Principe chromatographie pdf

The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area.

Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times Figure 3 [ 11 ]. Sephadeks G type is the most frequently used column material.

Besides, dextran, agorose, polyacrylamide are also used as column materials [ 12 ]. This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins [ 13 ].

A ligand which can make a complex with specific protein dextran, polyacrylamide, cellulose etc binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support matrix , and retained in the column, while free proteins leave the column.

Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution Figure 4 [ 14 ]. In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used.

In this method, the mobile phase travels upward through the stationary phase The solvent travels up the thin plate soaked with the solvent by means of capillary action.

During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent [ 16 ].

In cases where molecules of the sample are colorless, florescence, radioactivity or a specific chemical substance can be used to produce a visible coloured reactive product so as to identify their positions on the chromatogram. Formation of a visible colour can be observed under room light or UV light. The position of each molecule in the mixture can be measured by calculating the ratio between the the distances travelled by the molecule and the solvent.

This measurement value is called relative mobility, and expressed with a symbol R f. In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Its carrier phase consists of gases as He or N 2. Mobile phase which is an inert gas is passed through a column under high pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase.

The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes [ 18 ]. Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye [ 19 ].

The planar ring structure with negatively charged groups is analogous to the structure of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is 10—fold stronger rhat that of the affinity of other adsorbents.

Under appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange property of adsorbent, the adsorbed proteins are separated from the column [ 20 , 21 ]. In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used.

HIC technique is based on hydrophobic interactions between side chains bound to chromatography matrix [ 22 , 23 ]. Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography IMAC [ 24 ]. In this technique, use of small particles, and application of high presure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time.

Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued [ 25 ]. Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders.

Gas chromatography is used in laboratories to measure steroids, barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and proteins. Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments. With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method.

Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins. Besides, these methods are used to control purity of a protein. HPLC technique which has many superior features including especially its higher sensitivity, rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.

Conflict of Interest: None declared. Financial Disclosure: The authors declared that this study has received no financial support. National Center for Biotechnology Information , U. Journal List North Clin Istanb v. North Clin Istanb. Published online Nov Ozlem Coskun.

Author information Article notes Copyright and License information Disclaimer. Correspondence: Dr. Received Feb 17; Accepted Oct 1. This article has been cited by other articles in PMC. Abstract Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.

Keywords: Chromatography , column chromatography , protein purification. Types of chromatography Column chromatography Ion-exchange chromatography Gel-permeation molecular sieve chromatography Affinity chromatography Paper chromatography Thin-layer chromatography Gas chromatography Dye-ligand chromatography Hydrophobic interaction chromatography Pseudoaffinity chromatography High-pressure liquid chromatography HPLC.

Column chromatography Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Open in a separate window. Ion- exchange chromatography Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material matrix. Gel- permeation molecular sieve chromatography The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes.

Affinity chromatography This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins [ 13 ]. Paper chromatography In paper chromatography support material consists of a layer of cellulose highly saturated with water.

Gas chromatography In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Dye- ligand chromatography Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye [ 19 ]. Hydrophobic interaction chromatography HIC In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used.

Pseudoaffinity chromatography Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography IMAC [ 24 ].

Application areas of chromatography in medicine Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders. Conclusion Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments.

Footnotes Conflict of Interest: None declared. Selective enzyme purification by affinity chromatography. Porath J. From gel filtration to adsorptive size exclusion. J Protein Chem. Harris DC. Exploring chemical analysis. Preparative ion-exchange chromatography of proteins from dairy whey. J Chromatogr A. Introduction to organic laboratory techniques. Experimental organic chemistry:Principles and Practice. Buts de la chromatographie. On peut distinguer deux objectifs principaux: 2.

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