Moving Boundary & Isoelctric focusing(JEEV).pptx

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1. MOVING BOUNDARY AND ISO ELECTRIC FOCUSING ELECTROPHORESIS Presented By : Jeevitha K B M Pharm 1 st Sem Pharmaceutics Dept. Mallige College of Pharmacy 1

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3. The method allows the charged species to migrate in a free moving solution in absence of a supporting medium. No supporting medium is used. It is a technique for separation of chemical compounds by electrophoresis in a free solution. The moving boundary method involves measuring the speed of displacement of the boundary between two solutions due to an electric current. For the separation of different charged molecules in a mixture, sample is placed in glass, which is connected to the electrodes. On applying electric potential across the tube, charged molecule migrates towards one or another electrode. 3

4. INSTRUMENTATION Apparatus consists of U shaped glass cell of rectangular cross section with electrodes located at the two ends used to apply an electric field. An inlet for sample introduction is available at the bottom or sides of the U cell. The sample solution is introduced through the inlet. Care must be taken to minimize the disturbing effect of convection caused by an increase in temperature during the passage of current through the solution. For this purpose, the apparatus is placed in constant temperature bath at 40 degree C. Detection is done by measuring refractive index throughout the solution. 4

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6. In moving boundary electrophoresis Zones generally contain more ionic species of the sample Composition of the sample plays an important role in the determination of the concentrations, pH value and conductivities of different zones So effective mobilities can be measured by using moving boundary electrophoresis 6

7. FACTORS AFFECTING SEPARATION The rate of migration(Separation of particles) during electrophoresis will depend on the following factors : The Sample The Electric Field The Buffer Electrolysis 7

8. The Sample Charge/mass ratio of the sample determines its electrophoretic mobility. The mass considers not only the size but also the shape of the molecule. Charge : The higher the charge, greater is the electrophoretic mobility. The charge is dependent on pH. Size : Bigger molecules have small electrophoretic mobility. Shape : Globular protein will migrate faster than the fibrous protein. 8

9. 2) The Electric Field The rate of migration under unit potential gradient is referred to as “Mobility of the ion” An increase in potential gradient increases the rate of migration. 9

10. 3) The Buffer The buffer can affect the electrophoretic mobility of the sample in various ways. The electrophoretic mobility is greatly affected by the pH of the buffer, particularly when the sample is weak acid or a weak base, because the pH establishes its degree of ionization. In case of amphoteric substances such as proteins, the direction of migration depends upon whether the buffer pH is above or below the isoelectric point of the sample. a)Composition: Commonly used buffers are “ Formate ” “Acetate” “Citrate” “Phosphate” etc… 10

11. The choice of buffer depends upon the type of sample being electrophoresed. b)Ionic Strength: “Ionic Strength is a measure of the electrical environment of ions in a solution”. When increase ionic strength of the buffer means a larger share of the current being carried by the buffer ions & small quantity proportion carried by the sample ions. When decrease ionic strength, a larger share of the current being carried by the sample ions leading to a faster separation. Note: The ionic strength used is usually between 0.05 to 0.1M. 11

12. 4) ELECTROLYSIS Electrophoresis is accompanied by electrolysis which causes microscopic bubbles to accumulate on the electrodes. When a bubble is formed, electric contact with buffer is lost. Simultaneously, resistance of electrode gradually builds up. 12

13. Advantages Biologically active fractions can be removed without the use of denaturing agents. A reference method for measuring Electrophoretic mobilities. Minute concentration of the sample can be detected upto 0.05mg/ml by Interferometric optical system. 13

14. Disadvantages/Drawbacks The resolution of the technique is very low due to the mixing of the sample as well as over-lapping of the sample components. It is not good to separate and analyze the complex biological sample, instead it can be used to study the behavior of the molecule in an electric field. It is relatively costlier. 14

15. Applications Used for quantitative analysis of complex mixture of macromolecules. It is employed in biochemical and clinical fields i.e. in the study of protein mixtures such as blood serum, heamoglobins and in the study of antigen-antibody interactions. To separate proteins and peptides. It is also used in Protein research/purification. 15

16. ISO ELECTRIC FOCUSING ELECTROPHORESIS 16

17. INTRODUCTION IEF began in 1964. This technique was popularized by H.Svensson in Sweden. In Isoelectric : Iso means “same” and Electric means “charge”. Isoelectric focusing (IEF), is a technique for separating different molecules by based on their isoelectric point. The isoelectric point is the pH at which the net charge of the protein is zero. IEF is often used as part of the quality control testing of therapeutic biological products to demonstrate batch consistency. 17

18. PRINCIPLE IEF is performed in a pH gradient. Proteins are amphoteric molecules with acidic and basic buffering groups In basic environment, the acidic groups become negatively charged. In acidic environment, the basic groups become positively charged. Isoelectric point( pI ): the pH where the charge of a protein is zero. Proteins with same molecular weights will separate out by pH. Proteins are positively charged in solutions at pH values below pI and migrate towards cathode. Proteins are positively charged in solutions at pH values below pI and migrate towards cathode. 18

19. All proteins have an isoelectric pH. When the electrophoresis is run in a solution buffered at constant pH , proteins having a net charge will migrate towards the opposite electrode so long as the current flows. The use of pH gradient across the supporting medium causes each protein to migrate to an area of specific pH. The pH of thr protein equals the pH of the gradient, thus resulting in sharp well defined protein bands. A procedure to determine the isoelectric point (PI) of proteins thus, a mixture of proteins can be electrophorised through a solution having a stable pH gradient in from the anode to the cathode and a each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing. IEF is also known as Electrofocusing . 19

20. PROCEDURE Protein is loaded at the top of a column where pH is very high. Most of them are negatively charged at this pH. Proteins are stripped from residue side chain. Proteins move in the electric field toward the distant cathode and away from the nearby anode. As the protein move through the pH gradient, they gain positive charge and reach neutrality. At pH = pI , the proteins have no charge and stop. 20

21. Proteins stop exactly when pH= pI and the stained proteins are very visible. Highly stable ampholytes are molecules with specific pKa to give a specific and unchanging pH gradient. 21

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24. Separation is achieved by applying a potential difference across a gel that contains a pH gradient. Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel. Isoelectric focusing gels contain synthetic buffers called ampholytes that smooth the pH gradients. Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids. Commercially available ampholytes are:- BIO-LYTE PHARMALYTE 24

25. ADVANTAGES IEF’s greatest advantage is its high resolution, resulting in greater separation of solutes. IEF of serum proteins results in many more bands; these bands are sharper because each pH region is very narrow. Performing IEF is easier because the placement of sample application is not important. The sample and ampholytes can be mixed before application; the ampholytes will migrate, create the gradient, and then the proteins separate and migrate. 25

26. APPLICATIONS IEF is a highly sensitive analytical technique and is particularly useful for studying microheterogeneity in a protein. The method is useful for separating & identification of isoenzymes (which are different forms of the same enzyme often differing by only one or two amino acid residues) 2D Gel electrophoresis is an application of IEF. Protein is first separated based on pI and then based on molecular weight using SDS-PAGE(Sodium dodecyl- sulfate polyacrylamide gel electrophoresis) Widely used for separation and identification of serum proteins. Protein purification To measure pI values. 26