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Premium member Presentation Transcript ELECTROPHORESIS: ELECTROPHORESIS SVCP M.PHARMACY PH.ANALYSISCONTENTS: CONTENTS Introduction Principle And Theory Factors Classification Moving Boundary Electrophoresis Zone Electrophoresis Isoelectric FocussingINTRODUCTION: INTRODUCTION The word electrophoresis is derived from a Greek word, which means borne by electricity. It is a separation technique in which the components are separated due to their varying behaviour under the influence of applied electric field. It is defined as the migration of charged molecules under the influence of external electric field. The major requirement of the component to be subjected to electrophoresis is that the component should be charged.INTRODUCTION (cont..,): INTRODUCTION (cont..,) It is mostly used for the separation of complex biological substances such as: Proteins Polysaccharides Nucleic acids Peptides Aminoacids Oligosaccharides Nucleosides Organic acids Small anions and cations in body fluidsPRINCIPLE: PRINCIPLE This technique is mainly used for separation of complex mixtures of biological substances which possess ionisable functional groups. Therefore they can be made to exist as electrically charged species, either as cation/anion. Molecules with similar charges will have different m/e ratio. This forms basis for differential migration when these ions in solution are subjected to an electric field.PRINCIPLE (cont..,): PRINCIPLE (cont..,) Therefore electrophoresis can be applied to any mixture in which the components carry a charge & have differential mobilities in an electrical field. The migration in an electrophoretic system depends on properties of particle as well as instrumental system Based on Stoke’s law the mobility of particle (µ) can be calculated from µ = Q/ π r η Where, Q= charge of particle µ= mobility of ion r= radius of particle in cm η = viscosity of mediumTECHNIQUES OF ELECTROPHORESIS: TECHNIQUES OF ELECTROPHORESIS It can be carried out by using either: Low voltage High voltage Low voltage electrophoresis: It consists of two compartments to hold the buffer & electrodes & a suitable carrier for support medium, such that its ends are in contact with buffer compartmentsTECHNIQUES OF ELECTROPHORESIS (cont..,): TECHNIQUES OF ELECTROPHORESIS (cont..,) The design of carrier depends on the medium The medium doesn’t dip into electrode compartments, but into separate compartments connected by wicks with anode & cathode cells The apparatus is enclosed to avoid evaporation LVE can be used in principle to separate any ionic substances Its main application is examination of biological and clinical specimens for aminoacids and proteinsTECHNIQUES OF ELECTROPHORESIS (cont..,): TECHNIQUES OF ELECTROPHORESIS (cont..,) High voltage electrophoresis: The construction of apparatus is similar to that of LVE except that it contains additional cooling system. It was found that much reduced analysis time could be achieved by using high voltage gradient There are three approaches: Use of direct cooling systems in electrophoresis unit. Reduction in concentration of buffer solution. The sheet can be immersed in non conducting liquid and heat exchangers.CLASSIFICATION: CLASSIFICATION Moving boundary electrophoresis. Zone electrophoresis Isotachophoresis Isoelectric focussing Capillary electrophoresis Immuno electrophoresisMOVING BOUNDARY ELECTROPHORESIS: MOVING BOUNDARY ELECTROPHORESIS Principle: It allows charged species to migrate in a free moving solution, without the supporting medium The main features of this method are: The formation of sharp boundaries Large electrode vessels containing reversible electrodes An optical system for following movement of boundaries Thermostatic controlSlide 13: Construction & working: The apparatus consists of U-shaped glass cell of rectangular cross-section consisting of 2 parts, one of which can be made to slide over the other The lower part of cell is filled with lypophilic solution under examination sometimes the sample solution is introduced into bottom of U-tube through a capillary side arm, usually in buffer medium, while the upper part only buffer solution The 2 limbs are connected to 2 large electrode vesselsSlide 14: Care must be taken to minimize the disturbing effect of convection currents caused by an increase in temperature during the passage of current through the solution. For this purpose, the apparatus is placed in a constant temperature bath at 4˚c (at this temperature, the density of water is maximum, hence density differences and convection currents can be minimised)Slide 15: By applying current of suitable potential difference, the differential migration of charged particles, towards one or another electrodes is observed Separation occurs due to difference in mobility of molecules. Mobility is proportional to m/e ratio. The position of moving ions, which forms a boundary, which is detected by measuring the changes in refractive index throughout solution. The concentration gradients which are formed during electrophoresis are usually detected by Schleiren optical method.Slide 16: In Philpot-Stvenson or shadow method the boundary between the solute and buffer appears as dark line on light back ground. These are photographed by cylindrical lens, where boundaries are seen as peaks. The height of area under peak is proportional to amount of protein causing number of electrophoretically different components.ZONE ELECTROPHORESIS: ZONE ELECTROPHORESIS It involves migration of charged particles, which are supported on relatively inert and homogeneous solid or gel framework. In this method the separated components are distributed into discrete zones on stabilizing media. The zones are heterogeneous and physically separated from one another. It is classified based on supporting material used. They are: Paper electrophoresis Cellulose acetate electrophoresis Thin layer electrophoresis Gel electrophoresisSlide 18: Principle : Basically a supporting media is saturated with buffer solution and a small amount of sample solution is applied as narrow band. On application of potential difference between the ends of strip, each component migrates at a rate determined by its electrophoretic mobility.ADVANTAGES AND DISADVANTAGES: ADVANTAGES AND DISADVANTAGES Useful in biochemical investigation. Very small quantity of samples can be analysed. Useful to study both simple and complex mixtures equally. Equipment cost is low and maintenance is easy. Unsuitable for accurate mobility and isoelectric point determination. Complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced.GENERAL METHOD OF OPERATION: GENERAL METHOD OF OPERATION Saturation of medium: the supporting medium other than gel must be saturated with a buffer so that it can conduct current. Sample application: sample is applied as spot or streak. Electrophoretic separation: the power is switched on at required voltage. After completion of separation the power is switched off before supporting media is removed. Removal of supporting medium: paper, cellulose acetate strips and thin layer plate are removed and air dried or in oven. The gels are removed by forcing water from hypodermic syringe.INSTRUMENTATION: INSTRUMENTATION Electrophoretic chamber: It contains buffer solution. Electrodes : Ag/AgCl reversible electrodes can be used. Diffusion barriers: The electrode should be separated from the electrophoretic bed by a barrier such as gel, filter paper, sponge. Supporting media: It should have low resistance to electric current, inert to sample, electrolyte and developing reagents.PAPER ELECTROPHORESIS: PAPER ELECTROPHORESIS One of the simplest process in electrophoresis involves spotting a mixture of solute in middle of paper , moistening the paper with some electrolyte and placing it between two sheets of glass. The ends of paper strip extending beyond glass plate are immersed in beakers of electrolyte. A potential of 5V/cm of paper length is placed from a DC source. It is allowed to continue for a period of several hours.ADVANTAGES AND DISADVANTAGES: ADVANTAGES AND DISADVANTAGES It is economical and also easy to use. Some compounds such as proteins can not be adequately resolved. There are three types of paper electrophoresis: Horizontal Vertical and ContinuousCELLULOSE ACETATE ELECTROPHORESIS: CELLULOSE ACETATE ELECTROPHORESIS Cellulose acetate strips, which are used widely in clinical laboratories produce excellent separations of 7 to 9 protein fractions in a few hours. this material is exceedingly fine and homogeneous, and little tailing is encountered due to adsorption. It is especially useful for separating alpha immunoglobulins from albumin. It contains 2 to 3 acetyl groups per glucose unit and its absorption capacity is less than that of paper. GEL ELECTROPHORESIS: GEL ELECTROPHORESIS The separation here is brought about through molecular sieving technique, based on molecular size of substancesTHIN LAYER ELECTROPHORESIS: THIN LAYER ELECTROPHORESIS Electrophoretic studies can be also carried out on thin layers of silica, keisulghur,alumina. The studies with these materials offer advantages of speed and resolution when compared with paper. They have greatest application in combined electrophoretic-chromatography studies in two-dimensional study of proteins and nucleic acid hydrolysates.ISO ELECTRIC FOCUSSING: ISO ELECTRIC FOCUSSING It is mainly used for a separation of electrolytes such as proteins. When electrophoresis is run in solution buffered at constant pH, proteins having net charge will migrate towards opposite electrode. The use of pH gradient across supporting medium causes each protein to migrate to an area of specific pH. A sharp well defined protein bands occur at the point where iso electric point equals to pH of gradient.Slide 30: Separation is carried out on gels on which a stable a pH gradient has been established. An ampholytic compound has a pH at which it is neutral. The pH gradient is achieved by impregnating the gel with polyamino-polycarboxylic acids. When subjected to electric field these migrate and come to rest in order of their pH. Thus each ampholyte migrates in applied field until it reaches a position on the plate where pH of medium is equal to iso electric point.Slide 31: At this point ampholyte is in its zwitter ion form and is neutral. Thus it losses electrophoretic mobility and becomes focused in narrow zone at this point.Slide 32: Advantages: Spreading of bands is minimized. Proteins that differ by little as 0.01pH can be adequately resolved Disadvantages: As carrier ampholytes are used in high concentration, a high voltage power supply is necessary. As a result the electrophoretic matrix must be cooled.Slide 33: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.