Lecture 4 - IPC and GPC

Question 1

What is ion-pair chromatography? (also known as MP ion chromotography)

Answer 1

• Uses a reversed phase LC bonded column to separate organic ionic species
• Use non polar packing materials (C18)
• The selectivity of separation is based on composition of MP so both cationic and anionic can be separated by the same SP.
• Need to add a specific counter ion
• Done by partitioning

Question 2

What are the 2 models behind IPC?

Answer 2

1 - Exchange model
For example if a cationic species is added, an anionic surfactant will be added to the MP. This lodges into the SP and changes it into an ion exchanger. The hydrophobic tail of the surfactant can partition into the C18 SP. When the cations pass through they will associate with the negatively charged SP (surfactant anions) by electrostatic interaction. Then resolve based on their electrostatic affinity to the polar SP.
2 - The Ion-pair model
Analyte ions (organic bases) form neutral pairs with the counter ions in the MP to become neutral. The pair then partitions into non-polar SP (C18) and the analytes then resolve based on their partition coeffs into the SP.

Concentrations need to be carefully tuned for these.

Question 3

What does the retention factor depend on?

Answer 3

1- Concentration of counter ions - For the separation of cationic organic species (1st model) alkane sulfonic acid is added to MP.
To separate organic species (2nd model) quaternary ammonium compounds are added to MP.
Concentration of the counter ion cant be too high or the surface of the SP can become blocked due to undissociated molecules of ion-pair reagent and thus separation is impossible.

2 - Type of counter ion - depends on the analyte.
If they are hydrophobic (interact well with non-polar) then the counter anion will be hydrophilic.
If they are surface inactive ins (wont react well with non polar C18) a more hydrophobic counter ion is used.

Question 4

How does pH effect retention times?

Answer 4

• The retention will increase with increasing degree of dissociation , the pH can alter the degree of dissociation.

Question 5

What is molecular exclusion chromatography? (also known as gel permeation chromatography)

Answer 5

• The gel is made up of pores
• There are no ions involved
• The separation is based on the size, smaller analytes will fit into the pores and elute last. Larger molecules will elute first they are too big and pass by the pores.
• The pore has “slits” in it, small molecules will fit into these and stay inside the pore for longer.

Question 6

What is the GPC calibration curve?

Answer 6

• The log of mass (y axis) is plotted against the log of retention volume (x axis)
• Molecules which have a mw between M1 - M2 can resolve as they will take different paths as they partially enter pores eluting
  between Vm and Vm + Vi (=the calibration range)
• Large molecules with a mass (> M2), excluded from the pores elute first,
  unresolved, at the void volume Vm.
• All small molecules (< M1) fully enter the pores elute last, unresolved, at Vm + Vi.

Question 7

Overall summary of how molecules are eluted.

Answer 7

• If they are too small for the pore size they will be unresolved and elute last.
• If they are too big they will be unresolved and elute first.
• Any molecules within the pore range will be eluted based on size, largest first too smallest.