Separation Techniques

Question 1

What are the 2 characteristics that electrophoresis separates compounds based on?

Answer 1

1. Size

2. Net charge

Question 2

Does electrophoresis use an electrolytic or a galvanic cell? What does this mean for the anode and cathode?

Answer 2

Electrolytic
Anode +
Cathode -

Question 3

What are the 3 types of electrophoresis?

Answer 3

1. Native PAGE
2. SDS PAGE
3. Isoelectric focusing

Question 4

What is the purpose of Native PAGE?

Answer 4

analyze proteins in their native state and allows for complete protein to be recovered

Question 5

What is the purpose of SDS PAGE?

Answer 5

separates based on mass alone (the SDS gives them all negative charges)

Question 6

Which compounds go fast in electrophoresis? Which ones go slow?

Answer 6

fast: small and highly charged proteins
slow: large and slightly charged proteins

Question 7

When is chromato preferred to electrophoresis?

Answer 7

when large amounts of compounds are being separated

Question 8

Which compounds go fast in gel column chromato? Which ones go slow?

Answer 8

fast: nonpolar and large
slow: polar and small

Question 9

Which compounds go fast in ion-exchange chromato? Which ones go slow?

Answer 9

fast: same charge as the beads
slow: opposite charge as the beads

Question 10

Which compounds go fast in size exclusion chromato? Which ones go slow?

Answer 10

size-exclusion:

fast: large
slow: small

Question 11

Which compounds go fast in affinity chromato? Which ones go slow?

Answer 11

fast: no affinity
slow: high affinity

Question 12

What is an issue with affinity chromato?

Answer 12

hard to unbind the protein

Question 13

What is the purpose of centrifugation?

Answer 13

isolate proteins based on LARGE size differences (usually prior to isolation)

Question 14

What is ELISA?

Answer 14

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.

Question 15

What is the purpose of protein assay?

Answer 15

to determine protein concentrations

Question 16

How does the Bradford protein assay work?

Answer 16

mixes a protein in solution with Coomassie Brilliant Blue dye:

protonated: brown-green → low [protein]
deprotonated: blue → high [protein]

Question 17

What is the purpose of the Edman degradation?

Answer 17

uses cleavage to sequence proteins of up to 50 to 70 aas → removes the N-terminal

Question 18

Vacuum vs gravity filtration?

Answer 18

Vacuum: used when product is in the solid
Gravity: used when the product is in the filtrate

Question 19

What are the 3 types of distillations?

Answer 19

1. Simple: bps under 150 and 25 apart
2. Vacuum: bps are over 150
3. Fractional: bps are less than 25 apart

Question 20

What is the concept behind chromatography?

Answer 20

Partitioning

Question 21

Is the stationary phase polar or nonpolar?

Answer 21

Polar

Question 22

Is the mobile phase polar or nonpolar?

Answer 22

Nonpolar

Question 23

Is the mobile phase polar or nonpolar in reverse-phase chromato?

Answer 23

Polar

Question 24

Is the stationary phase polar or nonpolar in reverse-phase chromato?

Answer 24

Nonpolar

Question 25

When is gas chromatography used?

Answer 25

To separate vaporizable and volatile compounds

Question 26

What is HPLC?

Answer 26

Similar to comlumn chromato, but used if the sample is small or when capillary action will affect results

Question 27

What dissolves in the organic phase?

Answer 27

nonpolar compounds

Question 28

What dissolves in the aqueous phase?

Answer 28

polar compounds

Question 29

What is the equation to calculate the microgration velocity in electrophoresis?

Answer 29

v = E.z/f

E=electric field strength
z=net charge
f=frictional coefficient

Question 30

What is another word for gas chromatography?

Answer 30

Vapor-phase chromato

Question 31

In gel electrophoresis of DNA, what end of the DNA do the shorter DNA fragments represent?

Answer 31

the shortest fragments represent the 5’ end