Capillary Electroporesis
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Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility in a thin capillary tube when an electric field is applied. It allows for much faster separation than traditional electrophoresis in under 15 minutes. Capillary electrophoresis can sequence up to 750,000 nucleotides per day due to its high resolution, reproducibility, and ability to be automated. It works by applying an electric field that causes ions to migrate through the capillary at different rates depending on their size and charge, separating them into distinct bands that can then be detected.
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Capillary Electroporesis
- 1. Assignment -3 Presentation Prepared By, Sindhuamuthan Kathiravan.
- 2. Capillary Electrophoresis Presented by Sindhuamuthan Kathiravan
- 3. Introduction Endeavours in capillary electrophoresis (CE) began as early as the late 1800’s. Experiments began with the use of glass U tubes and trials of both gel and free solutions. In 1930, Arnes Tiselius first showed the capability of electrophoresis in an experiment that showed the separation of proteins in free solutions. His work had gone unnoticed until Hjerten introduced the use of capillaries in the 1960’s. However, their establishments were not widely recognized until Jorgenson and Lukacs published papers showing the ability of capillary electrophoresis to perform separations that seemed unachievable.
- 4. Principle Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius. The rate at which the particle moves is directly proportional to the applied electric field, the greater the field strength, the faster the mobility. Neutral species are not affected, only ions move with the electric field. If two ions are the same size, the one with greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate.
- 5. Why Capillary Electrophoresis Normal electrophoresis requires more than an hour to run and difficult to quantitate and automate Sequencing gels were of very thin and lengthy gel which can easily melt during electrophoresis because of heat formation Capillary electrophoresis is a technique in which electrophoresis is carried out in very thin capillary tubes which is made up of fused silica and it sometimes coated with polyimidi Capillaries rapidly dissipate heat and hence permit the use of high electric fields typically 100 to 300 V per centimetre, which reduces separation times to a few minutes. Minimize band broadening caused by diffusion, thereby yielding extremely sharp separations. It can sequence 750000 nucleotides per day. It can be automated
- 6. Basic Theory of CE Ionic Mobility Electro Osmotic Flow Electrophoretic Mobility Electro kinetic Loading
- 8. Instrumentation (Contd) Cathode (Negatively Charged Electrode) Anode (Positively Charged Electrode) Power Supply to generate Voltage/Current Catholyte (Buffer Solution at the Cathode End) Anolyte (Buffer Solution at the Anodic End) Capillary (25mm to 100mm ID) A Detection Method Data Acquisition Method
- 12. Disadvantages Cannot handle large volume of samples Arrival of Advancements in sequencing methodology
- 13. One Mark Number of nucleotides can be sequenced by a capillary gel electrophoresis per day A) 3.8*109 B)7.5*105 C)5.0*103 D)6.0*104
- 14. 2 Marks Advantage of CE? Migration within short time Reduced band broadening High resolution Reproducibility Ability to automation
- 15. 2 Marks Difference between electrophoretic mobility and electroosmotic flow Flow of buffer solution into capillaries is known as electroosmotic flow Flow of sample solution into capillaries is electrophoretic mobility followed by electro osmotic flow
- 16. 10 Marks Explain about capillary electrophoresis its instrumentation, Working and types?
- 17. Any Queries
- 18. References http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Ca pillary_Electrophoresis http://mtc-usa.com/cebasic.asp Principles of gene manipulation author Primrose, Chapter 7 ‘Sequencing and Mutagenesis’, Page 125 Biochemistry 4th edition Author Voet and Voet, Chapter 4 ‘Techniques for nucleotide and Protein separation’, Page 151-152.