chapter 26 other methods. ion-exchange chromatography the mechanism of separation will be the...
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Chapter 26
Other Methods
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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
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Polymerization
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These aromatic rings can be modified
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Or to make an anion exchanger
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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.
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Sephadex
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Ion Exchange Selectivity
Equilibrium system R-Na+ + Li+ = R-Li+ + Na
K = [R-Li+][Na+]/[R-Na+][Li+]
K is called the selectivity coefficient
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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.
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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.
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Ion Exchange
Types Resins Gels Inorganic exchangers (Zeolites)
Use a gradient to remove stronger bound ions.
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Separation of Lanthanides
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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
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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
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Examples
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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.
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Ion Pair Chromatography
Separate ions on a reverse phase column. (Ammonium ions)
Add a surfactant to the mobile phase. Such as sodium octane sulfonate.
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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.
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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
Vo is the elution volume for large molecules Vo + Vi is the elution volume for small molecules
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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.
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Stationary Phase
A solid support with internal volume of fixed size. There are many options available. Both low pressure and high pressure (HPLC)
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Determination of Molecular Weight
Plot Log (MW) vs elution volume
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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)
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Antibody IgG1 using Protein A
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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.
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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.
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Single Cell Analysis
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Benzyl Alcohol Separation
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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
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Stokes Equation
F = 6r is the measure of solution viscosity
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This allows ions to move, what about neutrals.
Electroosmosis
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Bulk Solution now flows toward the cathode.
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Electroosmotic Flow (EOF)
ueo = eoE Units of the electroosmotic mobility is
m2/[V.s]
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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
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Apparent Mobility
Two mechanisms for movement. Electrophoresis and Electroosmosis.
Can be going the same direction or the opposite.
app = ep + eo
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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.
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Electroosmotic Mobility
t
neutraldneutraleo
LVtL
E
u
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Separation is based on size and charge
• Bovine carbonic anhydrase – acetylated at the lysine residues R-NH2
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Plates and Resolution
N = Ld/2
Or N = appV/2D* Ld/Lt
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Resolution
Same as for GC or HPLC
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Resolution Improvement (Increase E)
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Injection
Two Modes Hydrodynamic Injection
Electrokinetic Injection
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Detection
UV is most common.
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UV Detection
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Electrochemical is also used
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Electrochemical Detection Example
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Indirect Detection of Ions
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Elution order
In CZE Cations – highest mobility first Neutrals – unresolved Anions – highest mobility last
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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