High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC) is a reference physicochemical analysis method for the separation, identification, and quantification of the components of complex mixtures. Thanks to its precision, reproducibility, and versatility, HPLC is used routinely and in development across numerous industrial sectors, including pharmaceuticals, cosmetics, food and beverage, chemicals, the environment, materials, and polymers.
What is HPLC?
chromatography (HPLC ) is based on the separation of analytes in a sample according to their interaction with a stationary phase contained in a column and a liquid mobile phase (solvent or buffered mixture). It allows for the identification and quantification of constituents at low concentrations, including trace amounts, using detectors adapted to the nature of the compounds.
An HPLC analysis results in a chromatogram , where the x-axis represents time and the y-axis represents the signal from the detector. Each separated compound appears as a peak, which can be used for identification and quantification.
Principle and steps of an HPLC analysis
During an analysis, the dissolved sample is injected into a mobile phase circuit set in motion by a high-pressure pump. As they pass through the column, the compounds separate according to their respective affinities for the stationary and mobile phases. After elution, the analytes are measured by the detector, and the data are then processed by a data acquisition system to obtain and interpret the chromatogram.
An HPLC analysis typically includes the following steps: circulation of the mobile phase, injection of the sample, separation in the column, detection of analytes, and then generation and processing of the chromatogram.
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: whether manual or automated, the injection must be precise and reproducible.
- Chromatographic column: the heart of the separation, filled with a stationary phase (modified silica, exchange resins…).
- Detector: UV/Vis, diode array (DAD), fluorescence, refractometry or coupled to mass spectrometry (HPLC-MS).
- Data acquisition and processing system: conversion of signals into usable chromatograms.
The choice of chromatographic method depends on the nature of the sample and the analytical objectives:
- Reversed phase (RP-HPLC) : the most common, with a hydrophobic stationary phase
- Normal phase , ion exchange , size exclusion , etc.
HPLC detectors and detection selection
The choice of detector depends on the chemical structure, the expected concentration level, and the matrix.
UV/Vis and DAD detection are suitable for many routine assays where compounds absorb in the UV range. Fluorescence offers high sensitivity for fluorescent or derivatized analytes. Refractive index is useful for compounds with low or no UV absorption, such as certain families of sugars, polyols, or soluble polymers. ELSD is suitable for non-volatile compounds with low UV absorption. Coupling with mass spectrometry, in LC-MS or LC-MS/MS, is recommended when identity confirmation, selectivity, and trace sensitivity are critical.
For which matrices?
High-performance liquid chromatography is suitable for a very wide range of matrices:
Technical characteristics of HPLC
- Pressure range : up to 400 bar
- Separation modes : isocratic or gradient
- Column types : C18 silica, C8, ion exchange, gel permeation…
- 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)
Main industrial applications
HPLC is used routinely or in development in many industrial sectors:
HPLC and UHPLC, what's the difference?
UHPLC, sometimes called UPLC, represents a technological advancement aimed at improving resolution and reducing analysis times through the use of finer particles and higher operating pressures. Depending on the complexity of the sample and the objectives, a UHPLC approach can be relevant for accelerating analyses while improving separation.
Perform an HPLC analysis in the laboratory with YesWeLab
YesWeLab helps you define an HPLC analysis strategy tailored to your objective, whether it's quality control, compliance, stability studies, non-conformity investigation, or method development. Depending on selectivity and sensitivity requirements, an HPLC method can be complemented by UHPLC or LC-MS/MS coupling.
After defining the scope of your project, YesWeLab selects the most suitable partner laboratory for your matrix, sector, and compound family, prioritizing accredited laboratories where relevant. You benefit from centralized information management, sample tracking, and results reporting.
Deliverables may include quantitative results, chromatograms, separation conditions and associated performance, depending on the specifications defined beforehand.
Léa Géréec
Technical and scientific expert
