Separation Techniques Flashcards Preview

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Flashcards in Separation Techniques Deck (31):
1

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

1. Size
2. Net charge

2

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

Electrolytic
Anode +
Cathode -

3

What are the 3 types of electrophoresis?

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

4

What is the purpose of Native PAGE?

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

5

What is the purpose of SDS PAGE?

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

6

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

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

7

When is chromato preferred to electrophoresis?

when large amounts of compounds are being separated

8

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

fast: nonpolar and large
slow: polar and small

9

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

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

10

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

size-exclusion:
fast: large
slow: small

11

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

fast: no affinity
slow: high affinity

12

What is an issue with affinity chromato?

hard to unbind the protein

13

What is the purpose of centrifugation?

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

14

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.

15

What is the purpose of protein assay?

to determine protein concentrations

16

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]

17

What is the purpose of the Edman degradation?

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

18

Vacuum vs gravity filtration?

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

19

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

20

What is the concept behind chromatography?

Partitioning

21

Is the stationary phase polar or nonpolar?

Polar

22

Is the mobile phase polar or nonpolar?

Nonpolar

23

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

Polar

24

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

Nonpolar

25

When is gas chromatography used?

To separate vaporizable and volatile compounds

26

What is HPLC?

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

27

What dissolves in the organic phase?

nonpolar compounds

28

What dissolves in the aqueous phase?

polar compounds

29

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

v = E.z/f

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

30

What is another word for gas chromatography?

Vapor-phase chromato

31

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

the shortest fragments represent the 5’ end