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After Mid 1st Lecture
By Sir:- Tanveer Khan

Published in: Technology


  1. 1. CHROMATOGRAPHY Muhammad Tanveer Khan
  2. 2. INTRODUCTION <ul><li>Chromatography is a combination of two words; </li></ul><ul><li>* Chromo – Meaning color </li></ul><ul><li>* Graphy – representation of something on paper </li></ul>
  3. 3. DEFINITION <ul><li>“ 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 . </li></ul>
  4. 4. CHROMATOGRAPHY TERMS <ul><li>Chromatogram: </li></ul><ul><li>It is the visual output of the chromatograph. </li></ul><ul><li>Chromatograph: </li></ul><ul><li>It is equipment that enables a sophisticated </li></ul><ul><li>Separation. </li></ul><ul><li>Stationary phase (bounded phase): </li></ul><ul><li>It is a phase that is covalently bonded to the </li></ul><ul><li>support particles or to the inside wall of the </li></ul><ul><li>column tubing. </li></ul>
  5. 5. CHROMATOGRAPHY TERMS <ul><li>Mobile phase: </li></ul><ul><li>It is the phase which moves in a definite direction. </li></ul><ul><li>Analyte (Sample): </li></ul><ul><li>It is the substance to be separated during </li></ul><ul><li>chromatography. </li></ul><ul><li>Eluate: </li></ul><ul><li>It is the mobile phase leaving the column. </li></ul>
  6. 6. CHROMATOGRAPHY TERMS <ul><li>Retention time: </li></ul><ul><li>It is the characteristic time it takes for a </li></ul><ul><li>particular analyte to pass through the system </li></ul><ul><li>(from the column inlet to the detector) under </li></ul><ul><li>set conditions. </li></ul><ul><li>Eluent: </li></ul><ul><li>It is the solvent that will carry the analyte. </li></ul>
  7. 7. CHROMATOGRAPHY TERMS <ul><li>Retardation factor  ( R ): </li></ul><ul><li>Fraction of an analyte in the mobile phase </li></ul><ul><li>of a chromatographic system. </li></ul>
  8. 8. CLASSIFICATION <ul><li>According to mechanism of separation </li></ul><ul><li>Ion-exchange chromatography </li></ul><ul><li>Affinity chromatography </li></ul><ul><li>Size-exclusion chromatography </li></ul><ul><li>Adsorption chromatography </li></ul><ul><li>Partition chromatography </li></ul>
  9. 9. ION-EXCHANGE CHROMATOGRAPHY <ul><li>It is a process that allows the separation </li></ul><ul><li>of ions and polar molecules based on their charge. </li></ul><ul><li>PRINCIPLE: </li></ul><ul><li>Ion - exchange chromatography retains sample </li></ul><ul><li>molecules on the column based on ionic </li></ul><ul><li>interactions. </li></ul><ul><li>The surface of stationary phase displays ionic </li></ul><ul><li>functional groups (R-X) that interact with analyte </li></ul><ul><li>ions of opposite charge. </li></ul>
  11. 11. ION-EXCHANGE CHROMATOGRAPHY <ul><li>TYPES: </li></ul><ul><li>Cation exchange chromatography: </li></ul><ul><li>Cation exchange chromatography retains positively </li></ul><ul><li>charged cations because the stationary phase </li></ul><ul><li>displays a negatively charged functional group </li></ul><ul><li>Anion exchange chromatography: </li></ul><ul><li>Anion exchange chromatography retains anions </li></ul><ul><li>using positively charged functional group </li></ul>
  12. 12. ION-EXCHANGE CHROMATOGRAPHY <ul><li>APPLICATION: </li></ul><ul><li>It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. </li></ul><ul><li>Protein purification </li></ul><ul><li>Water analysis </li></ul><ul><li>Quality control </li></ul>
  13. 13. AFFINITY CHROMATOGRAPHY <ul><li>The method was discovered and developed by  Pedro Cuatrecasas  and  Meir Wilchek  for which the  Wolf Prize  in  Medicine  was awarded in 1987 . </li></ul><ul><li>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. </li></ul>
  14. 14. AFFINITY CHROMATOGRAPHY <ul><li>PRINCIPLE: </li></ul><ul><li>The stationary phase is typically a gel matrix </li></ul><ul><li>(often agarose) .   </li></ul><ul><li>The molecule of interest has a known and defined </li></ul><ul><li>property.  </li></ul><ul><li>The process is an entrapment in which the target </li></ul><ul><li>molecule becomes trapped on stationary phase. The </li></ul><ul><li>Stationary phase can then be removed from the </li></ul><ul><li>mixture, washed and then target molecule is </li></ul><ul><li>released from the entrapment </li></ul>
  16. 16. AFFINITY CHROMATOGRAPHY <ul><li>APPLICATIONS: </li></ul><ul><li>Purify and concentrate an enzyme solution </li></ul><ul><li>Purification of recombinant proteins </li></ul><ul><li>Purification of antibodies </li></ul>
  17. 17. SIZE-EXCLUSION CHROMATOGRAPHY <ul><li>The technique was invented by Grant Henry Lathe and Colin R Ruthven. They later received the John Scott Award for this invention. </li></ul><ul><li>It is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. </li></ul><ul><li>It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.  </li></ul>
  18. 18. SIZE-EXCLUSION CHROMATOGRAPHY 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.
  20. 20. SIZE-EXCLUSION CHROMATOGRAPHY <ul><li>APPLICATIONS: </li></ul><ul><li>Purification and analysis of synthetic and biological polymers, such as; </li></ul><ul><li>Proteins </li></ul><ul><li>Polysaccharides </li></ul><ul><li>Nucleic acids </li></ul><ul><li>It is also useful for determining the tertiary structure and quaternary structure of purified proteins </li></ul>