Study Set Content:
TYPES OF HPLC SEPARATION
Normal phase HPLC (NP HPLC):
Reverse phase HPLC (RP-HPLC or RPC):
Ion exchange HPLC:
Size exclusion HPLC (SEC) or Gel Permeation or Filtration Chromatography:
Bioaffinity chromatography or Affinity chromatography
Column packing is polar (e.g silica) and the mobile phase is non-polar.
Normal phase HPLC (NP HPLC):
The column packing is non-polar (e.g C18), the mobile phase is water + miscible solvent (e.g
methanol).
Reverse phase HPLC (RP-HPLC or RPC):
It is used for water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral
compounds.
Normal phase HPLC (NP HPLC):
It can be used for polar, non-polar, ionizable, and ionic samples.
Reverse phase HPLC (RP-HPLC or RPC):
• Column packing contains ionic groups and the mobile phase is buffer.
• It is used to separate anions and cations.
Ion exchange HPLC:
Molecules diffuse into pores of a porous medium and are separated according to their relative
size to the pore size.
Size exclusion HPLC (SEC) or Gel Permeation or Filtration Chromatography:
• Large molecules elute () and smaller molecules elute ()
First, later
This process is used for the separation of biomolecules such as antigen and antibody, enzyme
and inhibitor, hormone and carrier, receptor and ligand, or protein and nucleic acid
Bioaffinity chromatography or Affinity chromatography
Normal Phase
Hydrophilicity(High Polarity)
Reverse Phase HPLC
Hydrophobicity(Low polarity)
Ion-Exchange HPLC
Electrostaticity charged ions
Size exclusion HPLC or Gel Permeation of Filtration Chromatography
Smaller molecular size
Saccharides
Normal Phase
Nuclear acids
Normal Phase
Inorganic ions
Ion-Exchange HPLC
Small molecule pharmaceuticals
Reverse Phase HPLC
Vitamins
Reverse Phase HPLC
Amino acids
Ion Exchange HPLC
Proteins
Ion Exchange HPLC
