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Rapid Separation Of Amino Acids Using Capillary Electrophoresis With Laser Induced Fluorescence
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Book Synopsis Rapid Separation of Amino Acids Using Capillary Electrophoresis with Laser-induced Fluorescence by : Stephanie Ann Cunningham
Download or read book Rapid Separation of Amino Acids Using Capillary Electrophoresis with Laser-induced Fluorescence written by Stephanie Ann Cunningham and published by . This book was released on 2007 with total page 138 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Nanoscale Protein Analysis Utilizing Capillary Electrophoresis and Laser-induced Fluorescence Detection by : Michael D. Harvey
Download or read book Nanoscale Protein Analysis Utilizing Capillary Electrophoresis and Laser-induced Fluorescence Detection written by Michael D. Harvey and published by . This book was released on 2001 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The trend towards high throughput applications and miniaturization necessitates approaches capable of microlitre volume sampling and low protein concentration detection. Furthermore, one of the major trends in high throughput screening is the growing replacement of technologies that depend on radioactivity to generate a signal with those that rely on fluorescence. This trend towards non-radioactive detection in general can be understood by some of the advantages inherent to these methods over radioactive modes. These include a significant reduction in safety concerns leading to a relaxation of strict laboratory procedures, elimination of expensive waste disposal, extended shelf-life of labeled reagents, and the possibility of acquiring multiplexed data through the spectral isolation of different wavelength signals. A variety of capillary electrophoretic (CE) approaches utilizing laser-induced fluorescence (LIF) have thus been developed, providing researchers with valuable tools in protein analysis. Various covalent and non-covalent fluorescent derivatization approaches have been investigated, with emphasis on biochemical and/or clinical applications. The non-covalent dye, NanoOrange, is used as a clinical diagnostic tool for early disease diagnosis, quantitating nanomolar concentrations of human serum albumin in solution, and obtaining fluorescence-based biofluid profiles. An alternate non-covalent labeling approach utilizing the fluorescent probe, Sypro Red, and capillary gel electrophoresis allows for rapid, sensitive analysis of protein sample purity as well as molecular weight determination. These two non-covalent approaches are complemented by the development of a fluorescent Insulin-Like Growth Factor-I (IGF-I) analog for use in bioanalytical applications. Specific derivatization reaction conditions were developed to selectively label the N-terminus of the analog hence preserve biological activity. High-performance liquid chromatography and electrospray mass spectrometry were used to confirm the extent of labeling and modification site. Antibody recognition of this fluorescent analog was evaluated using CE-LIF, illustrating the clinical utility of this diagnostic reagent. In addition to the above CE-LIF approaches, a fourth capillary electrophoretic tool is provided for the clinical chemist. Rapid analysis of biofluids is of significant importance in early disease diagnosis. As such, an extensive CE-based analysis of human seminal plasma is presented. Separation conditions, sample stability, and protein/non-protein zone identification issues are addressed. This study and the CE-LIF methodologies discussed above represent original approaches to nanoscale protein analysis.
Book Synopsis Analysis by Capillary Electrophoresis with Laser-induced Fluorescence Detection by : Tara Lee Massie
Download or read book Analysis by Capillary Electrophoresis with Laser-induced Fluorescence Detection written by Tara Lee Massie and published by . This book was released on 2010 with total page 289 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a versatile and sensitive analytical tool with many potential applications. Its advantages over other chromatographic methods include minimal solvent and sample requirements, low waste volumes, simple instrumentation, and diversity of analytes studied. CE is one of the most efficient separation techniques available for the analysis of both large and small molecules, but because few analytes are natively fluorescent, it is necessary to bind them with fluorescent tags if LIF detection is to be employed. These tags increase sensitivity and provide additional separation from components of the sample that do not bind the tags. In this dissertation, four applications of CE-LIF for the determination of tagged analytes are documented. First, a rapid CE-LIF method was developed for the determination of gamma-aminobutyric acid (GABA) and other important brain biogenic amines and amino acids present in biological samples at low levels. The high efficiency and sensitivity necessary to identify toxicologically relevant changes in neurotransmitter levels in brain tissue and microdialysates following Mn exposure was achieved, and the method required minimum sample work-up and quantities of sample for analysis. A linear relationship between the concentration of GABA and the CE-LIF peak height was established, with correlation coefficients of 0.989 or better for four neurotransmitters. Second, the use of two squarylium-based fluorescent dyes (bis-SQHN-4d and SQHN-3c) as noncovalent protein labels was investigated. The fluorescence emissions of both dyes were enhanced upon complexation with model proteins, demonstrating promise as noncovalent fluorescent protein labels. Third, the nature of interactions between two boronic acid-based probes (SQTM-BA1 and ANQW-BA1) and two model proteins (bovine serum albumin (BSA) and hemagglutinin (HA)) have been explored. The complex between SQTM-BA1 and BSA was the only complex to show increased fluorescence emission, and the role of electrostatic interactions between SQTM-BA1 and BSA in complex formation was explored. Lastly, CE-LIF methods were developed to label and quantify turnip yellow mosaic virus (TYMV). Noncovalent labels Red-1c and NN127 effectively labeled the surface proteins of the virus, and the labeled viruses were then analyzed by CE-LIF. The CE-LIF methods developed herein for the analysis of neurotransmitters, free solution proteins, and viruses demonstrate the versatility of the technique, especially when coupled with fluorescent labeling.
Book Synopsis Capillary Electrophoresis of Proteins and Peptides by : Mark A. Strege
Download or read book Capillary Electrophoresis of Proteins and Peptides written by Mark A. Strege and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt: Throughout the more than 20 years that have followed the beginnings of capillary electrophoresis (CE), its application to the analysis of proteins and peptides has continued to be reliable, versatile, and productive. Over time, CE has matured to become a superb complement to HLPC, and in many cases has also evolved as an automated and quantitative replacement for conventional slab gel electrophoresis methods such as SDS-PAGE and isoelectric focusing. Within Capillary Electrophoresis of Proteins and Peptides, we have assembled contributions from researchers who are applying state-of-the-art CE for protein and peptide analysis, including topics that we believe are of great potential both in the present and for the future. In comparison to traditional separation methods, CE represents a miniaturized analysis technique (especially in its microchip-based format) that is highly dependent upon the basic fundamentals of effective sample recovery and high sensitivity detection. With these issues in mind, Chapters 1–4 describe recently developed approaches for both capillary coatings and analyte detection via laser-induced fluorescence. Since the discipline of biotechnology has established itself as a primary platform for the application of CE to the analysis of proteins and peptides, Chapters 5–7 demonstrate a variety of examples of the specific techniques that have been applied for the development of biopharmaceuticals and their commercialization. The methods covered here include also the analysis of oligosaccharides from glycoproteins.
Book Synopsis The Analysis of Limited Quantities of Amino Acids and Peptides Using Capillary Electrophoresis and Laser Induced Fluorescence Detection by : Beverly Nickerson
Download or read book The Analysis of Limited Quantities of Amino Acids and Peptides Using Capillary Electrophoresis and Laser Induced Fluorescence Detection written by Beverly Nickerson and published by . This book was released on 1991 with total page 742 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Capillary Gel Electrophoresis by : Andras Guttman
Download or read book Capillary Gel Electrophoresis written by Andras Guttman and published by Newnes. This book was released on 2021-12-04 with total page 391 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary Gel Electrophoresis and Related Microseparation Techniques covers all theoretical and practical aspects of capillary gel electrophoresis. It also provides an excellent overview of the key application areas of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods. It not only gives readers a better understanding of how to utilize this technology, but also provides insights into how to determine which method will provide the best technical solutions to particular problems. This book can also serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses. Covers all theoretical and practical aspects of capillary gel electrophoresis Excellent overview of the key applications of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods Teaches readers how to use the technology and select methods that are ideal for fundamental problems Can serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses
Book Synopsis Capillary Electrophoresis with Laser-induced Fluorescence Detection for the Determination of Amino Acid Neurotransmitters in Cerebrospinal Fluid by : Murali Seenappa
Download or read book Capillary Electrophoresis with Laser-induced Fluorescence Detection for the Determination of Amino Acid Neurotransmitters in Cerebrospinal Fluid written by Murali Seenappa and published by . This book was released on 2004 with total page 182 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Enhancing Detection Sensitivity for Measuring Amino Neurotransmitters with Flow-gated Capillary Electrophoresis by : Naveen Maddukuri
Download or read book Enhancing Detection Sensitivity for Measuring Amino Neurotransmitters with Flow-gated Capillary Electrophoresis written by Naveen Maddukuri and published by . This book was released on 2020 with total page 116 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary electrophoresis (CE) commences as a promising technique for analyzing a variety of biological substances and has been used in bioanalytical and biopharmaceutical applications. Flow-gated CE has numerous advantages over conventional and microchip CE. It has the advantage of fresh background electrolyte, improved separation efficiency, and PDMS flow-gate is easy for visual troubleshooting. However, it fails to have enough sensitivity in numerous valuable applications. The goal of the present research is to enhance the detection sensitivity in flow-gated CE for the measurement of amino neurotransmitters by using an integrated microfluidic system to perform in vitro and in vivo analysis of biological samples. This dissertation focuses on the development of novel methods to enhance the signal sensitivity for the detection of important molecular components in biological samples using laser-induced fluorescence detection. First, an efficient tagging strategy was developed to tag amino acids by taking advantage of the fast kinetics of the derivatization of primary amines with ophthalaldehyde (OPA) in the presence of a highly fluorescent thiol. Experimental results show that this strategy was capable of performing rapid fluorescent derivatization, thus providing detection sensitivity, reproducibility, accuracy, and robustness. Second, an on-line sample preconcentration technique based on vacuum-assisted electrokinetic supercharging (EKS) was developed for flow-gated CE to enhance the signal sensitivity of low abundant amino neurotransmitters, such as gamma-aminobutyric acid (GABA), glutamate and aspartate. The developed vacuum-assisted EKS technique was used to determine the GABA concentration in cerebrospinal fluid (CSF) samples by using the one-point standard addition method. Both techniques are anticipated to be valuable for in vivo measurements of neurotransmitters in neuroscience research and drug development.
Book Synopsis Subattomole Amino Acid Analysis by Laser-Induced Fluorescence by : YF. Cheng
Download or read book Subattomole Amino Acid Analysis by Laser-Induced Fluorescence written by YF. Cheng and published by . This book was released on 1990 with total page 9 pages. Available in PDF, EPUB and Kindle. Book excerpt: Subattomole analysis of fluorescein isothiocyanate (FITC) derivatives of amino acids is produced by combining capillary zone electrophoresis for high-efficiency separation with laser-induced fluorescence for high sensitivity detection. To optimize the separation process, several buffer systems were investigated. A pH 10 carbonate buffer provided good separation of 14 FITC amino acids. Concentration detection limits range from 5 x 10-12M for alanine to 9 x 10-11M for lysine, injected onto the column; ~9 x 10-21 mol of alanine is contained within the ~1-nL injection volume at the detection limit. The alanine detection limit corresponds to less than 6000 molecules injected onto the column and represents a four order of magnitude improvement in the state of the art for fluorescent detection of amino acids and a six order of magnitude improvement in the state of the art for the detection limit for isothiocyanate derivatives of amino acids.
Book Synopsis Capillary Electrophoresis by : S.F.Y. Li
Download or read book Capillary Electrophoresis written by S.F.Y. Li and published by Elsevier. This book was released on 1992-07-31 with total page 615 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary Electrophoresis (CE) has had a very significant impact on the field of analytical chemistry in recent years as the technique is capable of very high resolution separations, requiring only small amounts of samples and reagents. Furthermore, it can be readily adapted to automatic sample handling and real time data processing. Many new methodologies based on CE have been reported. Rapid, reproducible separations of extremely small amounts of chemicals and biochemicals, including peptides, proteins, nucleotides, DNA, enantiomers, carbohydrates, vitamins, inorganic ions, pharmaceuticals and environmental pollutants have been demonstrated. A wide range of applications have been developed in greatly diverse fields, such as chemical, biotechnological, environmental and pharmaceutical analysis. This book covers all aspects of CE, from the principles and technical aspects to the most important applications. It is intended to meet the growing need for a thorough and balanced treatment of CE. The book will serve as a comprehensive reference work. Both the experienced analyst and the newcomer will find the text useful.
Book Synopsis Methods for High-sensitivity Separations of Proteins and Peptides Using Capillary Electrophoresis and Microcolumn Liquid Chromatography by : Kelly Ann Cobb
Download or read book Methods for High-sensitivity Separations of Proteins and Peptides Using Capillary Electrophoresis and Microcolumn Liquid Chromatography written by Kelly Ann Cobb and published by . This book was released on 1991 with total page 450 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Application of Capillary Electrophoresis with Laser-induced Fluorescence Detection in Quantifying the Endogenous Amino Acid Pool of Mouse Embryos by : Sau-lai Yik
Download or read book The Application of Capillary Electrophoresis with Laser-induced Fluorescence Detection in Quantifying the Endogenous Amino Acid Pool of Mouse Embryos written by Sau-lai Yik and published by . This book was released on 2000 with total page 248 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Application of Capillary Electrophoresis with Laser-induced Fluorescence Detection in Quantifying the Endogenous Amino Acid Pool of Mouse Embryos by :
Download or read book The Application of Capillary Electrophoresis with Laser-induced Fluorescence Detection in Quantifying the Endogenous Amino Acid Pool of Mouse Embryos written by and published by . This book was released on 2000 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Fast Methods for Analysis of Neurotransmitters from Single Cell and Monitoring Their Releases in Central Nervous System by Capillary Electrophoresis, Fluorescence Microscopy and Luminescence Imaging by :
Download or read book Fast Methods for Analysis of Neurotransmitters from Single Cell and Monitoring Their Releases in Central Nervous System by Capillary Electrophoresis, Fluorescence Microscopy and Luminescence Imaging written by and published by . This book was released on 1999 with total page 5 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10−8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.
Book Synopsis Open Tubular Capillary Electrochromatography-laser Induced Fluorescence for the Separation and Detection of Proteins and Amino Acids by : Kellie M. Davis
Download or read book Open Tubular Capillary Electrochromatography-laser Induced Fluorescence for the Separation and Detection of Proteins and Amino Acids written by Kellie M. Davis and published by . This book was released on 2007 with total page 69 pages. Available in PDF, EPUB and Kindle. Book excerpt: "With its high efficiency and reproducibility, open tubular capillary electrochromatography (OTCEC) is beginning to prove itself as a promising method for the separation of proteins, peptides and pharmaceuticals. OTCEC, when coupled to an absorbance-based detector, suffers from poor sensitivity and high concentration limits of detection. However, laser induced fluorescence coupled to CEC has shown picomolar limits of detections for some compounds.2 This thesis describes the coupling of OTCEC to a laser induced fluorescence (LIF) detector to optimize both the separation and detection aspects of protein and amino acid analysis. OTCEC capillaries were etched and silanized, and the 4,4' cyanopentoxy biphenyl phase was attached through a radical-initiated process. Such biphenyl phases have shown unique dependence on temperature and mobile phase composition and demonstrated higher selectivity for certain compounds3 than bare silica capillaries. Proteins and amino acids were modified using pre-column derivatization formats with two different fluorescein-based dyes (fluorescein isothiocyanate, FITC) and Atto-tag FQ (3-(2-furoyl)quinoline-2-carboxaldehyde). These conditions were varied to attempt consistent labeling conditions with varying protein size. Optimizations of the analyses were achieved by modifying conditions such as background electrolyte, pH, voltage, and percent organic modifiers. These modified conditions were used to achieve the highest resolution on protein and amino acid derivatives. Results determined that high sensitivity and efficiency are achieved when LIF detection is coupled to cyano pentoxy biphenyl coated capillaries using optimal conditions. These conditions include 1) the use of Atto-tag FQ derivatization and 2) an 80:20 acetonitrile: 50 mM phosphate buffer (pH 2.00) background electrolyte with a positive electrical current applied."--Abstract from author supplied metadata.
Book Synopsis Capillary Electrophoresis with Post-column Laser-induced Fluorescence Detection of Proteins by :
Download or read book Capillary Electrophoresis with Post-column Laser-induced Fluorescence Detection of Proteins written by and published by . This book was released on 1996 with total page 42 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is one of the alternative methods being developed for fast and sensitive assays of proteins and peptides. This report summarizes CE-LIF instrument development, efforts at labelling proteins, tests conducted to prove the applicability of CE-LIF to the analysis of samples, and results of a comparison of CE-LIF with some other techniques. Appendices include an instrument operating procedure and a paper that summarizes the experimental details of the protein labelling procedure and a CE-LIF technique for sub-nanomolar assay of proteins.
Book Synopsis Capillary Electrophoresis Equipped with Laser Induced Fluorescence by : Shokoufeh Ahmadi
Download or read book Capillary Electrophoresis Equipped with Laser Induced Fluorescence written by Shokoufeh Ahmadi and published by . This book was released on 2012 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The aim of this research was to evaluate the feasibility of capillary electrophoresis in micellar electrokinetic mode (MEKC) equipped with laser induced fluorescence (LIF) as a novel technique to study dyes extracted from valuable textiles. CE identification of dyes requires extraction of dyes from the substrate, which is one of the most challenging aspects in conservation science. Therefore, developing a technique with minimum destructive effect on objects is favorable. The most important advantages of using LIF are the low detection limits and high sensitivity which allow for the separation of very small quantities of sample, which makes it suitable for working with historical objects. Feasibility studies were performed to explore separation properties of a small number of historical dyes (alizarin, purpurin, carmine and morin) using: different concentrations of modifiers and borate buffer at different pH values, and applied voltages. By manipulating separation parameters the optimum separation condition for a mixture of 10 dyes was defined as: under 22 kV, 20 mM borate buffer at pH = 8.5; 20 mM SDS with 5% acetonitrile. The separation of flavonols and purpurin using CE at pH>9.2 appeared to be complicated by reproducibility in peak height and peak shape of purpurin. In this study the poor peak reproducibility of CE separations was attributed to photochemical properties of dyes. The 1st order rate constants ( for the decomposition of purpurin and selected flavonol dyes were determined under different pH conditions, for purpurin at pH=9.0 under dark condition was The stability of these dyes was greatly affected by pH, a modest decrease in pH (pH = 8.5) improved the stability and peak reproducibility of these dyes. To extract dyes from textiles, the existing extraction methods with focus on Hanson solubility parameters were evaluated. It appeared that methanol was the most suitable solvent and the dyes in the following samples were extracted by methanol and identified running CE-LIF: textiles from "Salt Man" (600 BC), Safavid dynasty and Chinese robe (1700 AD). CE-LIF was used successfully for separation and identification of small quantities of natural dyes extracted from less than 1 mg samples such as wool, silk and paper.
