electrophorosis (1).pptx

all about electrophoresis in summarize form.

1. Amity institutes of pharmacy PRESENTATION ON TOPIC : ELECTROPHORESIS Presented By : ANKIT RAJ M. PHARMA 1 st SEMESTER

2. CONTENT INTRODUCTION PRINCIPLE FACTOR AFFECTING ELECTROPHORESIS INSTRUMENTATION TYPES OF ELECTROPHORESIS : 1. PAPER ELECTOPHORESIS 2. GEL ELECTROPHORESIS 3. CAPILARY ELECTROPHORESIS 4. MOVING BOUNDARY ELECTROPHORESIS

3. INTRODUCTION: Electrophoresis is a  physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. 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 travel towards the electrodes that bears opposite charges. electrophoresis was first observed by Russian professors Peter I.S and Ferdinand Frederic Reuss in 1807 at Moscow University.

4. PRINCIPLE : electrophoresis is the existence of charge separation between the surface of a particle and the fluid immediately surrounding it. An applied electric field acts on the resulting charge density, causing the particle to migrate and the fluid around the particle to flow. Any charged ion  or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge, size, shape and the applied electric current. Can be represented by following eq. V=E*q/f v=  velocity of migration of the molecule. E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient

5. FACTORS AFFECTING ELECTROPHORESIS 1. Magnitude of the charge of a molecule 2. Charge density of the molecule 3. Molecular weight 4. Its shape 5. The pH of the solution 6. Electric field 7. The viscosity of the solution or agarose concentration 8. The temperature of operation

6. INSTRUMENTATION

7. TYPES OF ELECTROPHORESIS 1. PAPER ELECTOPHORESIS 2. GEL ELECTROPHORESIS 3. CAPILARY ELECTROPHORESIS 4. MOVING BOUNDARY ELECTROPHORESIS

8. 1. PAPER ELECTOPHORESIS In this type of electrophoresis, a filter paper (similar to chromatography paper) with a low adsorption capacity and homogeneous pore size serves as the supporting medium for sample separation under the effect of an applied electric field..

9. INSTRUMENTATION The sample are spotted at centre of paper ,which is dip into a buffer solution , high voltage is applied and spot is migrate according to their charge , after electrophoresis the seperated component can be detected by variety of staining technique, depending upon their chemical intity .

10. Application Seperation of amino acids Seperation of protein serums Study of sickle cell anaemia in hemoglobin Seperation of antibiotics can be carried out

11. 2. GEL ELECTROPHORESIS Gel electrophoresis is a technique for separating DNA fragments (or other macromolecules like RNA and proteins) depending on size and charge. Electrophoresis is the process of passing a current across a gel containing the molecules of interest. The molecules will go through the gel in different directions or at different speeds depending on their size and charge, allowing them to be segregated from one another . Principle of separation According to charge: When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. According to size: The smaller molecules move more swiftly than the larger sized ones, as the can travel through the pores more easily than the later.

12. INSTRUMENTATION Sample is loaded into the wells using a clean micropipette. Molecules migrate towards oppositely charged electrodes, thus DNA, being negatively charged, moves towards anode. The migration is visually tracked via dye. After the process ends, gel is stained and visualized using a gel imager instrument.

13. APPLICATION Separation of  Deoxyribonucleic acid Separation of ribonucleic acid Separation of protein molecules It may be used as preparative technique prior to use of other methods such as mass spectroscopy, cloning, DNA Sequences, Southern Blotting for further characterization. Separation of amino acid Separation of lipoproteins Separation of enzyme in blood

14. 3. CAPILARY ELECTROPHORESIS In this method, separation are carried out inside a capillary tube. • The capillary tube has a diameter of 50 μm to facilitate temperature control. • The length of the capillary is typically 20–50 cm. • The ends of the capillary are dipped into reservoirs filled with the electrolyte. • The capillary is filled with running buffer, one end is dipped into the sample, and an electric field is applied to introduce the sample inside the capillary. • Migration through the capillary is driven by application of a high-voltage current (5KV for 5 sec). 

15. INSTRUMENTATION A typical capillary electrophoresis system consists of: • A buffer solution (Sodium dihydrogen phosphate, Sodium tetraborate). • A high voltage supply (5 – 30 KV). • A sample introduction system or sample injection (by pressure/ vaccum ). • A capillary tube with internal diameter of 10-100mm and a length of 20-100cms. • A detector and an output device. • Some instruments include temperature control device, to ensure reproducible results.

16. APPLICATION Genetic Analysis. Analysis of Pharmaceuticals. Pharmaceuticals with Chiral Centers (Enantiomers). Counter-ion analysis in drug discovery. Protein Characterisation. Genetic Analysis DNA Fragment  Analysis

17. 4. MOVING BOUNDARY ELECTROPHORESIS Moving boundary electrophoresis is electrophoresis in free solution. It was developed by Arne Tiselius. The principle of moving boundary electrophoresis is that there is motion of charged particles through stationary liquid under the influence of an electric field . This method allows the charged species to migrate in a free moving solution in the absence of a supporting medium. Samples are fractioned in a U shaped tube that has been filled with unstabilized buffer. An electrical field is applied by means of electrodes at the ends of the U tube. Separation takes place as a result of difference in mobilities.

18.   INSTRUMENTATION • It consists  of a U shaped glass tube of rectangular cross section, with electrodes placed on each limb of the tube. • Sample solution is introduced through the side arm and the apparatus is placed in a constant temperature bath at 40° C . • Detection is done by measuring refractive index throughout the solution.

19. APPLICATIONS • To separate  proteins and peptides. • For research in enzymology, immunology. • Used for quantitative analysis of complex mixture of macromolecules.