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Chapter 23

Chapter 23. Chromatographic Methods & Capillary Electrophoresis. Capillary Electrophoresis in Medicine. Sequence Human Genome. 23-1 Ion-Exchange Chromatography. 1) Structures. Cation exchangers: -SO 3 - , -CO 2 - Anion exchanger: -NR 3 +. 23-1 Ion-Exchange Chromatography.

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Chapter 23

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  1. Chapter 23 Chromatographic Methods & Capillary Electrophoresis

  2. Capillary Electrophoresis in Medicine Sequence Human Genome

  3. 23-1 Ion-Exchange Chromatography 1) Structures Cation exchangers: -SO3-, -CO2- Anion exchanger: -NR3+

  4. 23-1 Ion-Exchange Chromatography (2) Ion-exchange selectivity • R-K+ + Li + R-Li+ + K+ selectivity coefficient:

  5. 23-1 Ion-Exchange Chromatography b) Hydrated radius : hydrated radius or Ionic charge  bind to ion-exchange resin order of selectivity:

  6. 23-1 Ion-Exchange Chromatography c) Gradient elutionwith increasing ionic strength (ionic concentration) to separate one ion from another by ion-exchange chromatography.

  7. 23-1 Ion-Exchange Chromatography -5 3) What is deionized water ? • Preconcentration trace analysis  [s] is extremely low important for environmental problem

  8. 23-1 Ion-Exchange Chromatograph Metals in natural waters can be preconcentrated with a cation-exchange column . The cations can then be displaced into a small volume of solution by eluting the column with concentrated acid

  9. 23-2 Ion Chromatography • a high-performance version of ion-exchange chromatography, with a key modification that removes eluent ions before detecting analyte ions. • in semiconductor industry: to monitor anions & cations level at 0.1-ppb levels in deionized water. • in environmental analysis:

  10. 23-2 Ion Chromatography (4) Anions are separated by ion exchange & detected by their electrical conductivity. Problem : It is difficult to detect the conductivity change when analyte ions are eluted. Suppressed-ion anion chromatography :Remove the unwanted electrolyte prior to conductivity measurement.

  11. 23-2 Ion Chromatography

  12. 23-3 Molecular Exclusion Chrom. Molecules are separated according to their“SIZE”.

  13. 23-4 Affinity Chromatography Isolate a single compound from a complex mixture. a. specific binding to s.p. b. unbind : change pH or change ionic strength • Interaction between: • enzymes / substrates • antibodies / antigens • receptors / hormones

  14. 23-5 What is Capillary Electrophoresis? a) Electrophoresis is the migration of ion in an electric field.

  15. 23-5 What is Capillary Electrophoresis? • The greater the charge on the ion, the faster it migrates. • The greater the size of the molecule, the slower it migrates. • Different ions migrate at different speeds, so they separate.

  16. Electropherogram Sensitivity

  17. 23-5 What is Capillary Electrophoresis? b) Capillary electrophoresis : extremely high resolution in a narrow capillary tube (only B term in the van Deemter eqn.) No s.p.  C = 0 Open tubular column  A = 0

  18. 23-6 How Capillary Electrophoresis Works (1) Two processes operate in capillary electrophoresis: (a) electrophoresis: the migration of ions in an electric field cation  cathode anion  anode (b) electroosmosis: pump the entire solution through the capillary anode  cathode

  19. 23-6 How Capillary Electrophoresis Works • (2) Why electroosmosis? • Wall is covered with silanol, • pH > 2, Si-OH  Si-O- Electric double layer (Diffuse part of the double layer ~1 nm) (b) Electric field  flow  (c)electroosmotic flow(electric field) v.s. hydrodynamic flow (pressure difference)

  20. Hydrodynamic flow induced by a pressure difference, and creates broad band P.521

  21. 23-6 How Capillary Electrophoresis Works • (d) ↓ at low pH, ∵Si-OΘ→Si-OH • cation↓ in the double layer • - at neutral pH or high pH electroosmosis > electrophoresis the net flow: anions → cathode • - at low pH anode ← anion • & may never reach the detector.

  22. 23-6 How Capillary Electrophoresis Works • (3) Detectors • UV-absorbance monitor • Fluorescence detector • Electrochemical detection • Mass spectrometer • Conductivity detection • Indirect detector

  23. 23-7 Types of capillary electrophoresis Crown ether has greater affinity for D-aa than L-aa P.524

  24. 23-7 Types of capillary electrophoresis (1) Capillary zone electrophoresis : cations > neutrals (unseparated) > anions (2) Micellar electrokinetic capillary electrophoresis separate neutral molecules as well as ions

  25. 23-7 Types of capillary electrophoresis What is a micelle ? are anions & behave like a pseudostationary phase c  0 in van deemter eqn.

  26. 23-7 Types of capillary electrophoresis Neutral molecules reach the detector at a time between tmc (the time for micelles to reach detector) and t0 (absence of micelles). soluble in the micelle   time inside the micelle   migration time , i.e. tr: nonpolar solutes > polar

  27. 23-7 Types of capillary electrophoresis (3) Capillary gel electrophoresis • Macromolecules are separated by sieving • Small molecules travel faster than large molecules through the gel. (opposite in molecular exclusion chromatography) • Is used to sequence DNA (opening at p510)

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