Separation takes place when the sample is passed through the column.
Proper or correct selection of the column packing and the mobile phase used are the two most important factors in successful separation of sample with HPLC
Normally, columns are filled with silica gel due to is particle shape, its surface properties and the structure of the silica gel which is porous , helps to get a good separation. This silica gel is wetted by the mobile phased used, this silica gel is inert to most of the compounds. This silica gel is used for the separation of wide varieties of sample components.
Generally column are protected by a guard column. This guard column also adsorb undesirable sample components.
Different types of column used as per their functions, so this column are classified as:
- Normal Phase: Mobile phase used over here is non- polar and the stationary phase is polar. In this phase the column packing is polar eg., silica gel, cyanopropyl-bonded, amino-bonded etc. and the mobile phase is non polar eg; hexane, isooctane, methylene chloride, chloroform, diethyl ether , ethyl acetate. In this type the retention is governed my the interaction of the the polar parts of the stationary phase and the non polar solute. This technique is useful for water sensitive compounds, geometric isomers, chiral compounds etc.
- Reverse Phase: In this phase the column packing or the stationary phase is non polar and the mobile phase is polar. Retention is due to the non polar stationary phase and the polar mobile phase. Non polar stationary phased are non polar hydrocarbons, waxy liquids or bonded hydrocarbons such as C18, C8 etc. and the solvents are polar such as aqueous-organic mixtures such as methanol or water or acetonitrile.
- Size exclusion : In this type of column molecules are separated according to the size of the sample component. This small molecules penetrate into the pores within the packing while larger molecule only partially penetrate the pores. The larger molecules elute before the smaller molecules.
- Ion Exchange: In this type of column the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase.In ion exchange, the column packing contains ionic groups(e.g. sulfonic, tetraalkylammonium) and the mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.).
These are some of the types of mechanism used to select different types of columns for high performance liquid chromatography (HPLC).