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HPLC columns
After the sample enters the flow path, the mobile phase carries the sample to the column, where the separation occurs.
Columns can function in ambient air but are generally thermostatted and housed within a temperature-controlled column compartment. Proper column temperature control is essential to conserving retention time precision, selectivity, and separation efficiency.
A general rule to remember is that as the column temperature increases, analyte retention decreases, leading to faster separation.
HPLC system components
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How HPLC columns work
Representation of a sample mixture eluting and separating on the column at different rates.
HPLC columns contain a stationary phase bonded to a support material, usually porous silica particles, to provide a large surface area. The stationary phase provides the basis for separating sample components.
Stationary phase chemistry dictates the affinity of the sample components to stick or retain on the column as the mobile phase moves the sample through the column. As a result, the sample components traverse the column and elute at different rates.
For example, you can visualize a mixture (A) containing multiple components represented as a grey band introduced at the front, or head, of the column.
As the mobile phase moves the mixture through the column, the red component (B) retains more strongly than the purple. As a result, the purple and blue components move through the column faster and are the first ‘bands’ to elute from the column (C). The green, yellow, and red bands retain longer and elute later (D).
How to choose the correct HPLC column
The physiochemical properties of a sample, stationary phase chemistry, mobile phase composition, flow rate, and column temperature determine the rate at which components travel through the column. Researchers can choose from various stationary phase chemistries and column dimensions like the length, inner diameter, and support particle sizes.
Column type | Stationary phase | Mobile phase | Applications |
Reversed Phase (RP) | Non-polar such as C18 or phenyl | Mixture of water and polar organic solvent | Accounts for a majority of HPLC separations |
Normal Phase (NP) | Polar such as unbound silica | Mixture of less-polar organic solvents | Water-insoluble samples and isomers |
Hydrophilic Interaction (HILIC) | Polar such as silica or amide-bonded phase | Mixture of water and non-polar organic solvent | Highly polar samples poorly retained by reverse phase liquid chromatography |
Ion Exchange (IEX) | Ionizable groups | Usually an aqueous solution of a salt plus a buffer | Ionizable samples and large biomolecules |
Ion Pair | Non-polar reversed phase columns | Non-polar conditions plus an ion pairing reagent | Acids or bases weakly retained by reversed phase |
Chiral | Chiral groups such as polysaccharides or cyclodextrins | Analyte and SP dependent, can be RP, NP, polar organic, etc | Separation/purification of enantiomers, such as racemic drug mixtures |
Hydrophobic Interaction (HIC) | Non-polar, short alkyl chains or phenyl | High salt buffer gradient, non-denaturing conditions | Proteins, often antibodies |
Size Exclusion (SEC) | An inert column such as a dextran polymer | Used with either an aqueous or organic mobile phase | Large biomolecules or synthetic polymers |
Learn more about the different HPLC column types and applications ›
Easily find the correct column with our digital LC column selection guide ›
FAQ
Most common HPLC columns are made from stainless steel and packed with porous silica particles that are typically modified, e.g., a C18 bonding is a common choice in reversed-phase HPLC. However, there is a high variety of HPLC column hardware and packing material.
Before beginning a new analysis, consider the physical and chemical properties of the analytes, the mode of analysis and how the analytes will interact with the surface of the chromatographic phase.
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