High-Performance Liquid Chromatography (HPLC)

HPLC is a cornerstone technique in analytical chemistry, particularly in pharmaceutical development, quality control, and biotechnology. It enables the separation of compounds based on their interactions with a stationary phase and a mobile phase under high pressure, allowing for high resolution and sensitivity.

How does HPLC work?

In HPLC, a sample is dissolved in a solvent (the mobile phase) and pumped through a column packed with a solid or liquid stationary phase. Different compounds in the mixture interact differently with the stationary phase, leading to varying retention times. Detectors, such as UV-Vis, fluorescence, or mass spectrometry (LC-MS), measure the eluting compounds, generating a chromatogram that identifies and quantifies each component.

What are the main types of HPLC?

Common HPLC modes include:

• Reversed-phase HPLC (RP-HPLC): Uses a non-polar stationary phase and polar mobile phase; ideal for separating non-polar to moderately polar compounds.
• Normal-phase HPLC: Features a polar stationary phase and non-polar mobile phase; useful for polar compounds.
• Ion-exchange HPLC: Separates ions based on charge; used in protein and nucleic acid analysis.
• Size-exclusion HPLC (SEC): Separates molecules by size; commonly used for polymers and large biomolecules.

HPLC is essential for ensuring product purity, consistency, and compliance with regulatory standards such as USP, EP, and BP.

Related concepts

HPLC is often coupled with mass spectrometry (LC-MS) for enhanced identification. It is supported by standards like ISO 17025 and regulated under REACH and GHS for safety. Quality control relies on HPLC data for CoA, SDS, and method validation.