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.
How HPLC columns work
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.
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)
Usually an aqueous solution of a salt plus a buffer
Ionizable samples and large biomolecules
Non-polar reversed phase columns
Non-polar conditions plus an ion pairing reagent
Acids or bases weakly retained by reversed phase
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
Easily find the correct column with our digital LC column selection guide ›
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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