Normal-phase HPLC
Normal phase HPLC is performed by eluting a
silica-packed column, which is hydrophilic, with a
non-polar mobile phase. The mobile phase is usually
hexane, to which is added one or more of the following,
in increasing order of polarity: chloroform
(CHC13), tetrahydrofuran (THF), acetonitrile
(ACN), isopropyl alcohol (IPA) or methanol
(MeOH). Separation is achieved by partition with
differential adsorption and desorption of both the
solute and solvents during passage down the
column. Polar solutes are retained, but more
lipophilic molecules are not. By increasing the polarity
of the mobile phase (e.g. by adding MeOH or
IPA), polar solutes are eluted more quickly, whereas
non-polar solutes are better retained and their order
of retention is changed. Decreasing solvent polarity
increases polar solute retention and facilitates the
elution of lipophilic molecules.
In general, normal-phase HPLC is used for moderately
polar solutes (freely soluble in methanol).
Non-polar hydrocarbon-soluble solutes are difficult
to retain and very polar and water-soluble solutes are
difficult to elute sufficiently.