Chapter 26

Other Methods

Ion-Exchange Chromatography

The mechanism of separation will be the exchange of

ions from the column to the solution.
Water softening exchange Na ions for Ca and Mg.
Water deionization exchange H ions for cations and OH
ions for anions. Leaving water.
Can be larger scale. The support is modified to allow for
the ion exchange equilibrium.
Can be natural materials or synthetic

Polymerization

These aromatic rings can be

modified

Or to make an anion exchanger

Gels vs Resins

Resins are firm and can stand greater

pressure.
Gels are softer have lower charge
densities and are made from polymeric
sugars.
Polyacrylamide can also be used a the
backbone.

Sephadex

Ion Exchange Selectivity

Equilibrium system

R-Na+ + Li+ = R-Li+ + Na

K = [R-Li+][Na+]/[R-Na+][Li+]

K is called the selectivity coefficient

Which ions have greater affinity

Higher charge, higher polarizability and

decreased hydrated radius.

Pu4+>>La3+>Ce3+>Pr3+>Eu3+>Y3+>Sc3+>Al3+ >>
Ba2+> Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+
> Co2+ >Zn2+ > Mg2+ > UO2+ >> Ti+> Ag+>
Rb+> K+ >NH4+> Na+> H+> Li+

Reconditioning by having higher concentration

of the less tightly held ion.

Donnan Equilibrium

Concentration of ions outside the resin

will be higher than the inside
concentration.
Cations will be excluded from the inside
of an anion exchanger. (Has same charge
as resin site)
Ion Exclusion Chromatography
Non charged species can migrate in but
not ions.

Ion Exchange

Types

Resins
Gels
Inorganic exchangers (Zeolites)

Use a gradient to remove stronger

bound ions.

Separation of Lanthanides

Applications

Preconcentration

Water deionization.

Pass much water over a resin and then elute

with a high concentration of acid.
Cation exchange to trap cations
Chelex -100 to trap transition metals.
Cation exchange from cation removal.
Anion exchange for anion removal.

Water softening

Ion Chromatography

HPLC ion exchange.

Detection is an issue. Ions do not absorb

uv/vis light.
Conduction is used to detect ions but the
mobile phase will have high electrolyte like
KOH
We use ion suppression

Examples

Unsuppressed Ion Chromatography

The ions have higher conductivity than the

eluent. Carboxylic acids used as eluent.
Indirect Detection. Mobile phase has a light
absorbing ion. Phthalate ion.

Ion Pair Chromatography

Separate ions on a reverse phase column.

(Ammonium ions)
Add a surfactant to the mobile phase.

Such as sodium octane sulfonate.

Molecular Exclusion Chromatography

Separation Based on Size Only

Gel Filtration
Gel Permeation

Large molecules can not get into the

internal diameter so the elute more
quickly.

Vt = Vo + Vi + Vg + Vec

Vt is the total volume of the system. If we

ignore volume outside the column then we
have
Vt = Vo + Vi + Vg

Vo is the elution volume for large molecules

Vo + Vi is the elution volume for small
molecules

Elution

Ve = Vo + KVi

Kave assumes that Vg is very small and I

suggest you not use it.
K will fall between 0 and 1 unless there
is another mechanism in the column.

Stationary Phase

A solid support with internal volume of

fixed size. There are many options
available. Both low pressure and high
pressure (HPLC)

Determination of Molecular Weight

Plot Log (MW) vs elution volume

Affinity Chromatography

Stationary phase is made so that it has

a very specific interaction that can
cause binding to a specific substrate.
Elution is carried out by disrupting this
interaction. (Change pH is an example)

Antibody IgG1 using Protein A

Capillary Electrophoresis

Motive force is no longer pressure but

electrical migration.

Cations migrate to the cathode

Anions migrate to the anode

High electric field place across a

capillary column.

CZE

Very high resolution due to the lack of

no packing or stationary phase, no A
term or c term in the van Deempter
equation.

H = A + B/ux + Cux

Just longitudinal diffusion plays a role.

Single Cell Analysis

Benzyl Alcohol Separation

Mobility

Ion of charge q will accelerate in the

potential field until the frictional force
counter balances it and it travels at
constant speed.
uep = q/f*E = epE
ep is electrophoretic mobility
Relates speed and charge
Directly related to charge, indirectly
related to size

Stokes Equation

F = 6r
is the measure of solution viscosity

This allows ions to move, what about

neutrals.

Electroosmosis

Bulk Solution now flows toward the

cathode.

Electroosmotic Flow (EOF)

ueo = eoE

Units of the electroosmotic mobility is m2/

[V.s]

Joule Heating

Capillary tubes must be narrow enough

to get rid of the excess heat. 50 m
tubes are ok but 1 mm would be a real
problem. Some are cooled.

Heat is related to I2R

Apparent Mobility

Two mechanisms for movement.

Electrophoresis and Electroosmosis.
Can be going the same direction or the
opposite.
app = ep + eo

Apparent Mobility

Speed divided by electric field.

app

Ld

unet
t

V
E
Lt

Ld is the length to the detector and Lt

is the total length.

Electroosmotic Mobility

uneutral Ld t neutral
eo

V
E
Lt

Separation is based on size and

charge

Bovine carbonic anhydrase acetylated at

the lysine residues R-NH2

Plates and Resolution

N = Ld/2

Or
N = appV/2D* Ld/Lt

Resolution

Same as for GC or
HPLC

Resolution Improvement (Increase E)

Injection

Two Modes

Hydrodynamic Injection

Electrokinetic Injection

Detection

UV is most common.

UV Detection

Electrochemical is also used

Electrochemical Detection Example

Indirect Detection of Ions

Elution order

In CZE

Cations highest mobility first

Neutrals unresolved
Anions highest mobility last

MEKC
Micellar Electrokinetic
Chromatography

Add a surfactant to the mobile phase.

Micelles form above the CMC
Neutral species will partition into the micelles and
flow at that rate