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ChromatographyChromatography
Russian Russian scientistscientist TswettTswett inin 19061906 usedused aa glassglass columns columns packedpacked withwith finelyfinely divideddivided CaCOCaCO 33 toto separate plant pigments extracted by hexane. The separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands pigments after separation appeared as colour bands that can come out of the column one by one. that can come out of the column one by one. Tswett Tswett waswas thethe firstfirst toto useuse thethe termterm "chromatography" "chromatography" derivedderived fromfrom twotwo GreekGreek wordswords "Chroma" meaning color and "graphein" "Chroma" meaning color and "graphein" meaning tomeaning to write. write.
Definition of chromatographyDefinition of chromatography
Tswett Tswett (1906)(1906) stated thatstated that " chromatography" chromatography is ais a method method in whichin which thethe componentscomponents ofof a mixturea mixture areare separated on adsorbent column in a flowing system”. separated on adsorbent column in a flowing system”. IUPAC IUPAC definitiondefinition (( InternationalInternational UnionUnion ofof purepure andand applied Chemistry) applied Chemistry) (1993):(1993): Chromatography is a physical method of separation in Chromatography is a physical method of separation in which the components to be separated are distributed which the components to be separated are distributed between two phases, one of which is stationary while the between two phases, one of which is stationary while the other moves in a definite direction. other moves in a definite direction. The The stationarystationary phasephase maymay bebe aa solid,solid, oror aa liquidliquid supported on a solid or gel, the mobile phase may be supported on a solid or gel, the mobile phase may be either a gas or a liquid. either a gas or a liquid.
Classification of Chromatographic methodsClassification of Chromatographic methods
Different methods were attempted for classification of Different methods were attempted for classification of chromatography: chromatography:
A – A – According to mechanismAccording to mechanism of separation:of separation:
The mechanism of separation depends mainly The mechanism of separation depends mainly
on the nature of the stationary phase. Based on on the nature of the stationary phase. Based on
separation separation mechanismsmechanisms chromatographychromatography cancan
be classified into: be classified into:
1- Adsorption Chromatography:1- Adsorption Chromatography: It It isis thethe oldestoldest andand mostmost commoncommon typetype ofof chromatography. The stationary phase is a solid chromatography. The stationary phase is a solid with adsorption power. Mixture components will with adsorption power. Mixture components will be adsorbed on the surface of the stationary be adsorbed on the surface of the stationary phase with different powers and that account for phase with different powers and that account for separation. separation. SilicaSilica gelgel isis thethe mostmost commoncommon stationary phase in adsorption chromatography. stationary phase in adsorption chromatography.
3- Ion Exchange Chromatography:3- Ion Exchange Chromatography:
It is used for separation of charged molecules. The stationary It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic phase is an ion exchange resin to which a cationic or anionic groups groups areare covalentlycovalently bonded.bonded. IonsIons ofof oppositeopposite chargescharges (counter ions) in the mobile phase will be attracted to the resin (counter ions) in the mobile phase will be attracted to the resin and and competecompete withwith thethe componentscomponents ofof thethe mixturemixture forfor thethe charged group on the resin. Both the mixture components and charged group on the resin. Both the mixture components and the the mobilemobile phasephase mustmust bebe charged.charged. MixtureMixture ofof AlkaloidsAlkaloids (compounds (compounds withwith positivepositive charges)charges) cancan bebe separatedseparated onon anionic exchanger, while mixture of organic acids (negative anionic exchanger, while mixture of organic acids (negative charges) can be separated using cationic exchanger. Both charges) can be separated using cationic exchanger. Both types are used for desalination of water. types are used for desalination of water.
COO
NH 4
Counter counter ions ions Anionic exchanger Cationic exchanger **H
H
OH
OH -**
It is a type of planar chromatography.It is a type of planar chromatography.
Stationary phase is a layer of gel Stationary phase is a layer of gel
moistened with buffer solution spread over moistened with buffer solution spread over
a glass plate. a glass plate.
Can separate positive and negative ions in Can separate positive and negative ions in
same process. same process.
4- Zone Electrophoresis:
Sample application Bands of negativey charged compounds Bands of postivey charged compounds
very large molecules eluted first without separation large molecules can enter some pores very small molecules enter all pores and eluted at last
6- Affinity Chromatography:6- Affinity Chromatography:
It uses the affinity It uses the affinity of proteins to specificof proteins to specific ligandsligands such as enzymes. The ligand is attached to suitable such as enzymes. The ligand is attached to suitable polysaccharide polysaccharide polymerpolymer suchsuch asas cellulosecellulose - - agarose – dextran. agarose – dextran.
7- Chiral Chromatography: 7- Chiral Chromatography:
In this type we can separate In this type we can separate enantiomers – we usedenantiomers – we used chiral chiral stationarystationary phasephase thatthat reactreact withwith oneone enantiomer more then the other so separation takes enantiomer more then the other so separation takes place. place.
C- ACCORDING TO THE TECHNIQUE (methods ofC- ACCORDING TO THE TECHNIQUE (methods of holding the stationary phase): holding the stationary phase): 1- Planar or Plane Chromatography: 1- Planar or Plane Chromatography: In this type of chromatography the stationary phase is used in the form of In this type of chromatography the stationary phase is used in the form of layer. Plane chromatography is further classified into: layer. Plane chromatography is further classified into: a- Thin Layer Chromatography (TLC): a- Thin Layer Chromatography (TLC): The stationary phase in the form of fine powder is spread on glass or The stationary phase in the form of fine powder is spread on glass or plastic or aluminum sheets. plastic or aluminum sheets. b- Paper Chromatography (PC): b- Paper Chromatography (PC): A specific type of papers is used as stationary phase in the form of sheets. A specific type of papers is used as stationary phase in the form of sheets. 2- Columnar or Column Chromatography (CC): 2- Columnar or Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal. The stationary phase is held in to a tube made of glass or metal.
D- ACCORDING TO PURPOSE OF USE:D- ACCORDING TO PURPOSE OF USE:
Chromatography can be used for analytical work and also to obtain Chromatography can be used for analytical work and also to obtain pure materials from mixtures. pure materials from mixtures. 1- Analytical Chromatography: 1- Analytical Chromatography: a- Qualitative Chromatography a- Qualitative Chromatography In this case Chromatography In this case Chromatography can be used to:can be used to: 1- Confirm the absence 1- Confirm the absence oror probableprobable presencepresence of certain constituentof certain constituent in the sample under investigation in the sample under investigation 2- Give an idea 2- Give an idea about the complexityabout the complexity ofof the mixturethe mixture and the leastand the least number of compounds present. number of compounds present. 3- Check purity and identity of any compound. 3- Check purity and identity of any compound. 4- Establish 4- Establish a (finger print ) pattern for extractsa (finger print ) pattern for extracts ,, volatilevolatile oilsoils oror pharmaceutical pharmaceutical preparations. These finger prints can be then used topreparations. These finger prints can be then used to check the identity and purity in the future. check the identity and purity in the future. 5- 5- MonitorMonitor bothboth columncolumn chromatographychromatography andand organicorganic chemicalchemical reactions. reactions.
2- Preparative application:2- Preparative application:
This This waswas thethe firstfirst andand isis thethe mainmain applicationapplication ofof chromatography. chromatography. TheThe techniquetechnique waswas developeddeveloped primarily for this purpose. primarily for this purpose. Chromatography Chromatography isis usedused toto obtainobtain reasonablereasonable quantities of pure compounds from mixtures. quantities of pure compounds from mixtures.
