Postcolumn concentration in liquid chromatography. On-line eluent evaporation and analyte postconcentration in ion chromatography

On-line sample concentration by evaporation through a narrow-bore membrane tube is described. The device can be deployed just prior to the detector or the sample may be concentrated prior to injection. As solution flows through a solvent-permeable membrane tube, (heated) drying gas (nitrogen/air) flows outside it to remove the solvent. The removal rate increases with increasing sample residence time, drying gas flow rate, and temperature. Various membranes and three concentrator designs (a rectangular maze, a serpentine and a filament-filled helix, the last performing the best) were fabricated and tested for post- and preseparation applications in suppressed anion chromatography. An order of magnitude concentration factors are readily obtained. The present system involves active mass transport radially outward through the walls of a tube. This is a system in which many of the traditional paradigms of flow through a tubular conduit no longer hold true. Because the flow rate continuously varies along the tube, residence time does not scale linearly with residence volume or conduit length. The effects of such mass transport on the parabolic velocity profile of laminar flow remain unknown.     

Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

pH- and concentration-programmable electrodialytic buffer generator

We have presented in a companion paper a suppressor-based electrodialytic buffer generator (EBG) that can produce programmable pH gradients. Here we demonstrate a three-electrode EBG. In this three-compartment flow-through device, the central compartment is separated from the outer compartments with a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM), respectively. One platinum electrode is disposed in each compartment. The flows through each compartment are independent. With appropriate solutions in each compartment, independent potentials are applied to the CEM and AEM electrodes with respect to the grounded central electrode. The CEM current and the AEM current can be independently manipulated to generate buffers with variable concentration and pH in the central compartment. Both the CEM and AEM currents can be positive or negative. For the CEM, a positive current (i(cat)(in)) indicates that cations are coming in from the CEM channel to the center. A negative current (i(cat)(out)) takes cations out of the center to the CEM channel. Similarly for the AEM, currents governing anion transport into the center channel from the AEM channel (AEM electrode negative) or the reverse (AEM electrode positive) are respectively denoted by i(an)(in) or i(an)(out). Most examples herein involve inward ion transport, referred to as the additive mode. Depending on whether i(cat)(in) i(an)(in), H(+)/O(2) and OH(-)/H(2) are respectively produced at the central electrode to maintain electroneutrality. Any gas formed is subsequently removed by a gas removal device. The pH of the central channel effluent is related to the ratio of the currents through the two membranes, while the generated concentration is controlled by the absolute value of the currents. The buffer concentration and pH can be varied in a controlled predictable manner. A pH span of 3-12 was attained and a phosphate buffer concentration up to 140 mM was generated. We demonstrate a variety of combined pH/concentration gradients from a mixture of ethylenediamine, citrate, and phosphate by manipulating i(cat)(in), which controls introduction of the ethylenediammonium ion, and i(an)(in), which controls the introduction of citrate and phosphate ions. We also demonstrate an additive-subtractive mode of operation where both inward and outward currents are used to add one type of ion while removing another type of ion to reproducibly generate pH/concentration gradients.     

Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography

Polymer-based strong cation-exchange monolithic capillary columns with different capacities were constructed for ion chromatography by radical polymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate in a 250-microm-i.d. fused-silica capillary and its subsequent sulfonation based on ring opening of epoxides with 1 M Na(2)SO(3). The cation-exchange capacities can easily and reproducibly be controlled in the range of up to 300 microequiv/mL by changing the immersion time of the epoxy-containing polymer in the Na(2)SO(3) solution. The chromatographic performance of the produced monolithic capillary columns was evaluated through the separation of a model mixture of common cations such as Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+). As an example, these cations could be well separated from one another on a 15-cm-long cation-exchange monolithic column (column volume, 7.4 microL) with a capacity of 150 microequiv/mL by elution with 10 mM CuSO(4). The pressure drop of this 15-cm column was approximately 1 MPa at a normal linear velocity of 1 mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for the cations were above 3000 plates/15 cm. This GMA-based cation-exchange monolithic column could withstand high linear velocities of at least 10 mm/s. Over a period of at least two weeks of continuous use, no significant changes in the selectivity and resolution were observed. The applicability of a flow rate gradient elution and the feasibility of direct injection determination of major cations in human saliva sample were also presented.     

On-line electrodialytic salt removal in electrospray ionization mass spectrometry of proteins

Salts and buffers, commonly used in isolation and stabilization of biological analytes, have a deleterious effect on electrospray ionization mass spectrometry (ESI-MS). Excessive concentrations of salts lead to ion suppression and adduct formation, which mask or complicate ion signals. In this work, we describe a salt remover (SR), configured as a three-compartment flow-through system, where the central compartment is separated from the outer compartments by a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM). One platinum electrode is placed in each of the outer compartments, where water or electrolyte is flowing. The CEM electrode is held at a negative potential relative to the AEM side; cations/anions migrate by electrophoresis to the CEM/AEM receiver liquids, respectively. Proteins have poorer electrophoretic mobility relative to the buffer components, permitting removal of the salt. The salt-free proteins proceed to the ESI source. The capillary scale SR (internal volume 2.5 μL) described here effectively desalted continuous flows of NaCl solutions (200 mequiv/L at 1 μL/min, equivalent to a flux of 200 nequiv/min with 88% efficiency) and achieved >99.8% salt removal with 154 mM NaCl (isotonic saline) at 1 μL/min. With optimized current, >80% of concurrently present 20 μM protein was transmitted. Desalting efficiency and analyte loss was evaluated with different salt concentration and flow rate combinations under different applied current. Good-quality ESI-MS spectra of cytochrome c, myoglobin, and lysozyme as test proteins in a saline solution, passed through the SR, are demonstrated.     

大型发电机定子局部放电在线监测系统的研究

局部放电测量是诊断定子绕组绝缘最为有效的方法的之一.结合以往的科研实践,且基于磁耦合原理及小波去噪技术,研制出了一套大型发电机定子绕组局部放电在线监测系统,该系统具有工作频带宽、抗干扰能力强、灵敏度高等特点.采用高压电机定子绕组对该系统进行测试,测试结果表明,该系统能准确提取出局部放电脉冲的特征量,满足定子绝缘状况诊断的要求.     

High-speed liquid chromatography by simultaneous optimization of temperature and eluent composition

Clearly, a major trend in liquid chromatography is to increase its speed to make it faster. Improving throughput for routine analysis of pharmaceutical release samples and stability assays are all key motivations for improving separation speed. Recent work has focused on the theoretical benefits of temperature on speed in liquid chromatography. We have shown that the 5-10-fold decrease in eluent viscosity that comes from a temperature increase of 175 degrees C over ambient, and the concomitant increase in analyte diffusitivity, act to dramatically decrease the time needed to generate a theoretical plate. Lower viscosities at elevated temperatures decrease the pressure drop across the column and allow the use of higher linear velocities as the pump pressure limit is approached. Simultaneously, faster analyte diffusion at higher column temperature improves efficiency at high eluent velocity conditions compared to the efficiency at lower temperatures at the same velocity. We find that higher temperature plays a central role in improving speed. In this work, we show that when the percent organic modifier in the eluent and column temperature are adjusted to keep retention factors fixed, highly efficient, subminute separations can be routinely achieved when a hot column is used at the maximum system back pressure. We find that the best way to facilitate such an optimization, assuming constant selectivity, is to use a very retentive column so that one can work at both high temperature and high volume fraction of organic modifier to achieve the lowest possible eluent viscosity. We have also analyzed the effect that key extracolumn contributions have on column selection and system design.     

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.     

On-line process control of liquid chromatography

Many analytical methods are based on liquid chromatography and typically the only measure of system stability is standards, injected repeatedly throughout the sequence. In this paper, a novel approach is presented, where the analytical run is treated as a process with the chromatographic data as the product. It is postulated that enhanced quality of the data can be obtained through monitoring the process, i.e., the chromatographic system, during the sequence. For this purpose, a liquid chromatography process control (LCPC) system has been developed. Here, several parameters, e.g., the pressure at the column and the injection valve, are monitored. Chemometrics is used for interpreting the data and producing multivariate statistical process control (MSPC) charts. The chromatographic run is divided into two parts: the dynamic injection phase and the static elution phase. Two principal component analysis (PCA) models, one for each phase, are continuously created and upgraded as the data are collected. The results of the PCA are shown in the MSPC charts, and when an error detection limit is exceeded, the analyst is promptly notified. LCPC, a continuous system suitability test, provides better control of the analysis, allowing a reduction in the number of standards and replicates. Furthermore, troubleshooting is facilitated.     

On-line fourier transform infrared detection in capillary electrophoresis

The coupling of Fourier transform infrared (FT-IR) spectroscopy as a new on-line detection principle in capillary electrophoresis (CE) is presented. To overcome the problem of total IR absorption by the fused-silica capillaries that are normally employed in CE separations, a micromachined IR-transparent flow cell was constructed. The cell consists of two IR-transparent CaF2 plates separated by a polymer coating and a titanium layer producing an IR detection window, 150 microm wide and 2 mm long, with a path length of 15 microm. The IR beam was focused on the detection window using an off-axis parabolic mirror in an optical device (made in-house) attached to an external optical port of the spectrometer. The connections between the fused-silica capillaries and the flow cell were made by a small O-ring of UV-curing epoxy adhesive on the sharply cut ends of the capillaries, allowing the capillaries to be easily replaced. Aqueous solutions comprising mixtures of adenosine, guanosine, and adenosine monophosphate were used to test the system's performance. Conventional on-line UV detection was employed to obtain reference measurements of analytes after the IR detection flow cell. The limit of FT-IR detection for all analytes (in absolute amounts) was in the nano- to picogram range corresponding to concentrations in the low-millimolar range.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

Aptamer-modified monolithic capillary chromatography for protein separation and detection

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation.     

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.     

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.     

Dynamic range expansion applied to mass spectrometry based on data-dependent selective ion ejection in capillary liquid chromatography fourier transform ion cyclotron resonance for enhanced proteome characterization.

The characterization of cellular proteomes is important for understanding biochemical processes ranging from cell differentiation to cancer development. In one highly promising approach, whole protein extracts or fractions are digested (e.g., with trypsin) and injected into a packed capillary column for subsequent separation. The separated peptides are then introduced on-line to an electrospray ionization source of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the detection of peptide accurate mass tags that serve as biomarkers for their parent proteins. In this work, we report the use of data-dependent selective external ion ejection in conjunction with FTICR and on-line capillary LC separations for the enhanced characterization of peptide mixtures and a yeast extract proteome. The number of peptides identified in an LC-FTICR analysis of a yeast proteome digest employing data-dependent rf-only dipolar ejection of the most abundant ion species prior to ion accumulation was 40% higher than that detected in a separate LC-FTICR analysis using conventional nonselective ion accumulation.     

Microchip for combining gas chromatography or capillary liquid chromatography with atmospheric pressure photoionization-mass spectrometry

We present a microfabricated nebulizer chip for combining atmospheric pressure photoionization-mass spectrometry (APPI-MS) with gas chromatography (GC) or capillary liquid chromatography (capLC). The chip consists of a silicon plate and a glass plate or two glass plates. The chip includes a sample inlet channel, auxiliary gas and dopant inlet, vaporizer channel, nozzle, and platinum heater. The sample eluted from the capLC or GC is mixed with auxiliary gas and dopant (toluene) in the heated vaporizer. The chip forms a confined jet of the sample vapor, which is photoionized as it exits the chip. The analytical performance of GC- and capLC-microchip APPI-MS was evaluated with some polycyclic aromatic hydrocarbons, amphetamines, and steroids. The GC-muAPPI-MS method provides high sensitivity down to 0.8 fmol, repeatability (RSD = 7.5-14%), and linearity (r = 0.9952-0.9987). The capLC-muAPPI-MS method shows high sensitivity down to 1 fmol, good repeatability (RSD = 3.6-8.1%), and linearity (r = 0.9989-0.9992).     

On-line coupling of microdialysis sampling with microchip-based capillary electrophoresis

Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 microL/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-beta-d-galactopyranoside (FMG) by beta-d-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.     

On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis

Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.