L12 - chromatography Flashcards
column chromatography
buffer + sample added to column
buffer continuously applied to the top of the column
ion exchange chromatography
ions that are electrostatically bound to an insoluble and chemically inert matrix are reversibly replaced by ions in solution
what determines the affinity of a protein to an ion exchanger
-identity of the other ions in solution
-pH
gel filtration (size exclusion) chromatography
-molecules are separated according to their size
-small molecules that can fit in the beads are eluted as a larger volume.
what is the elution volume (V๐)
the volume of the solvent required to elute the solute from the column
what is the void volume (V0)
the volume of the solvent space surrounding the beads
what is the relative elution volume
Vแต/v0
affinity chromatography
many proteins can bind specifically to other molecules, at a very high affinity, but not covalently. this property is exploited in affinity chromatography.
a ligand that bonds specifically to a protein of interest is attached to a resin
different types of affinity chromatography
immunoaffinity chromatography
glutathione-S-transferase - glutathione
insulin - insulin receptor
glucose - glucose binding protein
metal chelation: His-tag and divalent metal ions (Ni2+, Co2+, Zn2+)
reverse phase chromatography
liquid-liquid chromatography
- used to separate non polar substances. (denatured proteins)
what is retention time
time at which a compound emerges from the column
depends on:
hydrophobicity
number of C atoms
branching
saturation
functional groups
how to increase retention time
add salts to mobile phase
add more water to mobile phase
mobile phase pH
hydrophobic interaction chromatography
separates native protein on the basis of surface hydrophobicity
what is the difference between reverse phase and HIC
ligands in reverse phase are much more hydrophobic. HIC can use more moderate elution conditions
