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Chromatography Techniques

Key Concepts

Chromatography is an analytical technique used to determine the purity of a substance or to separate a mixture into its components.
The stationary phase refers to the solid or liquid to which components in a mixture bind or adsorb.
The mobile phase refers to the liquid or gas that moves the components in a mixture over the stationary phase.
Components in a mixture are separated based on their different abilities to bind or adsorb to the stationary phase, and on their different abilities to desorb or dissolve in the mobile phase.
In paper chromatography and thin layer chromatography the retardation factor, R_f, is a comparison of the distance travelled by a component to the distance travelled by the solvent.

Comparison of Chromatography Techniques

Type Features Uses

Paper

inexpensive
slow
often used for water soluble compounds
a qualitative technique
stationary phase: usually water (present in the paper);
mobile phase:pure solvent or solvent mixture
Retardation factor, R_f, measured

identification of colours in inks or foods
identification of amino acids
separation of pigments in plant leaves

Thin Layer (TLC)
inexpensive
faster than paper and more sensitive
used for less polar compounds
qualitative technique although components may be scraped off the plate & their masses measured
stationary phase: plate coated with silica gel or Al₂O₃;
mobile phase:pure solvent or solvent mixture
Retardation factor, R_f, measured

identification of biological sugars
identifying components of pain-relieving preparations
identifying components (eg, carboxylic acids) that might damage paper

Gas
(GC) area under the peak measures the amount of the component present

expensive
fast
used for gases or easily vaporised compounds
M_r < 300
both qualitative and quantitative technique
stationary phase:either a sold (eg, glass beads) or liquid (eg, long chain hydrocarbon);
mobile phase (carrier gas):helium, nitrogen, or some other inert gas
Retention time, R_t, measured

analysis of air-borne pollutants
analysis of athletes' urine samples for performance enhancing drugs
analysis of oil spills
analysis of essential oils in perfume preparation

High-performance Liquid
(HPLC) area under the peak measures the amount of the component present

expensive but less expensive than GC
fast but slower than GC
used for organic compounds that decompose if vaporised or compounds with M_r > 300
both a qualitative and a quantitative technique
stationary phase: small waxy solids;
mobile phase (eluent): pure solvent or solvent mixture
Retention time, R_t, measured

analysis of pharmaceuticals
cyanide analysis
analysis of organic pollutants in water
analysis of toxic compounds in shell fish
protein analysis