Chromatographic Methods

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Chapter 23

• Chromatographic Methods
• Capillary Electrophoresis

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Capillary Electrophoresis in Medicine
Sequence Human Genome
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23.1 Ion-Exchange Chromatography

• 1) Structures

Cation exchangers -SO3-, -CO2- Anion
exchanger -NR3
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23.1 Ion-Exchange Chromatography

• Ion-exchange selectivity
• R-K Li ? R-Li K
• selectivity coefficient

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23.1 Ion-Exchange Chromatography

• b) Hydrated radius
• hydrated radius?
• or Ionic charge?
• ? bind to ion-exchange resin?
• order of selectivity

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23.1 Ion-Exchange Chromatography

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

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23.1 Ion-Exchange Chromatography -5

• 3) What is deionized water ?
• Preconcentration
• trace analysis
• ? s is extremely low
• important for environmental problem

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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
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23.2 Ion Chromatography

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

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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.

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23.2 Ion Chromatography
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23.3 Molecular Exclusion Chrom.

• Molecules are separated according to their
  SIZE.

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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 antibodi
es / antigens receptors / hormones
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23.5 What is Capillary Electrophoresis?

• a) Electrophoresis is the migration of ion in
  an electric field.

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23.5 What is Capillary Electrophoresis?

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.

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• Electropherogram
• Sensitivity

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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

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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
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23.6 How Capillary Electrophoresis Works

• (2) Why electroosmosis?
• Wall is covered with silanol,
• pH gt 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)

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Hydrodynamic flow induced by a pressure
difference, and creates broad band
P.521
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23.6 How Capillary Electrophoresis Works
(d) ? at low pH, ?Si-OT?Si-OH
cation? in the double layer - at
neutral pH or high pH electroosmosis gt
electrophoresis the net flow anions ?
cathode - at low pH anode ? anion may
never reach the detector.
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23.6 How Capillary Electrophoresis Works

• (3) Detectors
• UV-absorbance monitor
• Fluorescence detector
• Electrochemical detection
• Mass spectrometer
• Conductivity detection
• Indirect detector

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

• (1) Capillary zone electrophoresis
• cations gt neutrals (unseparated) gt anions
• (2) Micellar electrokinetic
• capillary electrophoresis
• separate neutral molecules
• as well as ions

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

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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)