Analysis of Carbohydrates by Capillary Electrophoresis

ISBN-13: 9783322850225 / Angielski / Miękka / 2012 / 176 str.

Aran Paulus; Antje Klockow-Beck; Kevin D. Altria

Opis

Carbohydrates are ubiquitous. They come as monomers, oligomers and polymers with a startling variety of chemical, physical and material properties. They affect almost every as pect of human live. For example they are an essential part of our nutrition. Cellulose, a car bohydrate polymer, acts as a central material in civilization: as building material for housing and as paper for written communication. Scientifically, the interest in carbohydrates ranges from structural elucidation of simple carbohydrates to fundamental biochemical processes such as photosynthesis and glycolysis. Always, a deeper understanding of a particular aspect of carbohydrate chemistry was accompanied by new methods of measuring and characteri zing this class of molecules. During the last decades a better understanding of the roles of carbohydrates in biochemi cal pathways developed. It turned out, that carbohydrates attached to proteins in glycoprote ins are involved in a number of molecular recognition and targeting processes. This lead to a new research field: Glycobiology. However, unlike for proteins or nucleic acids, there is at this time no consistent theory how chemical composition and structure controls function and inherent information. In order to develop the database, on which new theories and scientific hypothesis are founded, analytical methods to separate compounds with an enormous struc tural diversity and only minor differences in chemical or physical properties has to be deve loped, preferably using only minute quantities."

Spis treści

1 Introduction.- References.- 2 Capillary electrophoresis, instrumentation and modes.- 2.1 Electrophoretic mobility.- 2.2 Fused silica surface and electroosmotic flow.- 2.3 Plate number, migration time and resolution.- 2.4 Instrumentation.- 2.4.1 Power supply and cooling system.- 2.4.2 Capillaries.- 2.4.3 Detection.- 2.4.3. l UV detection.- 2.4.3.2 LIF detection.- 2.4.3.3 Indirect optical detection methods.- 2.4.3.4 Electrochemical detection.- 2.4.3.5 Refractive index detection.- 2.4.3.6 Mass spectrometry detection.- 2.5 CE modes.- 2.5.1 Capillary Electro Chromatography.- 2.5.2 Micellar Electrokinetic Capillary Chromatography.- 2.5.3 Capillary Zone Electrophoresis.- 2.5.4 Isotachophoresis.- 2.5.5 Capillary Isoelectric Focusing.- 2.5.6 Capillary Gel Electrophoresis.- References.- 3 Structures and properties of carbohydrates.- 3.1 Monosaccharides.- 3.2 Oligosaccharides.- 3.3 Polysaccharides.- 3.4 Glycoproteins.- 3.5 Glycosaminoglycans and Proteoglycans.- 3.6 Glycolipids.- References.- 4 Separation and detection of carbohydrates in capillary electrophoresis.- 4.1 Separation and detection of non-derivatized carbohydrates.- 4.1.1 Electrophoresis of native carbohydrates with direct UV detection.- 4.1.1.1 CZE and MECC separation conditions.- 4.1.1.2 CZE after on-column complexation with borate.- 4.1.1.3 CZE of glycopeptides and glycoproteins.- 4.1.2 Electrophoresis of native carbohydrates at extreme alkaline pH.- 4.1.2.1 Electrophoresis of native carbohydrates at extreme alkaline pH with indirect detection.- 4.1.2.2 Electrophoresis of native carbohydrates at extreme alkaline pH with amperometric detection.- 4.1.2.3 Electrophoresis of native carbohydrates at extreme alkaline pH with refractive index detection.- 4.1.3 Concluding remarks.- 4.2 Separation and detection of derivatized carbohydrates.- 4.2.1 Labeling of carbohydrates.- 4.2.1.1 Reductive animation.- 4.2.1.2 Labeling by condensation with l-phenyl-3-methyl-5-pyrazolone.- 4.2.1.3 Labeling by condensation of acidic saccharides with amines.- 4.2.1.4 Esterfication of aminoalditols.- 4.2.2 Detection of labeled carbohydrates.- 4.2.2.1 Mass and concentration detection limits and minimal derivatization volume.- 4.2.2.2 Detection sensitivity of UV derivatized carbohydrates.- 4.2.2.3 Detection sensitivity of fluorescent derivatized carbohydrates.- 4.2.3 Separation of labeled carbohydrates.- 4.2.3.1 Separation in acidic electrolytes under low EOF conditions.- 4.2.3.2 Separation under high EOF conditions.- 4.2.3.3 MECC separations.- 4.2.3.4 Gel Separations.- References.- 5 Applications.- 5.1 Mono- and disaccharides.- 5.2 Oligosaccharides.- 5.2.1 Underivatized oligosaccharides.- 5.2.2 Derivatized oligosaccharides.- 5.2.3 Enzyme action on labeled oligosaccharides.- 5.2.4 Lipooligosaccharides.- 5.3 High molecular weight polysaccharides.- 5.4 Glycopeptides and Glycoproteins.- 5.4.1 Glycofbrms.- 5.4.1.1 Erythropoietin.- 5.4.1.2 Ribonuclease.- 5.4.1.3 Ovalbumin.- 5.4.1.4 Chorionic Gonadotropin.- 5.4.1.5 Tissue Plasminogen Activator.- 5.4.1.6 Transferrin.- 5.4.1.7 Interferon.- 5.4.1.8 Immunoglobulins.- 5.4.1.9 ?1-Acid Glycoprotein.- 5.4.1.10 Various glycoproteins.- 5.4.1.11 New approaches in CE glycoform analysis.- 5.4.1.12 CE-MS of glycoforms.- 5.4.2 Glycopeptides.- 5.4.3 Complex Oligosaccharides.- 5.4.3.1 UV detection of derivatized complex oligosaccharides.- 5.4.3.2 UV detection of underivatized complex oligosaccharides.- 5.4.3.3 Laser induced fluorescence detection (LIF) of complex oligosaccharides.- 5.5 Glycosaminoglycans.- 5.5.1 Heparins.- 5.5.2 Chondroitin and Dermatan.- 5.5.3 Hyaluronan.- 5.5.4 Derivatized Glycosaminoglycans.- 5.6 Glycolipids.- 5.7 Other glycoconjugates.- References.

O autorze

Dr. Aran Paulus arbeitet und forscht im Bereich Analytik/Chromatographie für die Firma Soane in Kalifornien, USA. Dr. Antje Klockow-Beck ist in gleicher Funktion bei der Firma R.W. Johnson in Schaffhausen, Schweiz, tätig.

Blurb

Carbohydrates are next to nucleic acids and proteins the third important class of biopolymers and we are just beginning to understand their role in biological systems on a molecular level. In recent years, the post-translational modification of proteins has obtained increased interest. Specifically the glycosylation of enzymes, receptors and antibodies is the focus of numerous research porjects as it becomes clear that carbohydrates are involved in cellular communication and immuno recognition processes. In order ot understand this "sweet sugar language" and consequently use it in proteomics and the development of biopharmaceuticals, suitable analytical techniques have to be developed to separate carbohydrates with high selectivity in complex biological matrices and quantify them in in minute quantities.

The present monograph covers all aspects of carbohydrate analysis by Capillary Electrophoresis including instrumental aspects, derivatization for high sensitivity applications, separation conditions and applications in various fields and industries. It will be a valuable source of information for everyone involved in carbohydrate research in life sciences including glycobiology, biotechnology and immunology and in the food industry.