Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.     

Sample gating in open tubular and packed capillaries for high-speed liquid chromatography

A new liquid chromatography instrument is described that employs small-diameter capillary columns and an optically controlled sample gating procedure to separate a mixture of fluorescein isothiocyanate (FITC) labeled amines in as few as 6 s. Efficiency of the separation, expressed as the number of theoretical plates, is observed to increase linearly with column length and thus analysis time. The combination of short analysis time and automated sample introduction allows signal averaging to be used to enhance the precision of the measurement.     

Isotachophoresis in free-flow using a miniaturized device

For the first time, we report a miniaturized approach for isotachophoresis employing the technique of free-flow electrophoresis. Using a micromachined separation chamber with a volume of 200 nL, a sample mixture of fluorescein, eosin G, and acetylsalicylic acid was separated, stacked, and concentrated in less than a minute. Additionally, an isotachophoretic separation of a reaction mixture of myoglobin and fluoresceinisothiocyanate as a fluorescence label has shown the potential of this method for on-line sample preparation.     

Improvements in polymer characterization by size-exclusion chromatography and liquid chromatography at the critical condition by using enhanced-fluidity liquid mobile phases with packed capillary columns

Microscale chromatography has found numerous applications in liquid chromatography. The combination of enhanced-fluidity liquid mobile phases with packed-capillary LC is evaluated for polymer characterization using size-exclusion chromatography (SEC) and liquid chromatography at the critical condition (LCCC) phase. Separations of polystyrene polymers and copolymers are completed using liquid chromatography at the critical condition. The critical conditions of polystyrene polymers were approached by changing the concentration of CO(2) in the mixture combined with temperature and pressure variation. Because the solvent strength of enhanced-fluidity liquid mixtures is affected by temperature and pressure variation, the solvent strength could be fine-tuned to accurately find the critical condition. Long packed capillaries could be used in this application because the enhanced-fluidity mobile phases have low viscosities. High efficiencies resulted. The performance of packed-capillary and analytical-scale analytical columns containing the same packing material was compared for a challenging separation at the critical condition.     

In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography

Fused-silica capillary columns packed with 1.0-microm nonporous C18 bonded particles are evaluated with isocratic ultrahigh-pressure liquid chromatography (UHPLC). Improved UHPLC techniques have demonstrated column efficiencies as high as 730 000 plates/m and run pressures over 6800 bar (100 000 psi) for packed 10-microm-inner diameter (i.d.) columns. Columns as large as 150 microm have been tested with UHPLC and show no flow-induced heating effects on separation efficiencies. van Deemter plot analysis for column i.d.s ranging from 10 to 150 microm shows an increase in column efficiency with a decrease in column i.d. Reduced parameter analysis further illustrates a decrease in reduced parameter A term and C term values with decreasing i.d. However, reduced parameter C term values for columns evaluated with UHPLC are an order of magnitude larger than C term values for larger particles at conventional pressures. Retention factors for moderately retained compounds are observed to increase with column i.d., suggesting an increase in packing density. Highly ordered packing arrangement at the column wall is seen for packed beds extruded from large-diameter columns.     

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.     

Determination of peroxide-based explosives using liquid chromatography with on-line infrared detection

A nondestructive analytical method for peroxide-based explosives determination in solid samples is described. Reversed-phase high-performance liquid chromatography in combination with on-line Fourier transform infrared (FT-IR) detection is used for the analysis of triacetonetriperoxide (TATP) and hexamethylenetriperoxide diamine (HMTD). In contrast to other liquid chromatographic methods with optical detection, no derivatization or decomposition of the peroxides is required. The peroxides are identified and quantified via their characteristic absorption spectra in the mid-infrared range of the electromagnetic spectrum. The detection limit of 0.5 mmol L-1 for HMTD and 1 mmol L-1 for TATP allows the identification of the explosives in complex matrixes.     

The XT-tube extractor: a hollow fiber-based supported liquid membrane extractor for bioanalytical sample preparation

A new supported liquid membrane extractor for bioanalytical sample preparation is presented. The extractor consists of a polypropylene hollow fiber mounted inside a PTFE tube by means of a cross-connector and a tee-connector. All parts are commercially available, inexpensive, and easily assembled. An organic solvent in the pores of the fiber forms a liquid membrane that separates the sample, which is pumped along the outside of the fiber, from the acceptor phase, which is pumped inside. The length of the hollow fiber may easily be varied to meet different demands on extractive surface and extract volumes. To test the system, the strongly acidic plasticizer/flame retardant metabolite diphenyl phosphate ester (DPhP), with a pKa value of 0.26, was extracted from urine. DPhP was protonated using 4 M hydrochloric acid and extracted into an acceptor phase at pH 9. Thirty extractions were made with the same liquid membrane without any decrease in extraction efficiency and with a relative standard deviation <7%. An analyte concentration enrichment of 5-10 times was achieved in the extraction step, giving a limit of detection (S/N = 3) of 0.014 microg/mL with LC/ESI-MS and 0.18 microg/mL with CE-UV. The effects on extraction efficiency using different sample pH, organic solvents, sample flow rates, and lengths of the fiber were evaluated.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

Recovery of phenol from aqueous solution by supported liquid membrane using vegetable oils as liquid membrane

The transport of phenol through a flat sheet supported liquid membrane (SLM) containing vegetable oil as liquid membrane (LM) has been investigated. The permeation of phenol was investigated by varying the experimental conditions like, selection of LM, support material, feed phase pH, stripping solution concentration, stirring speed and different initial concentration of phenol. It has been found that, each LM investigated in the present study shows the effective removal of phenol using polytetrafluoroethylene (PTFE) membrane and PP supported membrane as a solid support. Among the various oils tested, palm oil has chosen to be the best LM with permeability of 8.5x10(-6) m/s in acidic feed of pH 2.0 with 0.2 M sodium hydroxide as effective stripping agent. After 6 h all the phenol from the feed side gets transported to strip solution with an initial concentration of 100 mg/L. A concentration factor of five has been achieved in the present investigation easily with 0.2 M sodium hydroxide as stripping reagent. After 10 transport studies with one impregnation of LM, the LM showed no significant loss in the transport rate with average permeability of 7.9x10(-6) m/s with initial concentration 100 mg/L. Further study has also been attempted with cresols to explore the possibility of applying this to industrial wastewater under the optimized conditions for phenol. After 14 h of the transport studies in the phenol-formaldehyde industry wastewater, phenolic concentration in the feed solution was found to be below detectable level (1x10(-2) mg/L). For wood processing industry wastewater the transport takes place at the initial permeability of 7.1x10(-5) m/s. Thus it has been demonstrated the use of renewable, cheap, non toxic, naturally occurring vegetable oils as a novel, green liquid membrane for the recovery of phenol from aqueous solution in SLM, which has never been employed before in liquid membrane techniques.     

离子液体充填型支撑液膜分离乙醇／水混合物

使用离子液体"充填型"支撑液膜,进行乙醇/水混合物的蒸汽渗透膜分离实验,比较了原料浓度、操作温度对分离性能的影响.结果表明,离子液体支撑液膜的蒸汽渗透过程能够实现乙醇脱水,用于制备无水乙醇.经过140 h的实验测定,支撑液膜分离性能稳定.在50℃温度下,原料中含水摩尔分数0.024～0.24时,蒸汽渗透分离因子5.5～3.2,渗透通量55 g/(m2·h).离子液体支撑液膜的蒸汽渗透膜分离过程,有望用于醇类水溶液组成的恒沸体系脱水.     

Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry

We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.     

Permeation of a fully ionized species across a polarized supported liquid membrane

An analytical technique for the detection of permeation of a fully ionized analyte across a lipophilic membrane is reported. The system, which is comprised of two aqueous compartments (donor and acceptor) separated by a supported liquid membrane, is based on the parallel artificial membrane permeation assay (PAMPA), widely used in the drug discovery process to estimate permeability in vivo. The in situ spectroelectrochemical method developed here employs mechanical stirring of the solution phases on either side of the membrane, external polarization of the membrane, and in situ detection of the analyte via UV-vis spectrophotometry. The flux of the crystal violet cation across the membrane is simultaneously measured via UV-vis spectrophotometry and voltammetry/chronoamperometry as a function of applied potential. The relative contribution of two permeation modes, i.e., that due to naked ions and ion-pairs, is thereby quantified. The open circuit potential difference between the two aqueous compartments and the cyclic voltammetric response are also recorded as a function of time and compared with the predicted values.     

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposition. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capillary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configuration, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins.     

Sample preparation for MALDI mass spectrometry using an elastomeric device reversibly sealed on the MALDI target

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 microL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well.     

Exact mass measurements on-line with high-performance liquid chromatography on a quadrupole mass spectrometer

Exact mass measurements were performed on-line with high-performance liquid chromatography on a quadrupole mass spectrometer. Compounds with molecular weights from 98 to 797, mainly aromatic sulfonates and sulfonamides, were analyzed with electrospray ionization in positive or negative mode. Internal mass calibration compounds were continuously added after separation. A Gaussian fit of the mass errors of 808 individual measurements (concentrations of 1-10 mg/L, 20-200 ng absolute on column) resulted in a mean error of 0.1 mmu (0.45 ppm) and a standard deviation sigma of 1.5 mmu (5.4 ppm). The 99.7% confidence intervals (3sigma) were +/-4.5 mmu (+/-16.2 ppm) for single mass measurements. Averaging 10 measurements further reduced the errors to less than +/-1.5 mmu (+/-5 ppm). Isobaric interferences with ions resulting from the mass calibrants were avoided by the use of complementary mass calibrants. The results were verified (differences below +/-4.5 mmu) with a LC/ oa-TOFMS. Limited mass range chromatograms were used to enhance selectivity in the analysis of mixtures. The method was applied to determine the elemental composition of a potential dye metabolite detected in anaerobically treated textile wastewater.     

High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.