electrophoresis technique principle procedure

electrophorosis

1. By K. Lakshmi Bhavani Y11MPH425 I/II M.Pharmacy Pharmaceutical Analysis Chalapathi Institute of Pharmaceutical Sciences Instrumentation and applications of ELECTROPHORESIS 1

2. Introduction: It was introduced by the sweedish chemist Arne Tiselius. Electrophoresis involves the separation of charged species on the basis of their movement under the influence of an applied electric field. Molecules under study must have either a net positive or negative charge Principle: In electrophoresis under the influence of an applied electric field positively charged molecules will move towards the cathode while negatively charged molecules move towards the anode. A molecule with no net charge should remain stationary 2

3. In this molecules move in a liquid medium which is supported by an inert solid substance such as paper or a semisolid gel. The liquid serves as a conducting medium for the electric current generated by the application of an external voltage to the medium. Migration velocity α strength of the applied electric field v α E v = µE where µ is electrophoretic mobility When a molecule is suspended in liquid medium, the applied electric field exerts a force on the molecule which will helps to accelerate the charged species towards electrodes. 3

4. Elecric force (-QE) Electric force (+QE) +Q -Q Viscous drag Viscous drag + anode - cathode magnitude of force of viscous drag α viscosity of the medium, size and shape of the molecule When electric force = viscous drag molecule reaches steady state. Electri force = field strength(E)×size of the charge(Q) The molecule will be opposed by the frictional force of mediumm which is called as viscous drag 4

5. Instrumentation of electrophoresis include Electrophoretic chamber Supporting media Electrodes Source of current Electrolytes and buffers Sample origin Cloth or paper wick Sample origin Cloth or paper wick cathode anode Glass plate Buffer solution Supporting medium 5

6. Electrophoretic chamber made up of insulating material provision for measuring the voltage drop between the ends of the electrophoretic bed provision for preventing the evaporation of buffer from electrophoretic bed. adjusting the electrolyte to equal level there by preventing the siphoning action through bed. it should provide with continuous mixing and recycling of buffer in the chamber in order to neutralize the decomposition products. Destruction of buffer is minimised by interposing electrically permeable barriers between the electrode chambers and the bed 6

7. Supporting media : may be filter paper, cellulose acetate, gels Electrodes includes graphite rods, stainless steel, silver chloride and the noble metals like ‘Pt’ ‘Pt’ is best because it never be replaced Electrodes should not be too small, because of the following reason they may be polarized by becoming covered with gas bubbles or other products of electrolysis they also limit the amount of current that is allowed to flow between them. Source of current direct current range from 100 to 300 volts 7

8. Electrolytes/Buffers used the P H of the buffer to be used depends upon the types of compounds to be separated the ionic strength of the buffer affects the migration velocity of compounds. migration velocity is inversely proportional to ionic strength. most commonly used buffers 1. Barbitone buffer 0.07mol/lit P H 8.6 2. Tris - acetate buffer 0.07mol/lit P H 7.6 3. Citrate buffer 0.07mol/lit P H 3.0 other buffers of different P H can also be used for separation based on type of compound. 8

9. Different types of Electrophoretic system 1. moving boundary electrophoresis 2. zone electrophoresis 3. steady state electrophoresis a. I soelectric focussing b. I sotachophoresis 9

10. Moving boundary Electrophoresis Consists of U shaped vessel with rectangular cross section. Place protein solution and then buffer solution Draw backs slowest and most rapidly moving components of a mixture Can be obtained in pure form. Useful for determination of complexity Heterogenous samples 10

11. zone electrophoreis (gel) Most commonly used method. Sample is applied to the support medium as a spot or a narrow band. During run sample separate into bands which are kept distinct by the presence of the support medium. 11

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13. Isotachophoresis useful for the separation of charged molecules of Small relative molecular mass, such as certain drugs and polypeptides of medical interest. in isotachophoresis all charged components become stacked one behind. The other according to their electrophoretic mobilities . to separate two ions from each other by isotachophoresis their must be a difference of at least 10% between their electrophoretic mobilities . 13

14. Support media used in zone electrophoresis FILTER PAPER CELLULOSE ACETATE GEL MEDIA Starch gel Polyacrylamide gels Agarose gels Polyacrylamide-Agarose gels 14

15. Filter paper Filter paper is moistened then placed on a supporting rack Samples are applied with a capillary applicator as spots or streaks Used for separation of proteins, oligonucleotides, carbohydrates and lipids Sample origin paper cathode cover anode Compartment support divider and buffer solution 15

16. ADVANTAGES: Simple, quick and required relatively simple apparatus . DISADVANTAGES: If paper is too thin - tear easily too thick - boundaries between the zones become distorted proteins interact with OH groups in cellulose and leads to tailing and alter the separation characteristics of protein components of a mixture Electro-osmosis due to interaction between COOH and water molecules 16

17. Cellulose acetate Problem of tailing has been encountered by introducing cellulose acetate membrane. OH groups are esterified by acetylation Separation is quickly when compared to filterpaper . Care should be taken to select a solvent that will not damage the components of interest. Disadvantages Electro osmotic anode to cathode flow of water. Uses In clinical laboratories for separation and analysis of proteins in biological fluids by using small amount of sample and by short run times 17

18. Gel media Gels are cross linked polymers. Properties of gel include inert and not to interact with the molecules under study Does not have any ionisable groups Spaces between the gel molecules are the pores of the gel. Pore size depends on the concentration of polymer conc α 1/ poresize While molecules moving through the continuous gels experiences a frictional resistance to its movement. Frictional resistance depends on the poresize and the radius of the molecule. The bands obtained in electrophoresis doesnot have only one component 18

19. Starch gels Prepared from hydrolysed potato starch which is heated in electrophoresis buffer untill the suspension becomes transparent and poured into a mould. Advantages cheap,easy and quick technique. widely used for proteins Disadvantages Narrow range pore size Contains negatively charged side chains which may interact with proteins and leads to electro osmosis. Poly acrylamide gels By varying concentration of acrylamide pore size of gel varies Acrylamide + N N 1 -methylene catalyst polyacrylamide bisacrylamide initiator Initiators used are ammonia, potasssium persulphate , light and riboflavin. 19

20. Dyes used to visualise the mobility bromophenol blue anionic dye methylene blue cationic dye Advantages heat dissipation is better high resolving power A comb is inserted into the apparatus before the gel polymerizes and is removed after polymerization leaving sample wells in which samples are loaded. Uses For analysis of proteins, small RNA molecules, and fragmentation of DNA 20

21. Types of electrophoresis using polyacrylamide gel Disc electrophoresis SDS- polyacrylamide gel electrophoresis Disc Electrophoresis Disc is termed because of the formation of discoid zones. It has two gels Lower gel or running gel or upper gel or stacking or seiving gel or separating gel spacer gel Polyacrylamide-2.5% 15% P H , ionic strength- high low Pore size - small large 1 st lower layer gel then upper layer power is switch on dye is added Zones are separated Then sample with sucrose soln carefully layered on top of the gel 21

22. Uses for the determination of the purity of a protein sample and For analysis of mixtures containing many components. used to estimate the relative molecular masses of different proteins. D = a-blog 10 M D – distance migrated by a molecule m – molecular mass a, b – are constants of the electrophoresis system Agarose gels linear polymer of D- galactose and 3,6 anhydro galactose contains 0.04%sulphate ions agarose obtained from sea weeds. It dissolves in boiling water after cooling it gives gel Pore size depends upon the concentration of agarose and is large than poly acrylamide gel Electro osmotic flow is avoided because there is pretreatment with alkali, which hydrolyses the anionic groups. 22

23. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS for protein molecules of different net charges the difference in charge can be eliminated by complexing them with the anionic detergent sodium dodecyl sulphate at neutral P H in presence of 1%w/v SDS and 0.1M 2-mercapto ethanol, protein break down at disulphide bridges and binds to SDS through hydrophobic interactions. The sodium ion serve as a counter ion to the SDS- protein complex. Proteins have an identical charge to size ratio because the amount of SDS bound per unit mass of protein is constant Uses For detemination whether a protein is made up of subunits 23

24. Poly Acrylamide - Agarose gels combination of polyacrylamide and agarose produces relatively large pore sizes and good mechanical strength Uses For separation of nucleic acids,nucleoproteins , very large proteins Factors effecting Electrophoretic mobility 1 . viscosity of the medium ( η ) ν α 1/ η 2. strength of the applied electric field ν α E 3.net charge on the molecule(Q) ν α Q 4. the size and shape of the molecule ν α 1/r 5 . P H of buffer and Ionic strength migration of compounds is inversely proportional to the ionic strength. At low strength migartion is faster 24

25. Detection Of Sample Components Separated By Electrophoresis 1. optical methods : - by using UV-radiation: by photographing the electrophoretogram . - detection of local changes in refractive index and allows visualisation of the separating components. - these two methods depends upon the interaction of molecules with electromagnetic radiation. 25

26. UV absorption by separated components : proteins show at 280nm.due to resonance of peptidee bond. Molar absoptivity of proteins depends on th proportions of tyrosine, tryptophan and phenylalanine. polynucleotides show at 260nm due to resonance of heterocyclic ring. difficulty while using polyacrylamide gels because of amide bond resonance it absobs UV radiation 26

27. 2.staining: by staining with dye or stain we can detect the positions by absorption or fluorescence. selection of stain: - should be selective - product formed between the chemical and the substance of interest must be stable and insoluble during staining and destaining procedures - the reaction and its removal will be quickly. - the stained prodct should have a high molar absorptivity or fluorescence emission, to ensure high sensitivity. we can stain -before electrophoresis - after electrophoresis 27

28. Type of substance Staining reagent comments proteins Dansyl chloride 1-anilino-8-naphthalene sulphonic acid - fluorescamine Reacts with amino group Non fluorescent, but gives fluorescent product - Non fluorescent but gives a fluorescent product Polynucleotides , including RNA and DNA Acridine orange Double stranded poly nucleotides Ethidium bromide Very sensitive, widely used with aarose gels. Fluorescent stains : 28

29. Type of substance stained Staining reagent comments Aminoacids , peptides, proteins Nynhydrin Used afteer paper electrophoresis Proteins Amido black 10B - Coomassie brilliant blue Binds to cationic groups on protiens and adsorb onto cellulose giving high back ground sensitivity.destainig causes dehydration and shrinkage of poly acrylamide gels Binds to basic groups on protiens and also by non polar interactions. Glycoprotiens Alcian blue Stains sugar moiety Copper containing protiens Alizarin Blue S Specifically indicates the presence of ‘Cu’ Polynuceotides including RNA and DNA - Acridine orange - Pyronine Y Stained product assessed quantitatively. gives permanent staining, so electrophoretogram can be stored for several weeks. 29

30. Stains give either chromogenic or fluorescent product. When using fluorescent staining technique, the samle is usually treated with the dye before electrophoresis Radiochemical methods: in this substances are labeled with radio active isotopes. After elactrophoresis the radio activity is measured by - liquid scintillation counting - Auto radiography. Radio isotopes used are high energy γ -emitter - 125 I – for proteins high energy β -emitter - 32 P - for nucleic acids medium enrgy β -emitter- 14 C, 35 S low energy β -emitter - 3 H 30

31. Biological assay methods sensitive selective even detect small amount of the protein Detecting either enzymatic activity or antigenic activity 31

32. APPLICATIONS The applications of electrophoresis include separation of proteins Separation of polynucleotide's Analytical and preparative purpose Miscellaneous applications Vaccine analysis Determination of impurities Chiral analysis analysis of carbohydrates and macro molecules Analysis of inorganic anions/metal ions 32

33. Separation of PROTEINs Proteins are made up of amino acids. Amino acid has –NH 2 , -COOH groups. Some of them have more than 2groups -aspartic acid, glutamic acid - 2 COOH groups - lysine, arginine - 2 NH 2 groups Net charge depends on ionisation characteristics and P H of the solution. at low P H COOH unionised and the amino group protonates results in ‘+’ ve charge at high P H NH 2 group and COOH group gets ionise results in ‘-’ ve charge at isoelectic point no charge Protein molecules are needed by our body cells and may be analyzed, for instance by getting blood and urine samples The amount of proteins present in blood and urine samples are measured and compared to established normal values 33

34. Separation of poly nucleotides In case of RNA at low P H – insoluble due to unionised form at basic P H – soluble due to ionization so net ‘-’ ve charge In case of ds DNA purine , pyrimidine bases are not available for ionization. because they are involved in the internal hydrogen bonding of the double helix. DNA molecules are soluble in basic P H . they always have a negative charge nad well migrate towards the anode. DNA is the unique code of every individual Through electrophoresis specific DNA sequences can be analyzed, isolated and cloned. The analyzed DNA may be used in forensic investigations and paternity tests 34

35. Analytical and preparative electrophoresis Widely used for analysis of mixtures. Also used for preparative scale. Problems encoutered in the scale up of analytical electrophoretic techniques for their application to preparative work include Heat generated increases the dimensions of the system. Resolution loss due to changes in the support medium. Difficult to recovery the separated components. 35

36. Assay of drugs These are the drugs that have been successfully estimated by this process Atropine sulphate i.v solution Codeine phosphate syrup Ketamine HCl i.v solution Phenylephrine i.v solution VACCINE ANALYSIS There are several vaccines that have been purified, processed and analyzed through electrophoresis such as influenza vaccine hepatitis vaccine and polio vaccine 36

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38. References : 1. Instrumental methods of chemical analysis by B.K sharma pg.No 268-285 2. Electrophoresis by Mauren Melvin 3. www.google.com 38

39. Thank you 39