Capillary Electrophoresis Equipment Research Articles

Pediatric reference intervals of the hemoglobin fractions HbA2, HbF and HbA0 using high-performance liquid chromatography and capillary electrophoresis

Introductionβ-thalassemia is a common genetic disorder with an estimated prevalence of 80–90 million carriers worldwide. As elevated hemoglobin A2 (HbA2) is a primary feature of carriers, hemoglobin fraction analysis is a common technique used for initial screening. However, pediatric reference intervals (RIs) are scarce. Hence, the aim was to establish pediatric RIs of hemoglobin fractions using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). MethodsSamples were collected from assumed healthy children and adolescents of 1–18 years. Analyses were conducted using the Tosoh Automated Glycohemoglobin Analyzer HLC-723®G11 (Tosoh G11, HPLC) and the Capillarys 3 Octa (CE). Data were investigated for need of partitioning by both age (1–6 years vs. 6–18 years) and sex. ResultsIn total, 189 and 196 subjects were included in the statistical analysis of HPLC and CE, respectively. The 95% RI of HbA2 was 2.00–2.90% by HPLC and 2.2–3.0% by CE. Partitioning of data was not clinically relevant by HPLC. However, partitioning by age was suggested by CE. ConclusionRIs of hemoglobin fractions in individuals of 1–18 years using commercially available HPLC and CE equipment were reported. This is the first report of a pediatric RI of HbA2 using the Tosoh G11.

Study on development of an analytical procedure to quantify glyphosate by capillary electrophoresis method and its application for beverage

Nowadays, the issue of fresh food, drinks, and residue analysis of toxic compounds in food and drinks is increasingly interested in society. In this study, an analytical procedure for quantitative analysis of the glyphosate residue in beverages such as tea infusion and beer was developed based on the capillary electrophoresis method with capacitively-coupled contactless conductivity detector combined with sample preparation using solid phase extraction technique. The analytical conditions optimised for capillary electrophoresis equipment included: histidine/acetic acid background electrolyte solution with histidine concentration of 1 mM and pH value was adjusted to 2.75 by acetic acid; a separation voltage was 20 kV was applied and a high voltage injection at 20 kV for 10 seconds was chosen. The optimised analytical procedure has resulted in a low detection limit of glyphosate (0.42 μg/l), the repeatability and reproducibility expressed by the relative standard deviation of the peak area and migration time were both less than 10%, a wide linear range, and the obtained recovery efficiency of glyphosate on different drinks were achieved to values ranging from 88.7 to 96.3%.

3-(Methacryloxy)propyl]trimethoxysilane-based monolithic stationary phases for capillary-scale chromatography and their characterization by contactless conductivity detection and solid-state NMR spectroscopy

This work describes the preparation, characterization and application of monolithic stationary phases for capillary-scale chromatography. The columns were prepared by polymerization of [3-(methacryloxy)propyl]trimethoxysilane (MPTMS) monomer in two consecutive steps: i) sol–gel process and ii) photoinitiated radical addition. The photopolymerization was carried out in 100 μm i.d. UV-transparent coating fused silica capillaries. The columns were applied in the qualitative separation of five polycyclic aromatic hydrocarbons (PAH) by both capillary liquid chromatography (nano-LC) and capillary electrochromatography (CEC) in a capillary electrophoresis equipment. In an attempt to improve the separation of the PAHs, the stationary phase was modified by incorporating benzyl methacrylate (BM) as a comonomer in the MPTMS-based polymer structure, but no improvement was observed. The physical homogeneity of the columns was evaluated by using an optical microscope and a capacitively coupled contactless conductivity detector (C4D) as a non-invasive scanner moving longitudinally along the capillary. The chemical characterization of the polymer was done by both 13C and 29Si solid-state nuclear magnetic resonance spectroscopy (SSNMR). It was possible to propose a carbon chain structure and the substitution degree of the silyl group indicating the cross-linking of the polymer chains through the formation of dimers and trimers by condensation of the alkoxysilane groups.

Study of insertion-deletion polymorphisms (indels) in the UAE population

Insertion-deletion polymorphisms (indels) have been used less in forensic identification of individuals in comparison to short tandem repeat (STR) and single nucleotide polymorphism (SNP) systems. The use of indels has many advantages including low costs, simple typing methods as well as a small amplicon size of under 160 bp making them suitable for genotyping highly degraded DNA. They can also be valuable as a complementary tool to help resolve kinship cases, with the advantage of lower mutation rates when compared to STRs. The main objective of this study was to investigate the polymorphism of 30 indel loci in the UAE population and to evaluate its suitability for forensic applications. The Qiagen Investigator® DIPplex kit was used to amplify 30 biallellic indel loci and the amelogenin locus in 500 blood samples. This technique was robust, relatively simple, and the results were analysed using the same capillary electrophoresis equipment and software used for STR typing. Allele frequencies were evaluated and forensic parameters (match probability, power of discrimination, polymorphism information content, power of exclusion and typical paternity index) were estimated for the 30 autosomal indel loci. No significant departure from Hardy-Weinberg equilibrium (HWE) expectations were observed for most of the indel loci analyzed in the samples obtained from the UAE population. The combined power of discrimination (CPD) and combined match probability (CMP) values were 0.999999999999754 and 2.46 × 10−13, respectively, which indicated that the 30 indel loci are highly polymorphic and are appropriate for forensic human identification purposes. Even when the match probability of STRs was low, indels gave an acceptable value for forensic application.

Integration of three-phase microelectroextraction sample preparation into capillary electrophoresis

A major strength of capillary electrophoresis (CE) is its ability to inject small sample volumes. However, there is a great mismatch between injection volume (typically <100 nL) and sample volumes (typically 20–1500 µL). Electromigration-based sample preparation methods are based on similar principles as CE. The combination of these methods with capillary electrophoresis could tackle obstacles in the analysis of dilute samples.This study demonstrates coupling of three-phase microelectroextraction (3PEE) to CE for sample preparation and preconcentration of large volume samples while requiring minimal adaptation of CE equipment. In this set-up, electroextraction takes place from an aqueous phase, through an organic filter phase, into an aqueous droplet that is hanging at the capillary inlet. The first visual proof-of-concept for this set-up showed successful extraction using the cationic dye crystal violet (CV). The potential of 3PEE for bioanalysis was demonstrated by successful extraction of the biogenic amines serotonin (5-HT), tyrosine (Tyr) and tryptophan (Trp). Under optimized conditions limits of detection (LOD) were 15 nM and 33 nM for 5-HT and Tyr respectively (with Trp as an internal standard). These LODs are comparable to other similar preconcentration methods that have been reported in conjunction with CE. Good linearity (R2 > 0.9967) was observed for both model analytes. RSDs for peak areas in technical replicates, interday and intraday variability were all satisfactory, i.e., below 14%. 5-HT, Tyr and Trp spiked to human urine were successfully extracted and separated. These results underline the great potential of 3PEE as an integrated enrichment technique from biological samples and subsequent sensitive metabolomics analysis.

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors.

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE- immobilized microenzyme reactors (IMER) development, human flavin-containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in-line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE-IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE-IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.

STR Profiling of HTLV-1-Infected Cell Lines.

Many investigations of the replication and pathogenesis of human T-cell leukemia virus type 1 (HTLV-1) employ chronically infected cell lines, cell lines stabilized from primary adult T-cell leukemia cells, and noninfected T-cell lines. The validity of data obtained from such studies depends on the unambiguous identification of each cell line, which can be performed by short-tandem-repeat (STR) profiling (DNA fingerprinting). While kit-based profiling represents the standard method for cell line authentication, not all labs have ready access to the required capillary electrophoresis equipment, and the costs of such tests can become substantial, especially if the cell lines are to be tested frequently. We analyzed DNA from a panel of HTLV-1-infected cell lines and noninfected T-cell lines using a commercial STR kit and then analyzed the same DNA for individual STR markers followed by nondenaturing polyacrylamide gel electrophoresis. This simplified method should facilitate routine confirmation of cell line identity in diverse laboratory settings.

Research Article Genetics Analysis of the Biggest Cashew Tree in the World

This study performed a genetic study of several canopies of Anacardium occidentale L. specimen called Cashew King (Cajueiro da Praia-PI, Brazil) in order to assess whether all canopies belong to the same plant. Leaves were collected from different distant points, and preserved for analysis of DNA. The analysis was made from automated sequencing by capillary electrophoresis equipment ABI3500 Genetic Analyzer (Applied Biosystems) and alignment of nucleotide sequences generated with reference sequences deposited in GenBank. According to the analysis, it was shown that all canopies belong to the same plant. Thus, the Cashew King can be considered as a single specimen A. occidentale occupying an area of 8,834 m2. The length of the specimen indicates that the “Cashew King” is the largest cashew registered according to the literature and therefore should be preserved.

Soil characterisation by bacterial community analysis for forensic applications: A quantitative comparison of environmental technologies

The ubiquity and transferability of soil makes it a resource for the forensic investigator, as it can provide a link between agents and scenes. However, the information contained in soils, such as chemical compounds, physical particles or biological entities, is seldom used in forensic investigations; due mainly to the associated costs, lack of available expertise, and the lack of soil databases. The microbial DNA in soil is relatively easy to access and analyse, having thus the potential to provide a powerful means for discriminating soil samples or linking them to a common origin. We compared the effectiveness and reliability of multiple methods and genes for bacterial characterisation in the differentiation of soil samples: ribosomal intergenic spacer analysis (RISA), terminal restriction fragment length polymorphism (TRFLP) of the rpoB gene, and five methods using the 16S rRNA gene: phylogenetic microarrays, TRFLP, and high throughput sequencing with Roche 454, Illumina MiSeq and IonTorrent PGM platforms. All these methods were also compared to long-chain hydrocarbons (n-alkanes) and fatty alcohol profiling of the same soil samples. RISA, 16S TRFLP and MiSeq performed best, reliably and significantly discriminating between adjacent, similar soil types. As TRFLP employs the same capillary electrophoresis equipment and procedures used to analyse human DNA, it is readily available for use in most forensic laboratories. TRFLP was optimized for forensic usage in five parameters: choice of primer pair, fluorescent tagging, concentrating DNA after digestion, number of PCR amplifications per sample and number of capillary electrophoresis runs per PCR amplification. This study shows that molecular microbial ecology methodologies are robust in discriminating between soil samples, illustrating their potential usage as an evaluative forensic tool.

Recent Advances in Portable Analytical Electromigration Devices

This article presents an overview of recent advances in the field of portable capillary electrophoresis and microchip electrophoresis equipment during the period 2013–Mid 2015. Instrumental achievements in the separation as well as the detection part of the equipment are discussed. Several applications from a variety of fields are described.

Analysis of 30 Biallelic INDEL Markers Using the Investigator DIPplex(®) Kit.

Insertion/deletion polymorphisms (INDELs) are a relatively new class of a DNA marker to be used in forensic casework; used most commonly as a supplementary method to STR-based typing. INDELs, like SNPs, are particularly useful for the analysis of highly degraded DNA as the amplicon sizes are typically below 160 bp; they can also be valuable as an additional tool to help resolve kinship cases, with the advantage over STRs that they do not have high mutation rates. INDELs have an advantage over SNPs in that they are length polymorphisms and so can be analyzed by simply measuring the length of the allele(s). The Qiagen Investigator(®) DIPplex Kit is currently only one of two commercially available kits for the amplification of INDEL polymorphisms; it amplifies 30 biallelic INDEL loci and the amelogenin locus. The primers used are fluorescence labeled with 6-FAM, BTG, BTY, and BTR. This technique is robust, relatively simple, and the results are analyzed using the same capillary electrophoresis equipment and software as used for STR typing.

ICSH recommendations for assessing automated high‐performance liquid chromatography and capillary electrophoresis equipment for the quantitation of HbA2

Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of β-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.

Multiplex ligation-dependent probe amplification (MLPA) assay for blood group genotyping, copy number quantification, and analysis of RH variants

Abstract The blood group multiplex ligation-dependent probe amplification (MLPA) is a comprehensive assay, developed for genotyping the majority of clinically relevant blood group antigens in both patients and donors. The MLPA is an easy method to apply and only requires a thermal cycler and capillary electrophoresis equipment. Because the molecular basis of blood group antigens can be a single nucleotide polymorphism, an insertion/deletion polymorphism, or genetic recombination, a single assay such as the MLPA to facilitate these different types of genetic variation is a prerequisite in blood group typing. An MLPA assay allows the simultaneous detection of up to 50 polymorphisms in a single tube. The blood group MLPA currently consists of three separate probe pools targeting 104 different blood group alleles of 18 blood group systems. The assay is performed in a 96-well plate; therefore, a maximum of 32 genomic DNA samples can be processed simultaneously. Results are available within 24 hours, and software for analysis of the MLPA results is available free of charge. In addition to the analysis of genetic variation in blood group genes, a major advantage of the test is the ability to detect aberrations in gene copy numbers, which is especially useful for the determination of homo- or hemizygous status of RHD or other blood group genes and for detection of blood chimerism. A relatively large number of RH wild-type and mutation-specific probes are included in the assay, allowing an extensive analysis of RHD variants. In our reference lab in the Netherlands, the MLPA was validated to detect RH variants in patients, donors, and pregnant women. Furthermore, we have used the MLPA to provide comprehensive typing after blood transfusion of 52 blood group antigens simultaneously, in patients with red cell autoantibodies or patients with rare phenotypes. Immunohematology 2015;31:58–61.

Comparison of the first fully automated agarose gel electrophoresis system with the Capillarys electrophoresis method for the identification of monoclonal immunoglobulins

Serum protein electrophoresis (SPE), used to identify monoclonal gammopathies, is generally performed using the “gold standard” agarose gel as migration support (AGE). However, despite being semi-automated, this electrophoresis technique remains labour-intensive, meaning analytical performance and throughput are limited. Hence fully automated capillary electrophoresis (CE), which enables rapid separation, has been adapted for use in clinical laboratories within the last decade [1–5]. Any qualitative abnormalities suggestive of monoclonal immunoglobulin detected by either AGE or CE are further analysed by immunofixation electrophoresis (IFE), to confirm the presence of monoclonal bands and to identify the type of monoclonal component (MC) [6]. In order to increase throughput, a fully automated innovative AGE technique—Agarose Gel Easy Interlab G26 electrophoresis (Interlab, Rome, Italy)—has recently been launched, to be used with purpose-designed IFE kits. The aim of this study was to compare the AGE system with the Capillarys 2® CE equipment (Sebia, Evry, France) currently in use in our laboratory, to detect serum MCs subsequently confirmed, characterized and quantified by IFE on

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.

Interfacing commercially available capillary electrophoresis to sample preparation and/or detection systems to solve analytical problems

This review presents the state-of-the-art of interfacing sample preparation and detector systems with commercially available capillary electrophoresis (CE) equipment. The analytical potential of combined sample preparation units into commercially available CE equipment under integrated or coupling approaches, allowing at-line, on-line, and in-line arrangements to automate sample preparation is discussed. Some examples of the analytical applications are reported when using different sample treatment procedures, such as cleanup and preconcentration, extraction/filtering, gas extraction/diffusion, extraction with membrane and hollow fiber, and dialysis and extraction with supported liquid membrane. CE interfaced detectors are also discussed by distinguishing between commercially available CE equipment and those not commercially available and focusing on the latter. Interfaced CE approaches clearly expand the field of application of the CE and connect to the real world in routine analytical laboratories, avoiding the drawbacks and limitations of electrophoretic separations.

Photonic crystal fibres as efficient separation component in capillary electrophoresis

Telecommunication developments are leading to original and new fibre designs which indeed are being used in a huge variety of fields. In this manuscript we show that Photonic Crystal Fibres (PCFs) can be used as efficient chemical separation devices in commercial capillary electrophoresis (CE) equipment. A comparison of conventional capillaries, PCFs and new devices inspired on PCFs named Smart Micro-Structured Capillaries (SMSCs) is made. Results showed the usefulness and efficiency of these physical supports in CE through the detection of fluorescein. In addition, some considerations to take into account for SMSCs manufacturing are discussed. Further work should allow coupling the separation advantages achieved by these elements with the wide variety of detection techniques available in fibre-optics.

Design and Adaptation of an Interface for Commercial Capillary Electrophoresis—Evaporative Light Scattering Detection Coupling

In this technical note, an interface for coupling commercially available capillary electrophoresis (CE) equipment to an evaporative light scattering detector (ELSD) is described. The nebulization process was identified as the most critical parameter for performing the reliable coupling between both pieces of equipment. Therefore, appropriate modifications in the nebulization chamber and in the conventional nebulizer were brought to make ELSD fully compatible with CE. The impact of the customized interface on CE separation and detection was evaluated in terms of resolution and sensitivity. ELSD can be considered as an attractive alternative to other CE detection systems (e.g., UV-vis, fluorescence, electrochemical detection, or even MS detection), particularly those in which derivatization is needed. This advantage is due to the versatility and the quasi-universality of ELSD. Thus, sensitive and fast separations of several compounds were performed using this CE-ELSD customized arrangement, which opens up an interesting analytical potential for the determination of compounds not presenting sensitive UV-vis chormophore, fluorophore, or electroactive groups. Carbohydrates were selected in this work to demonstrate the applicability of CE-ELSD coupling.

Multiplex Ligation-dependent Probe Amplification (MLPA) in Tumor Diagnostics and Prognostics

The increasing knowledge about genetic alterations and molecular biomarkers in cancer initiation and progression opens new possibilities for the treatment of various types of cancer. This requires the inclusion of sensitive, and preferably multiplex, methods for the detection of molecular genetic alterations in the toolbox of classic pathology. Multiplex ligation-dependent probe amplification (MLPA) is a multiplex polymerase chain reaction-based method that can detect changes in the gene copy number status, DNA methylation, and point mutations simultaneously. MLPA probes recognize target sequences of only 50 to 100 nucleotides in length. This makes it possible to use MLPA even on highly fragmented DNA, and allows the detection of small deletions encompassing only a single exon. MLPA is a reliable, cost-effective, and robust method that can be performed using a standard thermocycler and capillary electrophoresis equipment, generating results within 24 hours with a short hands-on working time. Up to 50 different genomic locations can be tested in a single reaction, which can be sufficient to detect those genetic alterations that are of diagnostic and prognostic significance in a certain tumor entity. In the last years, MLPA has been used successfully in tumor diagnostics and in cancer research. This review gives an overview on the collected experience of MLPA applications on tumor DNA, about the advantages but also potential pitfalls and limitations of this technique.

Short communication: Identification of coagulase-negative staphylococcus species from goat milk with the API Staph identification test and with transfer RNA-intergenic spacer PCR combined with capillary electrophoresis

Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria from goat milk, but they have often been identified with phenotypic methods, which may have resulted in misclassification. The aims of this paper were to assess the amount of misclassification of a phenotypic test for identifying CNS species from goat milk compared with transfer RNA intergenic spacer PCR (tDNA-PCR) followed by capillary electrophoresis, and to apply the tDNA-PCR technique on different capillary electrophoresis equipment. Milk samples were collected from 416 does in 5 Californian dairy goat herds on 3 occasions during lactation. In total, 219 CNS isolates were identified at the species level with tDNA-PCR and subjected to the API 20 Staph identification test kit (API Staph; bioMérieux, Durham, NC). If the same species was isolated multiple times from the same udder gland, only the first isolate was used for further analyses, resulting in 115 unique CNS isolates. According to the tDNA-PCR test, the most prevalent CNS species were Staphylococcus epidermidis, Staphylococcus caprae, and Staphylococcus simulans. Typeability with API staph was low (72%). Although the API Staph test was capable of identifying the majority of Staph. epidermidis and Staph. caprae isolates, sensitivity for identification of Staph. simulans was low. The true positive fraction was high for the 3 most prevalent species. It was concluded that the overall performance of API Staph in differentiating CNS species from goat milk was moderate to low, mainly because of the low typeability, and that genotypic methods such as tDNA-PCR are preferred.