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Home , Affinity electrophoresis, Dielectrophoresis, Electroblotting

Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192

Pharma Science Monitor 7(2),Apr-Jun 2016

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com

REVIEW ON: : METHOD FOR SEPARATION ShindeDipa V.*, JasminaSurati Department of Quality Assurance, Shree NaranjibhaiLalbhai Patel College of Pharmacy,Umrakh -394 345,Bardoli, Gujarat, India.

ABSTRACT Electrophoresis is one of the widely used techniques in molecular , microbiology, biomedical research. It is a type of protein separation method .It is one of the highly efficient techniques of analysis and sole method for separation of for , RNA studies etc. It is a both qualitative and quantitative analysis technique. Separation depend upon electrophoretic mobility.Electrophoresis technique are of various type like Moving boundary electrophoresis ,Zone electrophoresis , ,Pulsed field electrophoresis ,.this technique mainly used in antibiotic analysis,vaccine analysis DNA analysis and protein analysis as well as fingerprint analysis. KEYWORDS:Electrophoresis, Electrophoretic mobility,Zone Electrophoresis, Moving boundary Electrophoresis, Dielectricphoresis.

INTRODUCTION Electrophoresis is a physical method of analysis based on the migration of electrically charged proteins, colloids, molecules or other particles dissolved or dispersed in an electrolyte solution in the direction of the electrode bearing the opposite polarity when an electric current is passed through it. Separations may be conducted in systems without support phases (such as free solution separation in capillary electrophoresis) or in stabilising media such as thin-later plates, filins or gels. The electrophoretic mobility is the rate of movement in metres per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second. For practical reasons it is given in squarecentimetres per volt second cm2 V-iS-I. The mobility is specific for a given electrolyte under precisely determined operational conditions. Depending on the method used, the electrophoretic mobility is either measured directly or compared with that of a reference substance.

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 193

Principle of Electrophoresis: The term Electrophoresis means Electro=electric field + Phoresis=migration. So as the name indicates, “Electrophoresis is a method of separation where in charged molecules migrate in differential speeds in an applied electric field.” The charged molecules under the influence of electric field migrate towards oppositely charged electrodes. Those molecules with +ve charge move towards cathode and -ve molecules move towards Anode. The migration is due to charge on the molecules and potential applied across the electrodes. The sample under test is placed at one end of the paper near one of electrodes. When electricity is applied, the molecules start moving to respective electrodes. But the movement is influenced by molecular weight of the molecule. So when a mixture isplaced on the electrophoresis paper or gel, different bands are seen along the paper after the process.This is due differentialrate of movement by molecules based on their weight.

Those molecules with higher molecular weight move slower. While those with small weight move faster. Also the size of the molecule also influences the movement. The bigger size molecule experience more friction than smaller ones in motion. These molecules migrate at different speed and to different lengths based on their charge, mass and shape. Based on type of apparatus used in electrophoretic method divided in two category: 1) Zone electrophoresis a) Paper electrophoresis b) c) Thin layer electrophoresis d) Cellulose acetate electrophoresis

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 194

2) Moving boundary electrophoresis a) Capillary electrophoresis b) Isoatchophoresis c) Isoelectric focussing d) Immuno electrophoresis. Also other type of electrophoresis is : a) Affinity electrophoresis b) Dielectrophoresis c) d) Plused field gel electrophoresis 1) ZONE ELECTROPHORESIS(4-5) : This method requires the use of small samples only.In zone electrophoresis, the sample is introduced as a narrow zone or spot in a column, slab or film of buffer. Migration of the components as narrow zones perrrrits their complete separation. Remixing of the separated zones by thermal convection is prevented by stabilising the electrolyte in a porous matrix such as powdered solid, or a fibrous material such as paper, foil or gel such as agar, starch or polyarcylamide. The rate of migration depends on four main factors viz. The mobility of the charged particle, the electro-endosmoticflow,the evaporation flow, and the strength of the field. It is necessary to operate under clearly defined experimental conditions and to use, wherever possible, reference substances. a) Paper electrophoresis : Paper electrophoresis is a techniques which employs a Whattman filter paper No.1 which is moistened by a buffer and then connected at two ends to two opposite charged electrodes. Then sample is applied on to one end and let for separation of components under electric field. After separation, the paper is dried and stained to get colored bands. These colored bands are recognized for the nature of sample by comparing with the standard. For a sample of serum, 5 bands of proteins can be separated by paper electrophoresis.

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 195

b) Gel electrophoresis (6) : Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry and to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of .

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 196

Type of gel gel electrophoresis are classified as 1) Agarose gel electrophoresis 2) Polyacrylamide gel electrophoresis 1) Agarose gel electrophoresis : Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix of agarose. The proteins may be separated by charge and/or size ( agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.[2] 2) Polyacrylamide gel electrophoresis : Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biologyand biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule. As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins,sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for instance many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS.[1]

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 197

2 )MOVING BOUNDARY ELECTROPHORESIS(7) :This method is chiefly employed with substances of high relative molecular weight and low diffusibility. In the method a buffered solution of proteins in a U-shaped is subjected to an electric current which causes the proteins to form series of layers in order of decreasing mobility, which are separated by boundaries. Only a part of the fastest moving protein is physically separated from the other proteins, but examination of the moving boundaries by physical processes such as refractometry or conductimetry helps to locate the boundaries and also provides data for calculation of mobilities and information on the qualitative and quantitative composition of the protein mixture. The operating conditions must be such as to make it possible to determine as many boundaries as there are components.

a) Capillary electrophoresis(8) : The capillary electrophoresis is an advanced methods of electrophoresis. This was developed with an intent to minimize the time taken for separation and analysis in slab electrophoresis. This capillary electrohporesis requires small sample in the range if 0.1 to 10 ηl while slab method requires in μl range. Also this method yields high speed and high resolution separations. Besides, the separated components which exit from one end of capillary, are immediately analysed by detectors fixed at the end of tubes.

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 198

b) : Isotachophoresis (ITP) is a technique in analytical chemistry used for selective separation and concentration of ionic analytes. It is a form of electrophoresis charged analytes are separated based on ionic mobility, a factor which tells how fast an ion migrates through an electric field. In conventional ITP separations, sample is introduced between a zone of "fast" leading electrolyte (LE) and a zone of "slow" terminating (or: trailing) electrolyte (TE). Usually, the LE and the TE have a common counterion, but the coions (having charges with the same sign as the analytes of interest) are different: the LE is defined by coions with high ionic mobility, while the TE is defined by coions with low ionic mobility. The analytes of interest have intermediate ionic mobility. Application of an electric potential results in a low electrical field in the leading electrolyte and a high electrical field in the terminating electrolyte. Analyte ions situated in the TE zone will migrate faster than the surrounding TE coions, while analyte ions situated in the LE will migrate slower; the result is that analytes are focused at the LE/TE interface. ITP is a displacement method: focusing ions of a certain kind displace other ions. If present in sufficient amounts, focusing analyte ions can displace all electrolyte coions, reaching a plateau concentration. Multiple analytes with sufficiently different ionic mobilities will form multiple plateau zones. Indeed, "plateau mode" ITP separations are readily recognized by stairlike profiles, each plateau of the "stair" representing an electrolyte or analyte zone having (from LE to TE) increasing electric fields and decreasing conductivities. In "peak mode" ITP, analytes amounts are insufficient to reach plateau concentrations, such analytes will focus in sharp Gaussian-like peaks. In peak mode ITP, analyte peaks will strongly overlap, unless so- called spacer compounds are added with intermediate ionic mobilities between those of the analytes; such spacer compounds are able to segregate adjacent analyte zones.

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A completed ITP separation is characterized by a dynamic equilibrium in which all coionic zones migrate with equal velocities. c) Isoelectric focusing : Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels. IPGs are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines. An Immobiline is a weak acid or base defined by its pK value. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode (negatively charged electrode). As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction towards either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands. Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a "positive" anode and "negative" cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. At this point the molecule no longer has a net (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel. The gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pI values to electrophoresis.

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 200

d) Immuno electrophoresis: This is the method with combination of principles of both electrophoresis with immune reactions. First the proteins are separated on to the electrophoresis paper. Then the are allowed to diffuse through the paper and react with separated protein molecules in bands. ELISA and RIA principle are based . Both of the methods are very specific and highly sensitive and widely used in microbiology. 1) Affinity electrophoresis : Affinity electrophoresis is a analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules (mainly macromolecules) through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in affinity electrophoresis or characterization of molecules with specific features like content or binding. For enzymes and other ligand-binding proteins, onedimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to by use of immobilized ligands. 2) Dielectrophoresis:

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 201

Dielectrophoresis (or DEP) is a phenomenon in which a is exerted on a particle when it is subjected to a non-uniform electric field. This force does not require the particle to be charged. All particles exhibit dielectrophoretic activity in the presence of electric fields. However, the strength of the force depends strongly on the medium and particles' electrical properties, on the particles' shape and size, as well as on the frequency of the electric field. Consequently, fields of a particular frequency can manipulate particles with great selectivity. This has allowed, for example, the separation of cells or the orientation and manipulation of nanoparticles and . 3) Electrobloting : Electroblotting is the method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands like , or stains. This method can be used with all polyacrylamide and agarose gels. An alternative technique for transferring proteins from a gel is capillary blotting. This technique relies upon current and a transfer buffer solution to drive proteins or nucleic acids onto a membrane. Following electrophoresis, a standard tank or semi-dry blotting transfer system is set up. A stack is put together in the following order from cathode to anode: sponge | three sheets of filter paper soaked in transfer buffer gel PVDF or nitrocellulose membrane | three sheets of filter paper soaked in transfer buffer | sponge. It is a necessity that the membrane is located between the gel and the positively charged anode, as the current and sample will be moving in that direction. Once the stack is prepared, it is placed in the transfer system, and a current of suitable magnitude is applied for a suitable period of time according to the materials being used. Typically the electrophoresis gel is stained with Coomassie Brilliant Blue following the transfer to ensure that a sufficient quantity of material has been transferred. Because the proteins may retain or regain part of their structure during blotting they may react with specific antibodies giving rise to the term immunoblotting. Alternatively the proteins may react with ligands likelectins giving rise to the term affinity blotting.

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4) Pulsed field electrophoresis(9) : Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction. The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely large molecules (up to around 2 Mb), switching-interval ramps can be used that increases the pulse time for each direction over the course of a number of hours—take, for instance, increasing the pulse linearly from 10 seconds at 0 hours to 60 seconds at 18 hours. REFERENCES 1. Indian pharmacopoeia;Govt. Of India,The Indian pharmacopoeialCommission,Ministry of Health and Family Welfare,Gaziabad,vol-II,2010, pp 122-123 2. Sharma B : Instrumental methods of chemical analysis. Goel publishing house ,first edition 1972. 3. Skoog : principle of instrumental analysis .Thomson books/cole ,fifth edition 1998. 4. Indian pharmacopoeia;Govt. Of India,The Indian pharmacopoeialCommission,Ministry of Health and Family Welfare,Gaziabad,vol-II,2010, pp 122-125 5. Sharma B : Instrumental methods of chemical analysis. Goel publishing house ,first edition 1972. 6. “Gel electrophoresis” 19th April 2016http.wikepediea .org 7. Indian pharmacopoeia;Govt. Of India,The Indian pharmacopoeialCommission,Ministry of Health and Family Welfare,Gaziabad,vol-II,2010, pp 123

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8. Lena ohannesian : Handbook of pharmaceutical analysis.CBS publisher and Distributors ,First Indian reprint 2005. 9. “Pulsed field gel electrophoresis” 19th April 2016http.wikepedia .org.

For Correspondence SHINDE DIPA Email: [email protected]

Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203