High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC) is a leading physicochemical analysis method for the separation, identification, and quantification of components in complex mixtures. Thanks to its precision, reproducibility, and versatility, HPLC has become an essential tool in the pharmaceutical, cosmetic, food, chemical, environmental, and materials industries.
What is HPLC?
HPLC (High Performance Liquid Chromatography) is a chromatography technique that relies on the separation of analytes in a sample based on their interaction with a stationary phase (column) and a mobile phase (liquid solvent). It allows the constituents of a mixture to be identified and quantified with great sensitivity, even at trace levels.
The sample is carried by a solvent under high pressure through a column containing a stationary phase. Each compound migrates at a different speed depending on its affinity, generating a chromatogram that can be analyzed by a detector (UV, fluorescence, MS, etc.).
How HPLC chromatography works
An HPLC system consists of several integrated modules:
- High pressure pump: allows the mobile phase to circulate through the column (up to 400 bars).
- Sample injector: manual or automated, injection must be precise and reproducible.
- Chromatographic column: heart of the separation, filled with a stationary phase (modified silica, exchange resins, etc.).
- Detector: UV/Vis, diode array (DAD), fluorescence, refractometry or coupled with mass spectrometry (HPLC-MS).
- Data acquisition and processing system: conversion of signals into usable chromatograms.
The choice of chromatographic mode depends on the nature of the sample and the analytical objectives:
- Reversed phase (RP-HPLC) : most common, with a hydrophobic stationary phase
- Normal phase , ion exchange , size exclusion , etc.
Technical characteristics of HPLC
- Pressure range : up to 400 bars
- Separation modes : isocratic or gradient
- Column types : C18 silica, C8, ion exchange, gel permeation, etc.
- Detectors : UV/Vis, DAD, fluorescence, RI, MS
- Sensitivity : Trace detection (ppm to ppb)
- Related standards : USP 621, Ph. Eur. 2.2.29, ICH Q2(R1)
For which matrices?
High-performance liquid chromatography is suitable for a very wide range of matrices:
Main industrial applications
HPLC is used routinely or in development in many industrial sectors:
Léa Géréec
Technical and scientific advisor
