CE Instrument Research Articles

White LEDs as broad spectrum light sources for spectrophotometry: Demonstration in the visible spectrum range in a diode‐array spectrophotometric detector

Although traditional lamps, such as deuterium lamps, are suitable for bench-top instrumentation, their compatibility with the requirements of modern miniaturized instrumentation is limited. This study investigates the option of utilizing solid-state light source technology, namely white LEDs, as a broad band spectrum source for spectrophotometry. Several white light LEDs of both RGB and white phosphorus have been characterized in terms of their emission spectra and energy output and a white phosphorus Luxeon LED was then chosen for demonstration as a light source for visible-spectrum spectrophotometry conducted in CE. The Luxeon LED was fixed onto the base of a dismounted deuterium (D(2) ) lamp so that the light-emitting spot was geometrically positioned exactly where the light-emitting spot of the original D(2) lamp is placed. In this manner, the detector of a commercial CE instrument equipped with a DAD was not modified in any way. As the detector hardware and electronics remained the same, the change of the deuterium lamp for the Luxeon white LED allowed a direct comparison of their performances. Several anionic dyes as model analytes with absorption maxima between 450 and 600 nm were separated by CE in an electrolyte of 0.01 mol/L sodium tetraborate. The absorbance baseline noise as the key parameter was 5 × lower for the white LED lamp, showing clearly superior performance to the deuterium lamp in the available, i.e. visible part of the spectrum.

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.

Cover Picture: Electrophoresis 15'2010

AbstractAs science develops and continues to influence our lives, new analytical challenges are presented. Specific needs for faster, simpler, more efficient, and less expensive methodologies abound in almost all fields. Although capillary electrophoresis and microchip capillary electrophoresis currently address many of these challenges, pertinent developments of novel and reliable instrumentation are critical. Addressing such demands, ELECTROPHORESIS has published an increasing number of papers over the past few years and has dedicated Issue no. 15 to “Instrumentation for CE and Microchip‐CE”. This special issue comprises 27 manuscripts distributed over 6 separate parts. Part I has 3 review articles on instrumentation for CE and microchip‐CE, toner and paper‐based fabrication for microfluidics applications, and liquid chromatography on chip. Part II consists of 3 articles on fabrication of microfluidics chips, while Part III is on sample preparation and separation that consist of 5 articles on fast serial analysis of single cells, in‐line extraction on functionalized magnetic particles, hydrodynamic injection, etc. Part IV assembles 8 articles on various detection approaches. Complete and hyphenated systems are described in Part V while applications are the subject of Part VI.

Instrumentation for CE and Microchip‐CE

ELECTROPHORESISVolume 31, Issue 15 p. 2467-2468 Editorial Instrumentation for CE and Microchip-CE First published: 23 July 2010 https://doi.org/10.1002/elps.201090062Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume31, Issue15Special Issue: INSTRUMENTATION FOR CE AND MICROCHIP-CEAugust 2010Pages 2467-2468 RelatedInformation

Call for Papers

ELECTROPHORESISVolume 31, Issue 15 Call for papers Call for Papers First published: 23 July 2010 https://doi.org/10.1002/elps.201090064Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume31, Issue15Special Issue: INSTRUMENTATION FOR CE AND MICROCHIP-CEAugust 2010 RelatedInformation

Meetings Diary

ELECTROPHORESISVolume 31, Issue 15 Meetings Diary Meetings Diary First published: 23 July 2010 https://doi.org/10.1002/elps.201090066Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume31, Issue15Special Issue: INSTRUMENTATION FOR CE AND MICROCHIP-CEAugust 2010 RelatedInformation

Recent developments in instrumentation for capillary electrophoresis and microchip-capillary electrophoresis.

Over the last years, there has been an explosion in the number of developments and applications of CE and microchip-CE. In part, this growth has been the direct consequence of recent developments in instrumentation associated with CE. This review, which is focused on the contributions published in the last 5 years, is intended to complement the articles presented in this special issue dedicated to instrumentation and to provide an overview of the general trends and some of the most remarkable developments published in the areas of high-voltage power supplies, detectors, auxiliary components, and compact systems. It also includes a few examples of alternative uses of and modifications to traditional CE instruments.

Decoupling CE and ESI for a more robust interface with MS

An interface for CE-ESI-MS that decouples both the electrical and the solution flow rate requirements of the separation and ionization processes is presented. The interface uses a tapered and beveled stainless steel hollow needle surrounding the separation capillary terminus so that the inside of the electrode acts as the CE outlet vial and the outside tip acts as the electrospray emitter. No capillary pre-treatment is required, enabling the use of capillaries with any type of surface modification. A chemical modifier solution is introduced through a second capillary connected to the needle via a tee junction and can be used to improve the compatibility of the CE BGE with electrospray. The flow rate of modifier solution can be as low as 0.1 microL/min, much less than that in a typical sheath-flow interface, thus minimizing dilution of the CE effluent in order to maximize sensitivity. The presence of the modifier solution also allows the use of neutral-coated capillaries for protein analysis by CE-MS without using an assisting pressure, despite the absence of EOF under these conditions. The interface is easily integrated into a commercial CE instrument, such that all operations can be carried out by the automated controls. Compared with a commercial sheath-flow CE-MS interface operating under optimized conditions, LODs for amino acids were, on average, improved fivefold.

Separation of DNA-containing organelles fromToxoplasma gondiiby CZE

Toxoplasma gondii and other members of the family Apicomplexa have two organelles, in addition to the nucleus, that contain DNA. Herein is reported the separation of the DNA-carrying organelles from T. gondii tachyzoites, i.e. the mitochondrion and the apicoplast, by CZE. The cells were stained with SYTO9, a dye that exhibit fluorescence when interacting with double stranded nucleic acids (e.g. DNA) and disrupted by nitrogen cavitation. Following careful removal of the heavier cellular material, the remaining lysate was injected on a CE instrument and the DNA-containing organelles were detected by LIF. The mitochondrion had longer migration time than the apicoplast, and the migration times were comparable in the replicates. This method should potentially also work for other members of the Apicomplexa including Plasmodium falciparum.

Electrically assisted capillary liquid chromatography using a silica monolithic column

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 μm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 μm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0–100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.

A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV-LIF (266 nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25 ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214 nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by beta-mercaptoethanol is reported, demonstrating the ease of performing the analysis.

On‐line combination of CE and microscopy: An insight into the migration of microorganisms

Identification of microbial contamination by CE has interested many researchers mainly because of the high speed of CE analysis. However, the CE separation of such big structures brings a lot of questions mainly about the behavior of microorganisms and about the mechanism of separation. In this work, we constructed a simple apparatus where a microscope was used as one detector and a UV detector was used as the second one and we made the comparison of three typical setups for CE of microorganisms. Saccharomyces cerevisiae was chosen as a model microorganism and was analyzed in bare fused-silica capillaries, covalently modified capillaries and dynamically modified capillaries by poly(ethylene oxide) or CTAB. Results showed that the use of CE instrument directly connected with a microscope could be advantageous for the study of separation mechanisms and/or migration behavior.

Determination of sulphide in the presence of sulphate in water by capillary electrophoresis with a high-sensitivity cell

A sensitive and selective capillary electrophoresis method with indirect UV-detection employing a high-sensitivity cell of special design was used for the determination of sulphide in the presence of sulphate in drinking and wastewaters. The running buffer consisted of chromate, tetradecyltrimethylammonium hydroxide, glutathione and polethylene glycol at pH 10.0. The use of a high-sensitivity cell and the addition of polyethylene glycol were the key factors of improving sensitivity and selectivity of the proposed method. The method was proved suitable for its intended use by applying the procedure starting from the calibration of the CE instrument to validation of all experimental parameters. The resolution factor between sulphide and sulphate under optimal conditions was found to be about 2.0. The limits of detection (LODs) of sulphide and sulphate were 2.0 and 0.3 ng/mL, respectively. Analysis of standard mixtures of sulphide and sulphate gave good linearity in the ranges of 5.0–500.0 and 2.0–160.0 ng/mL, respectively, with correlation coefficients ranging between 0.994 and 0.996 at 95% confidence level. Application of the proposed method to the analysis of water was successfully achieved and statistically confirmed. It is important to note that in drinking water ultra trace concentrations of sulphide ions were successfully detected by the current method.

Direct chiral analysis of primary amine drugs in human urine by single drop microextraction in‐line coupled to CE

Three-phase single drop microextraction (SDME) was in-line coupled to chiral CE of weakly basic amine compounds including amphetamine. SDME was used for the matrix isolation and sample preconcentration in order to directly analyze urine samples with the minimal pretreatment of adding NaOH. A small drop of an acidic aqueous acceptor phase covered with a thin layer of octanol was formed at the tip of a capillary by simple manipulation of the liquid handling functions of a commercial CE instrument. While the saline matrix of the urine sample was blocked by the octanol layer, the basic analytes in a basic aqueous donor phase were concentrated into the acidic acceptor drop through the octanol layer by the driving force of the pH difference between the two aqueous phases. The enantiomers of the enriched amines were resolved by using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector for the subsequent CE separation. From 10 min SDME with the agitation of the donor phase by a small stirrer retrofit to the CE instrument, enrichment factors were about a 1000-fold, yielding the LOD of 0.5 ng/mL for amphetamine. This low LOD value as well as the convenience of in-line coupled SDME make the proposed scheme well suited for the demanding chiral analysis of amphetamine-type stimulants.

Analysis of γ‐globulin mobility on routine clinical CE equipment: Exploring its molecular basis and potential clinical utility

A study was conducted on the variability of gamma-globulin mobility in serum protein electrophoresis and its molecular basis. We found that the migration time of gamma-globulins can be reproducibly determined (CV=1.1%) on clinical CE equipment. Moreover, we found a significant difference (p<0.001) in the migration of gamma-globulins between chronic liver disease patients (n=98) and a healthy reference group (n=47). Serum immunoglobulins were purified from these patients' sera using protein L-agarose and their glycosylation was studied using CE on a DNA sequencer. This glycomics approach revealed that several non-sialylated N-glycans show a moderate Pearson correlation coefficient (r=0.2-0.4) with the migration time of gamma-globulins. Their sialylated structures correlate negatively (r=-0.2 to -0.3). Immunoglobulins are significantly more sialylated in the healthy reference group compared with the patients (p<0.001). We estimated that sialylation heterogeneity contributes about 36% to the molecular variance (carbohydrates and amino acid composition) that affects the electrophoretic mobility of immunoglobulins. This is the first report on the migration time of gamma-globulins on a clinical CE instrument and its potential clinical value to the routinely analyzed serum protein CE profiles.

Integration of on‐line protein digestion by trypsin in CZE by means of electrophoretically mediated microanalysis

In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared with classical in-solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA-based method is rapid, can be automated and requires only a small amount of trypsin preparation. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CZE separation of peptides. In this set-up, a part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris-HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.

In‐capillary microextraction using monolithic polymers: Application to preconcentration of carbamate pesticides prior to their separation by MEKC

In the present work we report a novel procedure for in-capillary microextraction using a monolithic polymeric sorbent. In the proposed methodology, sample treatment takes place in the CE instrument but in a different capillary from that used for the electrophoretic separation. Polymers based on butyl methacrylate and divinylbenzene formed in situ inside a capillary column were assayed. The best results were found with the divinylbenzene-based polymers. The usefulness of the proposed procedure was checked for the determination of carbamate pesticides (Methomyl, Asulam, Carbendazim, Aldicarb, Carbetamide, Propoxur, Pirimicarb, Carbaryl, Carbofuran and Methiocarb) and three of their degradation compounds (Aldicarb sulphoxide, 2-isopropoxyphenol and alpha-naphthol) using MEKC. The optimization of the MEKC is reported, a good separation of the 13 analytes being obtained in less than 6 min. The analytical method using in-capillary microextraction was validated in terms of linearity, repeatability, precision (RSD< or =18% for 50 microg/L), and LODs (1-16 microg/L), and it revealed the usefulness of this in-capillary preconcentration procedure for the determination of analytes of intermediate polarity.

Recent significant developments in detection and method development for the determination of inorganic ions by CE

CE has been available as a tool for almost 20 years, but it is only in the past several years that it has been implemented widely. This has been the result of some significant advances in the technique. These fall in the areas of indirect photometric detection (through the use of dyes as probes and LEDs as light sources), the introduction and establishment of capacitively-coupled contactless conductivity detection (C4D) as a routine, sensitive and commercially available detection method, and in software capable of simulation of separations and the selection of optimal composition of the BGE. These developments are reviewed and their impact illustrated by reference to a case study involving the rapid separation and sensitive detection of 15 anions and 12 cations on a portable CE instrument. It is shown that C4D provides considerably superior detection sensitivity (by a factor of about 8 in comparison with optimised indirect photometry).

Separation of R‐form lipopolysaccharide and lipid A by CE–Fourier‐transform ion cyclotron resonance MS

Lipopolysaccharide (LPS) is the main component of the outer leaflet of the outer membrane of Gram-negative bacteria. Native isolates of LPS comprise a high inherent heterogeneity. This paper presents the first application of CE to separate and online mass analyze the different constituents of underivatized R-form LPS and lipid A with an FT ion cyclotron resonance mass spectrometer. A Beckman P/ACE MDQ and a home-made CE instrument were coupled to the mass spectrometer using a sheath liquid interface. An optimized CE electrolyte based on water, isopropyl alcohol, triethylamine and acetic acid at pH 8.9 allowed the separation of molecular species even of complex mixtures to a large extent with observed detection limits of single constituents of the samples in the low femtomol range. Comparison of CE-MS and MS measurements under equal conditions enabled the investigation of ion suppression effects in LPS samples.

Determination of the surface heat‐transfer coefficient in CE

A knowledge of the heat-transfer coefficient, h(s), for the external surface of the capillary or the overall heat coefficient, h(OA), is of great value in predicting the mean increase in temperature of the electrolyte, DeltaT(Mean), during electrokinetic separations. For CE, traditional indirect methods of determining h(s) were time-consuming and tended to overestimate cooling efficiency; a novel method is introduced, which is based on curve-fitting of plots of conductance versus voltage to calculate several important parameters including DeltaT(Mean), h(s), the conductance free of Joule heating effects (G(0)) and the voltage that causes autothermal runaway, V(lim). The new method is superior to previously published methods in that it can be performed more quickly and that it corrects for systematic errors in the measurement of electric current for voltages <5 kV. These errors tended to exaggerate the cooling efficiency of commercial instruments so that the calculated increases in electrolyte temperature were smaller than their actual values. Axially averaged values for h(s) were determined for three different commercial CE instruments ranging from 164 W m(-2) K(-1) for a passively cooled instrument in a drafty environment to 460 W m(-2) K(-1) for a liquid-cooled instrument.