Chapter 23 Chromatographic Methods & Capillary Electrophoresis

Capillary Electrophoresis in Biology

23.1 Ion-Exchange Chromatography -1 1)Structures Cation exchangers: -SO 3 -, -CO 2 - Anion exchanger: -NR 3 +

2)Ion-exchange selectivity a) R - Cs + + Li + R - Li + + Cs + selectivity coefficient: 23.1 Ion-Exchange Chromatography -2

b) Hydrated radius : hydrated radius  or Ionic charge   bind to ion-exchange resin  order of selectivity: p Ion-Exchange Chromatography -3

c) Gradient elution with increasing ionic strength (ionic concentration) to separate one ion from another by ion-exchange chromatography Ion-Exchange Chromatography -4

3)What is deionized water ? 4)Preconcentration trace analysis  [s] is extremely low important for environmental problem 23.1 Ion-Exchange Chromatography -5

Ex: 23.1 Ion-Exchange Chromatography -6

23.2 Ion Chromatography -1 (1) in semiconductor industry: to monitor anions & cations level at 0.1-ppb levels in deionized water. (2) in environmental anaylsis:

23.2 Ion Chromatography -2 (3) Anions are separated by ion exchange & detected by their electrical conductivity. (4) Problem : it is difficult to detect the conductivity change when analyte ions are eluted. (5) Suppressed-ion anion chromatography : remove the unwanted electrolyte prior to conductivity measurement.

23.2 Ion Chromatography -3

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

23.4 Affinity Chromatography -1 Isolate a single compound from a complex mixture. specific binding to s.p. ‚unbind : change pH or change ionic strength

23.4 Affinity Chromatography -2 enzymes / substrates antibodies / antigens receptors / hormones

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

(1)The greater the charge on the ion, the faster it migrates. (2)The greater the size of the molecule, the slower it migrates. (3)Different ions migrate at different speeds, so they separate What is Capillary Electrophoresis?

Electropherogram sensitivity

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 = What is Capillary Electrophoresis?

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? (a)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)

(d) ↓ at low pH, ∵ Si-O Θ →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 How Capillary Electrophoresis Works

(3) Detectors (a) UV-absorbance monitor (b) Fluorescence detector (c) Electrochemical detection (d) Mass spectrometer (e) Conductivity detection (f) Indirect detector

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

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

Neutral molecules reach the detector at a time between t mc (the time for micelles to reach detector) and t 0 (absence of micelles). soluble in the micelle   time inside the micelle   migration time , i.e. t r : nonpolar solutes > polar 23.7 Types of capillary electrophoresis

(3) Capillary gel electrophoresis (a)Macromolecules are separated by sieving (b)Small molecules travel faster than large molecules through the gel. (opposite in molecular exclusion chromatography) (c)Is used to sequence DNA (opening at p498) 23.7 Types of capillary electrophoresis