Chapter 23

Chromatographic Methods &

Capillary Electrophoresis

Capillary Electrophoresis in Biology

23.1 Ion-Exchange Chromatography -1

1) Structures

Cation exchangers: -SO3-, -CO2

-

Anion exchanger: -NR3+

2) Ion-exchange selectivity

a) R-Cs+ + Li + R-Li+ + Cs+

selectivity coefficient:

LiCsR

Cs LiRK

23.1 Ion-Exchange Chromatography -2

b) Hydrated radius :

hydrated radius or Ionic charge

bind to ion-exchange resin

order of selectivity: p.501

23.1 Ion-Exchange Chromatography -3

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

23.1 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 conductivit

y change when analyte ions are eluted.

(5) Suppressed-ion anion chromatography : remove the unwanted electrolyte pri

or to conductivity measurement.

23.2 Ion Chromatography -3HCl

cationseparator

co lumn(H +)

suppressorco lumn

O H -

rep lacesanion

[co

n]

time

?

time

?[co

n]

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.

① specific binding to s.p.

② 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.

23.5 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 = 0

23.5 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 pHelectroosmosis > electrophoresis

the net flow: anions → cathode

- at low pH anode ← anion

& may never reach the detector.

23.6 How Capillary Electrophoresis Works

23.6 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.

23.7 Types of capillary electrophoresisWhat is a micelle ?

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(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