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Liquid Chromatography Columns


Conventional liquid chromatography uses plastic or glass columns that can range from a few centimeters to several meters. The most common lengths are 10-100 cm, with the longer columns finding use for preparative-scale separations. High-performance liquid chromatography (HPLC) columns are stainless steel tubes, typically of 10-30 cm in length and 3-5 mm inner diameter. Short, fast analytical columns, and guard columns, which are placed before an analytical column to trap junk and extend the lifetime of the analytical column, are 3-10 cm long.

Picture of an HPLC column

Stationary Phases

Partition Chromatography

In partition chromatography the stationary phase is bonded to inert particles of 3-10 Ám of diameter, with the smaller sizes, 3-5 Ám, being used in analytical columns, and the larger particles being used in preparative-scale HPLC. Analytes separate as they travel through the column due to the differences in their partitioning between the mobile phase and the stationary phase.

Reverse-phase partition chromatography uses a relatively nonpolar stationary phase and a polar mobile phase, such as methanol, acetonitrile, water, or mixtures of these solvents. The most common bonded phases are n-octyldecyl (C18) and n-decyl (C8) chains, and phenyl groups. Reverse-phase chromatography is the most common form of liquid chromatography, primarily due to the wide range on analytes that can dissolve in the mobile phase.

Normal-phase partition chromatography uses a polar stationary phase and a nonpolar organic solvent, such as n-hexane, methylene chloride, or chloroform, as the mobile phase. The stationary phase is a bonded siloxane with a polar functional group. The most common functional groups in order of increasing polarity are:
cyano: -C2H4CN
amino: -C3H6NH2
dimethylamino: -C3H6N(CH3)2

Adsorption Chromatography

The stationary phase in adsorption chromatography are silica or alumina particles. Analytes are separated due to their varying degree of adsorption onto the solid surfaces. The main advantage of adsorption chromatography is in separating isomers, which can have very different physisorption characteristics due to steric effects in the molecules.

Chiral stationary phases

Separating enantiomeric mixtures is very challenging because the optical isomers are identical chemically. Chiral analytes can be separated by using a chiral stationary phase. One optical isomer of a chiral molecule is usually bonded to a polymer, which is then coated onto silica packing material. Separations occur because the chiral analytes will interact with the one isomer on the stationary phase differently.

Ion Chromatography

Cation and anion-exchange resins are described in the ion chromatography document.

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