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NATIONAL COLLEGE OF PHARMACYDEPT. OF PHARMACOGNOSYSHIMOGA -577201 An evaluation Seminar on Subject : Modern Pharmaceutical Analysis Topic: Electrophoresis Presented By: Neelanjan Chatterjee (I M.Pharm) Department of Pharmacognosy Under the guidence of:Mr. Manzoor Ahmed NCP, Shimoga.2010

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Electrophoresis Migration of charged solutes in a liquid medium under an electrical field Many biological molecules have ionisable groups eg. amino acids, proteins, nucleotides, nucleic acids Under an electric field -> charged particles migrate to anode (+) or cathode (-)

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Moving boundary electrophoresis : Density gradient electrophoresis Isotachophoresis Zone electrophoresis: Paper elecrophoresis Gel elecrophoresis Capillary zone elecrophoresis Immuno elecrophoresis Isoelectric focusing elecrophoresis:

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Moving boundary electrophoresis This method allows the charged species to migrate in a free moving solution in absence of a supporting medium. Samples are fractioned in a U shaped tube that has been filled with unstabilised buffer An electrical field is applied by means of electrodes at the ends of the tube Separation takes place as a result of difference in mobilities Bands are obtained which are located by optical systems

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Zone Electrophoresis Migration of charged molecules Support medium porous eg. CA or agarose can be dried & kept Same pH & field strength thru’out Separation based on electrophoretic mobility Separates macromolecular colloids eg. proteins in serum, urine, CSF, erythrocytes; nucleic acids

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Isotachophoresis Migration of small ions Discontinuous electrolyte system leading electrolyte (L- ions) & trailing electrolyte (T- ions) Apply sample solution at interphase of L & T Apply electric field -> each type of ion arrange between L and T ions -> discrete zones Separates small anions, cations, organic & amino acids, peptides, nucleotides, nucleosides, proteins

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Isoelectric Focusing To separate amphoteric cpds eg. proteins Proteins moves to zone where: pH medium = pI protein => charge = 0 pI of protein confined in narrow pH range -> sharp protein zones Method: use horizontal gels on glass/plastic sheets introduce ampholytes into gel -> create pH gradient

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apply a potential difference across gel anode -> area with lowest pH cathode -> area with highest pH proteins migrate until it arrives at pH = pI wash with fixing solution to remove ampholytes stain, destain, visualise Separations of proteins with 0.01 to 0.02pH unit differences

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IMMUNOELECTROPHORESIS: Immunoassay: use of antibodies to detect analyte Antibodies Immunoglobulins that bind to Antigens 5 classes: IgG, IgA, IgM, IgD, IgE Immunogen Protein or a substance coupled to a carrier When introduced into foreign host -> induce Ab to form Antigen Any material which can react with Ab May not induce Ab formation

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APPLICATIONS: Electrophoresis is employed in biochemical and clinical fields: in the study of mixtures such as blood, serum, urine, CSF etc Excellent method for fractionation of protein mixture without denaturation Analysis of lipoproteins Separation of hemoglobin mixtures Separation of mixtures of organic acids Separation of DNA fragments

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REFERENCES: Textbook of Pharmaceutical analysis- Dr S Ravisankar Pharmaceutical Biotechnology: Vyaas SP and Dixit VK http://www.molecular-plant-biotechnology.info/recombinant-DNA-technology/gel-electrophoresis.htm http://www.beckmancoulter.com/resourcecenter/labresources/ce/cedefinitionmodes.asp http://en.wikipedia.org/wiki/Electrophoresis http://www.jbc.org/content/240/1/148.full.pdf http://www.topac.com/isoelectric.html

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