Flashcards in Separation Techniques Deck (31):
What are the 2 characteristics that electrophoresis separates compounds based on?
2. Net charge
Does electrophoresis use an electrolytic or a galvanic cell? What does this mean for the anode and cathode?
What are the 3 types of electrophoresis?
1. Native PAGE
2. SDS PAGE
3. Isoelectric focusing
What is the purpose of Native PAGE?
analyze proteins in their native state and allows for complete protein to be recovered
What is the purpose of SDS PAGE?
separates based on mass alone (the SDS gives them all negative charges)
Which compounds go fast in electrophoresis? Which ones go slow?
fast: small and highly charged proteins
slow: large and slightly charged proteins
When is chromato preferred to electrophoresis?
when large amounts of compounds are being separated
Which compounds go fast in gel column chromato? Which ones go slow?
fast: nonpolar and large
slow: polar and small
Which compounds go fast in ion-exchange chromato? Which ones go slow?
fast: same charge as the beads
slow: opposite charge as the beads
Which compounds go fast in size exclusion chromato? Which ones go slow?
Which compounds go fast in affinity chromato? Which ones go slow?
fast: no affinity
slow: high affinity
What is an issue with affinity chromato?
hard to unbind the protein
What is the purpose of centrifugation?
isolate proteins based on LARGE size differences (usually prior to isolation)
What is ELISA?
The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution.
What is the purpose of protein assay?
to determine protein concentrations
How does the Bradford protein assay work?
mixes a protein in solution with Coomassie Brilliant Blue dye:
protonated: brown-green → low [protein]
deprotonated: blue → high [protein]
What is the purpose of the Edman degradation?
uses cleavage to sequence proteins of up to 50 to 70 aas → removes the N-terminal
Vacuum vs gravity filtration?
Vacuum: used when product is in the solid
Gravity: used when the product is in the filtrate
What are the 3 types of distillations?
1. Simple: bps under 150 and 25 apart
2. Vacuum: bps are over 150
3. Fractional: bps are less than 25 apart
What is the concept behind chromatography?
Is the stationary phase polar or nonpolar?
Is the mobile phase polar or nonpolar?
Is the mobile phase polar or nonpolar in reverse-phase chromato?
Is the stationary phase polar or nonpolar in reverse-phase chromato?
When is gas chromatography used?
To separate vaporizable and volatile compounds
What is HPLC?
Similar to comlumn chromato, but used if the sample is small or when capillary action will affect results
What dissolves in the organic phase?
What dissolves in the aqueous phase?
What is the equation to calculate the microgration velocity in electrophoresis?
v = E.z/f
E=electric field strength
What is another word for gas chromatography?