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Chromatography

Overview of Chromatography, For more details refer to "methods of purification of proteins" slides uploaded already.

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CHROMATOGRAPH
Y
(SHORT NOTES)
Pradeep Singh
prdnarwat@gmail.com
Introduction
■ Chromatography is a method of separation in which the components of a mixture to be
separated are distributed between two phases, one is called the stationary phase and
other is called mobile phase.
■ The components of a mixture redistribute themselves between two phases by a process
which may be adsorption, partition, Ion exchange or size exclusion.
■ The stationary phase can be solid or liquid and the mobile phase can be liquid, gas or
supercritical fluid.
■ Centrifugation involves a sample (Solute or analyte) being dissolved in a mobile phase.The
mobile phase is then forced through an immobile, immiscible stationary phase.
■ A component which is quite soluble in the stationary phase will take longer to travel
through it than a component which is not very soluble in the stationary phase but very
soluble is mobile phase.
Classification of Chromatography
Chromatography
On the basis of
shape of
chromatographic
bed
Two dimensional
Paper
chromatography
Thin layer
chromatography
Three
dimensional
Column
chromatography
On the basis of
physical state of
mobile phase
1. Liquid
chromatography
2. Gas
chromatography
3. Supercritical
fluid
chromatography
On the basis of
interaction of solute
to the stationary
phase
1. Adsorption
chromatography
2. Partition
chromatography
3. Ion-exchange
chromatography
4. Size exclusion
or Gel filtration
chromatography
On the basis of interaction of the
solute to the stationary phase
1. Adsorption Chromatography
Mobile Phase: Liquid or Gas
Stationary Phase: Solid
Principle: Adsorption chromatography ids a type of chromatography in which the separation of
components present in a mixture is abased on the relative difference in adsorption of components to the
stationary phase present in the chromatographic column.
The adsorption involves weak non-covalent interactions such as ionic interactions,Van der waals
interactions, hydrogen bonding and hydrophobic forces between the components of the mixture and the
stationary phase.
Polar compounds adsorb more strongly to the polar stationary phase while non-polar components adsorb
strongly to the non-polar stationary phase.
Due to difference in degree of adsorption, less tightly bound compounds elute out by the mobile phase
earlier than the tightly bound ones.
It includes: i) Column chromatography ii)Thin layer chromatography iii) Gas-solid chromatography
2. Partition chromatography
Mobile Phase: Liquid/Gas
Stationary Phase: Liquid (Immiscible in mobile phase)
Principle: Partition chromatography is a type of chromatography in which the components of
the mixture get distributed into two phases due to difference in their partition coefficient
(Kd).
The difference of solutes between two phases (mobile & stationary) is primarily based on
solubility difference between the two phases.
It includes:
i. Liquid-liquid chromatography
ii. Liquid-gas chromatography
3. Ion – Exchange Chromatography
Mobile Phase: Liquid
Stationary Phase: Solid phase of insoluble matrix with covalently attached ion
exchangers
Principle: Ion-exchange chromatography is a technique for separation of charged
molecules including proteins, nucleotides and amino acids. Ion-exchange chromatography
is based in the relative retention of the ions during their progress through an ion
exchange column which has functional group of opposite charge attached to its surface.
Solute ions of the opposite charge in the liquid mobile phase bind reversibly to the ion-
exchangers by electrostatic interactions.
The stronger the charge on the ion, the greater is the retention time in the column.The
bound solutes can be released by eluting the column with a buffer of increased ionic
strength or pH.
It includes: i) Cation exchange chromatography ii) Anion exchange chromatography

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Role of vitamin D supplements in prevention of hungry bone syndrome for prima...Role of vitamin D supplements in prevention of hungry bone syndrome for prima...
Role of vitamin D supplements in prevention of hungry bone syndrome for prima...
 

Chromatography

  • 2. Introduction ■ Chromatography is a method of separation in which the components of a mixture to be separated are distributed between two phases, one is called the stationary phase and other is called mobile phase. ■ The components of a mixture redistribute themselves between two phases by a process which may be adsorption, partition, Ion exchange or size exclusion. ■ The stationary phase can be solid or liquid and the mobile phase can be liquid, gas or supercritical fluid. ■ Centrifugation involves a sample (Solute or analyte) being dissolved in a mobile phase.The mobile phase is then forced through an immobile, immiscible stationary phase. ■ A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble is mobile phase.
  • 3. Classification of Chromatography Chromatography On the basis of shape of chromatographic bed Two dimensional Paper chromatography Thin layer chromatography Three dimensional Column chromatography On the basis of physical state of mobile phase 1. Liquid chromatography 2. Gas chromatography 3. Supercritical fluid chromatography On the basis of interaction of solute to the stationary phase 1. Adsorption chromatography 2. Partition chromatography 3. Ion-exchange chromatography 4. Size exclusion or Gel filtration chromatography
  • 4. On the basis of interaction of the solute to the stationary phase 1. Adsorption Chromatography Mobile Phase: Liquid or Gas Stationary Phase: Solid Principle: Adsorption chromatography ids a type of chromatography in which the separation of components present in a mixture is abased on the relative difference in adsorption of components to the stationary phase present in the chromatographic column. The adsorption involves weak non-covalent interactions such as ionic interactions,Van der waals interactions, hydrogen bonding and hydrophobic forces between the components of the mixture and the stationary phase. Polar compounds adsorb more strongly to the polar stationary phase while non-polar components adsorb strongly to the non-polar stationary phase. Due to difference in degree of adsorption, less tightly bound compounds elute out by the mobile phase earlier than the tightly bound ones. It includes: i) Column chromatography ii)Thin layer chromatography iii) Gas-solid chromatography
  • 5. 2. Partition chromatography Mobile Phase: Liquid/Gas Stationary Phase: Liquid (Immiscible in mobile phase) Principle: Partition chromatography is a type of chromatography in which the components of the mixture get distributed into two phases due to difference in their partition coefficient (Kd). The difference of solutes between two phases (mobile & stationary) is primarily based on solubility difference between the two phases. It includes: i. Liquid-liquid chromatography ii. Liquid-gas chromatography
  • 6. 3. Ion – Exchange Chromatography Mobile Phase: Liquid Stationary Phase: Solid phase of insoluble matrix with covalently attached ion exchangers Principle: Ion-exchange chromatography is a technique for separation of charged molecules including proteins, nucleotides and amino acids. Ion-exchange chromatography is based in the relative retention of the ions during their progress through an ion exchange column which has functional group of opposite charge attached to its surface. Solute ions of the opposite charge in the liquid mobile phase bind reversibly to the ion- exchangers by electrostatic interactions. The stronger the charge on the ion, the greater is the retention time in the column.The bound solutes can be released by eluting the column with a buffer of increased ionic strength or pH. It includes: i) Cation exchange chromatography ii) Anion exchange chromatography
  • 7. 4. Size Exclusion Chromatography Mobile Phase: Liquid Stationary Phase: Solid (Porous beads) Principle: Size exclusion chromatography or Molecular sieve chromatography or Gel filtration chromatography separates molecules on the basis of size and shape. A column matrix filled with porous gel beads made up of an insoluble and hydrated polymer such as polyacrylamide or dextran (Sephadex) or agarose (Sepharose) acts as stationary phase. If a solution containing molecules having different sizes is passed through the column, molecules whose diameter is smaller than the pores can enter the pores in the beads and traces a longer path whereas larger molecules cannot.Therefore, larger molecules moves faster and elute first followed by molecules with small diameter. It includes: i. Gel permeation chromatography  Uses organic mobile solvent ii. Gel filtration chromatography  Uses aqueous mobile solvent
  • 8. On the basis of chromatographic shape of chromatographic bed 1. Thin Layer Chromatography Mobile Phase: Liquid Stationary Phase: Solid Thin layer chromatography is a chromatography technique useful for separation of organic compounds. Principle: The separation of components present in a mixture depends on the relative affinity of components towards stationary phase and mobile phase. The components under the influence of mobile phase (driven by capillary action) travel over the surface of stationary phase. During this movement the compounds with higher affinity to stationary phase travel slowly while others move faster. Once separation is over, individual components are visualized as spots at respective level of travel on the plate.
  • 9. 2. Paper Chromatography Mobile Phase: Liquid Stationary Phase: Liquid Paper chromatography is used to separate coloured chemicals or substances such as amino acids. Principle: Stationary phase is water which is held in pores ofWhatman filter paper over which used liquid mobile phase moves. The components of mixture get separated on the stationary phase due to difference in their affinity towards water (stationary phase) and mobile phase solvent. The migration of substances usually expressed as RfValue. RfValue: Distance travelled by the solute Distance travelled by the solvent
  • 10.  On the basis of physical state of mobile phase Liquid Chromatography Liquid – liquid chromatography Eg: Paper chromatography Liquid – solid chromatography Eg:Thin Layer Chromatography 1. Liquid Chromatography
  • 11. HPLC (High Performance Liquid Chromatography) Mobile Phase: Liquid Stationary Phase: Solid or Liquid HPLC is a type of column chromatography that uses high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complete mixtures with high resolution. Principle: Separation of components occur on the basis of polarity and solubility.The interaction between stationary and mobile phase allow the separation of components in the mixture from each other (like dissolves like). On the basis of polarity of stationary phase and mobile phase, HPLC can be of two types: i) Normal Phase HPLC ii) Reverse Phase HPLC
  • 12. Normal Phase HPLC Reverse Phase HPLC Stationary Phase Polar Non-Polar Mobile Phase Non-Polar Polar In Normal phase HPLC, the mobile phase is non-polar and the stationary phase is polar & hydrophilic. Stationary phase column is filled with silica particles.The silica is polar, the polar molecules in the analyte binds and absorb it and non-polar molecules will pass more quickly the stationary phase. In Reverse phase HPLC, the mobile phase is polar and the stationary phase is non- polar.The silica particles present in the stationary phase column are modified to make it nonpolar (silicaC18 molecules).The non-polar molecules in the analyte bytes and absorb it and the polar molecules will pass more quickly the stationary phase.
  • 13. 2. Gas Chromatography Mobile Phase: Gas Stationary Phase: Liquid/Solid Gas chromatography is used to analyse volatile substances in the gas phase. Principle: Solute separation is based on the relative difference in the solutes vapour pressure and the interactions with the stationary phase Thus, more volatile solutes elute from the column before a less volatile one.
  • 14. 3. Affinity Chromatography Principle: The substance to be purified is specifically and reversibly absorbed to a ligand which is attached to an insoluble matrix which forms the stationary phase. Macromolecule + Ligand  Macromolecule-LigandComplex The operation of affinity chromatography involves the following steps: i. Choice of appropriate ligand ii. Immobilization of the ligand onto a solid support iii. Binding of the molecules of interest with the ligands iv. Removal of non-specifically bound molecules. v. Elution of the molecule of interest in the purified form. Types of ligand Target molecules Enzymes Substrate analog Antibody Antigen Poly (U) Poly (A) tail containing RNAs Glutathione Glutathione-S-transferase Metal ions Poly (His) fusion proteins
  • 15. LCMSVs GCMS ■ LCMS: Liquid chromatography followed by analysis of the desired substance by mass spectrometry. ■ GCMS: Gas chromatography followed by analysis of the desired substance by mass spectrometry.
  • 16. Biological Application of Chromatography 1. The separation and purification of proteins, peptides, nucleic acids and other charged molecules. 2. Purification of enzymes, immunoglobulins. 3. Isolation of m-RNA. 4. Separation of bound and unbound radioisotopes. 5. Detection of endogenous neuropeptides in brain, extercellular fluids. 6. Identification of counterfeit drug products. 7. Pharmaceutical quality control. 8. Identification of anabolic steroids in serum, urine, sweat and hair. 9. Shelf life determination of pharmaceutical products. 10. Forensic determination of textile dyes.