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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.

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Chapter 26

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Chapter 26

Chapter 26

Other Methods

Ion exchange chromatography

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



These aromatic rings can be modified

These aromatic rings can be modified

Or to make an anion exchanger

Or to make an anion exchanger

Gels vs resins

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.



Ion exchange selectivity

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

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

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

Ion Exchange

  • Types

    • Resins

    • Gels

    • Inorganic exchangers (Zeolites)

  • Use a gradient to remove stronger bound ions.

Separation of lanthanides

Separation of Lanthanides



  • 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

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



Unsuppressed ion chromatography

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

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

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.

Other methods

  • 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



  • 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

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

Determination of Molecular Weight

  • Plot Log (MW) vs elution volume

Affinity chromatography

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 igg 1 using protein a

Antibody IgG1 using Protein A

Capillary electrophoresis

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.

Other methods


  • 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

Single Cell Analysis

Benzyl alcohol separation

Benzyl Alcohol Separation



  • 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

Stokes Equation

  • F = 6phr

  • h is the measure of solution viscosity

This allows ions to move what about neutrals

This allows ions to move, what about neutrals.

  • Electroosmosis

Bulk solution now flows toward the cathode

Bulk Solution now flows toward the cathode.

Electroosmotic flow eof

Electroosmotic Flow (EOF)

  • ueo = meoE

    • Units of the electroosmotic mobility is m2/[V.s]

Joule heating

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

Apparent mobility

Apparent Mobility

  • Two mechanisms for movement. Electrophoresis and Electroosmosis.

  • Can be going the same direction or the opposite.

  • mapp = mep + meo

Apparent mobility1

Apparent Mobility

  • Speed divided by electric field.

Ld isthe length to the detector and Lt is the total length.

Electroosmotic mobility

Electroosmotic Mobility

Separation is based on size and charge

Separation is based on size and charge

  • Bovine carbonic anhydrase – acetylated at the lysine residues R-NH2

Plates and resolution

Plates and Resolution

  • N = Ld/s2

  • Or

  • N = mappV/2D* Ld/Lt



  • Same as for GC or HPLC

Resolution improvement increase e

Resolution Improvement (Increase E)



  • Two Modes

    • Hydrodynamic Injection

    • Electrokinetic Injection



  • UV is most common.

Uv detection

UV Detection

Electrochemical is also used

Electrochemical is also used

Electrochemical detection example

Electrochemical Detection Example

Indirect detection of ions

Indirect Detection of Ions

Elution order

Elution order

  • In CZE

    • Cations – highest mobility first

    • Neutrals – unresolved

    • Anions – highest mobility last

Mekc micellar electrokinetic chromatography

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

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