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 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.

Water deionization. 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.

t

dnet

app

LVt

L

E

u

Ld is the length to the detector and Lt

is the total length.

Electroosmotic Mobility

t

neutraldneutraleo

LVtL

E

u

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