EVALUATION SEMINARSUBJECT: MODERN PHARMACEUTICAL ANALYSISTOPIC: ELECTROPHORESIS Presented by DEVI. S I M. Pharm, Department of Pharmaceutics, M.S.R.C.P, Bangalore-54.
CONTENTS¤ Introduction¤ Definition and Principle¤ Theory¤ Factors affecting the migration of ions¤ Types of electrophoresis o Moving boundary electrophoresis o Zone electrophoresis
INTRODUCTION Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. ELECTROPHORESIS (Greek word)=BORNE BY ELECTRICITY It is a separation technique in which the components are separated due to their varying behavior under the influence of an applied electric field. The technique was pioneered in 1937 by the Swedish chemist Arne Tiselius for the separation of proteins.
DEFINITION AND PRINCIPLEElectrophoresis is defined as the “migration of charged molecules under the influence of an external electric field” 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.
THEORYIon migration velocity can be expressed as: V=µeEThe applied field (Fef) (driving force) : Fef=qEThe friction (Ffr) drag is given by: Ffr=6ήπrvAt equilibrium: Fef=FfrTherefore: qE=6ήπrv
FACTORS AFFECTING THE MIGRATION OFIONS Factors related to the sample Charge Size Shape Properties of electric field Potential difference Current Resistance Environmental characteristics pH Temperature Electrolyte concentration Composition and nature of supporting medium Buffer Supporting medium
TYPES OF ELECTROPHORESISFree solution / frontal / moving boundaryelectrophoresis: supporting medium is absentZone electrophoresis: supporting medium is present1) Paper electrophoresis2) Gel electrophoresis3) Capillary electrophoresis4) Continuous electrophoresis5) Isotachophoresis6) Iso electric focusing
MOVING BOUNDARY ELECTROPHORESIS
ZONE ELECTROPHORESIS Paper electrophoresisa) Open strip Paper Electrophoresis Horizontal Verticalb) Closed strip Paper Electrophoresis Horizontal Vertical
c. Immersed strip Paper Electrophoresis - + Cooling liquid Filter paper Bufferd. Enclosed strip Paper Electrophoresis
ISOTACHOPHORESIS Iso-equal , tachos- speed, phoresis-migration The technique of Isotachophoresis depends on the development of potential gradient.Principle: A leading electrolyte(chloride) with a higher mobility than the analytes and a trailing electrolyte(glycinate) with a lower mobility are used. After application of an electric potential a low electrical field is created in the leading electrolyte and a high electrical field in the terminating electrolyte. The pH at sample level is determined by the counter-ion of the leading electrolyte that migrates in the opposite direction.
ISO ELECTRIC FOCUSINNG(IEF)» Proteins carry both positive and negative charges, which is the PH when molecule has no net charge.» The pH which gives zero net charge is the isoelectric point or pH.» Generally proteins readily crystallize at the isoelectric point.» Most of the proteins have isoelectric point of 5-9.» When electrophoresis is run in a solution buffered at constant PH , proteins having a net charge will migrate towards the opposite electrode so long as the current flows.» The use of PH gradient across the supporting medium causes each protein to migrate to an area of specific PH» Proteins are found at the point of the gradient where they carry no net charge.» The PH of the protein equals the PH of the gradient, thus resulting in sharp well defined protein bands.
REFERENCES1. Instrumental methods of chemical analysis by B.K. sharma pg.no C269-c281.2. Electrophoresis by Melvin wiley publications.3. http://en.wikipedia.org/wiki/Electrophoresis.4. www.pharmainfo.net.5. Theory of Electrophoresis-K.S. Pitre, Dr. Harisingh Gour University, Sagar, India, Encyclopedia of Analytical Science.6. http://books.google.co.in7. Fundamentals of Analytical chemistry-Skoog (pg.no-1003)
‘winning doesn’t always mean being first, means ur doing better than you have done before’ Bonnie Blair