Chromatography in liquid phase

See also: CPL , LLC

The chromatography in liquid phase (CPL) or Liquid chromatography (LLC) is a technique of quantitative, qualitative and separative analysis mainly used in the field of the analytical Chimie like major scientific tool but also in fields varied such as the Organic chemistry and the Biochimie. It recovers the Thin layer chromatography (CCM), the Chromatographie on paper, the Chromatography in liquid phase in open column or with low pressure, and the Chromatography in liquid phase with high pressure (HPLC).
Ce standard of chromatography rests on the separation of made up in a liquid ( mobile phase ) which progresses in a tube ( column ) containing a material called stationary phase , or on a surface containing this stationary phase . Separation takes place according to the chemical or physical interactions analytes with the mobile phase like with the stationary phase.

Classification according to the interactions with the stationary phase

  • chromatography of adsorption
  • chromatography of division (normal phase or phase reverses)
  • chromatography of exchange of ions
  • chromatography of pairs of ions
  • chromatography of exchange of ligands
  • steric chromatography of exclusion (THESE or English SEC, one also speaks about permeation of freezing GPC)

Material

In the case of the CLHP, one (isocratic elution) or several (elution by gradient) pumps are used for the circulation of the phase known as " mobile" , i.e. liquid circulating in the chromatographic system. Tubes in Teflon, molten PEEK or Silice make it possible to connect the pumps to the chromatographic Injecteur.

The mobile phase is often made up of several solvents (methanol, acétonitrile, water…) and of salts (plugs, ionic reagent…). These solvents must be degassed (by neutral gas splashing in the reserves of mobile phase or by degassers on line) in order to withdraw from it the dissolved air which could form bubbles and obstruct the progression of the liquid in the tubes. In an elution with two pumps, a mixer is also necessary in order to obtain a homogeneous mobile phase.

After the injector is placed a chromatographic column (cylinder filled by the stationary Phase) which allows separation. The column is followed of a chromatographic Détecteur (UV, bar of diode, chemical,…). A computer generally supplements this device, for the ordering of the chromatographic system, as well as acquisition and the data processing.

In practice

Analysis LLC implements several stages:
  • preparation of the sample by the operator
  • chromatographic injection
  • separation
  • detection

In CLHP, the operator having a solution to analyze, prepares it in a mixture similar to that circulating in the system. He introduces a small quantity of this mixture into a loop of injection in order to avoid the phenomena of Band broadening, then begins acquisition. The loop is then connected in the injector to the remainder of the system, causing the passage of the molecules until at the head of chromatographic column. This column, selected among the very broad commercial range, is filled with a stationary phase, which will make it possible to separate the chemical molecules according to some their respective properties (size, polarity, hydrophily, affinity, contained out of metals…). The molecules will thus leave the column at various times called time of retention , according to their interactions with the stationary phase and the mobile phase, and will be detected, by the detector, there too, according to some their properties.

Nowadays, these simple techniques are used in relation to and recognition detector receiving sets of the molecules more powerful (ms (Spectrometry mass), NMR (nuclear Magnetic resonance) inter alia).

See too

External bonds

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