Electrophoresis Methods PDF

Modern Methods of Pharmaceutical Analysis Seminar On topic of By, Gokul Prasath. A B.Pharmacy...

Modern Methods of Pharmaceutical Analysis Seminar On topic of By, Gokul Prasath. A B.Pharmacy KMCH COP 1  ELECTROPHORESIS Greek word meaning “transport by electricity” (Electro – electricity, phoresis – movement) Electrophoresis is a physical analysis which involves the separation of the compounds that are capable of acquiring electric charge in conducting electrodes. 2  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 the two electrodes tends to travel towards the electrodes that bears opposite charge. 3  PRINCIPLE 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. v=Eq/F Where, v = velocity of the molecule E = electric field (Volt/cm) q = net charge on molecule F = frictional coefficient, which depends upon mass and shape of the molecule. 4 The rate of migration of an ion in an electrical field depends on, 1) Net charge of molecule 2) Size, mass and shape of the particle 3) Strength of electrical field 4) Properties of supporting medium 5) Temperature of operation 5  FACTORS AFFECTING ELECTROPHORESIS 1) SAMPLE a) Charge: rate of migration increases with increases in net charge b) Size: rate of migration decreases for larger molecules. It is due to frictional and electrostatic forces. c) Shape: molecules have similar charge but differ in shape exhibit different migration rate 2) ELECTRIC FIELD a) Voltage: increase in voltage leads to increase in rate of migration b) Current: increase in current leads to increase in voltage, migration also increases c) Resistance: resistance increases and migration decreases. 6 3) BUFFER a) Ionic strength: rate of migration increases in high ionic strength b) pH: ionization of organic acid increases as pH increases 4) SUPPORTING MEDIUM a) Adsorption: reduces both rate of migration and resolution of separation of molecule b) Electro-endosmosis: it is due to the presence of charged groups on the surface of the supporting medium. Ex: paper(carboxyl group), agarose (sulphate group). It will accelerate the movement of cations, but retard the anion movements. 7  TYPES OF ELECTROPHORESIS a) ZONE ELECTROPHORESIS: 1) Paper electrophoresis 2) Gel electrophoresis 3) Thin layer electrophoresis 4) Cellulose acetate electrophoresis b) MOVING BOUNDARY/FRONTAL ELECTROPHORESIS: 1) Capillary electrophoresis 2) Isotachophoresis 3) Isoelectric focussing 4) Immuno-electrophoresis 8  INSTRUMENTATION (General Apparatus) It consists basically of items, 1) Power pack 2) Electrophoresis unit Electrodes Buffer reservoir Support for electrolysis medium A transparent insulating cover 3) Sample injector 4) Detector 9  GENERAL METHOD OF OPERATION Saturation of the medium with the buffer ↓ Sample application ↓ Electrophoretic separation ↓ Detection 10  a) ZONE ELECTROPHORESIS It involves the migration of the charged particle on the supporting media. Paper, cellulose acetate membrane, starch gel, polyacrylamide are used. 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. 11 ADVANTAGES: 1) Useful in biochemical investigations. 2) Cost is low 3) Small quantity of sample can be analysed. 4) Easy maintenance DISADVANTAGES: 1) Unsuitable for accurate mobility and isoelectric point determination. 2) Due to the presence of supporting medium, technical complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced. 12  1) PAPER ELECTROPHORESIS It is the form of electrophoresis that is carried out on filter paper. This technique is useful for separation of small charged molecules such as amino acids and small proteins. 13  FILTER PAPER It is the stabilising medium. Whatman filter paper, cellulose acetate filter paper or chromatographic papers can be used. APPARATUS Power back, electrophoretic cell that contains electrodes, buffer reservoirs, support for paper, transparent insulating cover. SAMPLE APPLICATION The sample may be applied as a spot (about 0.5 cm in diameter) or as a uniform streak. 14  ELECTROPHORETIC RUN The current is switched on after the sample has been applied to the paper and the paper has been equilibrated with the buffer. The types of buffer used depends upon the type of separation. Once removed, the paper is dried in vacuum oven. DETECTION AND QUANTITATIVE ASSAY To identify unknown components in the resolved mixture the electrophoretogram may be compared with another electrophoretogram on which standard components have been electrophoresced under identical conditions. Physical properties like fluorescence, UV absorption or radioactivity are exploited for detection. 15  WORKING 1) In this sample is applied as a circular support on a strip of whatman filter paper moistened with the buffer solution. 2) The ends of the paper are immersed in separate reservoirs containing buffer and in which the electrodes are fitted. 3) Upon passing electric current, the ions in the sample migrate towards oppositely charged electrodes. 4) This method is suitable for low molecular weight compounds such as amino acids and nucleotides. 16  2) GEL ELECTROPHORESIS It is a technique used for the separation of DNA, RNA or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix. GEL: Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules. TYPES OF GEL 1) Agarose gel 2) Polyacrylamide gel 3) Sephadex gel 17  AGAROSE GEL ELECTROPHORESIS A highly purified uncharged polysaccharide derived form agar. Used to separate macromolecules such as nucleic acid, larger proteins and protein complexes. It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C. It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds. 18 19  SDS PAGE ELECTROPHORESIS Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis SDS is an anionic detergent which binds strongly to and denature proteins. Protein SDS complex carries net negative charges, hence move towards anode and the separation is based on the size of the particle and also be used for determining the relative molecular mass of a protein. APPLICATIONS: 1) Useful for protein purification. 2) Determine molecular weight of proteins. 3) Quantifying proteins 4) Blotting applications 20  PROCESS OF SDS PAGE 1) Boil the samples for 10 minutes to completely denatures the proteins. 2) Assembly the gel into the apparatus. 3) Pour the buffer solution into the chamber. 4) Load 20µL of samples into the well. 5) After that, run electrophoresis by connecting the current supplies. 6) Visualises the band under UV light or staining technique. 21  3) THIN LAYER ELECTROPHORESIS Electrophoretic studies can be carried out in thin layer of silica, keisulghur, alumina. ADVANTAGES: 1) Less time consuming 2) Good resolution APPLICATION: Used in combined electrophoretic-chromatography studies in two dimensional study of proteins and nucleic acid hydrolysates. 22 4) CELLULOSE ACETATE ELECTROPHORESIS It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper. It gives sharper bands. Provides a good background for staining glycoproteins. ADVANTAGES: 1) No tailing of proteins 2) Gives sharp bands and good resolution. DISADVANTAGES: 1) Expensive 2) Presence of carboxylic residue causes induced electro-osmosis during process. 23  PRINCIPLE In an alkaline pH (8.2-8.6) Haemoglobin is a negatively charged(-) molecule and will migrate toward the anode(+). The various Haemoglobin moves at different rates depending on their net (-) charge, which in turn is controlled by the composition of the Haemoglobin molecule. 24 b) MOVING BOUNDARY ELECTROPHORESIS PRINCIPLE: The moving boundary method allows the charged species to migrate in a free moving solution without the supporting medium. ADVANTAGES: 1) Biologically active fractions can be recovered without the use of denaturing agents. 2) Minute concentrations of the sample can be detected. DISADVANTAGES: 1) Costlier 2) Elaborate optical system are required. APPLICATIONS: 1) To study homogenecity of a macromolecular system. 2) Analysis of complex biological mixture. 25  INSTRUMENTATION Consists of a U shaped glass cell of rectangular cross section with electrodes placed on the one each of the limbs of the cell. Sample solution is introduced at the bottom or through the side arm and the apparatus is placed in a constant temp. bath at 40°C. Detection is done by measuring refractive index throughout the solution (Schileren optical system). 26  1) CAPILLARY ELECTROPHORESIS Capillary tube is placed between the two buffer reservoir and an electric field is applied, separation depends on electrophoretic mobility and electro-osmosis. Defined volume of analyte is introduced into the capillary by replacing one buffer reservoir with sample vial. Electrophoretic separation is measured by detector. Using narrow bore tubes, capillary electrophoresis removes the joule heating effect which decreases band broadening, giving faster separations than gel. CE uses tubes 20-100µm diameter and 20-100cm length. CE is used with/without gel. Longitudinal diffusion is the main source of band-broadening. 27  Higher electric fields result in high efficiency and narrow peaks (analyte migrates faster). All analytes travel the same distance, but the migration time (tm) for that distance is measured. Relate time to identity. Relate peak area or height to amount. 28  2) ISOTACHOPHORESIS Iso – equal, tachos – speed, phoresis – migration The technique of isotachophoresis depends on the development of potential gradient. Used for separation of smaller ionic substances. They migrate adjacent with contact one another, but not overlapping. The sample is not mixed with buffer prior to run. Hence current flow is carried entirely by the sample ions. APPLICATION: Separation of small anions and cations. 29  3) ISO ELECTRIC FOCUSSING Isoelectric focussing also known as electro-focussing is a technique for separating different molecules by differences in their isoelectric point. It is suitable for separation of amphoteric substances. It is a type usually performed on proteins in a gel, that takes advantages of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. Method has high resolution. APPLICATIONS: 1) Human genetic lab. 2) Useful for separating iso-enzymes. 3) Research in enzymology and immunology. 30  4) IMMUNO ELECTROPHORESIS When electrical field is applied to study of antigen- antibody reactions, it is called immuno-electrophoresis. The antibody are electrophoretically separated and antigens diffuse towards each other resulting in precipitin arcs where antigen antibody complexes form. It is used to detect the presence of antibodies. Used mainly to determine the blood levels of three major immunoglobulins :  IgM  IgG  IgA 31  PROCESS Ag molecules are separated according to differences in their electrical charge by electrophoresis. Antibody is placed in a trough cut in the agar. Antibody diffuse towards antigen → precipitin arcs. Interpretation – precipitin arc represent individual antigens. 32  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 ie., in the study of protein mixtures such as blood serum, haemoglobin and in the study of antigen-antibody interactions. 8) Electrophoresis in combination with auto-radiograhy is used to study the binding of iron to serum proteins. 33 9) Used for analysis of terpenoids, steroids and antibiotics. 10) For testing purity of thyroid hormones by zone electrophoresis. 11) Paper chromato-electrophoresis is used to separate free insulin form plasma proteins. 12) It is used for diagnosis of various disease of kidney, liver and CVS. 13) It is also used for separation of scopolamine and ephedrine using buffer at pH 4.2 14) It is also used for separation of carbohydrates and vitamins. 15) Quantitative separation of all fractions of cellular entities, antibiotics, RBC, enzymes, etc.., are possible. 34 35