Chromatography

Chromatography

1. Definition

   • Techniques to separate mixtures based on physical properties such as size or charge

2. 2 Phases

   • Stationary
     • A substance that the compounds to be separated pass by or interact with
   • Mobile
     • The carrier for the compounds to be separated

3. Mixed Samples

   • Need to have variable affinity
     • Some may have higher affinity in stationary/mobile phases.
       • Different Affinities= Different Rates

4. Basic Steps

   • Pouring
     • A glass tube with a valve at the bottom is filled with a mixture of stationary and mobile phases.
   • Packing
     • Opening the stopper and letting some liquid run out to make the stationary phase more densely and evenly packed.
   • Loading
     • The sample of interest is placed on top of the stationary phase.
   • Running/ Eluting
     • The sample flows into the column and more mobile phase is added on top to keep the column from drying out.
   • Collecting
     • Samples of defined volumes are caught into tubes at the bottom and saved/tested.

5. Size Exclusion Chromatography

   • Partition Coeffiecient
     • To get reproducible results from variable columns we need a way to standardize elution measurements.
       • Partition coeffiecient is the fraction of the volume of the column available to the sample.
         • We can use this to compare on experiment to another.
     • • Ve= Elution Volume
       • Vo= Void Volume
         • If Vo only, then Vt and Vo will be the same and Vt- Vo= 0
       • Vt= Total Volume
       • Always between 0 and 1
   • Sizes
     • Large proteins can not enter the beads so they will migrate around them.
     • Moderate Sized proteins enter beads some times which increases the volume available and move more slowly.
     • Small proteins do not interact with the beads and move the slowest.
   • Limitations
     • If Kav is >0.8 or <0.2 , poor separation of proteins.
     • Samples can become diluted as:
       • Turbulence from layering sample over gel.
       • Diffusion during travel
       • Friction differences along the glass tube wall.

6. Ion Exchange Chromatography

   • A column of positively or negatively charged beads forms the stationary phase.
     • Anion Exchange
       • Column is +, Targets are -
     • Cation Exchange
       • Column is -, Targets are +
     • Proteins in solution get carried down by gravity and will get slowed down by the beads proportionally based on charge
       • Specific combination of mobile and stationary phases chose to retain protein of interest.
       • Rates
         • Like charge or neutral molecules will elute quickly
         • The greater the charge on a protein, the slower it will travel down the column.
       • Stepwise/ Gradient Elution
         • Alter the pH to reduce the charge of the proteins bound to the column
         • Increase salt concentration to increase the competition for binding the stationary phase.
   • Give a fast/ clean method to extract DNA from cleared bacterial lysate.

7. Affinity Chromatography

   • Specific trap for protein of interest attached to stationary phase.
     • Enzyme
       • Substrate
     • Receptor
       • Hormone
     • Antigen
       • Antibody
   • High purity can be achieved.

8. How to Choose a Chromatography Method

   • Order of Specificity
     • Affinity
       • High Specificity
     • Ion Exchange
       • High Specificity
     • Size Exclusion
       • Simplest
   • For better yield and purity more specific methods should be chosen

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