Chromatographic Methods & Capillary Electrophoresis Chapter 23 Chromatographic Methods & Capillary Electrophoresis

Capillary Electrophoresis in Medicine Sequence Human Genome

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

23.1 Ion-Exchange Chromatography Ion-exchange selectivity R-K+ + Li +  R-Li+ + K+ selectivity coefficient:

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

23.1 Ion-Exchange Chromatography c) Gradient elution with increasing ionic strength (ionic concentration) to separate one ion from another by ion-exchange chromatography.

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

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

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:

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.

23.2 Ion Chromatography

23.3 Molecular Exclusion Chrom. Molecules are separated according to their “SIZE”.

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

23.5 What is Capillary Electrophoresis? a) Electrophoresis is the migration of ion in an electric field.

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.

Electropherogram Sensitivity

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

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

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)

Hydrodynamic flow induced by a pressure difference, and creates broad band

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.

23.6 How Capillary Electrophoresis Works (3) Detectors UV-absorbance monitor Fluorescence detector Electrochemical detection Mass spectrometer Conductivity detection Indirect detector

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

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

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

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

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)