Paper electrophoresis ppt

A detailed description of Paper electrophoresis has been discussed in this ppt

1. Paper Electrophoresis R Deepthi Assistant Professor Vignan Institute of Pharmaceutical Technology Visakhapatnam

2. Paper Electrophoresis Paper electrophoresis is the most extensively used technique for separation of substances like amino acids, peptides and proteins. Electrophoretic analysis (using paper) of plasma proteins is of clinical significance in the diagnosis of various diseases including multiple myeloma, cirrhosis and nephrosis. Advantages This technique is very simple and inexpensive . Low operational costs . Numerous samples can be simultaneously isolated on a single paper. Various substances including amino acids, proteins, peptides, antibiotics, alkaloids etc. can be easily isolated by this technique.

3. Principle The principle involved in paper electrophoresis is the separation of charged particles from the sample applied on a paper upon application of current using two electrodes. The rate of separation of particles is based on their mass to charge (m/e) ratios. As paper does not conduct electricity, it is wetted with a buffer for facilitating the transport of current. The particles or ions present in the sample move either towards the cathode or the anode depending on the charge they possess.

4. Migration of ions also depends on the following factors: 1. Charge of the Particle: Rate of migration of the ions is directly proportional to the charge on the molecule. Ex: X 2+ ions migrate faster when compared to X + ions. 2. Size and shape of the molecule: Rate of migration of ions is inversely proportional to their size, hence smaller ions migrate faster when compared to larger ions. According to Stoke’s law, electrophoretic mobility of ion is expressed as, µ = Q/6πrɳ Where, µ = Electrophoretic mobility of the ion Q = Charge of the ion R = Radius of the ion ɳ = Viscosity of the buffer solution

5. 3 . Viscosity of the Buffer solution: Electrophoretic mobility of ion is inversely proportional to viscosity of the buffer solution i.e., rate of migration of ion decreases with an increase in the viscosity of buffer solution. 4. Voltage applied: Rate of migration of ion is directly proportional to the voltage applied across the electrodes. Therefore, mobility of ions can be enhanced by increasing the voltage. Furthermore, application of high voltage produces sharp bands on paper which are easy to detect. However, care should be taken to minimize the evaporation of buffer solution due to high voltage.

6. 5. pH and ionic Strength of the Buffer solution: Rate of migration of ions is inversely proportional to the ionic strength of the buffer solution i.e., ionic mobility increases with decrease in ionic strength. Influence of pH on rate of migration of ions depends on whether pH of the buffer solution is above or below the isoelectric point of the sample to be analyzed.

7. Requirements of Paper Electrophoresis 1. Paper In paper electrophoresis, various types of papers are used as stabilizing agents including Whatmann 1,2,3; Eaton- Dikemann 301-85, 320, 352, Munketells 20/50, Schleicher and Schull 2040 A and B etc. Of all those mentioned above, Whatmann 1 is extensively used particularly for large samples greater than 20µl. For samples which react with paper, paper made up of borosilicate glass fibres like whatmann GF/B are used. In general, filter paper obtained from various manufacturers differ in terms of thickness, optical homogeneity and content of foreign materials. Hence, filter papers are 1 st washed with distilled water and then with 0.1M HCl or 0.01M EDTA to remove any impurities.

8. 2. Electrodes Graphite rods, stainless steel, silver chloride, platinum (Pt) are the most commonly used electrode materials in paper electrophoresis. Platinum electrode is most extensively used and is available in various forms such as fine wires, sheets of foil etc. Size of the electrodes should not be small as they possess greater risk of being polarized with gas bubbles or products of electrolysis and also limit the amount of current flow.

9. 3. Buffers In general, ionic strength of about 0.05-0.5M is commonly used in paper electrophoresis. Examples of buffering agents are used are barbitone buffer or veronal buffer at a concentration of 0.07 moles/ lt and pH 8.6, tris -acetate buffer at a concentration of 0.07mole/Lt and pH 7.6 and citrate buffer at a concentration of 0.07moles/Lt and pH 3.0 or 6.8.

10. Procedure In paper electrophoresis, the stabilizing medium i.e., filter paper is 1 st dipped into the buffer solution and excess buffer is removed by placing it on another sheet of paper. Prior wetting of the filter paper is essential as it ensures uniform distribution of the buffer. After removing excess buffer solution, the paper is laid across two beakers containing the buffer solution of known ionic strength and the ends of the paper strip are immersed into the beakers

11. Paper is then allowed to stand for some time and the apparatus is enclosed in an air tight chamber so as to prevent excessive loss of buffer solution due to evaporation. Sample is then introduced on paper as a band/spot at its centre with proper care. Voltage potential is then applied across the electrodes immersed in the beakers. Care should be taken to ensure that paper and electrodes are sufficiently isolated so as to prevent electrode reactions which occur due to changes in the composition of buffer solution.

12. When voltage is applied, the ions and ionizable substances present in the sample to be analyzed migrate towards their respective electrodes based on their charge. The spots/bands obtained on filter paper are detected using suitable visualizing agents as in paper chromatography. This technique is extensively used for both Qualitative and Quantitative analysis of the sample.

13. Types of Paper Electrophoresis 1 . Based on Voltage Potential: Depending upon the potential applied across the electrodes, paper electrophoresis is broadly classified into two types: i ) Low voltage paper Electrophoresis: In this type, voltage applied across the two electrodes ranges from 5-15V/cm or 100-300V/strip with a current of about 0.4mApm/cm or 1.5mAmp/strip. Low voltage paper electrophoresis is widely used for the separation of ions for laboratory purpose.

14. ii) High voltage Paper Electrophoresis: In this type, voltage applied across the two electrodes ranges from 50-215 V/cm or 10,000V/strip . Here, isolation of ions requires less time and sharp bands are obtained. Hence, similar compounds can be easily separated and more number of samples can be analyzed at the same time. However, it is very hazardous thus requiring proper care . Furthermore, a large amount of heat is produced due to high voltage as a result of which the paper may become dry when the buffer evaporates.

15. 2. Based on Design of the Instrument: Based on the design of the instrument, paper electrophoresis is broadly classified into three types. The principle involved in the operation of all the three types is same but the design of the instrument varies. Horizontal Type Paper Electrophoresis: In this type, Whatmann filter paper of suitable grade and dimensions is moistened with buffer solution of known ionic strength and pH. Excess buffer solution is removed and the sample is applied at the centre of the paper.

16. The paper is then supported horizontally between two glass or plastic plates so as to prevent the evaporation of buffer solution. When suitable potential is applied across electrodes, migration of ions to their respective electrodes takes place. Spots or bands obtained on the filter paper due to migration of ions are detected by using a suitable visualizing agent. In horizontal type, separation of ions takes place within 12-14 hrs .

17. Vertical Type Paper Electrophoresis: It is similar to horizontal type except that here the paper is supported at some angle between glass or plates. In this the rate of migration of ions depends upon gravity hence complete separation of ions takes place within 6-8hrs. Quantitative detection of bands/spots is carried out by densitometer.

18. iii) Continuous Paper Electrophoresis: In this type, buffer solution is made to flow over the paper bed at a constant rate while the sample stream flows across the bed in the same direction as that of buffer. When potential is applied, migration of ions to their respective electrodes takes place. Isolation of ions takes place vertically, owing to the differences in distribution ratio between the mobile and stationary phase. Thus, each band is made to fall down and pure compounds obtained are collected in separate beakers as shown in the figure below:

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