1. ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 23 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of chemistry Department of natural sciences Clayton state university

2. CHAPTER 23 CAPILLARY ELECTROPHORESIS

3. ELECTROPHORESIS - Migration of ions in an electric field - Cations are attracted to the negative electrode (cathode) - Anions are attracted to the positive electrode (anode)

4. ELECTROOSMOSIS - The application of electric field to drive fluid in a capillary tube from anode towards cathode Capillary Electrophoresis - Is a combination of electrophoresis and electroosmosis

5. CAPILLARY ELECTROPHORESIS - High-resolution separation method - Separates charged and neutral analytes in a narrow capillary tube - Capillary tube wall is usually fused silica Good technique for the separation of - Small ions (Na +, K + ) - Proteins - DNA

6. Background Electrolyte (run buffer) - A buffer solution in the electrode reserviors - Controls pH and ionic strength CAPILLARY ELECTROPHORESIS

7. - Both ends of the capillary tube is first immersed in a background electrolyte - One end of tube is dipped in vial containing the sample - Pressure or electric field is applied to introduce ~10 nL of sample into the capillary - Capillary is placed back into the electrolyte - 20 to 30 kV is applied and causes ions in capillary to migrate CAPILLARY ELECTROPHORESIS

8. - Different ions migrate at different speeds - This results in separation of ions - Ions reach a detector and an electropherogram (response versus time) is recorded - Very narrow bands are usually seen - Terms A and C in the van Deemter equation are reduced (no multiple paths and no stationary phase) CAPILLARY ELECTROPHORESIS

9. - Electroosmosis sweeps analyte molecules towards the detector - Detector is placed near the cathode - Detector is set to a wavelength of about 200 nm - Cations reach the detector first - Neutral molecules reach the detector after cations - Anions reach the detector after neutral molecules CAPILLARY ELECTROPHORESIS

10. At High pH - Electroosmosis is faster than electrophoresis - Net flow of anions is towards the cathode At Neutral pH - Electroosmosis is faster than electrophoresis - Net flow of anions is towards the cathode CAPILLARY ELECTROPHORESIS

11. At Low pH - Electroosmosis is slower than electrophoresis - Net flow of anions is towards the anode - Anions may not reach the detector - Polarity can be reversed to separate anions CAPILLARY ELECTROPHORESIS

12. Detectors - Ultraviolet absorbance (most common) - Conductivity - Electrochemical - Fluorescence CAPILLARY ELECTROPHORESIS

13. Types Capillary Ozone Electrophoresis - Separation is based on different velocities of different ions Micellar Electrokinetic Capillary Chromatography - Used to separate different neutral molecules - Also used for ions - Micelles are added to the background electrolyte - An example is sodium dodecyl sulfate (a surfactant) CAPILLARY ELECTROPHORESIS

14. Types Capillary Gel Electrophoresis - Separation is based on size of molecules - Molecules are separated upon migrating through a gel in the capillary column - The process is called sieving - Small molecules travel faster than large molecules CAPILLARY ELECTROPHORESIS