171x Filetype PPTX File size 2.46 MB Source: www.agilent.com
Agilent Technologies is committed to the educational community and is willing to provide access to company-owned material contained herein. This slide set is created by Agilent Technologies. The usage of the slides is limited to teaching purpose only. These materials and the information contained herein are accepted “as is” and Agilent makes no representations or warranties of any kind with respect to the materials and disclaims any responsibility for them as may be used or reproduced by you. Agilent will not be liable for any damages resulting from or in connection with your use, copying or disclosure of the materials contained herein. You agree to indemnify and hold Agilent harmless for any claims incurred by Agilent as a result of your use or reproduction of these materials. In case pictures, sketches or drawings should be used for any other purpose please contact Agilent Technologies a priori. For teaching purpose only 9/3/22 5991-8063EN © Agilent Technologies, Inc. 2016 2 Introduction Electrophoresis has been defined as the differential movement of charged species (ions) by attraction or repulsion in an electric field. Electrophoresis as a separation technique was introduced by Arne Tiselius (Sweden, 1902-1971) in 1937. Placing protein mixtures between buffer solutions in a tube and applying an electric field, he found that sample components migrated in a direction and at a rate determined by their charge and mobility. For his work in separation science Tiselius was awarded a the Nobel Prize for Chemistry (1948). For teaching purpose only. 9/3/22 5991-8063EN © Agilent Technologies, Inc. 2016 3 Table of Contents (ToC) Introduction • How does it work? Principles • Electrophoresis - Ion velocity • Electrophoresis - Forces • Electrophoresis - Mobility • Electroosmotic flow • Mobility and migration time • Resolution Comparison of techniques • Chromatography vs Electrophoresis Characteristics of capillary electrophoresis Further Information For teaching purpose only. 9/3/22 5991-8063EN © Agilent Technologies, Inc. 2016 4 Introduction How does it work? Electro (electric) + Phoresis (migration) Negative electrode Simplified process: • Samples are loaded into wells and n o & i charged molecules start migrating t d c l e e r i i through the gel when voltage is f d c n i r o t i applied. c t e a l r E g i • Molecules with smaller molecular m weight move faster then molecuels with higher molecular weight. Positive electrode ToC For teaching purpose only. 9/3/22 5991-8063EN © Agilent Technologies, Inc. 2016 5 Introduction How does it work? Separation efficiency in free solution, as performed by Tiselius, was limited (a) (b) by thermal diffusion and convection. For this reason, electrophoresis traditionally has been performed in anti-convective media, such as polyacrylamide or agarose gels. Primary application area: • Size-dependent separation of biological macromolecules such as nucleic acids (RNA/DNA) and proteins. Comparison of gel used for slab gel electrophoresis (a) and capillary for Capillary electrophoresis (b). ToC For teaching purpose only. 9/3/22 5991-8063EN © Agilent Technologies, Inc. 2016 6
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