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btech chp 3 - Coggle Diagram

- - - - column volume: Vc=cross sectional area*height
      - void volume(V0)=volume outside solid particles
      - bed volume=void volume(for non porous )
      - total liquid volume(Vt, bed volume for porous), diff from geometric Vc
      - elution volume(Ve)-volume of mobile phase needed for sample component to be eluted from column
- - - - resolving gel: higher % of acrylamide, higher ionic strength, alkaline pH, proteins movesolwer for separation
    - - isoelectric focusing: proteins start migrating across the gel to the point at which their pI is equal to pH of the gel(pH gradient on the gel)
        
        followed by 2d gel electrophoresis using SDS-PAGE, which separates the protein
- - - - anion(use positively charged resin to bind to anion) and cation-exchange(use negatively charged resin to bind to cation) chromatography
        
        anion exchangers will carry positively charged groups on its surface
        
        cation exchangers will carry negatively charged groups on its surface
        
        for protein to get charged, at pH <pI value of protein, protein has more +ve charged groups on its surface. at pH >pI value of protein, protein has more -vely charged groups on its surface
        
        exhibit net +ve charge and -ve charge
      - during binding, solution has low ioninc strength(low salt conc etc.)
        
        sample components eluted by changing pH/ioninc strength of mobile phase
        
        salt gradients are used to elute proteins from ion exchangers
        
        bound proteins are displaced from resin
        
        protein molecules with weak interaction with resin will be eluted first, increasing conc, of ions of salt gradients will displace protein with strong interactions
      - well-suited for large-scale application due to high capacity, selectivity and concentrating effect
    - - high specificity(ligands bind to very specific groups on biomolecule
      - e.g.:enzyme, antibody, lectin, nucleic acid, hormone, vitamin
      - useful in isolating pure substance at very low conc. in large volumes of culture media, can remove specific contaminants, can separate biomolecules from their denatured or diff-functioning forms
      - PROCESS: sample applied, target molecule bind to ligand, wash to remove unbound materials, recover target molecule by changing conditions to elute, re-equilibrate to restore column
- - - - does not rely on adsorption or attraction of protein to solid phase
      - molecules to separated diffuse in random pattern into and out of pores
        
        molecules that are smaller are able to diffuse deeper into pores and spend more time within gel matric, will be retarded most in their passage
        
        intermediate sized molecules enter pores but spend less time there than small molecules(chance)
        
        molecules of very large size are completely excluded and move thru column rapidly
      - used to desalt protein solution: eg to separate protein from ammonium sulphate salt