Recent advances in nanomaterial‐based capillary electrophoresis

This review gives a summary of applications of different nanomateials, such as gold nanoparticles (AuNPs), carbon‐based nanoparticles, magnetic nanoparticles (MNPs), and nano‐sized metal organic frameworks (MOFs), in electrophoretic separations. This review also emphasizes the recent works in which nanoparticles (NPs) are used as pseudostationary phase (PSP) or immobilized on the capillary surface for enhancement of separation in CE, CEC, and microchips electrophoresis.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

On-column capture of a specific protein in capillary electrophoresis using magnetic beads

A method for capturing specific molecules separated by CE has been explored. To demonstrate on-column capture of migrating analyte molecules, two detection windows were fabricated on a capillary. Magnetic beads containing immobilized molecules that react with the specific molecules under study were placed between the detection windows in the capillary using magnets. Molecules in a sample solution injected into the capillary were separated and detected at the first detection window. After passing through the first detection window, the separated molecules encountered the magnetic beads, where the specific analyte was captured. As a result, the peak area for those analyte molecules decreased or disappeared completely at the second detection window. Rabbit IgG and carbonic anhydrase were employed to demonstrate on-column capture of a specific molecule. For rabbit IgG, magnetic beads containing the immobilized antibody (anti-rabbit IgG) were used. Rabbit IgG molecules were captured on the magnetic beads during CE migration. Furthermore, the capture of carbonic anhydrase was demonstrated by the reaction between magnetic beads (containing immobilized anti-rabbit IgG) and anti-carbonic anhydrase (rabbit IgG), before the beads were packed in the capillary. After packing the magnetic beads in the capillary, a mixture of two proteins was injected into the capillary. Two proteins were detected at the first detection window, while the peak corresponding to carbonic anhydrase disappeared at the second detection window. The results show that using an appropriate antibody, the present technique would be applicable to any proteins.     

Rapid analysis of inflammatory cytokines in cerebrospinal fluid using chip-based immunoaffinity electrophoresis

A chip-based capillary electrophoresis system has been designed for rapidly measuring the concentrations of inflammatory cytokines in the cerebrospinal fluid of patients with head trauma. Isolation of the reactive cytokines was achieved by immunoaffinity capture using a panel of six immobilized antibodies, directly attached to the injection port of the chip. The captured cytokines were labeled in situ with a red light-emitting laser dye and electroeluted into the separation channel. Separation of the isolated cytokines was achieved by electrophoresis in under 2 min with quantification of the resolved peaks being achieved by on-line laser-induced fluorescence and integration of each peak area. Comparison of the results to commercially available high-sensitivity immunoassays demonstrates that the chip-based assay provides a fast, accurate procedure for studying the concentrations of these analytes in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays, the system being able to analyze between 10-12 samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.     

Protein digestion and phosphopeptide enrichment on a glass microchip

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.     

Cover Picture: Electrophoresis 16'2011

Issue no. 16 is a regular issue with “Emphasis on Sensitivity Enhancement and Detection” consisting of 18 contributions distributed over 5 distinct parts and a Fast Track paper. The Fast Track paper is on “Barcoding of Giardia duodenalis isolates and derived lines from an established cryobank by a mutation scanning‐based approach”. The remaining 18 papers are grouped into 5 different parts. Part I and Part II represent the emphasis of this issue which involves “Sample Extraction and Enrichment and Sensitivity Enhancement” and “Detection Approaches” based on coupling CE with EC, ECL and MS. Part I has a series of 6 research papers on multifunctional magnetic nanoparticles for the enrichment of proteins, magnetic microspheres solid phase extraction of eight illegal drugs in human urine, hollow‐fiber liquid phase microextraction of nonsteroidal anti‐inflammatory drugs in wastewater, solid phase extraction to enhance sensitivity of CE for the determination of pharmaceuticals in river water, in‐line preconcentration CZE for the analysis of haloacetic acids in water, and dispersive liquid‐liquid microextraction coupled with CE for the determination of sulfonamides. Part II has 5 papers concerned with CE coupled with EC and ECL detection for the analysis of beta‐blockers, determination of nicotine and its metabolite cotinine in urine and cigarette samples by CE coupled with ECL, CE‐ECL detection for the analysis of ibandronate in drug formulation and human urine, CE‐ESI‐MS method for carbohydrate analysis, and analysis of phospholipids using MIP‐OTC in CEC‐ESI‐MS. Part III has 3 contributions on binding interaction and affinity capillary electrophoresis involving mobility shift assay for binding of DNA with NFAT3, rapid CE‐UV binding tests of environmentally hazardous compounds with polymer‐modified magnetic nanoparticles, and quantitative evaluation of lectin‐reactive glycoforms of alpha1‐acid glycoprotein using affinity CE with fluorescence detection. Part IV is on protein analysis by gel electrophoresis and has 2 contributions while Part V has 2 research papers on rice genotyping and determination of contrast agents by MEKC in urine and serum samples. Featured articles include: FAST TRACK: Barcoding of Giardia duodenalis isolates and derived lines from an established cryobank by a mutation scanning‐based approach. (( 10.1002/elps.201100283 )) Applications of multifunctional magnetic nanoparticles for the enrichment of proteins for PAGE separation. (( 10.1002/elps.201000657 )) Dispersive liquid‐liquid microextraction coupled with capillary electrophoresis for simultaneous determination of sulfonamides with the aid of experimental design. (( 10.1002/elps.201100142 )) Carbohydrate analysis by capillary electrophoresis‐microelectrospray ionization‐mass spectrometry. (( 10.1002/elps.201100027 )) Quantitative evaluation of lectin‐reactive glycoforms of α1‐acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection. (( 10.1002/elps.201100146 )) High throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.). (( 10.1002/elps.201100196 ))     

Immobilizing sulfotransferase 1A1 on magnetic microparticles and their evaluation using capillary electrophoresis

Sulfotransferases are categorized as phase II metabolic enzymes. Human sulfotransferase 1A1 (SULT1A1) is involved in the sulfonation of xenobiotics with aid from the cofactor 3'‐phosphoadenosine‐5'‐phosphosulfate that acts as a sulfonate donor. In this study, we have attempted to immobilize SULT1A1 on magnetic microparticles (MMs). Different functionalized MMs were used to immobilize SULT1A1 and their enzyme activity was compared to the control (enzyme in solution). Paracetamol was used as model substrate. Separation of paracetamol and paracetamol sulfate by CE‐UV was optimized and validated. MMs with epoxy based immobilization of SULT1A1 showed better enzyme activity. Hence, they were tested for repeated usage to allow their implementation for the development of a CE immobilized micro enzyme reactor.     

Measurement of neuropeptides in clinical samples using chip-based immunoaffinity capillary electrophoresis

The current interest in micro-fabrication has extended to the clinical arena where there is a growing lobby for promoting these for point-of-care purposes. The advantages of such devices are their relative speed of analysis, lower reagent costs, and their application to clinical screening and diagnosis. Two chip-based capillary electrophoresis systems have been designed and their performance evaluated for rapidly measuring the concentrations of inflammatory neuropeptides in tissue fluids of patients with neuropeptide-associated muscle pain. Both chips were manufactured to fit a commercially available chip electrophoresis system. One chip was designed to perform electrokinetic flow immunoassays while the other utilized an immunoaffinity port, containing an array of immobilized antibodies, to capture the analytes of interest. Comparison of the results to commercially available high-sensitivity immunoassays demonstrated that both chip-based systems could provide a relatively fast, accurate procedure for studying inflammatory biomarkers in complex biological fluids. However, the immunoaffinity capture system proved the superior of the two chips. Using this system, twelve different inflammation-associated mediators could be determined in approximately 2 min as compared to 30 min when using the flow immunoassay chip. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.     

Cover Picture: Electrophoresis 14'2011

Issue no. 14 is a regular issue consisting of 17 contributions distributed over 3 distinct parts. Part I has 7 articles describing studies on proteins and proteomics including measuring protein mobility using laser Doppler electrophoresis, high sensitivity protein analysis by FESI‐CE‐MALDI‐MS, protocol for SDS‐PAGE separation of myosin heavy chain isoforms, proteomics analysis of a spring wheat cultivar in response to prolonged cold stress, analysis of changes in the 20S proteasome, treatment of acute lymphoblastic leukemia with L‐asparaginase, and improved method for immunostaining of mucin. Part II is on nucleic acids and has 4 contributions on multiplex PCR‐CE analysis, copy number variation, ethnic difference, SNPs, CE‐SSCP, allelic ladder and SNaPshot technique. Part III has 6 articles on various fundamentals and methodologies, including nanofluidics, nanopore sensing, sequential injection setup for CIEF combined with MS detection, determination of imatinib mesylate in chronic myeloid leukemia patients by CE, UV detection of plasma malondialdehyde using CE‐FASI, enantioseparation of basic drugs by CE using clarithromycin lactobionate chiral selector and CE determination of bioactive constituents in Herba Houttuyniae. Featured articles include: Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique (( 10.1002/elps.201100108 )) Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure (( 10.1002/elps.201000608 )) Strategy for high‐fidelity multiplex DNA copy number assay system using capillary electrophoresis devices (( 10.1002/elps.201100093 )) Electrokinetic particle translocation through a nanopore containing a floating electrode (( 10.1002/elps.201100050 )) Sequential injection setup for capillary isoelectric focusing combined with MS detection (( 10.1002/elps.201100012 ))     

Development of a sheathless CE‐ESI‐MS interface

A sheath‐flow interface is the most common ionization technique in CE‐ESI‐MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE‐MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath‐flow CE‐MS. Because the interface is easy to construct, it is cost‐effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE‐MS‐based metabolomic analysis.     

The application of normal phase liquid chromatography/mass spectrometry by using coaxial continuous flow fast atom bombardment

This article describes the interfacing of a normal phase fused silica capillary high performance liquid chromatography system to a magnetic sector mass spectrometer by using continuous flow fast atom bombardment (CFFAB). While the performance of CFFAB using reversed phase techniques is well understood, there is very little if any documentation on interfacing nonaqueous normal phase systems with CFFAB. This article describes the use of packed fused silica capillary liquid chromatography columns and the corresponding normal phase solvent systems. The experimental parameters required with nonaqueous solvent systems differ significantly from those of aqueous solvent systems. Ditallowdimethylammonium chloride (DTDMAC), a cationic surfactant commonly used as the active ingredient in fabric softener products, was chosen as a model compound to demonstrate the technique. DTDMAC was identified in a commercially available fabric softener product by using on-line normal phase liquid chromatography/mass spectrometry with accurate mass and tandem mass spectrometry.     

Cover Picture: Electrophoresis 20'2010

Issue no. 20 is a regular issue comprising 17 contributions distributed over 7 distinct parts. Part I has 3 research articles on particle analysis. Part II has 2 research articles dealing with hyphenated techniques such as CITP‐CZE and CE‐ESI‐MS. Part III reports a variety of methodologies and systems for proteins and proteomics that are described in 4 research articles. Gel DNA: theory and visualization are treated in 2 research articles making up part IV. Part V has 2 contributions that describe CE systems for water analysis. The following Part VI is on affinity and Immunoaffinity CE and has 2 research articles. The last 2 articles in this issue (Part VII) are on MEEKC and free flow electrophoresis. Featured articles include: Dielectrophoretically patterned carbon nanotubes to sort microparticles ((doi: 10.1002/elps.201000104 )) Analysis of 5‐methyltetrahydrofolate in human blood, serum and urine by on‐line coupling of capillary isotachophoresis and zone electrophoresis ((doi: 10.1002/elps.201000193 ))     

Joule heating and determination of temperature in capillary electrophoresis and capillary electrochromatography columns

This article reviews the progress that has taken place in the past decade on the topic of estimation of Joule heating and temperature inside an open or packed capillary in electro-driven separation techniques of capillary electrophoresis (CE) and capillary electrochromatography (CEC), respectively. Developments in theoretical modeling of the heat transfer in the capillary systems have focused on attempts to apply the existing models on newer techniques such as CEC and chip-based CE. However, the advent of novel analytical tools such as pulsed magnetic field gradient nuclear magnetic resonance (NMR), NMR thermometry, and Raman spectroscopy, have led to a revolution in the area of experimental estimation of Joule heating and temperature inside the capillary via the various noninvasive techniques. This review attempts to capture the major findings that have been reported in the past decade.     

Separation of aminoalkanephosphonic acid enantiomers by indirect UV detection capillary electrophoresis with application of cyclodextrins

Indirect UV detection capillary electrophoresis (CE) was used for the separation of aminoalkanephosphonic acid (AP) enantiomers by applying commercially available cyclodextrins as chiral discriminators. The results show that the separation of the enantiomers depends on pH of the background electrolyte, the molar ratio of cyclodextrin to aminophosphonic acid, and on the type of the applied chiral selector. Optimization of process conditions allowed enantiomeric baseline separation or partial separation of 12 out of 14 alpha-aminophosphonic acids studied. This type of CE might therefore be successfully used for routine determination of enantiomeric purity of aminophosphonic acids.     

High Electric Field Strengths in Micellar Electrokinetic Capillary Electrophoresis with Ionic Liquids as Modifiers

Abstract

In this study, a method for the separation and determination of basic analytes in aqueous capillary electrophoresis (CE) was developed based on high electric field strengths and ionic liquids (ILs). The resulting electric field strengths ranged from 500 to 1000 V/cm. Trishydroxymethylaminomethane (Tris) and sodium cholate (SC) were used as main electrolytes. The ionic liquids 1‐ethyl‐3‐methylimidazoium tetrafluoroborate (1E‐3MI‐TFB) and 1‐butyl‐3‐methylimidazoium tetrafluoroborate (1B‐3MI‐TFB) were used as modifiers to improve the separation efficiency and selectivity. It was shown that increasing the applied electric field strengths not only caused short analysis time, but also did not induce excessive Joule heating in the capillary when ionic liquids were used as modifiers. The susceptibility to high electric field of separation efficiency in capillary electrophoresis, with the effect of ionic liquids, was subsequently discussed, and the developed method was used to analyze three model analytes in Sinacalia tangutica. The accurate results illustrated that high electric field strength with the ionic liquids was feasible in CE.     

On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 microm ID) in the PDMS structure, enabling independent flow characterization.     

An economic approach for construction of a multiplexed capillary electrophoresis system

We describe in this report an economic approach to construct a multiplexed capillary electrophoresis (CE) system with optic-fiber UV detection. To demonstrate the feasibility of this approach, a seven-lane CE setup is built. The system has a background noise of ∼10⁻⁵ absorbance unit, a limit of detection of 3-4 μM for uracil at 254 nm, and a linear dynamic range of 2.5 orders of magnitude. The apparatus consists of three major components: a UV light source, a photodiode array (PDA) and optical module assembly, and a computer. Compared to the recently reported 96-lane capillary electrophoresis system with UV-vis absorbance detection, the cost of goods is reduced by more than 10-fold, and the noise level is improved by 2 to 3 folds. Parallel CE separations of a protein mixture have been performed on this apparatus, and reproducible and high resolution separation results have been obtained. Detailed construction and characterization of the system have also been discussed.     

Is capillary electrophoresis on microchip devices able to genotype uridine diphosphate glucuronosyltransferase 1A1 TATA‐box polymorphisms?

In this commentary, we focused our attention on capillary electrophoresis. It achieves the efficient separation of molecular species by the application of high voltages to samples in solution. Actually, capillary electrophoresis can be performed on microchip devices, based on an automated and miniaturized electrophoresis system, based on lab‐on‐a‐chip technology. By this technology it is possible to separate nucleic acid fragments (DNA or RNA) with respect to sizing accuracy and sizing resolution. Currently, two automated capillary electrophoresis on microchips devices are available: the Agilent 2100 Bioanalyzer and the Experion? Automated Electrophoresis System. In this study, we evaluated if the CE is able to distinguish the three uridine diphosphate glucuronosyltransferase 1A1 TATA‐box genotypes.     

Rapid determination of 4‐aminobutyric acid and L‐glutamic acid in biological decarboxylation process by capillary electrophoresis‐mass spectrometry

4‐Aminobutylic acid (GABA) is a monomer of plastic polyamide 4. Bio‐based polyamide 4 can be produced by using GABA obtained from biomass. The production of L ‐glutamic acid (Glu) from biomass has been established. GABA is produced by decarboxylation of Glu in biological process. High‐performance liquid chromatography (HPLC) with derivatization is generally used to determine the concentration of GABA and Glu in reacted solution samples for the efficient production of GABA. In this study, we have investigated the rapid determination of GABA and Glu by capillary electrophoresis‐mass spectrometry (CE‐MS) without derivatization. The determination was achieved with the use of a shortened capillary, a new internal standard for GABA, and optimization of sheath liquid composition. Determined concentrations of GABA and Glu by CE‐MS were compared with those by pre‐column derivatization HPLC with phenylisothiocyanate. The determined values by CE‐MS were close to those by HPLC with pre‐column derivatization. These results suggest that the determination of GABA and Glu in reacted solution is rapid and simplified by the use of CE‐MS.     

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.