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Liquid samples were introduced into an open tubular column supercritical fluid chromatograph (SFC) using a short packed capillary column as a solute focusing device after a typical sample loop valve injector. The packing materials were held inside the capillary by porous ceramic frits. Excess solvent was vented into the atmosphere through the packed capillary. Since the exit end of the packed capillary column was at atmospheric pressure, a density gradient was established along the length of the column and solutes precipitated at the point where the density dropped below the carbon dioxide solvating density for these compounds

Sulfur chemiluminescence detection (SCD) for the determination of sulfur-containing compounds in gas chromatography (GC) and supercritical fluid chromatography (SFC) is described. The detector is based on the chemiluminescence reaction of ozone with sulfur monoxide which is produced in an H2/O2 reducing flame from sulfur-containing analytes. High selectivity over nonsulfur-containing species has been achieved for a number of mercaptans and sulfur-containing polycyclic aromatic hydrocarbons. Minimum detectable quantities of 23 and 26 pg S (S/N=3) for dibenzothiophene and benzo[b]naphtho[1,2-d]thiophene, respectively, have been obtained using the SFC/SCD system. It was found that connecting a buffer volume flask to the vent of the vacuum pump that draws the effluents and ozone gas into the chemiluminescence reaction chamber, increases the sensitivity of the SCD.

A highly efficient cross-linked polyacrylamide capillary coating has been developed for capillary electrophoresis of proteins. The fused silica capillary tubing was pretreated with 7-oct-1-enyltrimethoxysilane and then statically coated with the polymerization reagents. The copolymerization reaction was performed at 120°C when using a free-radical initiator. Coated columns were evaluated for the separation of both basic and acidic proteins. Methylene bisacrylamide was added in the preparation of the cross-linked polyacrylamide coating, and the cross-linking effects on column performance were evaluated. Both linear and cross-linked columns gave high separation efficiencies and good reproducibilities for proteins, and electroosmotic flow was eliminated. It was found that the cross-linked polyacrylamide coatings produced nearly double the efficiencies and only one third the relative standard deviations in migration times for proteins when compared to linear polyacrylamide coatings. Highly efficient and reproducible separations of proteins in human serum samples were achieved using the cross-linked column.

A coupled supercritical fluid extraction (SFE) and high-speed gas chromatography (GC) system was developed which permits simultaneous sample extraction and analysis. A thermal desorption modulator at the head of a 50-μm i.d. capillary column was used as an interface between the SFE and GC. The extraction fluid was first split between the modulator and a waste collection vessel. The substances introduced into the modulator were concentrated in the stationary phase and then released as a sharp injection pulse to the analytical column by electrical resistance heated thermal desorption. The modulator was operated at 10-s time intervals, which generated consecutive concentration pulses to the column. These concentration pulses were separated into individual chromatograms by high-speed GC. A set of chromatograms could be generated while the extraction proceeded. With this system, the extractant was sampled continuously so that the SFE process could be monitored in real time for selected components. Quantitative results could be extrapolated before extraction was complete from curves obtained by signal averaging of segmented chromatograms from the set.

A procedure for bonding polyethylene glycol (PEG) and polyethylene imine (PEI) onto the capillary surface through a highly crosslinked polysiloxane layer containing epoxy groups has been developed. Systematic variation of the coatings can be used to (a) allow separations over a pH range of 3 to 9, (b) provide for adjustment of electro-osmotic flow, and (c) reduce the adsorption of peptides and proteins. The variation of electroosmotic flow on these coated columns at different operational conditions can be explained using electrical double layer theory. Separations of substituted aromatic compounds and peptides on these coated columns were much improved over similar separations on untreated columns. The results indicate that PEI coated columns are suitable for the separation of basic proteins and PEG coated columns are suitable for acidic proteins. Separations of simple oligonucleotides which differ by only one base were also achieved.

In supercritical fluid chromatography (SFC), cosolvents (modifiers) have been added to CO2 in order to deactivate the analytical column and to improve the solvating power of CO2 for extending the utility of SFC to polar and high molecular weight solutes. However, the use of modifiers has proven to be difficult, and irreproducibility in retention and selectivity is often observed. This comprehensive review focuses on the phase behavior of analytically useful CO2-based mixed mobile phases in SFC. All of the CO2-based mixed mobile phases which have been used in packed or open tubular column SFC are reviewed. All of the available fluid phase equilibria data for such binary fluids are discussed, and the P-T-x region which is applicable to SFC is compiled in both graphs and tables. Single phase regions (pressure and temperature) are listed by mol% modifier. However, only mixtures of CO2 + benzene, + ethanol, + hexane, + methanol, or + toluene have enough P-T-x data points to enable extrapolation to any other conditions used in conventional SFC (0–20 mol% modifier, 40–400 atm, and 0–200°C). Problems associated with estimating the critical parameters of the pure components or the mixtures are reviewed. Phase transitions in the syringe pump, transfer lines, injector, column, and restrictor are delineated. Equilibration problems associated with the preparation of mixed mobile phases (premixed cylinders, liquid or gas phase saturation, and dual pumps with on-column mixing) and phase transitions during the operation of SFC instrumentation are discussed. Finally, future research needs are outlined.

A selective, low temperature depolymerization procedure has been applied to four coals of different rank to obtain products that are representative of the original structural units (monoclusters) of the coal macromolecular component, and that are amenable to Chromatographic analysis. The monocluster products of this depolymerization procedure retain most of their original aromatic-hydroaromatic structures, whereas functional groups serving as intercluster linkages, e.g. alkylene and etheric groups, undergo predictable modification. A comparison of liquid 13C n.m.r. spectra of the products and solid state 13C n.m.r. spectra of the original coals showed only minor changes in the aromaticities between the solid and depolymerized products, but some loss of carbonyl carbons was detected in all of the coals. Both the tetrahydrofuran pre-extract and the depolymerized products of the four coals were separated into chemical classes by adsorption chromatography. Two of these fractions were found to consist predominantly of aliphatic hydrocarbons (paraffins and naphthenes) and alkylsubstituted bi-, tri- and (some) tetracyclic arenes. The fractions were analysed using gas chromatography-mass spectrometry. Structural identifications were based on a combination of Chromatographic retention and mass spectral fragmentation data. For the lower rank coals, the compositions of the pre-extracts were quite different from the corresponding depolymerized coal products, and contained various molecular biological markers. The compositions of the pre-extracts became closer to those of the depolymerized coal products as rank increased.

On-line bioassay-directed fractionation of environmental samples is important because of the need to assign toxicological activity to certain classes of chemicals. Supercritical fluid chromatography (SFC) was used to fractionate a coal tar standard (SRM 1597) and these fractions were successfully directed into the Salmonella microsuspension mutagenicity assay. The reverse-mutation assay used in these experiments measures mutagenicity in Salmonella typhimurium TA 98 and was performed in a total volume of approximately 200 μl. Coupling of SFC with the microsuspension assay yielded quantitative recovery of the coal tar mutagenic activity, and the SFC chromatogram was compared to the mutagenicity profile to determine the activity of each fraction. This method of coupling SFC with bioassays may have broad applicability for fractionating various environmental samples and for identifying a wide range of biological activities in such samples.

Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monoliths were synthesized inside 75 mum i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The effect of gradient rate and initial salt concentration on the retention of proteins were investigated. Mass recovery was found to be greater than 96%, indicating the biocompatibility of this monolith. The monolith was mechanically stable and showed nearly no swelling or shrinking in different polarity solvents. The preparation of this in situ polymerized acrylate monolithic column was highly reproducible. The run-to-run and column-to-column reproducibilities were less than 2.0% relative standard deviation (RSD) on the basis of the retention times of protein standards. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles.

A simple device for field sampling and concentration of analytes for subsequent introduction into an injection port for gas chromatographic (GC) analysis has been developed. It consists of a tiny, coiled platinum wire filament (CWF) that is attached to a retractable plunger wire, which fits inside a syringe needle housing. Sampling is accomplished by dipping the end of the CWF in a liquid sample, which is drawn into the wire coil by capillary action, and introducing it into the injection port either before or after allowing the solvent to evaporate. The CWF can be used with or without a nonvolatile chemical coating. A major advantage of this sampling device is that nonvolatile sample matrix components remain on the wire coil, reducing the required injection port and liner cleaning frequency and contamination of the head of the chromatographic column. The coil itself can be easily cleaned between analyses by rinsing and/or burning off residual material in a small flame. The sampling coil facilitates specifically designed chemical reactions in the injection port, such as thermochemolysis and methylation. Applications demonstrated in this work include: (1) direct introduction of samples with little or no pre-treatment, (2) simultaneous thermochemolysis and methylation of lipid-containing samples such as bacteria and bacterial endospores for analysis of biomarkers, and (3) solid phase micro-extraction (SPME) using temporary wire coatings. The CWF allowed for significant reduction in sample preparation time, in most cases to less than a few minutes. The peak shapes examined for polycyclic aromatic hydrocarbon analytes (PAHs) were significantly better (asymmetry factors1.3) when using the CWF sampling technique compared to splitless and on-column injection techniques (asymmetry factors1.3). Extraction efficiencies for SPME (especially for high boiling point components such as PAHs) improved by an average of 2.5 times when using the CWF compared to the performance of commercially available SPME fibers. Coiled wire filaments and GC injection port liners were used for more than 100 Bacillus endospore thermochemolysis methylation analyses without the need for cleaning or replacement.

Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 microm i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (

Electric field gradient focusing (EFGF) uses an electric field gradient and a hydrodynamic counter flow to simultaneously separate and focus charged analytes in a channel. Previously, most EFGF devices were designed to form a linear field gradient in the channel. However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE. Dynamic improvement of peak capacity in EFGF can be achieved by using a nonlinear gradient. Numerical simulation results indicate that the peak capacity in a 4-cm long channel can be increased from 20 to 150 when changing from a linear to convex bilinear gradient. To demonstrate the increased capacity experimentally, an EFGF device with convex bilinear gradient was fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic copolymers. The desired gradient profile was confirmed by measuring the focusing positions of a standard protein for different counter flow rates at constant voltage. Dynamically controlled elution of analytes was demonstrated using a monolith-filled bilinear EFGF channel. By increasing the flow rate, stacked proteins that were ordered but not resolved after focusing in the steep gradient segment were moved into the shallow gradient segment, where the analyte peak resolution increased significantly. In this way, the nonlinear field gradient was used to realize a dynamic increase in the peak capacity of the EFGF method.

The use of small internal diameter precolumns (11–25-μm i. d.) helped solute focussing when using solvent vent sample introduction in open tubular column supercritical fluid chromatography (SFC). Column efficiencies were similar to those obtained with split injection (3000–5000 plates m−1). Trace analysis in SFC was improved because large volumes (1 μL) could be introduced for analysis with little sacrifice in column efficiency.

Solute focusing is an important topic in chromatography, but it has not been discussed extensively in the recent supercritical fluid chromatography literature. The purpose of this paper is to review the different methods that have been used to focus solutes in supercritical fluid chromatography. The theoretical basis for phase ratio and partition coefficient focusing is described. Different solute focusing devices that are used in on-line supercritical fluid extraction and chromatography are shown. The methods and principles of removing the solvent from the solutes prior to the analytical column, and hence reducing band broadening in the column due to the solvent, are discussed in context with the use of a retention gap or a mixing chamber, and in solvent vent injection.

Alternatives to conventional gas chromatographic simulated distillation methods for high boiling materials are reviewed. Operational parameters for the use of supercritical fluid chromatography for simulated distillation are explored and examples for the analysis of both petroleum and coal-derived samples are compared to conventional gas chromatographic methods and thermogravimetric analysis. Extension of the boiling range determined by simulated distillation to over 800°C is demonstrated. An n- octylpolysiloxane stationary phase minimized the aliphatic versus aromatic boiling point discrepancy common to other chromatographic methods.

Eight hydroxylated carbazoles and dibenzothiophenes were tested for mutagenic activity using the CHO/HGPRT mutation assay for thioguanine resistance. All showed increased activity over carbazole and dibenzothiophene. Some of the hydroxycarbazoles showed slightly higher activity than the corresponding hydroxy-dibenzothiophenes (71.4 ± 5.2 mutants/106 survivors for 1-hydroxycarbazoles compared to 21.8 ± 6.1 mutants/106 survivors for 4-hydroxydibenzothiophene at 5 μg/ml, and 32.8 ± 3.3 mutants/106 survivors at 5 μg/ml for 4-hydroxycarbazole as compared to 16.4 ± 0.9 mutants/106 survivors for 1-hydroxydibenzothiophene at 5 μg/ml). The most mutagenic compound was 1-hydroxycarbazole, which also exhibited cytotoxicity. The relative order of mutagenic activity of the hydroxycarbazoles was 1 ≥ 4 > 2 = 3 > carbazole, and the relative activity of the hydroxydibenzothiophenes was 2 ≥ 3 > 1 = 4 > dibenzothiophene. Overall, the mutagenic activity of the hydroxycarbazoles and hydroxydibenzothiophenes was dependent on the structural position of the hydroxy group.

A technique was developed for large volume sample introduction in capillary supercritical fluid chromatography. A 20-cm length of 200-μm i.d. capillary tubing was used as precolumn. The precolumn temperature could be easily controlled by passing an electrical current through an electrically conductive paint coated on its outer surface. During injection, the sample solvent was vented from the precolumn with CO2 (gas) at 32 atm, while the precolumn was kept at room temperature. Solutes were transferred onto the head of the analytical column as a narrow band by density gradient focussing, which was established with (a) a temperature gradient along the precolumn, (b) a rapid expansion of CO2 from supercritical fluid to gas, and (c) a temperature difference between the precolumn and the analytical column. This injection approach minimized solute mass discrimination and could be easily performed.

The phase behavior (P-T-x) of propylene carbonate was studied using a variable volume view cell for the regions applicable to supercritical fluid chromatography (SFC) and extraction (SFE). Type 5 phase behavior was observed. Phase separation was shown to significantly alter retention and selectivity in the analysis of coal extracts by capillary SFC. Propylene carbonate was found to have limited use as a modifier in capillary SFC. With 5.1 mol% propylene carbonate at 60°C, a single phase is maintained only at pressures above 250 atm. Twenty-two methods used to estimate critical pressure (Pc), twenty-seven methods used to estimate critical temperature (Tc), and thirty methods used to estimate critical volume (Vc) were evaluated for various modifiers employed in supercritical fluid chromatography (SFC). None of the methods could predict Pc, Tc, or Vc to within a 5% relative error for all of the modifiers tested. However, several of the methods were successful in predicting Pc, Tc, or Vc to within 5% for select chemical classes of compounds. Four methods used to estimate the vapor-liquid critical loci of binary mixtures not only failed to accurately predict vapor-liquid separation, but failed to predict the type of phase separation occurring. None of the above methods could reliably estimate the critrical parameters of propylene carbonate or the critical parameters of the mixture.

A new class of chiral copolymers consisting of chiral organic and dimethylsiloxane units is described. One example of such copolymers, containing alternating diamide and dimethylsiloxane units, has been synthesized. Using these chiral materials as stationary phases in capillary columns, chiral diols can be efficiently resolved by supercritical fluid chromatography.

A new polar polysiloxane stationary phase for capillary column gas chromatography containing 4-(methylsulfonyl)phenyl substitution was synthesized and compared to a cyanophenylpolysiloxane, OV-225. The sulfonylphenylpolysiloxane phase was useful and stable over a wide temperature range from 70°C to 320°C, and it exhibited similar overall polarity to OV-225. However, because of the specific chemical properties of the 4-(methylsulfonyl)phenyl group, this phase demonstrated unique selectivities for certain classes of compounds including basic nitrogen heterocycles and polychlorinated biphenyls.

Mixed mobile phases have often been used improperly in supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE). Phase separation is unavoidable at certain conditions where conventional SFC and SFE are performed, resulting in inhomogeneous mobile phases. We describe here a relatively rapid and accurate approach to determining transition pressures at different temperatures and modifier concentrations. There is close agreement between the experimental values determined in this work and those published by others for methanol and carbon dioxide. The results have been used to test the predictions of several common methods for estimating the critical pressures of mixtures. The methods failed to predict the critical pressure to within 20 atm between approximately 4–20 mol % of modifier. Chromatographic performance was shown to be severely degraded when the pressure in the column is below the gas-liquid transition pressure. The effect of pressure drop in the column on phase behavior is discussed.

As in the case of all chromatographic methods, the ideal sample introduction method for supercritical fluid chromatography (SFC) would provide a narrow and sharp solvent peak (or no solvent peak), quantitative transfer of the sample into the column, and high reproducibility. Elimination of the solvent before the solutes enter the column would be preferred; however, this practice is always associated with the risk of partial solute elimination. In open tubular column SFC, samples are normally introduced using a smallvolume internal loop valve. These valves can be operated such that none, some, or all of the solvent is eliminated. Several approaches were quantitatively evaluted, and optimized conditions were determined. Particular attention was given to trace analysis. The minimum detectable levels (S/N >4) achievable using a flame ionization detector were determined to be approximately 30 ng (150 ppm) for split injection, 4 ng (20 ppm) for delayed split injection, and 1 ng (5 ppm) for direct injection.

An on-line two-dimensional open tubular column supercritical fluid chromatograph was constructed and evaluated. A rotary valve interface allowed independent flow control of two 50-μm i.d. open tubular columns, providing maximum versatility for heartcutting. A solvent-venting injection technique was incorporated in the system that enabled single or multiple 2.0-μL volumes to be injected into an uncoated, yet deactivated, length of capillary precolumn without flooding of the analytical column. A cold trap was employed to refocussolutes from single or multiple fractional cuts after being transferred to the second dimension. The performance of the system was demonstrated with the analysis of mixtures of polycyclic aromatic compounds and steroids. Efficiencies of 4,500 plates m−1 were preserved in the second column after heartcutting.

Radial diffusion of solutes in the mobile phase limits the practical linear velocities that can be used to increase speed of analysis in conventional open tubular column chromatography. Several methods have been devised to enhance radial dispersion that do not depend only on molecular radial diffusion. In this review, the methods of enhancing dispersion found in the chromatographic literature are discussed. These methods include the use of deformed columns, and spinning band and static mixer columns. These techniques have been found to be of little value in analytical chromatography. Electroosmotic flow has been used to generate a plug-like flow profile in LC and generates one to two orders of magnitude lower plate height than produced at laminar flow conditions. Turbulent and secondary flow have been used to enhance radial diffusion in open tubular columns for gas, liquid, and supercritical fluid chromatography. These techniques are described from their theoretical and practical aspects. Turbulent flow has been investigated as a means for enhancing radial dispersion in open tubular column chromatography for several years. At low linear velocities (Reynolds numbers < 2000), the Golay equation accurately predicts that increasing velocities generate higher plate heights. However, turbulent flow experimental results have shown that unretained solutes have decreasing plate heights with increasing velocity after a critical velocity is achieved. Up to this time, turbulent flow chromatography has not been shown to be useful for retained solutes, and applications in analytical separations are absent in the literature. Secondary flow has also been studied by several investigators for the enhancement of radial dispersion. A circular, radial flow profile can be generated by centrifugal forces in tightly coiled columns, and after a critical velocity is achieved, columns coiled to appropriate aspect ratios yield lower plate heights than straight columns for unretained solutes as the velocity is increased. Although retained solutes yield higher plate heights in coiled rather than conventional straight columns in some cases due to the resistance to mass transfer at the interphase region, higher speeds of analysis are obtained. Secondary flow chromatography has been successfully applied in gas and supercritical fluid chromatography for the rapid separation of alkanes having low retention. Nevertheless, applications using secondary flow in chromatographic separations are limited.

The synthesis of 2-methyl-1-anthracenamine (2), 3-methyl-1-anthracenamine (7), 4-methyl-1-anthracenamine (14), 1-methyl-2-anthracenamine (20), 3-methyl-2-anthracenamine (26), and 1-methyl-3-anthracenamine (30) from the corresponding 9,10-anthraquinones in order to confirm their presence in coal-derived products is reported.

A series of polysiloxane stationary phases containing 4-cyanophenyl, 4-nitrophenyl, 2,4-dinitrophenyl or 8-quinolinyl units at the ends of diethylene oxide groups has been prepared by hydrosilylating the appropriate polar alkene onto a well-define polymethylhydrosiloxane polymer. The polar alkenes were prepared by connecting together eugenol, 2-chloroethyl ether and the polar-substituted phenol or 8-hydroxyquinoline. These stationary phases with silicone backbone and differently substituted eugenol moieties were found to possess both polar and polarizable characteristics. The properties of the phases could be tuned by varying the end groups of the polysiloxane side arms, thereby gaining unique selectivities.

Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris–HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm 2 for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 μg/mL).

In-channel atom transfer radical polymerization (ATRP) was used to graft a PEG layer on the surface of microchannels formed in poly(glycidyl methacrylate)-co-(methyl methacrylate) (PGMAMMA) microfluidic devices. The patterned and cover plates were first anchored with ATRP initiator and then thermally bonded together, followed by pumping a solution containing monomer, catalyst, and ligand into the channel to perform ATRP. A PEG-functionalized layer was grafted on the microchannel wall, which resists protein adsorption. X-ray photoelectron spectroscopy (XPS) was used to investigate the initiator-bound surface, and EOF was measured to evaluate the PEG-grafted PGMAMMA microchannel. Fast, efficient, and reproducible separations of amino acids, peptides, and proteins were obtained using the resultant microdevices. Separation efficiencies were higher than 1.0 x 10 4 plates for a 3.5 cm separation microchannel. Compared with microdevices modified using a previously reported ATRP technique, these in-channel modified microdevices demonstrated better long-term stability.

Electric field gradient focusing (EFGF) methods have received increased attention in recent years, with potential applications demonstrated by several research groups. In order to move EFGF from the research stage to routine use in application areas, a more detailed understanding of practical aspects of device performance is required. Useful theoretical models for EFGF are available but have not been verified through systematic checks under a variety of conditions. In this paper, we compare modeled and experimental results for an EFGF device with the goal of optimizing the time sequence of voltages applied to the device for maximum resolution of analytes with close electrophoretic mobilities. Measured peak profiles depend strongly on the sequence of voltages applied to the device. We investigate the characteristic behavior of the elution profile under various voltage programs. Rapid voltage drops lead to fast elution of closely spaced protein peaks with narrow widths, whereas a carefully designed voltage program can be used to increase the separation between analytes and achieve higher resolution. Simulated and experimental results demonstrate that the behavior of analyte diffusion at an electric field singularity associated with the transition from the EFGF device to elution capillary can be used to separate analyte peaks which may not be resolved within the EFGF device itself, thereby increasing the achievable resolution of the EFGF technique.

Fabrication of microfluidic systems from polymeric materials is attractive because of simplicity and low cost. Unfortunately, the surfaces of many polymeric materials can adsorb biological samples. Therefore, it is necessary to modify their surfaces before these polymeric materials can be used for separation and analysis. Oftentimes it is difficult to modify polymeric surfaces because of their resistance to chemical reaction. Recently, we introduced a surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), which can be modified easily and is suitable for fabrication of microfluidic devices. Epoxy groups on the surface can be activated by air plasma treatment, hydrolysis, or aminolysis. In this work, the resulting hydroxyl or amino groups were reacted with 2-bromoisobutylryl bromide to introduce an initiator for surface-initiated atom-transfer radical polymerization (SI-ATRP). Polyethylene glycol (PEG) layers grown on the surface using this method were uniform, hydrophilic, stable, and resistant to protein adsorption. Contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to characterize activated polymer surfaces, initiator-bound surfaces, and PEG-grafted surfaces. We obtained excellent capillary electrophoresis (CE) separations of proteins and peptides with the PEG-modified microchips. A separation efficiency of 4.4 x 10(4) plates for a 3.5 cm long separation channel was obtained.

Detailed identification of sulfur-containing polycyclic aromatic compounds (S-PAC) was accomplished for solvent extracts of five different coals. The S-PAC were isolated by ligand-exchange chromatography. Due to the great complexities of these samples, capillary gas chromatography (GC) was employed for their analysis. An element selective radio frequency plasma detector was used to provide sulfur selective detection. Gas chromatography-mass spectrometry was used to identify individual compounds in isolated sulfur fractions. Condensed thiophenic compounds were found to be the major constituents in all five S-PAC fractions. Diaryl sulfides were also detected in the high volatile bituminous Illinois #6 coal. The prevalence of various S-PAC in different coal extracts was observed as a function of their rank.

Dichlorofluoromethane, trichlorofluoromethane and tetramethylsilane were used in the static coating of small diameter capillary columns (5 to 50 μm I.D.) in order to obtain highly efficient columns for gas and supercritical fluid chromatography. Capillary columns of 5-, 10-, 25-, and 50-μm I.D. were coated with stationary phase films of SE-33, SE-54, OV-215, 50% n-octyl, 45% phenoxypolyethyl ether, 50% liquid crystal, 25% biphenyl, 50% pentafluorophenyl and 50% cyanopropyl polysiloxane stationary phases. Resultant evaluations of these columns in gas chromatography gave approximately 9000, 66 000, 45 000, and 19 000 plates m−1, respectively, for the different internal diameters. Important parameters which affect coating efficiency are identified and discussed in detail.

A new method for the simultaneous deactivation and coating of porous silica particles for supercritical fluid chromatography (SFC) has been developed. This method is based on a dehydrocondensation reaction between polymeric silicon hydride reagents and the silanol groups on the surface of the particles. The procedure produces a less active surface than conventional silica packings, which results in less adsorption and improved peak shapes for polar analytes. In SFC, more polar analytes can be chromatographed without the need for mobile phase modifiers. Furthermore, the sensitive and universal flame ionization detector (FID) can be used, since modifiers are not necessary. To avoid splitting of the column effluent before FID detection, packed capillary columns were utilized in this study. The ability to use packed capillary columns for the analysis of polar compounds, while at the same time allowing the use of a wide range of detection methods, serves to expand the number of useful applications for packed column SFC.

A radio-frequency plasma source has been characterized for use as an element-selective detector with capillary gas chromatography using atomic emission in the near-infrared region of the spectrum. With selected compounds introduced at rates similar to those encountered during a chromatographic run, the performance of the plasma has been characterized as a function of several interdependent variables, including plasma makeup gas flow, electrode spacing, and applied power. These settings have been optimized, and detection limits for several nonmetallic elements determined. Detection limits range from 0.2 to 50 pg/s.

Solanesol, a trisesquiterpenoid alcohol, has been shown to be present in environmental tobacco smoke. Results from the determination of particulate-phase solanesol in environmental tobacco smoke in both chamber and indoor environments show that solanesol is a suitable tracer for the particulate phase of environmental tobacco smoke

A mobile phase elimination interface and a microscope accessory are used to combine capillary supercritical fluid chromatography with Fourier-transform infrared spectrometry for analyzing carboxylic acids. Separations of a mixture of oligomers prepared by a self-condensation polymerization of 12-hydroxystearic acid and of a mixture of 16 phenolic carboxylic acids are achieved by using a well-deactivated fused-silica capillary column coated with an oligoethyleneoxide substituted methylpolysiloxane (glyme) stationary phase and carbon dioxide as the mobile phase. Components requiring identification are deposited, with elimination of the carbon dioxide, onto potassium bromide discs as compact spots, from the end of a heated restrictor, which is attached at the end of the column. Unique spectroscopic information is obtaned by positioning the spots in the microscope and measuring their infrared spectra. Phenolic acids are unambiguously identified and the degree of polymerization of oligomers in the 12-hydroxysteric acid reaction mixture is indicated from the ratio of ester carbonyl to acid carbonyl absorptions.

The application of mass spectrometry in the negative ion mode with capillary column supercritical fluid chromatography is described. The supercritical fluid chromatography mobile phase (CO2) was used as the primary reagent gas to generate thermalized electrons and reagent anions (such as O− and CO2−). When trace amounts of CH4 or NH3 gas were also introduced into the chemical ionization source, the mass spectrometer sensitivity was increased. Detection limits (S/N = 5) for heptachlor were determined to be 20 pg and 200 fg for full scan and selected ion monitoring modes, respectively. Mass spectra of a cyclic peptide, valinomycin, obtained with these mixed reagents (CH4 + CO2 and NH3 + CO2) gave the molecular anion at m/z 1110 as the most abundant ion and additional fragment ions that yielded peptide sequence information.

The alkaloid content was determined, mainly by GCMS, for three rare western United States species of the Papaveraceae: Arctomecon californica, A. humilis and A. merriamii . Allocryptopine and protopine were major alkaloids in all species, along with lesser quantities of a new alkaloid, 12-methoxyallocryptopine. Eleven other alkaloids, especially benzophenanthridines, were detected in minor amounts. The pattern of alkaloid structures in Arctomecon , one of the few remaining genera of the Papaveraceae left to be studied in detail, is briefly discussed.

Two polymer monoliths were designed and synthesized from commercially available monomers with an attempt to decrease hydrophobicity for strong cation-exchange chromatography. One was prepared from the copolymerization of sulfoethyl methacrylate and poly(ethylene glycol) diacrylate, and the other was synthesized from vinylsulfonic acid and poly(ethylene glycol) diacrylate. Both of the monoliths were synthesized inside 75-microm i.d., UV-transparent fused-silica capillaries by photopolymerization. The hydrophobicities of the two monoliths were systematically evaluated using standard synthetic undecapeptides under ion-exchange conditions and propyl paraben under reversed-phase conditions. The poly(sulfoethyl methacrylate) monolith demonstrated similar hydrophobicity as a monolith prepared from copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid and poly(ethylene glycol) diacrylate, and 40% acetonitrile was required to suppress any hydrophobic interactions with peptides under ion-exchange conditions. However, with the use of vinylsulfonic acid as the functional monomer, a monolith with very low hydrophobicity was obtained, making it suitable for strong cation-exchange liquid chromatography of both peptides and proteins. It was found that monolith hydrophobicity could be adjusted by selection of monomers that differ in hydrocarbon content and type of vinyl group. Finally, excellent separations of model protein standards and high-density lipoproteins were achieved using the poly(vinylsulfonic acid) monolith. Five subclasses of high-density lipoproteins were resolved using a simple linear NaCl gradient.

Electroosmotic flow was studied in thin film microchannels with silicon dioxide and silicon nitride sidewalls formed using plasma-enhanced chemical vapor deposition (PECVD). A sacrificial etching process was employed for channel fabrication allowing for cross-sections with heights of 3 mum, ranging from 2 mum to 50 mum in width. Flow rates were measured for single channels and multichannel electroosmotic pump structures for pH levels ranging from 2.6 to 8.3, and zeta potentials were calculated for both silicon dioxide and silicon nitride surfaces. Flow rates as high as 0.086 muLmin were measured for nitride multichannel pumps at applied electric fields of 300 Vmm. The surface characteristics of PECVD nitride were analyzed and compared to more well-known oxide surfaces to determine the density of amine sites compared to silanol sites.

We describe a novel radio frequency ion trap mass analyzer based on toroidal trapping geometry and microfabrication technology. The device, called the halo ion trap, consists of two parallel ceramic plates, the facing surfaces of which are imprinted with sets of concentric ring electrodes. Radii of the imprinted rings range from 5 to 12 mm, and the spacing between the plates is 4 mm. Unlike conventional ion traps, in which hyperbolic metal electrodes establish equipotential boundary conditions, electric fields in the halo ion trap are established by applying different radio frequency potentials to each ring. The potential on each ring can be independently optimized to provide the best trapping field. The halo ion trap features an open structure, allowing easy access for in situ ionization. The toroidal geometry provides a large trapping and analyzing volume, increasing the number of ions that can be stored and reducing the effects of space-charge on mass analysis. Preliminary mass spectra show resolution (m/Deltam) of 60-75 when the trap is operated at 1.9 MHz and 500 Vp-p.

A gas chromatographic method that uses solid-phase microextraction for analysis of organic marker compounds in fine particulate matter (PM2.5) is reported. The target marker compounds were selected for specificity toward emission from wood smoke, diesel or gasoline combustion, or meat cooking. Temperature-programmed volatilization analysis was used to characterize the thermal stabilities and volatile properties of the compounds of interest. The compounds were thermally evaporated from a quartz filter, sorbed to a solid phase microextraction (SPME) fiber, and thermally desorbed at 280 degrees C in a gas chromatograph injection port connected via a DB 1701 capillary separating column. Various experimental parameters (fiber type, time, and temperature of sorption) were optimized. It was found that high extraction yield could be achieved using a polyacrylate fiber for polar substances, such as levoglucosan, and a 7-microm polydimethylsiloxane (PDMS)-coated fiber for nonpolar compounds, such as hopanes and polyaromatic hydrocarbon. A compromise was made by selecting a carboxen/PDMS fiber, which can simultaneously extract all of the analytes of interest with moderate-to-high efficiency at 180 degrees C within a 30-min accumulation period. The optimized method was applied to the determination of levoglucosan in pine wood combustion emissions. The simplicity, rapidity, and selectivity of sample collection with a polymer-coated SPME coupled to capillary gas chromatography (GC) made this method potentially useful for atmospheric chemistry studies.

Over the past 15 years, research in the field of microfluidics has experienced rapid growth due to significant potential advantages such as low cost, short analysis times, and elimination of sources of contamination. Although etched and thermally bonded glass substrates have seen widespread use and offer solid performance, device fabrication still remains cumbersome. Recent advances in sacrificial layer microfabrication methods for microfluidics have overcome many disadvantages of conventional fabrication approaches. Phase-changing sacrificial layers have been implemented in making inexpensive and high-performance polymer microchips for electrophoretic analysis, protein focusing, and sample preconcentration. In addition, novel channel fabrication methods based on standard thin-film processes, which are readily integratable with microfabrication techniques used for electrical components, are being applied increasingly for the creation of microfluidic devices. These new sacrificial layer fabrication approaches will be instrumental in making low-cost and high-quality polymer microchips, and in interfacing electrical and fluidic systems on glass or semiconductor substrates.

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.

Because of their surface heterogeneity, proteins readily adsorb on polymeric substrates via various interactions, which adversely affects the performance of polymeric microfluidic devices in electrophoresis-based protein/peptide analysis. Therefore, it is necessary to use surface modification techniques such as dynamic coating or more complicated permanent surface modification, which has broader application and better performance, to render the polymeric microchannels protein-resistant. This manuscript is a review of the surface chemistry of microfluidic devices used for electrophoretic separations of proteins and peptides. The structural complexity of proteins as it relates to adsorption is described, followed by a review of the mechanisms and structural characteristics of protein-resistant surfaces. Permanent surface modification techniques used in grafting protein-resistant materials onto the surfaces of electrophoresis microchannels fabricated from polymer substrates are summarized and successful examples are presented.

Electro-osmotic flow (EOF) pumps are attractive for fluid manipulation in microfluidic channels. Open channel EOF pumps can produce high pressures and flow rates, and are relatively easy to fabricate on-chip or integrate with other microfluidic or electrical components. An EOF pump design that is conducive to on-chip fabrication consists of multiple small channel arms feeding into a larger flow channel. We have fabricated this type of pump design using a thin film deposition process that avoids wafer bonding. We have evaluated pumps fabricated on both silicon and glass substrates. Consistent flow rate versus electric field were obtained. For the range of 40–400 V, flow rates of 0.19–2.30 μL∕min were measured. Theoretical calculations of pump efficiency were made, as well as calculations of the mechanical power generated by various pump shapes, to investigate design parameters that should improve future pumps.

A stable poly[2-acrylamido-2-methyl-1-propanesulfonic acid-co-poly(ethylene glycol) diacrylate] monolith was synthesized inside a 75-microm-i.d. capillary by photoinitiated copolymerization with water, methanol, and ethyl ether as porogens. The resulting monolith was evaluated for strong cation-exchange capillary liquid chromatography of both synthetic and natural peptides. Although the monolith possessed relatively strong hydrophobicity due to the use of 2-acrylamido-2-methyl-1-propanesulfonic acid as one monomer, the monolith had a high dynamic binding capacity of 157 microequiv of peptide/mL, or 332 mg of cytochrome c/mL. Exceptionally high resolution resulting from extremely narrow peaks was obtained, resulting in a peak capacity of 179 when using a shallow salt elution gradient. Although a second, naturally formed gradient might contribute to the sharp peaks obtained, high efficiency was mainly due to the use of poly(ethylene glycol) diacrylate as a biocompatible cross-linker.