EVALUATION SEMINAR

SUBJECT: M O D E R N P H A R M A C E U T I C A L A N A LY S I S

TOPIC: ELECTROPHORESIS

Presented by DEVI. SI 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 electrophoresiso 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 PRINCIPLE

Electrophoresis 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.

THEORY

Ion migration velocity can be expressed as: V=µeE

The applied field (Fef) (driving force) : Fef=qE

The friction (Ffr) drag is given by: Ffr=6ήπrv

At equilibrium: Fef=Ffr

Therefore: qE=6ήπrv

FACTORS AFFECTING THE MIGRATION OF IONS Factors related to the sampleChargeSizeShape Properties of electric fieldPotential differenceCurrent Resistance Environmental characteristicspHTemperatureElectrolyte concentration Composition and nature of supporting mediumBufferSupporting medium

TYPES OF ELECTROPHORESIS

Free solution / frontal / moving boundary

electrophoresis: supporting medium is absent

Zone electrophoresis: supporting medium is present

1) Paper electrophoresis

2) Gel electrophoresis

3) Capillary electrophoresis

4) Continuous electrophoresis

5) Isotachophoresis

6) Iso electric focusing

MOVING BOUNDARY ELECTROPHORESIS

ZONE ELECTROPHORESISPaper electrophoresis

a) Open strip Paper Electrophoresis

Horizontal

Vertical b) Closed strip Paper Electrophoresis

Horizontal Vertical

c. Immersed strip Paper Electrophoresis

d. Enclosed strip Paper Electrophoresis

+-

Buffer

Filter paper

Cooling liquid

GEL ELECTROPHORESIS

CAPILLLARY ELECTROPHORESIS

CONTINUOUS 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.in

7.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