Chromatography - PowerPoint by 6x8AT7jQ

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									                                         Chromatography




Biophysical Tools '04 - Chromatography        10/9/2012
                              Partition chromatography
•   redistribution (partition) of the molecules between the stationary and
    mobile phase




                                               Plate model of chromatography of mixture of
                                               compounds partitioning 1:1 (bold) and 1:3
                                               (italics) between the mobile and stationary
                                               phase.


                                              •larger number of plates – better
     Biophysical Tools '04 - Chromatography   separation
                                                10/9/2012
                                               Adsorption

 •    stationary phases: alumina, activated carbon, hydroxyapatite

Column Support
     Application                      mesh size

     Preparative                         50-100

     Analytical                         100-200

 high resolution                        200-400

       HPLC                               >400




      Biophysical Tools '04 - Chromatography        10/9/2012
  TLC and GLC

 Thin layer chromatography




Gas-liquid chromatography




  Biophysical Tools '04 - Chromatography   10/9/2012
                         Kinds of chromatography




Biophysical Tools '04 - Chromatography   10/9/2012
                                         Gel exclusion




Biophysical Tools '04 - Chromatography          10/9/2012
            Molecular Weight by Gel Exclusion

   Veluate  Vvoid
K
   Vtotal  Vvoid

       gel 1




K   gel 2




                       log M




    Biophysical Tools '04 - Chromatography   10/9/2012
                                         Optimization


                                                            • rate
                                                            • column height




Biophysical Tools '04 - Chromatography          10/9/2012
                                         Sample size




Biophysical Tools '04 - Chromatography         10/9/2012
                                   Ion-Exchange chromatography




                                                           attached to
charge on molecule




                                                           anion
                                                           exchangers


                                                                 pH
                     attached to
                     cation
                     exchangers



                                   isoelectric point


                       Biophysical Tools '04 - Chromatography            10/9/2012
                                  Elution gradient




                                                salt gradients or pH

Biophysical Tools '04 - Chromatography     10/9/2012
                                     pH of the elution




Biophysical Tools '04 - Chromatography        10/9/2012
                                             HPLC

•   Extremely large number of plates  high pressure

                                                           rate of elution




    Biophysical Tools '04 - Chromatography     10/9/2012
             Other types of chromatography:

•   Affinity
     (couple ligand to CNBr-activated
        Sepharose)
•   Hydrophobic
     (low pressure RP-HPLC equivalent)




    Biophysical Tools '04 - Chromatography   10/9/2012
                           Filtration and Dialysis
 Dialysis
                                                       change buffer often




Reverse dialysis




                                                        •   Filtration
                                                             – nitrocellulose &
                                                               fiberglass
  Biophysical Tools '04 - Chromatography   10/9/2012         – Centricon series

								
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