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HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

 

In biochemistry, some of the main aims are to understand and describe the structural basis of biological molecules as they relate to function. The diversity of life is reflected by the enormous number of molecules found in living organisms. If these molecules are to be studied they must be purified. One of the biggest breakthroughs in biochemistry was the introduction of chromatography which made separation of molecules possible in an fast and efficient manner.

Chromatography is the science of separation which uses a diverse group of methods to separate closely related components of complex mixtures.It ıs a physical separation of two or more compounds based on their differential distribution between two phases.

There are millions of proteins found in living organisms which makes the isolation of a single protein molecule a tedious and time consuming task.

Protein molecules are separated according to their physical properties such as their size, shape, charge, hydrophobicity, and affinity for other molecules. The term High Performance Liquid Chromatography (HPLC) was coined to describe the separation of molecules under high pressure in a stainless steel column filled with a matrix. In HPLC, both pump control and data acquisition can be under the control of the same computer.

 

Types of HPLC

 

There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified.

In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption-desorption steps.

 

In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.

 

In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a "gel".

Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography.

  • In normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.
  • Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained.
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hplcandgc.rar
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Mutlu Demirel,
16 Oca 2009 06:00
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