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The authors used multiplexed microemulsion electrokinetic chromatography with ultraviolet absorbance detection to develop a rapid approach for obtaining log P octanol-water (log [P.sub.ow]) values of neutral and basic compounds. By [...]

Molecularly imprinted polymers (MIPs) have been used in a wide range of analytical applications in particular in chromatography and sensing. However, the binding properties in MIPs are typically measured only in a narrow concentration range, which corresponds to only a subset of the sites in MIPs. This limited analytical window and binding site heterogeneity of MIPs leads to inaccuracies and inconsistencies in the estimation of their binding properties. This has hampered the characterization and optimization of MIPs for analytical applications. In this study, the origins of the molecular imprinting effect were studied using the newly developed Freundlich isothermaffinity distribution (FIAD) analysis. The analysis is able to readily calculate an affinity distribution for MIPs from the limited analytical window. The FIAD analysis also yields an estimate of number, affinity, and heterogeneity for this subset of binding sites. Consistent with previous studies, MIPs were found to have higher capacities than the corresponding nonimprinted polymers (NIPs). Interestingly, MIPs were also found to be more heterogeneous than NIPs. Examination of variables in the imprinting process including temperature, template concentration, and cross-linking percentages further confirmed these trends. Based on these observations, a model for the imprinting effect was developed. The larger population of high-affinity sites in MIPs appears to arise from a broadening of the heterogeneous distribution. This suggests that noncovalent MIPs may be ill-suited for chromatographic applications and other applications that are detrimentally affected by binding site heterogeneity and better suited to applications that are less affected by heterogeneity such as sensing.

Forty years ago Helfferich and Peterson published an article in Science regarding a 'paradoxical' behavior in nonlinear chromatography (Helfferich, F.; Peterson, D. L. Science 1963, 142, 661-662). They theoretically predicted that when an excess of sample molecules is injected into a chromatographic column that is equilibrated with a constant stream of identical molecules, the observed peak will not contain the injected molecules. Instead the observed peak will only contain molecules from the stream whereas the injected molecules will exit the column in a slower moving, 'invisible' peak. They considered it paradoxical that a single injection in a single-component system could cause the successive elution of two peaks (Helfferich, F. J. Chem. Educ. 1964, 41,410-413). In this study, the paradox is experimentally proven for the first time. Two different strategies were employed: (i) a radiochemical approach and (ii) a method based on the use of two enantiomers in a nonchiral separation system. The experiments were compared with computer simulations.

Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [[M + H].sup.+] m/z values of expected metabolites allows for the construction of user-defined M[S.sup.n] protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional 'survey scans' (e.g., precursor ion and constant neural loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/M[S.sup.] data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 'hits', pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/M[S.sup.] analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/M[S.sup.n] run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.

The coupling of a rotation planar preparative thin-layer chromatography system on-line with mass spectrometry is demonstrated using a simple plumbing scheme and a self-aspirating heated nebulizer probe of a corona discharge atmospheric pressure chemical ionization source. The self-aspiration of the heated nebulizer delivers ~20 [micro]L/min of the 3.0 mL/min eluate stream to the mass spectrometer, eliminating the need for an external pump in the system. The viability of the coupling is demonstrated with a three-dye mixture composed of fat red 7B, solvent green 3, and solvent blue 35 separated and eluted from a silica gel-coated rotor using toluene. The real-time characterization of the dyes eluting from the rotor is illustrated in positive ion full-scan mode. Other selfaspirating ion source systems including atmospheric pressure photoionization, electrospray ionization, and inductively coupled plasma ionization, for example, might be configured and used in a similar manner coupled to the chromatograph to expand the types of analytes that could be ionized, detected, and characterized effectively.

For the first time, on-chip planar hydrodynamic chromatography is combined with UV absorption detection. This technique is suitable for size characterization of synthetic polymers, biopolymers, and particles. Possible advantages of an on-chip hydrodynamic chromatography system over conventional techniques, such as size exclusion chromatography, and field-flow fractionation are fast analysis, high efficiency, reduced solvent consumption, and easy temperature control. The hydrodynamic separations are performed in a planar configuration realized in fused silica using a mixture of fluorescent and nonfluorescent polystyrene particles with sizes ranging from 26 to 155 nm. The planar chip configuration consists of a 1-[micro]m-high, 0.5-mm-wide, and 69-mm-long channel, an integrated 150-pL injection structure, and a 30-[micro]m-deep and 30-[micro]m-wide detection cell, suitable for UV absorption detection. By combination of the separation data obtained in the new fused-silica chip with those obtained using a previously presented planar hydrodynamic chromatography chip, which was realized using silicon and glass microtechnology, a description of the retention and dispersion behavior of planar hydrodynamic chromatography is obtained. Especially the influence of the sidewalls on the dispersion is investigated. Furthermore a hydrodynamic separation within 70 s of several biopolymers is shown in the glass--silicon chip.

Frontal affinity chromatography (FAC) is a simple but powerful method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of retardation of the elution front. By combination of FAC with a PE-Mariner electrospray ionization mass spectrometry, a very efficient and straightforward procedure was developed herein for analyzing the binding properties of different inhibitors of the epidermal growth factor receptor (EGFR). In this study, a polyclonal antibody prepared with a known anti-EGFR inhibitor coupled with bovine serum albumin was adopted as the stationary phase in the FAC system. Using the antibody to mimic the receptor, other different anti-EGFR inhibitors as well as the small-molecule half-antigen itself were recognized directly from the crude extract of herb, which afforded us a novel promising approach for the efficient screening of lead compounds or drug candidates from natural resources.

A chromatographic system was developed to separate oxygen from nitrogen, argon, carbon dioxide, and water vapor mixture for the determination of precise isotopic ratio measurements of oxygen in dissolved air. This system separates oxygen not only quantitatively but also rapidly as well; typical oxygen separation takes about 30 min. Fractionation of oxygen between liquid and gas phase was found to be similar to that of earlier reports.

The chromatographic separation of a highly water-soluble dendritic monoadduct methano[60]fullerene octadecaacid (dendrofullerene) with octadecylsilica bonded phases has been studied. It has been found that the RP-HPLC behavior of this dendrofullerene obeys the general rules of stationary-phase and mobile-phase selection for controlling the separation of usually acidic compounds. An RP-HPLC-ESI-MS analysis confirms the identity of the dendrofullerene and allows characterization of the molecular weights of the main impurities contained in the sample. The described methods can control the synthesis and efficiently purify this fullerene derivative, which has been previously shown to be active against mutant infectious clones of HIV-1, which are resistant to AZT and 3TC, drugs that are widely used in AIDS therapy.

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-[micro]m channels)for implementing the on-line coupling of capillary LC columns operated at 10 000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of ~1000 for capillaries having inner diameters between 15 and 150 [micro]m. The on-line coupled SPE columns increased the sample processing capacity by ~400-fold for sample solution volume and ~10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated ~98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.

A method to determine five postharvest fungicides (dichloran, flutriafol, o-phenylphenol, prochloraz, tolclofos methyl) in fruits (cherries, lemons, oranges, peaches) has been developed using solid-phase microextraction (SPME) coupled to liquid chromatography (LC) with photodiode array (DAD), mass spectrometry (MS), or tandem mass spectrometry (MS/MS) with ion trap detection. Extraction involved sample homogenization with an acetone/water solution (5:1), filtration, and acetone evaporation prior to fiber extraction. The pesticides were isolated with a fused-silica fiber coated with 50-[micro]m Carbowax/template resin. The effects of pH, ion strength, sample volume, and extraction time were investigated, and their impact on the SPME-LC/MS was studied. Dynamic and static modes of desorption were compared and the variables affecting desorption processes in SPME-LC optimized. Static desorption provided the best recoveries and peak shapes. Recoveries at the limit of quantification (LOQ) levels were between 10% for prochloraz and 60% for o-phenylphenol, with relative standard deviations from 13.6% for prochloraz to 3.1% for o-phenylphenol. The versatility of the method was also exhibited by its excellent linearity in the concentration intervals between 0.0005 and 5 mg [kg.sup.-1] for dichloran and 0.01-10 mg [kg.sup.-1] for tolclofos methyl and prochloraz. LOQs ranged from 0.25 to 1 [micro]g [g.sup.-1] using DAD, from 0.002 to 0.01 [micro]g [g.sup.-1] using LC/MS, and from 0.0005 to 0.01 to [micro]g [g.sup.-1] using LC/MS/MS. LOQs obtained in the present study using LC/MS and LC/MS/ MS are lower than maximum residue limits established for all the fungicides in any matrix studied. The method enables to determine polar pesticides at low-microgram per gram levels in fruits.

Liquid films composed of binary mixtures of carbon dioxide and methanol were created in empty capillary columns to produce effective stationary phases for chromatography. Under certain conditions of temperature, pressure, and stoichiometric composition, a binary mobile phase composed of C[O.sub.2] and an organic liquid, such as methanol, can form two immiscible (gas and liquid) phases within a chromatographic column. The two phases can coexist in dynamic equilibrium with the liquid phase migrating through the column at a slower velocity than the gas phase. The liquid phase, composed of methanol saturated with carbon dioxide, acted as a chromatographic stationary phase while the gas phase, composed of carbon dioxide saturated with methanol, acted as a chromatographic mobile phase. The exact conditions necessary for the formation of two phase systems were determined from three-dimensional (P, T, XY) phase diagrams calculated from the Peng-Robinson cubic equation-of-state using one-parameter mixing rules. Separations of simple hydrocarbon mixtures are illustrated under various experimental conditions.

A phenomenon was found whereby the application of an alternating voltage to a capillary column can vary the capacity factor of a sample solute. The alternating voltage-induced variation in the capacity factor was studied using an anion exchange mini-bed (a short capillary column 12 mm long). The capacity factor varied according to both the amplitude and frequency of an applied alternating voltage. The variation greatly depended on the kinds of sample solutes and packing materials. A new separation mode for capillary electrochromatography using an alternating voltage, that is, alternating voltage capillary electrochromatography (AV-CEC), was proposed as an application of this phenomenon to control the retention of a sample solute. The chromatographic behavior of three organic acids (benzoic acid, phthalic acid, and salicylic acid) was studied in AV-CEC using an anion exchange column.

A sol-gel chemistry-based method was developed for the preparation of highly stable capillary gas chromatography (GC) columns with surface-bonded poly(ethylene glycol) (PEG) stationary phase. Through a single-step procedure, it concurrently provided column deactivation, stationary-phase coating, and chemical immobilization of the coated film. Sol-gel reactions were carried out within fused-silica capillaries that were filled with properly designed sol solutions containing two sol-gel precursors, two different triethoxysilyl-derivatized poly(ethylene glycol)s, two sol-gel catalysts, and a deactivation reagent. Hydrolytic polycondensation reactions led to the formation of a sol-gel coating chemically bonded to the inner walls of the capillary. A number of sol-gel coated fused-silica capillary columns were prepared using sol-gel-active PEG derivatives. These columns demonstrated many inherent advantages, the main being the strong anchoring of the coating to the capillary wall resulting from chemical bonding with the silanol groups on the fused-silica capillary inner surface. This chemical bonding yielded strongly immobilized PEG coatings with outstanding thermal stability (up to 320 [degrees]C). To our knowledge, such a high thermal stability has not been achieved so far on conventionally prepared PEG GC columns. Sol-gel PEG columns provided excellent chromatographic performances: high number of theoretical plates, excellent run-to-run and column-to-column reproducibility, and pronounced selectivity for a wide range of test solutes. Using n-octadecane as a test solute (k = 7.14), an efficiency value of 3200 theoretical plates/m was obtained on a 10 m x 0.25 mm i.d. fused-silica capillary column. Five sol-gel PEG columns provided RSD values of 1.09% for column efficiency (solute, n-octadecane), 1.37% for retention factor (solute, n-octadecane), and 0.9% for separation factor (for solute pair o- and p-xylene). In five replicate measurements using the same column, RSD values of less than 0.50% for the retention time and 1.36% for retention factor (k) were obtained.

A protocol combining immobilized metal ion affinity chromatography and [beta]-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. [beta]-Elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and [beta]-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to [beta]-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.

A simple and rapid method able to determine residues of 12 sulfonamide (SAs) antibacterials in cattle and trout muscle tissues is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS instrumentation was equipped with an electrospray source and a single quadrupole. After 0.8 g of a flesh sample containing the analytes is deposited on sand (crystobalite), this material is packed into an extraction cell. SAs are extracted by flowing 4 mL of water through the cell heated at 80[degrees]C. A 0.5-mL aliquot of the bovine tissue extract is then directly injected into the LC column, while the fish tissue extract is filtered prior to LC-MS analysis. MS data acquisition was performed in the positive-ion mode and monitoring at least three ions for each target compound. Confirmatory ions were produced by the in-source collision-induced dissociation process. At the tolerance levels issued by the EU and U.S. Food and Drug Administration, i.e., 100 ppb, recovery of the analytes in bovine and trout muscle tissues was 75-98% with RSDs ranging between 1 and 8%. Estimated limits of quantification (S/N = 10) were 6-15 ppb for SAs in bovine muscle tissue and 3-13 ppb in trout fillet. When trying to reduce the analysis time by using a short chromatographic run time, severe ion signal suppression was experienced for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was removed by simply adopting more selective chromatographic conditions.

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between [1:10.sup.5] and [1:10.sup.6]. In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of ~0.5 fmol/[micro]L in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.

A monolithic silica column high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the high-speed direct simultaneous determination of a drug discovery compound and its major circulating metabolite (M-72) in rat plasma. This methodology makes use of flow programming and an alkyl-bonded silica rod column for fast macromolecule removal and chromatographic separation without the need for significant sample preparation. The matrix ionization suppression effect on the monolithic column HPLC-MS/MS system was investigated using the postcolumn infusion technique. After 200 plasma injections on a 50 x 4.6 mm monolithic silica column, consistent column efficiency of close to 39 000 theoretical plates/m and reproducible retention times for the analytes were observed. The apparent on-column recoveries of 12 test compounds in rat plasma samples were greater than 90%. The proposed fast direct plasma injection method was tested over a 3-day period with the interday coefficient of variation less than 15% for both analytes.

This "Milestones in Chromatography" column discusses the beginnings of gas chromatography (GC) in the United Kingdom and the first two international symposia that played an important role in the dissemination [...]

Bioanalytical methods are used in human clinical pharmacology, bioavailability, and bioequivalence studies that require pharmacokinetic evaluation in support of various drug applications to regulatory agencies such as the U.S. Food [...]

Stir-bar sorptive extraction is a new solventless sample preparation method for the extraction and enrichment of organic compounds from aqueous matrices. The method is based on the same principles as [...]

This month in "GC Connections," John Hinshaw examines basic laboratory heating, ventilation, cooling, electrical, and space requirements for the successful installation of a gas chromatograph. Modern gas chromatographs, and in [...]

Guest author Craig Young and column editor John Dolan provide some tips and techniques to get the most out of evaporative light-scattering detection. During the past 20 years, evaporative light-scattering [...]

A new chromatofocusing technique called gradient chromatofocusing is characterized. Gradient chromatofocusing generates linear pH gradients on anion-exchange columns with inexpensive low molecular mass buffer components via HPLC gradient mixing. Gradient chromatofocusing results are compared with that of conventional chromatofocusing in the chromatography of several proteins on a Mono P column, including [beta]-lactoglobulin A and B, ovalbumin, BSA, and conalbumin. Gradient chromatofocusing shows superior performance, with resolution increases greater than 3-fold being realized for the entire protein mixture and up to 25-fold for a particular protein pair. This performance superiority arises from inherent advantages in the gradient chromatofocusing technique in optimizing conditions pertinent to separation, including buffer concentration and pH gradient slope. These resolution gains arise from both increases in separation factor and decreases in peak width achieved with the pH gradient chromatofocusing technique through the manipulation of buffer concentration and the pH gradient profile. Gradient chromatofocusing is also compared with conventional NaCl gradient ion-exchange chromatography using the same Mono P column, demonstrating 3-fold resolution gains, resulting from a 3-fold decrease in peak width. The present work demonstrates the significantly improved performance that gradient chromatofocusing has in protein separations compared to other ion-exchange chromatographic techniques. Mechanisms for the various effects are discussed.

Chromatographers today rely upon computerized data-handling systems to record and analyze chromatograms. The ubiquitous PC-based data systems used today have changed completely the ways in which chromatographers record and reduce [...]

This paper discusses the use of pulse techniques for analysis of zonal elution data for the determination of mass-transfer and axial dispersion constants for porous support materials with adsorption to the surface or to a surface-bonded phase. As an example, this paper considers the case of controlled pore glass (CPG) with a bonded phase that is used with microcolmnns and a flow injection analysis system. For the CPG, axial dispersion in the form of eddy mixing can be described by (mathematical expression not reproducible in ASCII) = 0.203, and the overall mass-transfer term, K(sub OL) = 3.9 x 10(super -6) cm/s. Additionally, an affinity chromatography model was adapted to effectively describe systems employing CPG as the support material through modification of equations describing typical affinity chromatography systems and by inclusion of an axial dispersion term in the calculation of N. This model was used to predict breakthrough curves for cadmium adsorption by 8-hydroxyquinolinol immobilized on CPG packed in microcolumns. In general, the information from the model can be used to extract equilibrium-based constants (binding strengths and site capacities) from a nonequilibrium flow system. The data and model can also be employed in determining the performance for scaled-up extraction systems. The modified model is available in EXCEL spreadsheet format as Supporting Information.

Separation of cholesterol and its ester derivatives using micellar electrokinetic chromatography is a challenge due to the extreme hydrophobicity of these compounds. In this work, an isocratic capillary electrochromatography (CEC) method has been developed to separate a complex mixture of cholesterol and its 12-ester derivatives. The proportions of mobile phase (tetrahydrofuran, acetonitrile, water), as well as the effects of acid modifiers, buffer concentrations, voltage, and temperature on the separation of cholesterol derivatives were investigated. Addition of a polymeric surfactant, poly(sodium N-undecanoyl-L-glycinate), to the mobile phase reduced migration time and improved resolution of the analytes. The CEC method developed allows baseline separation of a complex mixture of cholesterol and 12 ester derivatives in less than 40 min. Finally, the method is applied to the characterization of cholesterol, cholesterol linoleate, and cholesterol oleate extracted from atherosclerotic plaque deposits in the arterial walls of a human aorta.

A strategy for highly sensitive metabolite screening by liquid chromatography-electrospray ionization (ESI) mass spectrometry with the negative-ion mode that involves the use of a reversed-phase column in gradient-elution mode and postcolumn addition of 2-(2-methoxyethoxy)ethanol (2-MEE), a novel signal-enhancing modifier, has been described. When a mobile phase of 50 mM ammonium acetate/acetic acid buffer (pH 4.4) at a flow rate of 100 (mu)L/min was employed, poor ESI response of ibuprofen as a model drug, probably due to both the high surface tension of the mobile phase and the ion-suppression effect of acetate anion in the mobile phase, was observed. On the other hand, the postcolumn addition of 2-MEE (50 (mu)L/min) into the mobile phase counteracted the ion suppression as well as the surface tension problem, resulting in approximately 100-fold signal enhancement of the analyte. The metabolite screening of ibuprofen in human urine was subsequently carried out comparing the results with and without postcolumn addition of 2-MEE. The results indicated that the postcolumn addition of 2-MEE dramatically improved the ESI responses of all urinary metabolites detected without affecting the chromatographic separation.

A novel approach for sample enrichment, namely, equilibrium sorptive enrichment (FSE), is presented. A packed bed of sorption (or partitioning) material is used to enrich volatiles from gaseous samples. Normally, air sampling is stopped before breakthrough occurs, but this approach is not very successful for weakly retained compounds (volatiles) as early breakthrough occurs. In ESE, sampling is continued until all compounds of interest are in equilibrium with the sorptive material. This allows accurate sampling of volatiles and enrichment at the maximum attainable sensitivity. However, due to the equilibrium nature of ESE, it is limited to samples with a constant concentration over the sampling time. This requirement is easily met for those compounds with short equilibration times (volatiles). Because of the nature of the sorption mechanism, which is basically dissolution, all compounds partition independently into the sorbent (stationary phase) and displacement effects do not occur. This is a great advantage over adsorption materials. Additionally, theory allows the calculation of enrichment factors from literature retention indexes. Moreover, ESE also benefits from the features of sorption materials such as a very high inertness and interference-free blanks. The performance of ESE is illustrated with the analysis of several analytes including the epoxides ethylene oxide and epichlorohydrin in real-life air sampling.

A derivatization reaction for the pyrolysis gas chromatography/mass spectrometry qualitative and quantitative analysis of polymers based on the reaction between methylamine and acetonylacetone has been developed. Chromatographic and spectroscopic data about the reaction affirm that the derivatized polymer undergoes a unique thermal degradation mechanism (TDM), which leads to the production of a major fragment or a set of fragment patterns. A study of the TDM of polyallylamine and its derivatized form showed the successful generation of a major fragment, 1,2,5-trimethylpyrrole, in its pyrolysis.

Partially ionized anionic-neutral compounds undergoing capillary electrochromatographic analysis exhibit unusually high efficiencies, reaching up to 2.5 million plates m(super -1), under reversed-phase conditions. The abnormally high efficiencies are noted when the migration time of the analyte and the elution time of sample-induced discontinuities in the mobile phase are closely matched. The phenomenon is explained in terms of nonequilibrium conditions due to pulses of stronger or weaker solvent arising from the sample. The increased efficiencies are also able to cause concurrent electrophoretic effects during the analysis of charged species, thus leading to even higher efficiencies.

A new tool for imaging both scalar transport and velocity fields in liquid flows through microscale structures is described. The technique employs an ultraviolet laser pulse to write a pattern into the flow by uncaging a fluorescent dye. This is followed, at selected time delays, by flood illumination with a pulse of visible light which excites the uncaged dye. The resulting fluorescence image is collected onto a sensitive CCD camera. The instrument is designed as an oil immersion microscope to minimize beam steering effects. The caged fluorescent dye is seeded in trace quantifies throughout the active fluid, thus images with high contrast and minimal distortion due to any molecular diffusion history can be obtained at any point within the microchannel by selectively activating the dye in the immediate region of interest. We report images of pressure- and electrokinetically driven steady flow within round cross section capillaries having micrometer scale inner diameters. We also demonstrate the ability to recover the velocity profile from a time sequence of these scalar images by direct inversion of the conserved scalar advection-convection equation.

The pore structures of cross-linked polyacrylamide gels can be altered by polymerizing in the presence of high concentrations of unreactive, micellar surfactant cosolutes which act as 'templates'. Removal of surfactant after polymerization is expected to leave pores with the approximate shape and dimensions of the surfactant micelles. A simple model was developed to simulate gel permeation chromatography (GPC) separations of globular proteins on templated gels. The model assumes that the partition coefficient for sieving of a protein is equal to the fraction of gel volume accessible to a sphere with a radius equal to the protein Stokes radius. The total gel volume is considered to include a fraction that is a conventional, random gel matrix and a remaining fraction contributed by templated pores. The pore size distribution of the conventional gel was estimated using the Ogston equation, which approximates the matrix as a random collection of long, thin, rigid fibers. Templated pores were assumed to have a Gaussian distribution of radii centered about some mean determined by the micelle radius. In comparison to conventional media, gels with templated pores are predicted to exhibit more sharply defined exclusion limits and improved resolution over a narrow size range centered on the mean templated pore size. Selectivity and resolution are expected to increase as the volume fraction of templated pores is increased and as the dispersion of templated pore radius is decreased. Small changes in template radius lead to large changes in the molecular weight range of optimal separation of globular proteins. It should be possible to create a series of GPC media that collectively offer high resolution over the molecular weight range of most globular proteins of interest.

A simple, inexpensive, fast, and sensitive method for improving detection limits using a novel sample concentrator is described. Analytes are concentrated on the tip of a GC syringe by evaporating the solvent with a flow of nitrogen. The increased sensitivity of the concentrator permits extraction with lower volumes, minimizes disposal costs, and simplifies sample preparation. The concentrator improved sensitivities for selected drugs (7.6-12.8 times), pesticides (12-17 times), and [C.sub.11]-[C.sub.16] alkanes (1.5-6 times). With or without use of the concentrator, relative standard deviations of peak areas were less than 10%. Diverse GC and GC/MS applications ranging from forensic toxicology to environmental analysis can benefit from the improved detection limits provided by this concentrator design.

In capillary electrophoresis, head-column field-amplified sample stacking (FASS) provides the largest sensitivity enhancement of all electrokinetic concentration techniques (Zhang, C.-X.; Thormann, W. Anal. Chem. 1996, 68, 2523). Application of head-column FASS to the analysis of closely related opioids by capillary zone electrophoresis in binary systems with ethylene glycol is described. It is shown that sample condensation is further increased about 2-fold by introduction of a preinjection plug, i.e., introduction of a short solution plug of high conductivity, high pH, and high viscosity at the capillary tip prior to injection. The preinjection plug acts as a temporary trap for solutes. Its effective length is shown to be limited to a few millimeters. The highest sample stacking efficiency in head-column FASS is obtained via optimization of the electric field strength and the effective electrophoretic mobility of the solutes within the sample and the adjacent zones and is thus strongly dependent on the compositions of both the sample matrix and the preinjection plug. Binary sample solutions of low conductivity and low viscosity containing small amounts of a weak acid are demonstrated to be most effective for the stacking of positively charged opioids. The procedure developed for capillary zone electrophoresis of opioids in binary systems with ethylene glycol and UV absorbance detection is documented to provide a 3 orders of magnitude sensitivity enhancement, exhaustive sample injection from an external reservoir of up to 20 [[micro]liter] (i.e., from a volume that is more than 20-fold the volume of the capillary employed), and a lowest detectable concentration of 0.1 ng/mL (S/N = 3). Using internal calibration, typical intraday and interday imprecisions of solute concentrations between 3 and 10 ng/mL and between 0.5 and 1.5 ng/mL are [less than or equal to]5% and [less than or equal to]15%, respectively. The stacking approach has been successfully applied to the analysis of dihydrocodeine in extracts of 20 [[micro]liter] of human plasma and is shown to permit the precise (imprecision [less than or equal to]10%) determination of dihydrocodeine plasma levels of pharmacological interest (3-300 ng/mL or 10-1000 nM). Furthermore, using a modified protocol for micellar electrokinetic chromatography, head-column FASS is shown to provide a 400-fold sensitivity enhancement for a number of opioids. The stacking procedure is based on insertion of a surfactant-free preinjection plug and temporary application of reversed voltage after electro-injection.

Tandem-in-time mass spectrometry, as implemented on an ion-trap detector (ITD), is the process whereby precursor ions are created, stored in a radio frequency (rf) trapping field, and then sequentially fragmented to form product ions by application of additional rf waveforms. As with any form of tandem mass spectrometry (MS/MS), tandem-in-time MS is highly selective, by virtue of both mass discrimination and specific gas-phase chemistry. Beyond this, however, tandem-in-time MS offers ion throughput efficiency and cost advantages over either quadrupole or sector instruments. This paper will describe the use of capillary gas chromatography combined with tandem-in-time mass spectrometry to quantify a novel therapeutic agent extracted from human plasma. For an example compound, a quantitation limit of 25 pg/mL (S/N [approximately equal to] 10, 15 fmol on-column) was attained out of plasma. The interday imprecision was [less than or equal to]12.2% over a dynamic range extending to 10 ng/mL. Due to favorable ionization conditions for the test analytes, electron ionization resulted in formation of [M.sup.+] ions, with very little fragmentation, allowing for maximum assay sensitivity. Although method characterization and validation demonstrated adequate instrumental performance, some lack of ruggedness was encountered during routine application.

A one-step method is described for in situ preparation of macroporous polymeric matrixes to be used as stationary phases for capillary electrochromatography. The monomers (acrylamide, bisacrylamide, and acrylic or vinylsulfonic acid), including hydrophobic ligands ([C.sub.4], [C.sub.6], or [C.sub.12]), and poly(ethylene glycol) have been polymerized in formamide (or N-methylformamide) aqueous solutions inside the capillary. The capillary wall had been activated first by a bifunctional reagent, to couple covalently the resulting gel inside the fused-silica tubing. Thus, no frit is necessary to keep the stationary phase in place. High efficiencies were obtained for a mixture of alkyl phenones (up to 398 000 plates/m). Good separations are achieved in less than 5 min. The migration time reproducibility is better than 1% (RSD) from run to run and 2.5% from day to day. The gel is stable up to at least 50% acetonitrile used as a mobile phase. On-column UV and fluorescence detection can readily be employed. Applications to peptides and carbohydrates are also shown.

A family of models is proposed for the description of skewed chromatographic peaks, based on the modification of the standard deviation of a pure Gaussian peak, by the use of a polynomial function, h(t) = [He.sup.-[(1/2)([t-[t.sub.R]]/[[s.sub.0]+[s.sub.1](t-[t.sub.R])+[ s.sub.2][(t-[t.sub.R]).sup.2]+...]).sup.2], where H and [t.sub.R] are the height and time at the peak maximum, respectively. The model has demonstrated a high flexibility with peaks of a wide range of asymmetry and can be used to accurately predict the profile of asymmetrical peaks, using the values of efficiency and asymmetry factor measured on experimental chromatograms. This possibility permits the simulation of chromatograms and the optimization of the separation of mixtures of compounds producing skewed peaks, where both the position and peak shape are considered. A first-degree polynomial was adequate for peaks of moderate asymmetry, but higher degree polynomials were preferable for peaks showing a high asymmetry, including those with negative skewness. The proposed models can be employed in the resolution of overlapped peaks in binary and ternary mixtures of compounds, or to improve the accuracy in the evaluation of peak shape parameters. The results obtained with the proposed models were comparable or even superior to those achieved with the exponentially modified Gaussian model.

A modified advection-dispersion transport model was utilized to determine the influence of anionic monorhamnolipid biosurfactant on the irreversible adsorption of various Pseudomonas (P.) aeruginosa strains. Analysis of the bacterial cell breakthrough curves of of P. aeruginosa ATCC 9027, ATCC 27853 and ATCC 15442 in glass chromatography columns indicated the role of rhamnolipid in stimulating cell adsorption. Rhamnolipid also enhanced the negative surface charge density of the glass chromatography column without altering its surface charge density.

A novel method of sample introduction into an isotope ratio mass spectrometer (IRMS) is described. The technique uses the chemical reaction interface (CRI) to convert samples coming from a gas chromatograph (GC) or high-performance liquid chromatograph (HPLC) into C[O.sub.2] using a microwave-induced helium plasma. Optimization parameters for both GC/CRI/IRMS and HPLC/CRI/IRMS are described. In both modes of operation, it was possible to obtain 13C[O.sub.2]/12C[O.sub.2] ratios With standard deviations less than 1[per thousands]. Investigation of HPLC/CRI/ IRMS performance at low and high concentrations (0.5-10 [[micro]gram]) resulted in no significant deviations of the isotope ratios. The ability to differentiate samples of different biological origins was illustrated using chlorophyll a from spinach and algae, where a large difference was observed but good precision was maintained (SD < 0.60[per thousands]).

Pattern recognition methods have been used to classify high-speed gas chromatograms of weathered and unweathered jet fuels. A total of 228 neat jet fuel samples representing common aviation fuels sold in the United States were characterized by 85-peak gas chromatograms. Discriminants were developed by parametric and non-parametric pattern recognition procedures that correctly classified the gas chromatograms of neat jet fuels according to fuel type (JP-4, Jet-A, JP-7, JPTS, or JP-5), and these discriminant functions were successfully used to classify gas chromatograms of jet fuels which had undergone weathering in a subsurface environment. This approach for identification of weathered fuels was taken because the physical and chemical interactions of jet fuel components with the subsurface environment are not yet fully understood.

The inherent instability of active esters provides a chromatographic challenge. A normal phase HPLC gradient was developed using nonprotic solvents and a diol stationary phase to determine the impurity profile of a mesylate which, under protic conditions, undergoes intramolecular ring closure to a cyclic ether. On-column cyclization of the analyte was found to be extensive at room temperature but insignificant at -30 [degrees] C. The on-column reaction rate was determined as a function of column temperature, and an Arrhenius activation energy was calculated.

The polyacrylamide gels described here have the ability to separate uncharged substances of comparatively low molecular weight due to the built-in, negatively charged functional groups. The functional groups permit the development of electroosmotic flow under the influence of a high voltage, which makes possible capillary electrochromatographic separations of uncharged compounds. The effects of gel compositions on the column performance were examined. Migration time reproducibilities of 0.43% RSD or less were found with the gel-filled capillary columns. Possible insight into the separation mechanisms is discussed.

A capillary electrokinetic chromatography technique is described that employs neutral cyclodextrins (CDs) as a primary phase, transported with electroosmotic flow, and charged CDs as an electrophoretically mediated secondary phase. Neutral, hydrophobic solutes are separated on the basis of their differential distribution between these CD phases. The technique resembles micellar electrokinetic capillary chromatography (MECC) with regard to instrumentation and the fundamental relationships for resolution and capacity factor, which are influenced by the existence of a finite elution window. Conversely, the CD technique offers unique and beneficial characteristics when compared to MECC. Efficiency, selectivity, and system retention are evaluated on the basis of separations of polyaromatic hydrocarbons (PAHs). Efficiency is comparable to that of MECC (>[10.sup.5] plates/m). The specificity associated with solute-CD inclusion complexation provides elution orders for PAHs that do not follow the hydrophobicity trends of MECC. Moreover, since the CD phases are largely noninteractive, complex CD systems can be used to enhance selectivity. Capacity factors can be altered in a convenient and predictable fashion simply by changing the CD phase ratio. The technique is rather robust with regard to the use of running buffers containing organic solvents; the effects or organic modifier and pH on system retention are demonstrated.

Electrochromatography is utilized to separate a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs). Fused-silica capillary columns ranging in size from 50 to 150 [[micro]meter] i.d. were packed (20-40-cm sections) with 3-[[micro]meter] octadecylsilica particles. A potential of 15-30 kV is applied across the 30-50-cm total length capillary column to generate electroosmotic flow that carries the PAlls through the stationary phase. An intracavity-doubled argon ion laser operating at 257 nm is used to detect the PAHs by laser-induced fluorescence. Efficiencies up to 400 000 theoretical plates/m are obtained when detection is performed within the column packing and up to 150 000 theoretical plates/m when detection is performed following a frit (used to hold the packing). The reproducibility of the peak retention times is better than 2% (RSD). The limits of detection for individual PAHs range between [10.sup.-17] and [10.sup.-20] mol ([10.sup.-9]-[10.sup.-11] M), with a linear response spanning 4 orders of magnitude in concentration.

Reactive porous rods of poly[(chloromethyl)styrene-co-divinylbenzene] have been prepared for the first time by copolymerization of the monomers in the presence of a porogenic diluent. A two-step modification process involving reaction with ethylenediamine followed by reaction with [Gamma]-gluconolactone leads to a porous medium that has highly hydrophilic surface functionalities. Preliminary chromatographic evaluation with both small molecules such as alkylbenzenes in reversed-phase mode and large proteins in hydrophobic adsorption and ion-exchange modes demonstrates that the surface of the modified rods possesses a hydrophilicity that is comparable to that of the best hydrophilic HPLC packings.

High-performance thin-layer chromatography with automated multiple development (AMD-HPTLC) was used to screen water samples for pesticides. A universal gradient based on dichloromethane was employed to check for the presence of pesticides from different classes such as phenylureas, carbamates, triazines, phenoxycarboxylic acids, and others. In total, 283 pesticides were analyzed applying this gradient. The data for migration distances, the UV spectra, and the instrumental detection limits were compiled. Eighteen pesticides show an instrumental detection limit of more than 100 ng applied and can therefore not be analyzed from 1 L drinking water samples with AMD-HPTLC without further treatment. One liter drinking water samples were extracted by means of solid-phase extraction using RP-C18 material or liquid/liquid extraction with dichloromethane and screened with the universal gradient. The confirmation of suspect positive pesticide residues was performed with a second analysis applying special gradients optimized for the individual pesticide classes. Two examples of drinking water spiked at the 100 ng/L level are presented to demonstrate the merits of the method. In the first sample, the two triazine herbicides cyanazine and atrazine, forming a critical pair with identical reflectance spectra, were separated in a confirmatory run applying a smoother gradient. In the second sample, acidic herbicides were unsatisfactorily resolved with the universal gradient but were separated with a confirmatory gradient of different composition, allowing easy identification of picloram, acifluorfen, haloxyfop acid, and MCPA.

When chromatographic analysis involves the use of two or more chromatograms, e.g., for performing calibration or for assessing composition changes among different samples, optimization based solely on chromatographic resolution, [R.sub.s], may not yield the most rapid or precise analyses. A simple model is developed that predicts quantitation precision for analysis of ill-resolved peaks as a function of retention time precision and [R.sub.s]. This model implies that use of shorter columns can provide rapid and precise quantitation. Short-column analyses have improved retention time precision and S/N ratio, which offset the detrimental quantitation effects of decreased [R.sub.s]. A quantitation precision study for liquid chromatography was done beginning with well-resolved peaks having [R.sub.s] = 1.06. Quantitation precision as percentage relative standard deviation (%RSD) was theoretically calculated and experimentally measured for two diverse experimental paths in which [R.sub.s] was diminished by decreasing either the selectivity ratio or the column length. The quantitation method chosen was deconvolution of mixture chromatograms by performing a classical least-squares fit to chromatograms of pure standards. The quantitation precision model agreed with calculated and measured %RSD values to within 3 percentage points. The short-column analyses yielded improved quantitation precision and shorter analysis time at equal [R.sub.s] compared to selectivity-limited analyses. For an analyte having a peak height ratio of 1:2.5 and a peak width ratio of 1:1.2 relative to an adjacent overlapping peak, similar quantitation precision as %RSD was obtained at equal selectivity ratio for a 50 cm column (3.1%) and a 7.5 cm column (4.3%), despite the decrease in [R.sub.s] from 1.06 to 0.27. The short-column advantages are also applicable to gas and supercritical fluid chromatographies and possibly to capillary electrophoresis.

The critical roles of dead time and void volume in chromatographic measurements are discussed. Void volume measurements are particularly important for chromatographic systems in which one or more components of the mobile phase (eluent) form an integral part of the stationary phase. Such systems include HPLC, SFC (especially with polar modifiers), and GC with adsorbable components in the carrier gas. In each of these cases, the measured column volumes determine the exact type of capacity factor obtained, the type of adsorption measured, and the experimentally determined volume of the adsorbed phase. Concise definitions are presented for the different types of void volumes and adsorbed phase volume of chromatographic systems along with a detailed discussion of 'excess' and 'total' adsorption. Experimental methods are also discussed for the accurate determination of the entire amount or volume of eluent in a column, the void volume, the total amount of material adsorbed, and the excess amount of material adsorbed. The effects of these parameters upon the type of capacity factor, equilibrium constant, and free energy are examined in depth.