Capillary Electrophoresis Instrument Research Articles

A Novel Method to Quantify Bioavailable Elements and Mobile ATP on Rock Surfaces and Lichens

The quantification of mobile ions on rock surfaces is essential for the investigation of mineral weathering. A need for such measurements arises from the study of initial soil formation in pioneering environments, the biogeochemical weathering of monuments and buildings, and the chemical reactivity of minerals in general. In the case of mineral surfaces covered by lichens, the quantification of adenosine triphosphate (ATP) is a measure of the vitality of the organisms. To date, non-destructive investigations of rock surfaces and growth of biofilms have generally been limited to visual methods. We evaluated a new technique for the analysis of readily available ions and ATP. For this, a single drop of pure water is spread on bare mineral surfaces or rock-based crustose lichens. The solution is recollected and analyzed for dissolved ions and ATP using a portable capillary electrophoresis instrument and a luminometer, respectively. We illustrate the natural heterogeneity of available ions on freshly broken granite surfaces and the effects of subsequent wetting, freezing, and thawing. In addition, the influence of humidity and age of crustose lichens is demonstrated by ion and ATP analysis.

Portable capillary electrophoresis instrument with contactless conductivity detection for on-site analysis of small volumes of biological fluids

A novel, easy to use and portable capillary electrophoretic instrument for injection of small volumes of biological fluids equipped with contactless conductivity detection was constructed. The instrument is lightweight (<5 kg), all necessary parts including a tablet computer are accommodated in a plastic briefcase with dimensions 20cm×33cm×17cm (w×l×h), allows hydrodynamic injection of small sample volumes and can continuously operate for at least 10hours. The semi-automated hydrodynamic sample injection is accomplished via a specially designed PMMA interface that is able to repeatedly inject sample aliquots from a sample volume as low as 10μL, with repeatability of peak areas below 5%. The developed interface and the instrument were optimized for the injection of biological fluids. Practical utility was demonstrated on the determination of formate in blood serum samples from acute methanol intoxication patients and on the analysis of ionic profile (nitrosative stress markers, including nitrite and nitrate) in the exhaled breath condensate from one single exhalation.

Headspace in-tube microextraction coupled with micellar electrokinetic chromatography of neutral aromatic compounds

Headspace (HS) extraction can be carried out easily and aptly via single drop microextraction coupled with capillary electrophoresis (CE). However, one drawback is the difficulty of keeping the single drop stably at the capillary tip. To solve this problem, we have recently demonstrated HS in-tube microextraction (ITME) of acidic compounds such as chlrophenols in an acidic sample using a basic run buffer plug in the separation capillary for CE as an acceptor phase. In this report, an organic acceptor plug in a capillary was used to extract neutral organic volatile pollutants such as BTEX (benzene, toluene, ethylbenzene, and m-xylene). After extraction, the analytes enriched in the organic acceptor plug were analyzed with micellar electrokinetic chromatography (MEKC). The enrichment factors for BTEX in a standard solution were up to 350 under an optimal condition of 25°C for 20min. As an application, BTEX spiked into bottled water were analyzed with HS-ITME-MEKC, and the enrichment factors for BTEX were up to 320. The limits of detections were 1–4ppb, which are at least 200 times lower than the US Environmental Protection Agency guidelines for drinking water, except benzene. The entire procedure of HS-ITME-MEKC was carried out automatically using a commercial CE instrument.

Screening determination of four amphetamine-type drugs in street-grade illegal tablets and urine samples by portable capillary electrophoresis with contactless conductivity detection

A simple and inexpensive method for the identification of four substituted amphetamines, namely, 3,4-methylenedioxy methamphetamine (MDMA), methamphetamine (MA), 3,4-methylenedioxy amphetamine (MDA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) was developed using an in-house constructed semi-automated portable capillary electrophoresis instrument (CE) with capacitively coupled contactless conductivity detection (C4D). Arginine 10mM adjusted to pH4.5 with acetic acid was found to be the optimal background electrolyte for the CE-C4D determination of these compounds. The best detection limits achieved with and without a sample preconcentration process were 10ppb and 500ppb, respectively. Substituted amphetamines were found in different seized illicit club drug tablets and urine samples collected from different suspected users. Good agreement between results from CE-C4D and those with the confirmation method (GC-MS) was achieved, with correlation coefficients for the two pairs of data of more than 0.99.

Increasing Allele Detection by Altering the Quantity of Internal Lane Standard.

Electrokinetic injection (EI) is the primary method used in forensic laboratories to load amplified PCR product in capillary electrophoresis for short tandem repeat (STR) fragment separation. Because all samples subjected to capillary electrophoresis use internal lane standard (ILS), this study investigated the consequence of varying the volume of ILS and its effects on allele peak heights and number of alleles detected. Results demonstrated that when the volume of ILS is reduced, the average peak height and number of alleles increased, thereby increasing the sensitivity of the detection method. Sizing anomalies were observed; however, they did not adversely affect accuracy and precision. The method developed in this study offers a simple and universal procedure to increase the alleles detected in forensic STR analysis. Reducing the volume of ILS to achieve greater sensitivity is applicable to all STR amplification kits and capillary electrophoresis instruments currently used in forensic DNA analysis.

Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).

Abstract 4933: Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel

Abstract The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research. The Ion Ampliseq™ cancer hotspot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable mutation targets present in 50 genes and the more comprehensive Ion Oncomine™ cancer panel (OCP) contains over 2000 mutations. A hallmark of these Ion Torrent Ampliseq cancer panels is the low amount of input DNA needed which is critical when the clinical specimen material is limited such as with fine needle biopsy or FFPE samples. Typically, 10 ng of DNA obtained from these sources is sufficient to produce informative sequencing data. Often, cancer-causing or promoting mutations are detected at relatively low allele frequencies like 10-20% compared to the major normal allele. Many researchers wish to verify these findings of low frequency mutations by an orthologous method such as traditional dye-fluorescent Sanger sequencing on a capillary electrophoresis (CE) instrument such as the Applied Biosystems 3500 genetic analyzer. To that end, we have developed a workflow that enables the amplification and traditional Sanger sequencing of individual Ion AmpliSeq targets directly from the AmpliSeq library starting material. The method requires a retainer of 1 μl (∼ 5%) of the original AmpliSeq preamplification material. A dilution of this aliquot is used as template source for individualized PCR/sequencing reactions. We show that a random selection of 48 targets from the CHPv2 panel could be successfully amplified and Sanger-sequenced from an Ion Torrent Ampliseq library originally prepared from 10 ng of FFPE DNA. Furthermore, we show the successful Sanger-re-sequencing of all individual 24 targets covering the TP53 exons from the same sample processed and pre-amplified with the OncoMine AmpliSeq panel. Taken together, this method will enable researchers to reflex-test potential mutations of interest from very material-limited specimen using Sanger CE sequencing. Citation Format: Edgar H. Schreiber, Adam Broomer, Mark Andersen, Kamini Varma. Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4933. doi:10.1158/1538-7445.AM2015-4933

Estimating allele dropout probabilities by logistic regression: Assessments using Applied Biosystems 3500xL and 3130xl Genetic Analyzers with various commercially available human identification kits.

Phenomena called allele dropouts are often observed in crime stain profiles. Allele dropouts are generated because one of a pair of heterozygous alleles is underrepresented by stochastic influences and is indicated by a low peak detection threshold. Therefore, it is important that such risks are statistically evaluated. In recent years, attempts to interpret allele dropout probabilities by logistic regression using the information on peak heights have been reported. However, these previous studies are limited to the use of a human identification kit and fragment analyzer. In the present study, we calculated allele dropout probabilities by logistic regression using contemporary capillary electrophoresis instruments, 3500xL Genetic Analyzer and 3130xl Genetic Analyzer with various commercially available human identification kits such as AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. Furthermore, the differences in logistic curves between peak detection thresholds using analytical threshold (AT) and values recommended by the manufacturer were compared. The standard logistic curves for calculating allele dropout probabilities from the peak height of sister alleles were characterized. The present study confirmed that ATs were lower than the values recommended by the manufacturer in human identification kits; therefore, it is possible to reduce allele dropout probabilities and obtain more information using AT as the peak detection threshold.

Stacking in a continuous sample flow interface in capillary electrophoresis

Using a tee connector in a commercial capillary electrophoresis instrument, the effect of field amplified sample injection from both flowing and static sample volumes was investigated. It is shown that under identical conditions (40min electrokinetic injection at 5kV from a sample volume of 295μL) the limit of detection using the continuous sample flow interface is 4 times lower than from a static vial. The relationship between different flow rates and injection voltages on the injected sample amount was also investigated using a 2D axisymmetric simulation (COMSOL 4.3b) and verified experimentally, confirming conditions under which there is near-quantitative injection of the sample target ions. Using electrokinetic injection at 30kV and a flow rate of 558nL/s the same enhancement from an even smaller volume of 184μL could be achieved in 5.5min than could be achieved from 295μL and a 40min injection. This sensitivity enhancement factor corresponded to four orders of magnitude improvement compared to a hydrodynamic injection. This is the first report showing that a continuous sample flow interface combined with stacking methods under conditions approaching quantitative injection from the entire sample volume has the potential to be more sensitive than a static system.

Correlating molar masses of nitrocelluloses with their intrinsic viscosities measured using capillary electrophoresis instrumentation

Specific viscosities for a set of six nitrocellulose (NC) standards comprising three different mass-average molar masses (between 20,000 and 300,000gmol−1) of two different nitrogen contents (11.2 and 12.1%) were measured at 20°C in tetrahydrofuran, using capillary electrophoresis instrumentation as a bench-top viscometer in frontal mode. Intrinsic viscosities were derived applying Huggins’ and Kraemer's models, showing excellent convergence of both models at infinitely diluted polymer concentration. Good overall consistency was shown between viscosity data experimentally acquired by this new protocol and the mass-average molar masses provided by the manufacturers. This simple protocol should be of interest for a better understanding of the solvent interaction given by this complex polymer, and beyond this, for tailoring NC solutions devoted to film deposition, and for the determination of mass-average molar masses of unknown NC samples.

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%-100%) and 99.6% specificity (95% CI, 98.5%-99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates post-PCR handling for all non-expanded samples, and exemplifies a truly efficient screening procedure.

In-house-made capillary electrophoresis instruments coupled with contactless conductivity detection as a simple and inexpensive solution for water analysis: a case study in Vietnam.

A simple and inexpensive method for the determination of various ionic species in different water matrices is discussed in this study. The approach is based on the employment of in-house-made capillary electrophoresis (CE) instruments with capacitively coupled contactless conductivity detection (C(4)D), which can be realized even when only a modest financial budget and limited expertise are available. Advantageous features and considerations of these instruments are detailed following their pilot deployment in Vietnam. Different categories of ionic species, namely major inorganic cations (K(+), Na(+), Ca(2+), Mg(2+), and NH4(+)) and major inorganic anions (Cl(-), NO3(-), NO2(-), SO4(2-), and phosphate), in different water matrices in Vietnam were determined using these in-house fabricated instruments. Inorganic trivalent arsenic (As(iii)), which is the most abundant form of arsenic in reducing groundwater, was determined by CE-C(4)D. The effect of some interfering ions in groundwater on the analytical performance was investigated and is highlighted. The results from in-house-made CE-C(4)D-instruments were cross-checked with those obtained using the standard methods (AAS, AES, UV and IC), with correlation coefficients r(2) ≥ 0.9 and deviations from the referenced results less than 15%.

Highly-sensitive amplification-free analysis of multiple miRNAs by capillary electrophoresis.

Sets of deregulated microRNAs (miRNAs), termed miRNA signatures, are promising biomarkers for cancer. Validation of miRNA signatures requires a technique that is accurate, sensitive, capable of detecting multiple miRNAs, fast, robust, and not cost-prohibitive. Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a capillary electrophoresis (CE)-based hybridization assay that was suggested as a methodological platform for validation and clinical use of miRNA signatures. While satisfying the other requirements, DQAMmiR is not sufficiently sensitive to detect low-abundance miRNAs. Here, we solve this problem by combining DQAMmiR with the preconcentration technique, isotachophoresis (ITP). The sensitivity improved 100 times (to 1 pM) allowing us to detect low-abundance miRNAs in an RNA extract. Importantly, ITP-DQAMmiR can be performed in a fully automated mode using a commercial CE instrument making it suitable for practical applications.

Concurrent determination of anions and cations in consumer fireworks with a portable dual-capillary electrophoresis system

A new automated portable dual-channel capillary electrophoresis instrument was built and applied to the concurrent determination of cations and anions. The system uses a single buffer and hydrodynamic injection of the sample is performed autonomously. A novel engraved flow-cell interface is used at the injection ends of the capillaries allowing the autonomous operation of the system. The engraved flow-cell replaces traditionally used split injectors in purpose made capillary electrophoresis systems and makes the system design easier. A new software package with graphical user interface was employed to control the system, making its operation simple and increasing its versatility. The electrophoretic method was optimized to allow the baseline separation of 12 cations and anions commonly found in fireworks. The system was proven to be useful for the analysis of consumer fireworks, saving time and expenses compared to separate analyses for anions and cations. This is the first time that cationic and anionic compositions of fireworks are investigated together. The analysis of samples revealed several inaccuracies between the declared compositions for the fireworks and the obtained results, which could be attributed to cross-contamination during their manufacture or to a transfer between other components of the pyrotechnic item. The presence of certain unexpected peaks, however, had no apparent reason and might represent an irregularity in the manufacture of some devices.

Generalized polymer effective charge measurement by capillary isotachophoresis

In this work, we have generalized the use of capillary isotachophoresis as a universal method for determination of effective charge of anionic and cationic (co)polymers on ordinary capillary electrophoresis instruments. This method is applicable to a broad range of strong or weak polyelectrolytes with good repeatability. Experimental parameters (components and concentrations of leading and terminating electrolytes, capillary diameters, constant electric current intensity) were optimized for implementation in 100μm i.d. capillaries for both polyanions and polycations. Determined values of polymer effective charge were in a very good agreement with those obtained by capillary electrophoresis with indirect UV detection. Uncertainty of the effective charge measurement using isotachophoresis was addressed and estimated to be ∼5–10% for solutes with mobilities in the 20–50×10−9m2V−1s−1 range.

ChemInform Abstract: On‐line Preconcentration in Capillary Electrophoresis for Analysis of Agrochemical Residues

AbstractReview: 175 refs.

Editorial Board: Electrophoresis 16'14

ELECTROPHORESISVolume 35, Issue 16 Editorial BoardFree Access Editorial Board: Electrophoresis 16'14 First published: 11 August 2014 https://doi.org/10.1002/elps.201470141AboutPDF 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. Volume35, Issue16Special Issue: Instrumentation for Capillary Electrophoresis and Microchip ElectrophoresisAugust 2014 RelatedInformation

Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of β-agonists in pharmaceutical and pig-feed samples

An inexpensive, robust and easy to use portable capillary electrophoresis instrument with miniaturized high-voltage capacitively coupled contactless conductivity detection was developed. The system utilizes pneumatic operation to manipulate the solutions for all flushing steps. The different operations, i.e. capillary flushing, interface rinsing, and electrophoretic separation, are easily activated by turning an electronic switch. To allow the analysis of samples with limited available volume, and to render the construction less complicated compared to a computer-controlled counterpart, sample injection is carried out hydrodynamically directly from the sample vial into the capillary by manual syphoning. The system is a well performing solution where the financial means for the highly expensive commercial instruments are not available and where the in-house construction of a sophisticated automated instrument is not possible due to limited mechanical and electronic workshop facilities and software programming expertise. For demonstration, the system was employed successfully for the determination of some β-agonists, namely salbutamol, metoprolol and ractopamine down to 0.7ppm in pharmaceutical and pig-feed sample matrices in Vietnam.

High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation

Viscosity characterization of protein formulations is of utmost importance for the development of subcutaneously administered formulations. However, viscosity determinations are time-consuming and require large sample volumes in the range of hundreds of microliters to a few milliliters, depending on the method used. In this article, an automated, high-throughput method is described to determine dynamic viscosity of Newtonian fluids using standard capillary electrophoresis (CE) equipment. CE is an analytical method routinely used for the separation and characterization of proteins. In our set-up, the capillary is filled with the test sample, and a constant pressure is applied. A small aliquot of riboflavin is subsequently loaded into the capillary and used as a dye to monitor movement of protein samples. Migration time of the riboflavin peak moving through the filled capillary is converted to the viscosity by applying the Hagen–Poiseuille's law. The instrument is operated without using an electrical field. Repeatability, robustness, linearity, and reproducibility were demonstrated for different capillary lots and instruments, as well as for different capillary lengths and diameters. Accuracy was verified by comparing the viscosity data obtained by CE instrumentation with those obtained by plate/cone rheometry. The suitability of the method for protein formulations was demonstrated, and limitations were discussed. Typical viscosities in the range of 5–40mPas were reliably measured with this method. Advantages of the CE instrumentation-based method included short measurement times (1–15min), small sample volumes (few microliters) for a capillary with a diameter of 50μm and a length of 20.5cm as well as potential to be suitable for high-throughput measurements.

Automated dual capillary electrophoresis system with hydrodynamic injection for the concurrent determination of cations and anions

The capillary electrophoresis instrument developed for the concurrent determination of cations and anions features two separate capillaries and individual detectors to allow independent optimization for each group of ions. The capillaries are joined in a common injector block. The sample is drawn into the injector with a small membrane pump and automated simultaneous injection into both capillaries is achieved by pressurization of the fluid with compressed air. Flushing of the injector and of the capillaries with the background electrolyte is also carried out automatically by the same means. The buffer consisted of 12mM histidine and 2mM 18-crown-6 adjusted to pH 4 with acetic acid and was suitable for the contactless conductivity detection employed. The system was optimized for the determination of cationic NH4+ and anionic NO3− and NO2−, and linear calibration curves from about 20μM up to about 1.5mM were obtained for these ions. In a test run over 8h, the reproducibility for the peak areas was within ±7%. For demonstration, the instrument was successfully applied to the concurrent monitoring of the concentrations of the three ions during the biological removal of ammonium from contaminated groundwater in a sequencing batch reactor, where NO3− and NO2− are formed as intermediate products.