Electrophoretic Separation Systems Research Articles

Deep eutectic solvents combined with beta-cyclodextrin derivatives for chiral separation of typical adrenergic receptor agonists by capillary electrophoresis with amperometric detection

Enantioseparation has always been one of the research hotspots and difficulties in the field of modern separation science. In this work, a binary chiral electrophoretic separation system was constructed using deep eutectic solvents (DESs) coupled with beta-cyclodextrin derivatives based on capillary electrophoresis with amperometric detection system, and five groups of typical adrenergic receptor agonists (adrenaline, salbutamol, isoproterenol, norepinephrine and terbutaline) were selected as the model enantiomers. The effects of additive types and contents of DESs and cyclodextrins, and the pH value and concentration of the running buffer on the resolution of the selected chiral compounds were investigated in detail. The mechanism of DESs improving separation was explored preliminarily by means of UV spectrophotometry, which was further verified based on the comparison of single and mixed components of choline chlorine-urea DES. Under the optimum conditions, the relative standard deviations for inter-day and intra-day repeatability of the migration time, peak area and resolution for adrenaline and salbutamol were within 8.7%, and the limits of detection reached 0.030 μg mL−1 (S/N = 3). The recovery data were in the range of 96.3–118.7%. The developed methods have been applied for the analyses of (+)-adrenaline hydrochloride injection and (±)-salbutamol aerosol. This binary chiral electrophoretic separation system by CE-AD has high detection sensitivity and low analytical cost, providing an alternative for the separation and analysis of chiral drugs.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Development of Sperm Separation System Using Electrical Current for Bull

A novel electrophoretic separation system has been successfully applied for the preparation of human sperm prior to the execution of assisted reproductive techniques (ARTs). This new system is designed to overcome the generation of reactive oxygen species (ROS) through centrifugation in conventional sperm preparation. Since the previous study showed favorable outcomes in humans, this study intends to implement this new system for animal sperm preparation particularly in bull. Fresh semen from adult bulls were used. Optimization of the electrophoretic system for optimum bull sperm separation involved different strength of voltage and separation time. The voltages applied were 10V, 20V, 30V, 40V, 50V, and 60V. For each voltage applied, the system was operated for a duration of 12 min . An average of 10 µl fractionalized semen was taken out at the collection site at every 2- min interval. Every fractionated sperm was then evaluated for percentage of viability, motility, and DNA damage assessment. Result showed that electrophoresis at 20V and 6 min yielded more than 80% viable and more than 70% motile sperm population with the lowest DNA damage. In conclusion, the system was able to fractionate high quality bull sperm at 20V and 6 min . DOI : http://dx.doi.org./10.17576/JSKM-2019-1701-13

Enhancement of the conductivity detection signal in capillary electrophoresis systems using neutral cyclodextrins as sweeping agents.

Conductivity detection is a universal detection technique often encountered in electrophoretic separation systems, especially in modern chip-electrophoresis based devices. On the other hand, it is sparsely combined with another contemporary trend of enhancing limits of detection by means of various preconcentration strategies. This can be attributed to the fact that a preconcentration experimental setup usually brings about disturbances in a conductivity baseline. Sweeping with a neutral sweeping agent seems a good candidate for overcoming this problem. A neutral sweeping agent does not hinder the conductivity detection while a charged analyte may preconcentrate on its boundary due to a decrease in its effective mobility. This study investigates such sweeping systems theoretically, by means of computer simulations, and experimentally. A formula is provided for the reliable estimation of the preconcentration factor. Additionally, it is demonstrated that the conductivity signal can significantly benefit from slowing down the analyte and thus the overall signal enhancement can easily overweight amplification caused solely by the sweeping process. The overall enhancement factor can be deduced a priori from the linearized theory of electrophoresis implemented in the PeakMaster freeware. Sweeping by neutral cyclodextrin is demonstrated on an amplification of a conductivity signal of flurbiprofen in a real drug sample. Finally, a possible formation of unexpected system peaks in systems with a neutral sweeping agent is revealed by the computer simulation and confirmed experimentally.

Electroosmotic Flow Dispersion of Large Molecules in Electrokinetic Migration.

It is a common knowledge that large molecules have small diffusion coefficients. Therefore, it was thought that these molecules should generate very narrow peaks in electropherograms, because longitudinal diffusion was thought to be the only factor that contributes to band broadening in electrophoretic separation systems in capillaries and microfluidics. However, it is also common to observe that large molecules such as proteins and polymers often produce wider peaks than most small molecules. It is demonstrated in this work that dispersion of analytes during electrokinetic migration is also the results of Taylor dispersion. Similar to conventional Taylor dispersion, peak broadening is more pronounced for particles with smaller diffusion coefficients. The theoretical description of band broadening caused by electroosmotic flow dispersion (EOFD) and the experimental verification of this phenomenon are presented in this paper. The dispersion of various sized molecules, from small peptides to large proteins, in capillary electrophoresis is used as examples in this study.

The ePrep-System: A new electrophoretic approach for DNA isolation from biological samples.

We present a new free-flow electrophoretic separation system that extends the established concepts of nucleic acid migration in an electric field to a broadly applicable preparative scale. The system comprises a disposable flow tube in which the target nucleic acids are separated from impurities by a balanced combination of electrophoretic migration and counter-streaming electroosmotic flow under the influence of an applied external electric field. Despite the complex theoretical background the introduced electrophoretic technology offers simple hardware setup and handling protocols. A variable number of small and disposable flow tubes can be processed in parallel, which largely eliminates the cumulative increase in extraction times inherent to batch processing methods and allows faster throughput of intermediate sample numbers. We demonstrate easy isolation of nucleic acids without user interaction during the run by using existing and well established lysis chemistries. Sample loading is realized by concentrated transfer of DNA-loaded magnetic beads from a lysis reaction into the extraction flow tube. The present study centers on the development of a functional model for the device and the flow tube as well as a preliminary standard extraction protocol. The system is compatible with a broad range of sample types and we present proof of principle data demonstrating its suitability for biomarker detection in translational research applications.

Biochemical characterization of stallion prostasomes and comparison to their human counterparts

Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins.

Column coupling isotachophoresis–capillary electrophoresis with mass spectrometric detection: Characterization and optimization of microfluidic interfaces

Two-dimensional electrophoretic separations are one of the most promising tools for the continuously growing needs of different bioanalytical fields such as proteomics and metabolomics. In this work we present the design and the implementation of a two-dimensional electrophoretic separation coupled to mass spectrometry. We started our work studying the sample transfer characteristics of different microfluidic interfaces compatible with capillary coupling for two-dimensional electrophoretic separations. These junctions are aimed at method decoupling and sample transfer in a modular two-dimensional electrophoretic separation system. In order to perform the characterization of the interfaces, we carried out capillary electrophoresis experiments and numerical simulations using three cationic compounds under different flow conditions. The comparison of the experimental and simulation results enables us to clearly define the desirable characteristics of interfaces in order to achieve method orthogonality with lossless sample transfer in a two-dimensional separation system. Finally, we present a glass microfluidic chip as interface for the implementation of a novel hybrid modular system for performing two-dimensional electrophoretic separations involving isotachophoresis and capillary electrophoresis. In this setup we include mass spectrometric and contactless capacitively coupled conductivity detection to monitor the separation process. We demonstrate the ability of the setup to be used as a flexible analysis tool by performing preconcentration, separation, detection and identification of four different human angiotensin peptides.

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

Eighth Polish Conference on Analytical Chemistry

The Polish Conference on Analytical Chemistry has a long and rich history. The first such scientific forum took place in Warsaw in 1981. Since 1995, Polish analysts have had a chance to meet every 5 years in Gdansk, Gliwice, Torun, and—in 2010—in Krakow. The VIIIth Polish Conference on Analytical Chemistry was held on July 4–9. It was organized by the Department of Analytical Chemistry, Jagiellonian University, in cooperation with the Polish Committee of Analytical Chemistry of the Polish Academy of Sciences. The meeting attracted 570 participants, who presented their works in the Auditorium Maximum—the main conference center in Krakow. The conference was opened by Adam Hulanicki—a famous and distinguished scientist, and a patron and tutor to several generations of Polish analysts. In his lecture, he recounted the history of Polish analytical conferences, including many interesting and little-known details. Another impressive presentation was given by a special guest of the conference—Georges Guiochon from the University of Tennessee, Knoxville, USA, who is a specialist in chromatographic analysis. He acquainted listeners with the latest developments in this analytical domain, placing emphasis on methodological and instrumental advances. The regular scientific program of the conference encompassed several plenary lectures, as well as oral and poster sessions. Eminent Polish scientists, working mostly in foreign research centers, were invited to deliver the plenary lectures. They were Anna Brajter-Toth (University of Florida, USA), Ewa Cukrowska (University of the Witwatersrand, South Africa), Pawel Ciborowski (University of Nebraska, USA), Tadeusz Gorecki (University of Waterloo, Canada), Andrzej Lewenstam (Abo Akademi University, Finland), Ryszard Łobinski (National Center for Scientific Research, France), and Marek Trojanowicz (Warsaw University, Poland). In their fascinating speeches, they outlined the current state and future directions of different analytical fields, providing a broad overview of the progress in modern analytical chemistry. The oral and poster presentations were given in 12 separate topical sessions, encompassing all the main analytical domains. A unique feature of this conference was a session titled “Criminalistic Analytics” reflecting the strong scientific collaboration between two Cracovian centers: Jagiellonian University and the Institute of Forensic Research. Another session was devoted to the Symposium on Flow Analysis, which has traditionally been organized in Krakow—for the seventh time in 2010. The thematic sessions revealed that Polish analytical chemistry is developing systematically in terms of methodological, instrumental, and applicative aspects, which are being introduced into all branches of our lives. Protection of human health, the environment, and cultural heritage is still of great importance to Polish analysts. Owing to the creation and application of analytical procedures, methods, and instruments of increasing sophistication, it has become possible to identify and determine ever-decreasing amounts of analytes in various forms and of various levels of threat to our environment. Novel techniques for preparation of biological samples together with chromatographic and electrophoretic separation systems and with mass spectrometry as a detection system have opened up new analytical possibilities in clinical, pharmaceutical, and forensic toxicology. The growing interest in exploitation of nondestructive analytical methods, flow techniques, and integrated and miniaturized instrumental systems for different purposes is also worth mentioning. All the above is evidence that analytical chemistry is increasingly being introduced into our everyday lives, helping to make them healthier, safer, and more comfortable. The papers presented in this special issue of Analytical and Bioanalytical Chemistry are representative of the discussion of this tendency at the conference. In addition to the scientific program, there was a wide range of social activities. A Chopin concert, which was held in the courtyard of Wawel Royal Castle, made a great impression on listeners. The conference banquet took place in one of the halls of the Wieliczka Salt Mine, situated within the Krakow metropolitan area—the mining company is believed to be the world's 14th oldest company still in operation today. During the meeting in Zalesie Inn, guests experienced the atmosphere, folklore, and food of the Cracovian region. Finally, all attendees were invited to sightsee Krakow. First on the list was, of course, the Old Town, with the largest mediaeval market square in Europe—the colorful and crowded Main Market with its pigeons and florists and assorted market stalls. Mariacki Church, from the highest tower of which a bugle-call is sounded every hour, and the museum of the famous Jagiellonian University—Alma Mater Cracoviensis—were also visited. The next Polish Conference on Analytical Chemistry will be held in Poznan in 2015.

Diagnosis of transient electrokinetic flow in microfluidic channels

Measuring the electro-osmotic velocity distributions in microchannels is usually performed for steady-state electrokinetic flows. Characterizing time-dependent electrokinetic flows is of importance to the development of microfluidic devices such as rapid capillary electrophoretic separation system, ac pumps, novel micromixers, etc. In this paper, we use a micron-resolution particle image velocimetry (micro-PIV) based phase locking technique with an ordinary PIV charge coupled device (CCD) camera to carry out an experimental study of the transient electrokinetic flow in microchannels by synchronizing different trigger signals for the laser, CCD camera, and in-house designed high-voltage switch. With the transient micro-PIV technique, we further propose a method to decouple the particle electrophoretic velocity from the micro-PIV measured velocity and to determine the zeta potential of the channel wall. The time evolution of the full-field, electro-osmotic velocity distributions in both open- and closed-end rectangular microchannels is obtained. Using the slip velocity approach and the measured channel zeta potential, the theoretical predictions of the transient electro-osmotic flow in the open- and closed-end microchannels are obtained, and they are found to be in good agreement with the experimental results.

Analysis of aminoimidazole ribosides by capillary electrophoresis — Diagnosing defects in second part of purine biosynthetic pathway

BackgroundOnly three inherited metabolic defects have been identified in purine de novo synthesis (PDNS). We present here CE methods for diagnosing defects in the second half of PDNS (from sixth to tenth enzymatic conversion) based on analysis of aminoimidazole ribosides – dephosphorylated intermediates – in urine. MethodsAssays were performed in an uncoated fused-silica capillary using two electrophoretic separation systems: 60 mmol/l borate — 2-amino-2-methyl-1-propanol–80 mmol/l sodium dodecylsulfate (pH 9.6) and 200 mmol/l phosphate — sodium (pH 1.8). ResultsThe reported conditions allowed separation of all metabolites from major urinary constituents with analysis time less than 10 min and separation efficiency of 220 and 350 thousands theoretical plates per meter for borate and phosphate system, respectively. The intra- and interday imprecisions were less than 4.4% and 9.9% CV. Potential usefulness of the methods was demonstrated on samples from a patient with adenylosuccinate lyase deficiency and Chinese hamster ovary cell lines defective in PDNS. ConclusionsCE is a useful and effective tool in the analysis of aminoimidazole ribosides which enables diagnosis of known as well as not so far identified inherited defects of PDNS pathway.

A simple microfluidic system for efficient capillary electrophoretic separation and sensitive fluorimetric detection of DNA fragments using light-emitting diode and liquid-core waveguide techniques

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.

DNA sequencing and genotyping in miniaturized electrophoresis systems

Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.

Design and Optimization of Compact Microscale Electrophoretic Separation Systems

The ability to manipulate and synthesize chemical species on microchip devices provides access to a new and exciting field. These technologies have several advantages throughout the industrial and scientific communities, especially in the emerging areas of biomedical engineering and the life sciences. Recent advances in microscale mixing, reaction, separation, and fluid handling have opened new areas in which process systems engineering techniques can be applied. Here we discuss our initial efforts at creating automatic synthesis methods for the design of microchip-based electrophoretic separation systems that occupy minimal areas. We use piecewise algebraic and logic models to compare the conflicting design goals of maximum system performance and minimum device area. We have implemented both heuristic and numerical optimization design techniques. The long-term goal of our work is the development of methodologies for the design of complete lab-on-a-chip devices.

Effect of Joule heating on efficiency and performance for microchip-based and capillary-based electrophoretic separation systems: a closer look.

An attempt is made to revisit the main theoretical considerations concerning temperature effects ("Joule heating") in electro-driven separation systems, in particular lab-on-a-chip systems. Measurements of efficiencies in microfabricated devices under different Joule heating conditions are evaluated and compared to both theoretical models and measurements performed on conventional capillary systems. The widely accepted notion that planar microdevices are less susceptible to Joule heating effects is largely confirmed. The heat dissipation from a nonthermostatically controlled glass microdevice was found to be comparable to that from a liquid-cooled-fused silica capillary. Using typically dimensioned glass and glass/silicon microdevices, the experimental results indicate that 5-10 times higher electric field strengths can be applied than on conventional capillaries, before detrimental effects on the separation efficiency occur. The main influence of Joule heating on efficiency is via the establishment of a radial temperature profile across the lumen of the capillary or channel. An overall temperature increase of the buffer solution has only little influence on the quality of the separation. Still, active temperature control (cooling, thermostatting) can help prevent boiling of the buffer and increase the reproducibility of the results.

Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture.

In vitro liver preparations are being used increasingly to study various aspects of chemical hepatotoxicity and thus have become powerful alternatives to in vivo toxicologic models. Primary hepatocyte culture systems are especially useful in screening cytotoxic and genotoxic compounds and assessing biochemical lesions associated with chemical exposure. We have begun to use this approach in combination with proteomic analysis to construct a molecular "toxicoproteomic" test system for a broad range of relevant and potentially toxic chemicals. Using a highly parallel two-dimensional electrophoretic (2-DE) protein separation system to analyze cells from culture systems, we previously observed significant variations in protein expression that were unrelated to chemical exposure. We hypothesized these artifactual protein alterations were the result of the variations in the culture conditions or cell manipulations, or both. Therefore, we conducted a study to assess the expression of hepatocyte proteins cultured on 6-well plates and recovered for analysis either by scraping/pelleting or direct in-well solubilization. Following incubation of 1.2 x 10(6) hepatocytes in six-well plate, recovery and solubilization of the cells and 2-DE of the solubilized lysates of 100 000 cells, we detected 1388 proteins in the in-well solubilized samples compared to 899 proteins in the washed/scraped/pelleted cell samples, a loss of 35%. Based on protein identification by peptide mass fingerprinting, the subcellular location of nearly all of the proteins whose abundance decreased were cytosolic and those few that increased were either microsomal, mitochondrial, or cytoskeletal proteins. These results emphasize the variation introduced by cell-handling during recovery of hepatocytes from culture plates and may explain at least some of the artifactual differences observed in earlier in vitro experiments.

Coupling of a Gas-Phase Chemiluminescence Nitrogen Detector and a Capillary Electrophoretic System

A capillary electrophoretic system has been successfully connected to a gas-phase chemiluminescence nitrogen detector via a pneumatic nebulizer interface. The interface, built in-house, consists of a nebulizing gas delivery subsystem, a sheath liquid subsystem, a short spray tube, and a liquid gap. The liquid gap is formed at the point where the spray tube, the separation capillary, and the sheath liquid subsystem meet. The sheath liquid subsystem consists of a grounding connection to complete the electric circuit for the electrophoretic system, a sheath liquid delivery pump, a sheath liquid overflow outlet maintained at atmospheric pressure, and a sheath liquid conduit with a hydrodynamic resistance that is much smaller than that of the separation capillary. The interface operates at the natural self-aspiration rate of the short spray tube. The design ensures that the natural self-aspiration rate of the nebulizer is higher than the maximum electroosmotic flow rate that can be produced in the separation capillary. The flow difference is made up by the sheath liquid which, due to the hydrodynamic resistance differences, is sucked into the liquid gap preferentially from the sheath liquid conduit. Thus, the spray tube and the separation capillary are decoupled from each other hydrodynamically, and any laminar flow-induced additional band broadening in the separation capillary is avoided. Using the combined electrophoretic separation and gas-phase chemiluminescence nitrogen detector system, the mass detection limit for five nucleotide bases used as test compounds was found to be about 10 pmol nitrogen.

Rapid and accurate determination of (CAG) n repeats in the androgen receptor gene using polymerase chain reaction and automated fragment analysis

Objectives: To develop and evaluate a new method for determination of the CAG repeat length in Exon 1 of the androgen receptor gene. Design and Methods: The method is based on PCR amplification of a DNA region encompassing the repeats and analysis of the length of the PCR product on a sequencing gel. One of the PCR primers was labeled with Cy5.5 fluorescent dye to facilitate detection after laser excitation. We used a fully automated system for electrophoretic separation of the PCR product and accurate sizing of the length of the PCR product using fragment analysis. Results: The major advantages of the new technique are its simplicity, speed, accuracy, and reproducibility. Analysis of the CAG repeats in genomic DNAs from 18 males indicated that they were all hemizygous with a mean CAG repeat number of 22 (range 20–30 repeats). Among 60 DNAs from females, 16 were homozygous and 44 were heterozygous. The repeat length ranged from 17–30 with a mean of 22. In both males and females, the distribution of CAG repeats was bimodal. Conclusion: We anticipate that this improved method for CAG repeat analysis will find applications in clinical studies involving prostate and breast cancer patients.

Effects of separation potential, hydrostatic pressure and auxiliary electroosmotic pumping on a suppressed conductometric capillary electrophoresis separation system

The separation potential, hydrostatic pressure and an auxiliary electroosmotic pump can all affect the migration time of an analyte in a capillary electrophoretic system. We report here a systematic study of the effects of variations in these parameters upon practical ionic separations, especially as applied to a suppressed conductometric capillary electrophoretic separation system (SuCCESS). Under conditions that are held constant during a run, auxiliary electroosmotic pumping generally produces the best results. When the parameter is changed during the run in a manner reminiscent of gradient elution in chromatography, under the optimum condition for each, gradient separation potential and auxiliary electroosmotic pumping produces comparable results except for late-eluting ions for which the latter technique clearly yields higher efficiencies.