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Electrophoresis

Eletrophoresis

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ELECTROPHORESIS
TECHNIQUES
S.ALEXANDAR
DEPT. OF PHARMACEUTICAL ANALYSIS
VINAYAKA MISSION’S COLLEGE OG
PHARMACY
SALEM
THEORY OF ELECTROPHORESIS
DEFINITION:
•Electrophoresis may be defined as the migration of the charged particle
through a solution under the influence of an external electrical field.
•Ions that are suspended between two electrodes tends to trave3 towards the
electrodes that bears opposite charges.
Depending on kind of charge the molecule carry, they move towards either
To cathode
Or to Anode
An ampholyte become positively charged in acidic condition and migrate to
cathode, in alkaline condition they become negatively charge and migrate to anode.
The rate of migration of an ion in electrical field
depend on factors,
1.Net charge of molecule
2.Size and shape of particle
3.Strength of electrical field
4.Properties of supporting medium
5.Temperature of operation
Conventional
electrophoresisInstrumentation :
Two reservoir for the
buffer
Power supply and
Electrodes
Separation medium
10
At any given PH, exist in a solution as electrically charged
species either as a cation (+) or anion(-).
Under the influence of an electric field these charged particles
will migrate either to cathode or anode, depending on the nature
of their net charged.
The equipment required for electrophoresis consist basically of
two items,
_power pack
Supplies a direct current between electrode in the electrophoresis
unit.
_electrophoresis unit
Available for running either vertical or horizontal
Gel system.

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Electrophoresis

  • 1. ELECTROPHORESIS TECHNIQUES S.ALEXANDAR DEPT. OF PHARMACEUTICAL ANALYSIS VINAYAKA MISSION’S COLLEGE OG PHARMACY SALEM
  • 2. THEORY OF ELECTROPHORESIS DEFINITION: •Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. •Ions that are suspended between two electrodes tends to trave3 towards the electrodes that bears opposite charges. Depending on kind of charge the molecule carry, they move towards either To cathode Or to Anode An ampholyte become positively charged in acidic condition and migrate to cathode, in alkaline condition they become negatively charge and migrate to anode.
  • 3. The rate of migration of an ion in electrical field depend on factors, 1.Net charge of molecule 2.Size and shape of particle 3.Strength of electrical field 4.Properties of supporting medium 5.Temperature of operation
  • 4. Conventional electrophoresisInstrumentation : Two reservoir for the buffer Power supply and Electrodes Separation medium
  • 5. 10
  • 6. At any given PH, exist in a solution as electrically charged species either as a cation (+) or anion(-). Under the influence of an electric field these charged particles will migrate either to cathode or anode, depending on the nature of their net charged. The equipment required for electrophoresis consist basically of two items, _power pack Supplies a direct current between electrode in the electrophoresis unit. _electrophoresis unit Available for running either vertical or horizontal Gel system.
  • 8. Buffer The buffer in electrophoresis has two purpose: Carry applied electrical current They set the pH as which electrophoresis is carried out.  Thus they determine; Type of charge on solute. Extent of ionization of solute Electrode towards which the solute will migrate.  The buffer ionic strength will determine the thickness of the ionic cloud.
  • 10. Supporting mediumSupporting medium is an matrix in which the protein separation takes place. Various type has been used for the separation either on slab or capillary form. Separation is based on to the charge to mass ratio of protein depending on the pore size of the medium, possibly the molecular size.
  • 11. TYPES OF ELECTROPHORESIS 1) Zone Electrophoresisa) Paper Electrophoresis b) Gel Electrophoresis c) Thin Layer Electrophoresis d) Cellulose acetate Electrophoresis 2)Moving Boundary Electrophoresis a) Capillary Electrophoresis b) Isotachophoresis c) Isoelectric Focussingd) Immuno Electrophoresis 4
  • 12. ZONE ELECTROPHORESIS  It involves the migration of the charged particle on the supporting media.  Paper, Cellulose acetate membrane, Starch Gel, Poly acrylamide.  Components separated are distributed into discrete zone on the support media.  Supporting media is saturated with buffer solution, small volume of the sample is applied as narrow band.  On application of PD at the ends of a strip components migrates at a rate determined by its electrophoretic mobility. ADVANTAGES:  Useful in biochemical investigations.  Small quanity of sample can be analysed.  Cost is low and easy maintenance. DISADVANTAGES:  Unsuitable for accurate mobility and isoelectric point determination. 5  Due to the presence of supporting medium, technical complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced.
  • 13. GENERAL METHOD OF OPERATIONSaturation of the medium with the buffer. Sample application. Electrophoretic separation. Removal of the supporting media. INSTRUMENTATION 6  Electrophoretic chamber.  Electrodes.  Diffusion barriers.  Supporting/ Stabilizing media. (inert to sample and to any developing reagents).
  • 14. PAPER ELECTROPHORESIS 1. Horizontal paper Electrophoresis 2. Vertical paper Electrophoresis 7
  • 16. GEL ELECTROPHORESIS Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.  Gel is a colloid in a solid form (99% is water).  It is important that the support media is electrically neutral.  Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.  A porous gel acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely. 11
  • 17. GEL ELECTROPHORESIS Gel Electrophoresis is caried out in two methods: •Vertical starch gel electrophoresis •Horizontal starch gel electrophoresis 12
  • 18. AGAR AND AGAROSE GEL  Agar is a mixture of poly saccharides extracted from sea weeds.  Agarose is a highly purified uncharged polysaccharide derived from agar.  Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L-galactose.  The pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes. ADVANTAGES: Easy to prepare and small concentration of agar is required. Resolution is superior to that of filter paper. Large quantities of proteins can be separated and recovered. Adsorption of negatively charged protein molecule is negligible. It adsorbs proteins relatively less when compared to other medium. Sharp zones are obtained due to less adsorption. Recovery of protein is good, good method for preparative purpose. 13 DISADVANTAGES  Electro osmosis is high.  Resolution is less compared to polyacrylamide gels.  Different sources and batches of agar tend to give different results and purification is often necessary.
  • 19. •Polyacrylamide gels are tougher than agarose gels. •It is thermo stable, transparent, strong and relatively chemically inert. •Gels are uncharged and are prepared in a variety of pore sizes. •Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving. • Gels are stable over wide range of pH and temperature. • Gels of different pore size can be formed. • Simple and separation speed is good comparatively. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) 15 ADVANTAGES:
  • 20. PAGE-PROCEDURE  The gel of different pore sizes is cast into a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom.  Microgram quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such a pH so as to change sample components into anions.  The foot of the gel column is made to dip in the same buffer in the bottom reservoir.  Cathode and anode are kept above and below the column to impose an electric field through the column.  Macromolecular anions move towards the anode down the gel column.  There is no external solvent space, all the migratory particles have to pass through the gel pores.  Rate of migration depends on the charge to mass ratio.  Different sample components get separated into discrete migratory bands along the gel 17column on the basis of electrophoretic mobility and gel filtration effect.
  • 21. Polyacrylamide (PAGE) a) The gel Gel Electrophoresis is poured vertically between two glass plates. b.) Protein bands are separated on th1e8 basis of relative molecular weight and visualized with stains. SLAB PAGEPAGE PROCEDURE
  • 22. VISUALIZATION  After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.  Ethidium bromide, silver, or coomassie blue dye may be used for this process.  If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioac1t9ivity added for visibility, an autoradiogram can be recorded of the gel.
  • 23. APPLICATIONS OF ELECTROPHORESIS 1. DNA Sequencing 2. Medical Research 3. Protein research/purification 4. Agricultural testing 5. Separation of organic acid, alkaloids,carbohydrates, amino acids, alcohols, phenols, nucleic acids, insulin. 6. In food industry 7. It is employed in biochemical and clinical fields i.e. in the study of protein mixtures such as blood serum, haemoglobins and in the study of antigen- antibody interactions. 8. Electrophoresis is also used for separation of carbohydrates and vitamins. 9. Quantitative separation of all fractions of cellular entities, antibiotics, RBC, Enzymes etc is possible. 10.Quantitative separation of all fractions of cellular entities, antibiotics, RBC, Enzymes etc is possible. 45
  • 24. 1. Electrophoresis in combination with autoradiography is used to study the binding of iron to serum 2. proteins. 3. Used for analysis of terpenoids , steroids and antibiotics. 4. For testing purity of thyroid hormones by zone electrophoresis. 5. Paper chromato-electrophoresis is used to separate free Insulin from plasma proteins. 6. It is used for diagnosis of various diseases of kidney , liver and CVS. 7. It is also used for separation of Scopolamine and Ephedrine using buffer at PH 4.2.