Capillary electrophoresis

capillary electrophoresis is simply separation of charged and non charged particles in capillary tube

1. CAPILLARY ELECTROPHORESIS Presented by: A. Sireesha Bai M. P harmacy ( firs t year ) 19HC1S0701 Department Of Pharmaceutical Analysis Santhiram college of pharmacy

2. What is Electrophoresis Migration of charged particles or molecules in a medium under the influence of an applied electric field

3. Purpose For Carrying Electrophoresis To determine number, amount and mobility of components in a given sample or to separate them. To obtain information about the electric double layers surrounding the particles. Determination of molecular weight of proteins and DNA sequences

4. Types of electrophoresis Paper electrophoresis Gel electrophoresis Capillary electrophoresis Iso - electric focusing electrophoresis Thin layer electrophoresis

5. Capillary Electrophoresis It is relatively new separation technique compared to the traditional agarose gel electrophoresis Separation of analyte ions is performed in an electrolyte solution present in a narrow capillary one of the major advantage of CE over other separation techniques is the ability to separate both charged and non charged molecules

6. Principle CE is an analytical technique that separates ions based on their electrophoretic mobility and electrosmotic flow The rate at which the particles moves is directly proportional to the applied electric field Neutral species are not affected only ions move with electric field

7. If 2 ions are of same size the one with greater charge will move fastest. For the ions of same charge, the smaller particles has less friction and overall faster migration rate. CE is mostly used because it gives fast results and provides high resolution separation and has large range of detection methods

8. Instrumentation The CE system consists of Sample injection system Buffer solution Capillary tube Power supply Detector Output device

9. Sample injection system It is done by either one of the methods Electro kinetic injection The sample is introduced by inserting the anode end of the capillary into the sample vial and then applying electric field to the sample vial Hydrodynamic injection The sample is introduced by inserting the anode end of the capillary into the sample vial and then applying pressure on the vial

10. Capillary Tube As the name suggests the separation is carried in a narrow capillary tube Length-30 to 100 cm Fused silica capillary Polyamide coating external ED-200 to 400 micrometer ID-50 to 100 micrometer Packed with buffer in use

11. Power supply High strength electric field of 20 to 100 kv required for separation Detectors

12. Advantages High separation efficiency Short analysis time Low sample and electrolyte consumption Low waste generation Easy of operation Disadvantages Due to small diameter of the capillary tube heat is dissipated that causes increased diffusion Due to this the resolution is not always proper

13. Modes of CE Capillary Zone Electrophoresis Capillary Gel Electrophoresis Capillary Iso Electric Focusing Capillary Isotachophoresis

14. Capillary Zone Electrophoresis (CZE ) Capillary Zone Electrophoresis (CZE), also known as Free Solution Capillary Electrophoresis .It is the most commonly used technique. Electrophoretic Mobility α Charge on t he Molecule Velocity of the ion moves α electrophoretic mobility Analytes move in the EOF but separates into bands because of differences in their electrophoretic mobility (µ). Difference in µ make each analyte overall migration velocity slightly different and difference in migration.

15. Capillary Gel Electrophoresis (CGE ) In CGE gels are contained inside the capillary. CGE uses separation based on the difference in solute size as the particles migrate through the gel . Gels are useful because they minimize solute diffusion that causes zone broadening, prevent the capillary walls from absorbing the solute, and limit the heat transfer by slowing down the molecules.

16. Capillary Isoelectric Focusing (CIEF ) During a CIEF separation, the capillary is filled with the sample in solution and typically no EOF is used (EOF is removed by using a coated capillary ). When the voltage is applied, the ions will migrate to a region where they become neutral (pH= pI ). The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). Compounds of equal isoelectric points are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection .

17. Capillary Isotachorphoresis (CITP ) CITP is the only method to be used in a discontinuous system i.e., leading electrolyte and trailing electrolyte When the current is applied the leading ions will move towards appropriate electrode. The sample ions will follow in order of their mobilites . The trailing will follow the sample ions Once equilibrium is achieved ions will move at same speed in discrete bands in order of their mobilities . The analyte migrates in consecutive zones and each zone length can be measured to find the quantity of sample present .

18. Factors Affecting Charge Mass and shape of the particle pH of the medium Strength of electric field Temperature

19. Applications Proteins Peptides DNA fragments Nucleic acid Amino acids Drugs or other metals Used to study

20. Thank you