Chapter 26
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Chapter 26 PowerPoint PPT Presentation

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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

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


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.


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


  • 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


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


  • 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.


  • 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


  • 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

  • F = 6phr

  • h 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 = meoE

    • 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 mm 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.

  • mapp = mep + meo

Apparent Mobility

  • Speed divided by electric field.

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

Electroosmotic Mobility

Separation is based on size and charge

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

Plates and Resolution

  • N = Ld/s2

  • Or

  • N = mappV/2D* Ld/Lt


  • Same as for GC or HPLC

Resolution Improvement (Increase E)


  • Two Modes

    • Hydrodynamic Injection

    • Electrokinetic Injection


  • 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

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