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

7. 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.

8. Sephadex

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

13. Which ions have greater affinity Higher charge, higher polarizability and decreased hydrated radius. Pu 4+ >>La 3+ >Ce 3+ >Pr 3+ >Eu 3+> Y 3+> Sc 3+> Al 3+ >> Ba 2+> Pb 2+ > Sr 2+ > Ca 2+ > Ni 2+ > Cd 2+ > Cu 2+ > Co 2+ >Zn 2+ > Mg 2+ > UO 2+ >> Ti + > Ag + > Rb + > K + >NH 4 + > Na + > H + > Li + Reconditioning by having higher concentration of the less tightly held ion.

14. 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.

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

16. Separation of Lanthanides

17. 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

18. 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

20. Examples

21. 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.

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

24. 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.

25. V t = V o + V i + V g + V ec V t is the total volume of the system. If we ignore volume outside the column then we have V t ’ = V o + V i + V g Vo is the elution volume for large molecules Vo + Vi is the elution volume for small molecules

26. Elution V e = V o + KV i K ave assumes that V g 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.

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

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

34. 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)

35. Antibody IgG 1 using Protein A

36. 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.

38. 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/u x + Cu x Just longitudinal diffusion plays a role.

39. Single Cell Analysis

40. Benzyl Alcohol Separation

41. Mobility Ion of charge q will accelerate in the potential field until the frictional force counter balances it and it travels at constant speed. u ep = q/f*E =  ep E  ep is electrophoretic mobility Relates speed and charge Directly related to charge, indirectly related to size

42. Stokes Equation F = 6  r  is the measure of solution viscosity

43. This allows ions to move, what about neutrals. Electroosmosis

44. Bulk Solution now flows toward the cathode.

45. Electroosmotic Flow (EOF) u eo =  eo E Units of the electroosmotic mobility is m 2 /[V. s]

46. 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 I 2 R

47. Apparent Mobility Two mechanisms for movement. Electrophoresis and Electroosmosis. Can be going the same direction or the opposite.  app =  ep +  eo

48. Apparent Mobility Speed divided by electric field. L d is the length to the detector and L t is the total length.

49. Electroosmotic Mobility

50. Separation is based on size and charge Bovine carbonic anhydrase – acetylated at the lysine residues R-NH 2

51. Plates and Resolution N = L d /  2 Or N =  app V/2D* L d /L t

52. Resolution Same as for GC or HPLC

53. Resolution Improvement (Increase E)

54. Injection Two Modes Hydrodynamic Injection Electrokinetic Injection

55. Detection UV is most common.

56. UV Detection

57. Electrochemical is also used

58. Electrochemical Detection Example

59. Indirect Detection of Ions

60. Elution order In CZE Cations – highest mobility first Neutrals – unresolved Anions – highest mobility last

61. 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