Lecture

High Performance Liquid Chromatography: Basics

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In course

CH-301: Analytical separation methods

Les étudiants comprennent les bases physico-chimiques des méthodes de séparation chromatographiques et électrophorétiques. ...

Description

This lecture introduces the basics of High Performance Liquid Chromatography (HPLC), a method used for separating molecules based on their molar mass. HPLC is preferred over column chromatography for its improved selectivity and resolution, especially for thermally unstable or non-volatile molecules. The lecture covers the principles of HPLC chromatography, including the role of eluents, pumps, injectors, and chromatographic columns. It explains the factors influencing solute retention, such as column temperature, particle diameter, and eluent polarity. Different modes of elution, isocratic and gradient, are discussed, along with their impact on separation efficiency and resolution.

Instructor

Christophe Roussel

Habilitation à Diriger les Recherches (HDR) in chemistry at the Ecole Normale Supérieure Paris-Saclay (Cachan, France)PhD Thesis in physical organic chemistry at the University of Bourgogne Franche-Comté (Besançon, France) / Ecole Normale Supérieure Paris-Saclay (Cachan, France)

Official source

Related concepts (19)

Column chromatography

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents (normal phase, reversed phase, or otherwise) can be used with a wide range of solvents.

Chromatography

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the mobile phase, which carries it through a system (a column, a capillary tube, a plate, or a sheet) on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate.

Elution

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions. In a liquid chromatography experiment, for example, an analyte is generally adsorbed ("bound to") an adsorbent in a liquid chromatography column. The adsorbent, a solid phase (stationary phase), is a powder which is coated onto a solid support.

Gas chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gas–liquid partition chromatography (GLPC).

Liquid chromatography–mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify (or confirm the suspected identity of) each separated component.

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