1. Chromatographic Methods & Capillary Electrophoresis
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
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 elution with 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)

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

22. 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. 23.6 How Capillary Electrophoresis Works
(3) Detectors
UV-absorbance monitor
Fluorescence detector
Electrochemical detection
Mass spectrometer
Conductivity detection
Indirect detector

24. 23.7 Types of capillary electrophoresis
Crown ether has greater affinity for D-aa than L-aa
P.524

25. 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

26. 23.7 Types of capillary electrophoresis
What is a micelle ?
are anions & behave like a pseudostationary phase c  0 in van deemter eqn.

27. 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

28. 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)