Chapter 26
Other Methods
Ion-Exchange Chromatography
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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
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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
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Equilibrium system
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R-Na+ + Li+ = R-Li+ + Na
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K = [R-Li+][Na+]/[R-Na+][Li+]
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K is called the selectivity coefficient
Which ions have greater affinity
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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
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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
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Types
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Resins
Gels
Inorganic exchangers (Zeolites)
Use a gradient to remove stronger bound
ions.
Separation of Lanthanides
Applications
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Preconcentration
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Water deionization.
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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
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HPLC ion exchange.
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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
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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
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Separate ions on a reverse phase column. (Ammonium
ions)
Add a surfactant to the mobile phase.
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Such as sodium octane sulfonate.
Molecular Exclusion Chromatography
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Separation Based on Size Only
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Gel Filtration
Gel Permeation
Large molecules can not get into the internal
diameter so the elute more quickly.
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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
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Vo is the elution volume for large molecules
Vo + Vi is the elution volume for small molecules
Elution
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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
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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
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Plot Log (MW) vs elution volume
Affinity Chromatography
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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
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Motive force is no longer pressure but
electrical migration.
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Cations migrate to the cathode
Anions migrate to the anode
High electric field place across a capillary
column.
CZE
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Very high resolution due to the lack of no
packing or stationary phase, no A term or c
term in the van Deempter equation.
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H = A + B/ux + Cux
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Just longitudinal diffusion plays a role.
Single Cell Analysis
Benzyl Alcohol Separation
Mobility
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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 = mepE
mep is electrophoretic mobility
Relates speed and charge
Directly related to charge, indirectly related to
size
Stokes Equation
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F = 6phr
h is the measure of solution viscosity
This allows ions to move, what about
neutrals.
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Electroosmosis
Bulk Solution now flows toward the
cathode.
Electroosmotic Flow (EOF)
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ueo = meoE
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Units of the electroosmotic mobility is m2/[V.s]
Joule Heating
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Capillary tubes must be narrow enough to get
rid of the excess heat. 50 mm tubes are ok
but 1 mm would be a real problem. Some
are cooled.
Heat is related to I2R
Apparent Mobility
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Two mechanisms for movement.
Electrophoresis and Electroosmosis.
Can be going the same direction or the
opposite.
mapp = mep + meo
Apparent Mobility
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Speed divided by electric field.
m app
Ld
u net
t
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V
E
Lt
Ld is the length to the detector and Lt is
the total length.
Electroosmotic Mobility
uneutral Ld tneutral
meo 

V
E
Lt
Separation is based on size and charge
•
Bovine carbonic anhydrase – acetylated at the
lysine residues R-NH2
Plates and Resolution
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N = Ld/s2
Or
N = mappV/2D* Ld/Lt
Resolution
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Same as for GC or HPLC
Resolution Improvement (Increase E)
Injection
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Two Modes
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Hydrodynamic Injection
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Electrokinetic Injection
Detection
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UV is most common.
UV Detection
Electrochemical is also used
Electrochemical Detection Example
Indirect Detection of Ions
Elution order
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In CZE
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Cations – highest mobility first
Neutrals – unresolved
Anions – highest mobility last
MEKC –
Micellar Electrokinetic Chromatography
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Add a surfactant to the mobile phase.
Micelles form above the CMC
Neutral species will partition into the micelles and flow at
that rate