Introduction
to HPLC
High performance liquid
chromatography (HPLC) is an important analytical tool for separating and
quantifying components in complex liquid mixtures. By choosing the appropriate equipment (i.e.
column and detector), this method is applicable to samples with components
ranging from small organic and inorganic molecules and ions to polymers and
proteins with high molecular weights.
The various types of HPLC and their characteristics are summarized in
the table below. In this experiment, we
will use reversed-phase partition chromatography.
|
TYPE
|
SAMPLE POLARITY
|
MOLECULAR
|
STATIONARY PHASE
|
MOBILE PHASE
|
|
Adsorption
|
non-polar to somewhat polar
|
100 - 104
|
silica or alumina
|
non-polar to polar
|
|
Partition (reversed-phase)
|
non-polar to somewhat polar
|
100 - 104
|
non-polar liquid adsorbed or chemically bonded to the packing
material
|
relatively polar
|
|
Partition (normal-phase)
|
somewhat polar to highly polar
|
100 - 104
|
highly polar liquid adsorbed or chemically bonded to the packing
material
|
relatively non-polar
|
|
Ion Exchange
|
highly polar to ionic
|
100 - 104
|
ion-exchange resins made of insoluble, high-molecular weight solids
functionalized typically with sulfonic acid (cationic exchange) or amine
(anionic exchange) groups
|
aqueous buffers with added organic solvents to moderate solvent
strength
|
|
Size-Exclusion
|
non-polar to ionic
|
103 – 106
|
small, porous, silica or polymeric particles
|
polar to non-polar
|
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