4×10−8 m2 V−1 s−1 electroosmotic mobility) the column lengths for open-tubular and packed columns should be less than 120 and 60 cm, respectively. Capillary CEC columns are generally classified into three types: packed, open-tubular, and continuous-bed or monolithic. The various column preparation procedures and the advantages and disadvantages of each column type are discussed in detail.

Three Argonne Premium Coals (Upper-Freeport, Pittsburgh #8, and Lewiston-Stockton) were extracted with benzene in both Soxhlet and pressurized fluid extraction (PFE) systems. The extracts were compared on the basis of dry mass yield and hydrocarbon profiles as obtained by gas chromatography/mass spectrometry. The dry mass yields (wt%, daf) indicated no difference by either method for the Upper-Freeport coal, but statistically different yields were obtained for the Pittsburgh #8 and Lewiston-Stockton coals. In this study, PFE required ∼90 vol.% less solvent compared to Soxhlet extraction. The gas chromatograms obtained from the Soxhlet extracts contained solvent contaminant peaks as dominant peaks, which were not observed in chromatograms of the PFE extracts. No thermal degradation, as indicated by excess phenolic compound concentrations, was noted in any of the PFE extracts.

Continuous-bed columns containing sol-gel bonded 3 microm silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups (ODS/SCX) were prepared by first packing the ODS/SCX particles into a fused-silica capillary, then filling the packed capillary with a siliceous sol-gel, curing the sol-gel, and finally drying the column with supercritical carbon dioxide. The performance of the sol-gel bonded ODS/SCX columns was evaluated for capillary electrochromatography using acetonitrile aqueous mobile phase containing phosphate buffer. The columns were mechanically strong and permeable. Both EOF velocity and current increased linearly with elevation of the applied electric field strength. The EOF velocity was high at low pH and nearly constant over a range of pH 2-9. Higher buffer concentration resulted in higher current and lower EOF velocity. The acetonitrile content had no significant effect on the EOF. Without thermosetting the column, no bubble formation was noticed with currents up to 2.5 microA. The minimum plate height of a 25/34 cm x 75 microm I.D. sol-gel bonded 3 microm ODS/SCX column was 5.7 microm (1.75 x 10(5) plates per meter) at an optimum EOF velocity of 0.92 mm s(-1). Mixtures of test aromatic compounds and aromatic hydrocarbon homologues gave symmetrical peaks when using a low pH mobile phase. The retention and elution order of aromatic compounds represented a typical reversed-phase separation mechanism similar to conventional ODS columns. The run-to-run and column-to-column retention factor reproducibility was better than 2.5% and 8.0% RSD, respectively.

Kinetic data from the literature were used to predict formation rates and product yields of oil and gas at typical low-temperature conditions of coal maturation. These data indicate that gas formation rates from hydrocarbon thermolysis are several orders of magnitude too low to have generated known coal-seam natural gas reserves, assuming bulk first-order kinetics defined by a single activation energy and preexponential factor. By assuming distributed activation energies, thermal cracking of liquid hydrocarbons and coal kerogen to methane can occur at sufficiently high rates to produce commercial quantities over long periods of geologic time. Acid-mineral-catalyzed cracking, transition-metal-catalyzed hydrogenolysis of liquid hydrocarbons, and transition-metal-catalyzed CO2 hydrogenation form gas at very high rates at geologic temperatures. Rates of gas production in these reactions are orders of magnitude higher than those predicted from thermolysis; moreover, the gaseous products for metal-catalyzed hyd...

Hydrocarbon hydrogenolysis and CO2 hydrogenation in the presence of Fe/SiO2 and Ni/SiO2 catalysts were evaluated as potential mechanisms contributing to natural gas formation in coalbeds. The hydrocarbons used as reactants in hydrogenolysis included butane, octane, 1-octene, and 1-dodecene. The reactions carried out in a laboratory batch reactor produced gas that contained methane concentrations greater than 90%, which resembles the composition of natural gas. Reaction temperatures were selected to resemble natural coalbed conditions. Evidence is presented to show that iron and nickel minerals, which can be present in coals at levels of 2000 and 10 ppm, respectively, can become active under geologic conditions. The oxides (Fe2O3 and NiO) used as precursors of the active catalysts (Fe and Ni metals) were reduced at 200°C under a hydrogen atmosphere. Mossbauer spectroscopy showed that ca. 6% of the iron oxide was converted to the metal; in the case of nickel, oxygen titration showed that the extent of reduction to the metal was ca. 29%. The resultant fractions of the active metals in coals are adequate to catalyze generation of appreciable amounts of methane over geologic time.

Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.