Other Methods

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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-LiNa/R-NaLi
• K is called the selectivity coefficient

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Which ions have greater affinity

• Higher charge, higher polarizability and
  decreased hydrated radius.
• Pu4gtgtLa3gtCe3gtPr3gtEu3gtY3gtSc3gtAl3 gtgt Ba2gt
  Pb2 gt Sr2 gt Ca2 gt Ni2 gt Cd2 gt Cu2 gt Co2
  gtZn2 gt Mg2 gt UO2 gtgt Tigt Aggt Rbgt K gtNH4gt
  Nagt Hgt 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/fE mepE
• mep is electrophoretic mobility
• Relates speed and charge
• Directly related to charge, indirectly related to
  size

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Stokes Equation

• F 6phr
• h 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 meoE
• 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 mm 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.
• mapp mep meo

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Apparent Mobility

• Speed divided by electric field.

Ld is the length to the detector and Lt is the
total length.
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Electroosmotic Mobility
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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/s2
• Or
• N mappV/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