Separations and Purifications

thin layer chromatography

• stationary phase: polar, silica gel

• mobile phase: nonpolar solvent

• large Rf: molecule is more similar to the mobile phase (solvent)

• small Rf: molecule is more similar to the stationary phase (polar)

• things like the solvent (np) elute 1st

liquid-liquid extraction

• organic layer: contains non-polar molecules, usually the top layer (less dense & no charge)

• aqueous layer: contains charged or polar molecules

• acids are extracted with weak bases

• bases are extracted with HCl (strong acid)

• goal: give target molecule a charge to move to the aqueous layer

• more effective to do multiple extractions with small amount of solvent

simple distillation

• separates compounds by boiling points

• limitations

  • boiling points of liquids must be under 150 Celcius

  • boiling points must be greater than 25 Celsius apart

• low BP elute first

fractional distillation

• used to separate mixtures whose boiling points are within 25 celsius of each other

vacuum distillation

• separates compounds with boiling points over 150 celsius

lower pH than pI =

proteins are positively charged

cation exchange

higher pH than pI =

proteins are negatively charged

anion exchange

salt concentrations for anion and cation exchange

• low salt: elutes proteins with small negative/+ charge

• high salt: elutes proteins with large negative/+ charge

increasing pH in cation exchange causes

• proteins with small pIs elute 1st

decreasing pH in anion exchange causes

• proteins with large pIs elute 1st