What is reversed-phase chromatography?

It is the most common HPLC mode, using a non-polar stationary phase and a polar mobile phase, so more hydrophobic analytes are retained longer; it suits a very wide range of organic and bioorganic compounds.

How does HPLC differ from gas chromatography?

HPLC uses a liquid mobile phase and works at near-ambient temperatures, so it handles non-volatile, polar, and thermally fragile compounds that cannot be analyzed by gas chromatography.

High-Performance Liquid Chromatography

High-performance liquid chromatography separates non-volatile and thermally labile compounds by pumping a liquid mobile phase through a column of fine packing under high pressure.

Definition

High-performance liquid chromatography is a separation technique in which a liquid mobile phase is forced under high pressure through a column of fine stationary-phase particles, separating analytes by their differential interaction with the two phases.

Scope

This topic covers liquid chromatography in its high-performance form: pumps and gradient systems, columns packed with small porous particles, the principal separation modes—reversed-phase, normal-phase, ion-exchange, and size-exclusion—and detectors including ultraviolet, diode-array, fluorescence, and mass spectrometric. It addresses method development, the role of mobile-phase composition in selectivity, and quantitation from peak areas.

Core questions

How do separation modes such as reversed-phase and ion-exchange differ in their retention mechanism?
How does mobile-phase composition, including gradient elution, control selectivity and run time?
Why do smaller stationary-phase particles improve efficiency, and at what cost?
How are HPLC detectors chosen for sensitivity, selectivity, and identification?

Key theories

Reversed-phase retention: In the dominant reversed-phase mode, a non-polar stationary phase retains analytes in proportion to their hydrophobicity from a polar aqueous–organic mobile phase; increasing the organic fraction reduces retention, giving a tunable, broadly applicable separation of organic compounds.

Mechanisms

A high-pressure pump drives a liquid mobile phase through a column packed with porous particles bearing a bonded stationary phase. A sample injected into the flow separates as each analyte partitions between mobile and stationary phases according to its chemistry; changing the mobile-phase composition during the run, gradient elution, sharpens and speeds the separation. Eluting analytes pass through a detector whose peak areas quantify them against calibration standards.

Clinical relevance

High-performance liquid chromatography is the dominant method for pharmaceutical assay and impurity profiling, therapeutic drug and metabolite measurement, food and natural-product analysis, and biomolecule characterization, and is the leading front end for liquid chromatography–mass spectrometry.

History

Modern liquid chromatography emerged in the late 1960s and 1970s as pumps, fine particle packings, and sensitive detectors allowed liquid separations under high pressure. Reversed-phase columns with bonded silica became the workhorse mode, and the later move to sub-two-micrometre particles produced ultra-high-performance systems with faster, more efficient separations.

Key figures

Lloyd Snyder
Joseph Jack Kirkland
Csaba Horváth

Seminal works

snyder2010
skoog2017

Frequently asked questions

What is reversed-phase chromatography?: It is the most common HPLC mode, using a non-polar stationary phase and a polar mobile phase, so more hydrophobic analytes are retained longer; it suits a very wide range of organic and bioorganic compounds.
How does HPLC differ from gas chromatography?: HPLC uses a liquid mobile phase and works at near-ambient temperatures, so it handles non-volatile, polar, and thermally fragile compounds that cannot be analyzed by gas chromatography.