Electro phoresis sud mpharm pdf


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Electro phoresis sud mpharm pdf

  1. 1. ELECTROPHORESIS Sudheer kumar kamarapu Assistant professorSri Shivani college of Pharmacy 1 sudheerkumar kamarapu
  2. 2. CONTENTS Introduction Principle And Theory Factors Classification Capillary electrophoresis Zone Electrophoresis2 sudheerkumar kamarapu
  3. 3. 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.3 sudheerkumar kamarapu
  4. 4. 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 fluids 4 sudheerkumar kamarapu
  5. 5. Cont... Separation Scientists work in many different disciplines, some of these include:  Analytical Chemistry  Biochemistry  Biotechnology  Forensic Science  Food Science  Clinical Science  Neuro-Science  Medical Research and Production  Pharmaceutical Science  Other Disciplines5 sudheerkumar kamarapu
  6. 6. 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.6 sudheerkumar kamarapu
  7. 7. 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 ηWhere, Q= charge of particle µ= mobility of ion r= radius of particle in cm η = viscosity of medium7 sudheerkumar kamarapu
  8. 8. TECHNIQUES OF ELECTROPHORESIS It can be carried out by using either:1. Low voltage2. High voltageLow 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 compartments8 sudheerkumar kamarapu
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  10. 10. 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 proteins10 sudheerkumar kamarapu
  11. 11. 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 gradient11 sudheerkumar kamarapu
  12. 12. CLASSIFICATIONA. Capillary electrophoresisB. Zone electrophoresis 1. Paper electrophoresis 2. Cellulose acetate electrophoresis 3. Thin layer electrophoresis 4. Gel electrophoresis12 sudheerkumar kamarapu
  13. 13. CapillaryElectrophoresis 13 sudheerkumar kamarapu
  14. 14.  In practical terms, a positive (anode) and negative (cathode) electrode are placed in a solution containing ions.  Then, when a voltage is applied across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive), will move through the solution towards the electrode of opposite charge.  Capillary electrophoresis, then, is the technique of performing electrophoresis in buffer-filled, narrow-bore capillaries, normally from 25 to 100 pm in internal diameter (ID).14 sudheerkumar kamarapu
  15. 15.  Definition: The differential movement for migration of ions by attraction or repulsion in an electric field.  Separation of components of a mixture using an electric field v=Eq/f  v = velocity of molecule  E = electric field  q = net charge of molecule  f = friction coefficient 15 sudheerkumar kamarapu
  16. 16. Capillary Electrophoresis – The Basics Of Instrumentation Electrophoresis in a buffer filled, narrow-bore capillaries Each capillary is about 25-100 μm in internal diameter When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge Depending on the charge, the molecules move through at different speeds  Separation is achieved 16 sudheerkumar kamarapu
  17. 17. Basics cont. A photocathode is then used to measure the absorbencies of the molecules as they pass through the solution The absorbencies are analyzed by a computer and they are represented graphically sudheerkumar kamarapu 17
  18. 18. Equipment  Capillary tube  Varied length but normally 25- 50 cm  Small bore and thickness of the silica play a role  Using a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage18 sudheerkumar kamarapu
  19. 19. Equipment Cont….  Detector  UV/Visible absorption  Fluorescence  Radiometric (for radioactive substances)  Mass Spec.19 sudheerkumar kamarapu
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  21. 21. Capillary Electrophoresis Apparatus21 sudheerkumar kamarapu
  22. 22. The Electropherogram The data output from CE is presented in the form of an electropherogram, which is analogous to a chromatogram. An electropherogram is a plot of migration time vs. detector response. The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence. The appearance of a typical electropherogram is shown in Figure for the separation of a three component mixture of cationic, neutral and anionic solutes. 22 sudheerkumar kamarapu
  23. 23. ZONEElectrophoresis 23 sudheerkumar kamarapu
  24. 24. 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:1. Paper electrophoresis2. Cellulose acetate electrophoresis3. Thin layer electrophoresis4. Gel electrophoresis24 sudheerkumar kamarapu
  25. 25. 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.25 sudheerkumar kamarapu
  26. 26. 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.26 sudheerkumar kamarapu
  27. 27. 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. 27 sudheerkumar kamarapu
  28. 28. INSTRUMENTATION1. Electrophoretic chamber: It contains buffer solution.2. Electrodes : Ag/AgCl reversible electrodes can be used.3. Diffusion barriers: The electrode should be separated from the electrophoretic bed by a barrier such as gel, filter paper, sponge.4. Supporting media: It should have low resistance to electric current, inert to sample, electrolyte and developing reagents.28 sudheerkumar kamarapu
  29. 29. 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.29 sudheerkumar kamarapu
  30. 30. 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:1. Horizontal2. Vertical and3. Continuous30 sudheerkumar kamarapu
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  33. 33. CELLULOSE ACETATEELECTROPHORESIS1. Cellulose acetate strips, which are used widely in clinical laboratories produce excellent separations of 7 to 9 protein fractions in a few hours.2. this material is exceedingly fine and homogeneous, and little tailing is encountered due to adsorption.3. It is especially useful for separating alpha immunoglobulins from albumin.4. It contains 2 to 3 acetyl groups per glucose unit and its absorption capacity is less than that of paper.33 sudheerkumar kamarapu
  34. 34. GEL ELECTROPHORESISThe separation here is brought about through molecular sieving technique, based on molecular size of substances 34 sudheerkumar kamarapu
  35. 35. THIN LAYER ELECTROPHORESIS1. Electrophoretic studies can be also carried out on thin layers of silica, keisulghur,alumina.2. The studies with these materials offer advantages of speed and resolution when compared with paper.3. They have greatest application in combined electrophoretic-chromatography studies in two- dimensional study of proteins and nucleic acid hydrolysates.35 sudheerkumar kamarapu
  36. 36. sudheerkumar kamarapuTHANK YOU 36