LC modes

Question 1

Normal phase (NP)

Answer 1

SP = polar (silica, alumina)
MP = non-polar (organic)
Retention: polar interaction analytes + SP
Reduce retention: more polar MP

Question 2

Reversed phase (RP)

Answer 2

SP = non-polar (hydrophobic) (bpnded silica: C18, C8)
MP = polar (aqueous) (water/buffer + modifier [MeOH, MeCN])
Retention: solvophobic effect
Reduce retention: add modifier
Small molecules and polypeptides
Isocratic + gradient running

Question 3

Hydrophilic interaction (HILIC)

Answer 3

SP = polar (~ NP) (silica, polyamide, zwitterionic)
MP = aqueous organic (MeCN + increasing H2O)
Retention: H2O molecules MP interact with SP --> stagnant layer --> analytes: layer  bulk MP
Hydrophilic analytes (log P < 0)

Question 4

Hydrophobic interaction (HIC)

Answer 4

SP = non-polar (~RP)
MP = extremely polar (decreasing salt gradient)
Separation of proteins under mild conditions

Question 5

Ion-exchange

Answer 5

SP = charged groups bound to polysterene beads
Retention: electrostatic interaction ions + SP
Different columns (4)

Question 6

Size exclusion

Answer 6

Separation based on size of molecules 
No interaction analytes + column
No retention: column volume = separation volume
Effective range is based on pore size
(Bio)polymers
Large molecules are fastest

Question 7

Affinity

Answer 7

Separating 1 type of compound

Abs - antigens, cation - organic phosphates, receptor/enzymes - ligands/substrates