Chromatography
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Chromatography

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After Mid 1st Lecture
By Sir:- Tanveer Khan

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    Chromatography Chromatography Presentation Transcript

    • CHROMATOGRAPHY Muhammad Tanveer Khan
    • INTRODUCTION
      • Chromatography 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 sophisticated
      • Separation.
      • Stationary phase (bounded phase):
      • It is a phase that is covalently bonded to the
      • support particles or to the inside wall of the
      • column tubing.
    • CHROMATOGRAPHY TERMS
      • Mobile phase:
      • It is the phase which moves in a definite direction.
      • Analyte (Sample):
      • It is the substance to be separated during
      • chromatography.
      • Eluate:
      • It is the mobile phase leaving the column.
    • CHROMATOGRAPHY TERMS
      • Retention time:
      • It is the characteristic time it takes for a
      • particular analyte to pass through the system
      • (from the column inlet to the detector) under
      • set conditions.
      • Eluent:
      • It is the solvent that will carry the analyte.
    • CHROMATOGRAPHY TERMS
      • Retardation factor  ( R ):
      • Fraction of an analyte in the mobile phase
      • of a chromatographic system.
    • CLASSIFICATION
      • According to mechanism of separation
      • Ion-exchange chromatography
      • Affinity chromatography
      • Size-exclusion chromatography
      • Adsorption chromatography
      • Partition chromatography
    • ION-EXCHANGE CHROMATOGRAPHY
      • It is a process that allows the separation
      • of ions and polar molecules based on their charge.
      • PRINCIPLE:
      • Ion - exchange chromatography retains sample
      • molecules on the column based on ionic
      • interactions.
      • The surface of stationary phase displays ionic
      • functional groups (R-X) that interact with analyte
      • ions of opposite charge.
    • ION-EXCHANGE CHROMATOGRAPHY
    • ION-EXCHANGE CHROMATOGRAPHY
      • TYPES:
      • Cation exchange chromatography:
      • Cation exchange chromatography retains positively
      • charged cations because the stationary phase
      • displays a negatively charged functional group
      • Anion exchange chromatography:
      • Anion exchange chromatography retains anions
      • using positively charged functional group
    • ION-EXCHANGE CHROMATOGRAPHY
      • APPLICATION:
      • It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
      • Protein purification
      • Water analysis
      • Quality 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.
    • AFFINITY CHROMATOGRAPHY
      • PRINCIPLE:
      • The stationary phase is typically a gel matrix
      • (often agarose) .  
      • The molecule of interest has a known and defined
      • property. 
      • The process is an entrapment in which the target
      • molecule becomes trapped on stationary phase. The
      • Stationary phase can then be removed from the
      • mixture, washed and then target molecule is
      • released from the entrapment
    • AFFINITY CHROMATOGRAPHY
    • AFFINITY CHROMATOGRAPHY
      • 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. 
    • 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.
    • SIZE-EXCLUSION CHROMATOGRAPHY
    • SIZE-EXCLUSION CHROMATOGRAPHY
      • 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