Chromatography

CHROMATOGRAPHY

Muhammad Tanveer Khan

INTRODUCTIONChromatography is a combination of two words;* Chromo – Meaning color* Graphy – representation of something on paper

DEFINITION

“It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.

CHROMATOGRAPHY TERMS

Chromatogram:It is the visual output of the chromatograph.

Chromatograph:It is equipment that enables a sophisticatedSeparation.

Stationary phase (bounded phase):It is a phase that is covalently bonded to thesupport particles or to the inside wall of thecolumn tubing.


Mobile phase:It is the phase which moves in a definite

direction.

Analyte (Sample): It is the substance to be separated duringchromatography.

Eluate:It is the mobile phase leaving the column.


Retention time:It is the characteristic time it takes for aparticular analyte to pass through the system(from the column inlet to the detector) underset conditions.

Eluent:It is the solvent that will carry the analyte.


Retardation factor (R):Fraction of an analyte in the mobile phaseof a chromatographic system.

CLASSIFICATION

According to mechanism of separation

Ion-exchange chromatographyAffinity chromatographySize-exclusion chromatography Adsorption chromatographyPartition chromatography

ION-EXCHANGE CHROMATOGRAPHY

It is a process that allows the separationof ions and polar molecules based on their charge.

PRINCIPLE:Ion-exchange chromatography retains samplemolecules on the column based on ionicinteractions.

The surface of stationary phase displays ionicfunctional groups (R-X) that interact with analyteions of opposite charge.

ION-EXCHANGE CHROMATOGRAPHYTYPES:

Cation exchange chromatography:Cation exchange chromatography retains

positivelycharged cations because the stationary phasedisplays a negatively charged functional group

Anion exchange chromatography:Anion exchange chromatography retains anionsusing positively charged functional group


APPLICATION:It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Protein purificationWater analysisQuality control

AFFINITY CHROMATOGRAPHY

The method was discovered and developed by Pedro Cuatrecasas and Meir Wilchek for which the Wolf Prize in Medicine was awarded in 1987.

It is a method of separating biochemical mixtures and based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.


PRINCIPLE:The stationary phase is typically a gel matrix(often agarose). The molecule of interest has a known and definedproperty. The process is an entrapment in which the targetmolecule becomes trapped on stationary phase.

TheStationary phase can then be removed from themixture, washed and then target molecule isreleased from the entrapment


APPLICATIONS: Purify and concentrate an enzyme solution Purification of recombinant proteins Purification of antibodies

SIZE-EXCLUSION CHROMATOGRAPHY

The technique was invented by Grant Henry Lathe and Colin R Ruthven. They later received the John Scott Award for this invention.

It is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight.

It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.


PRINCIPLE:Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase.

The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded.


APPLICATIONS:Purification and analysis of synthetic and biological polymers, such as;

Proteins Polysaccharides Nucleic acids It is also useful for determining the

tertiary structure and quaternary structure of purified proteins

Chromatography

Education

Transcript of Chromatography