High pressure liquid chromatography (HPLC)

High pressure liquid chromatography (HPLC)

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Types of HPLC Columns - Part 2

Exclusion chromatographic columns

Exclusion chromatography (also known as gel permeation chromatography) requires special columns. They are intended to enable the sample molecules to be separated according to their size. The molecular size is understood to mean the volume of the swollen, ball-shaped molecule (= effective molecule size in the solvent). The separation takes place with the help of the special columns made of styrene-divinylbenzene copolymers via the pore size. Chemico-physical interactions with the stationary phase are not desirable. The solvent used stays the longest because it is the smallest molecule and can get deepest into the pores. It is shown by an injection peak.

Exclusion chromatography is often used to determine the molecular weight of polymers, since the effective molecule size in solution within a group of substances is proportional to the molecular weight.

Ion chromatographic columns

The columns for ion chromatography are used to separate ionic compounds (inorganic and organic), so the eluents are predominantly aqueous, with added buffers and other modifiers. Plastic exchange resins are used as the column material.

Tab. 1
Example for ion exchangers
purposeActive groupstructure
Anion exchangequaternary ammonium groups, primary ammonium groups-N (CH3)3+OH--NH3+OH-
Cation exchangeSulfonic acid groupcarboxylic acid group-SO3-H+-CO2-H+

Affinity chromatographic columns

This type of pillars is one of the specific pillars. Biomolecules (proteins), which can only interact with very special components of the sample, are fixed on a carrier material. Only those analytes that find a suitable interaction environment in the active site of the proteins will remain on the column. Everything else flows past. By changing the eluent, e.g. pH-Value change, this captured analyte is then released again.

Video: Sjøholt Gry - Væskekromatografi (May 2022).