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Chapter 26

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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  1. Chapter 26 Other Methods

  2. 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

  3. Polymerization

  4. These aromatic rings can be modified

  5. Or to make an anion exchanger

  6. 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.

  7. Sephadex

  8. Ion Exchange Selectivity • Equilibrium system • R-Na+ + Li+ = R-Li+ + Na • K = [R-Li+][Na+]/[R-Na+][Li+] • K is called the selectivity coefficient

  9. 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.

  10. 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.

  11. Ion Exchange • Types • Resins • Gels • Inorganic exchangers (Zeolites) • Use a gradient to remove stronger bound ions.

  12. Separation of Lanthanides

  13. 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

  14. 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

  15. Examples

  16. 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.

  17. Ion Pair Chromatography • Separate ions on a reverse phase column. (Ammonium ions) • Add a surfactant to the mobile phase. • Such as sodium octane sulfonate.

  18. 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.

  19. 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

  20. 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.

  21. Stationary Phase • A solid support with internal volume of fixed size. There are many options available. Both low pressure and high pressure (HPLC)

  22. Determination of Molecular Weight • Plot Log (MW) vs elution volume

  23. 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)

  24. Antibody IgG1 using Protein A

  25. 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.

  26. 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.

  27. Single Cell Analysis

  28. Benzyl Alcohol Separation

  29. 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 = mepE • mep is electrophoretic mobility • Relates speed and charge • Directly related to charge, indirectly related to size

  30. Stokes Equation • F = 6phr • h is the measure of solution viscosity

  31. This allows ions to move, what about neutrals. • Electroosmosis

  32. Bulk Solution now flows toward the cathode.

  33. Electroosmotic Flow (EOF) • ueo = meoE • Units of the electroosmotic mobility is m2/[V.s]

  34. 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

  35. Apparent Mobility • Two mechanisms for movement. Electrophoresis and Electroosmosis. • Can be going the same direction or the opposite. • mapp = mep + meo

  36. Apparent Mobility • Speed divided by electric field. Ld isthe length to the detector and Lt is the total length.

  37. Electroosmotic Mobility

  38. Separation is based on size and charge • Bovine carbonic anhydrase – acetylated at the lysine residues R-NH2

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