Chromatography

Question 1

Give the definition of chromatography

Answer 1

Lab technique used to separate mixtures of substances into their components

Question 2

Why is chromatography important?

Answer 2

Isolates one protein from thousands to study its properties

Question 3

What are the two phases in chromatography?

Answer 3

Mobile phases (solvent)
Stationary phase (paper)

Question 4

What are the types of properties of proteins that chromatography utilises?

Answer 4

Charge distribution
Molecular size
Solubility
Binding properties

Question 5

What is salting in?

Answer 5

When low concs of salt are added the protein solubilities increase

Question 6

How do salt molecules stabilise protein molecules?

Answer 6

Decreasing the electrostatic energy between the protein molecules, which increase the solubility of proteins

Question 7

How do salt molecules increase the solubility of proteins?

Answer 7

The salt molecules compete with the protein molecules in binding with water

Question 8

Why do protein molecules tend to associate with each other instead of salt molecules?

Answer 8

Protein-protein reactions become energetically more favourable than protein-solvent reactions

Question 9

How does column chromatography work?

Answer 9

The compound mixture moves along with the mobile phase through stationary phase and it separates depending on the affinity/degree of adhesion of each component

Question 10

What does ion exchange chromatography (IEX) do?

Answer 10

Separates larger amounts of materials than gel filtration

Question 11

What is the IEX stationary phase?

Answer 11

A polymer (matrix/resin) attached with charged group

Question 12

What do the acidic groups of resin interact with in IEX?

Answer 12

Positively charged proteins and are called cation exchangers

Question 13

What do cation exchangers bind to in IEX?

Answer 13

Proteins with positive charges

Question 14

Give an example of cation exchangers in IEX

Answer 14

CM cellulose canon exchangers

Question 15

What happens in IEX if groups are basic in nature?

Answer 15

They interact with negatively charged molecules called anion exchangers

Question 16

What is an example of basic groups in IEX?

Answer 16

Diethylaminoethyl (DEAE) cellulose

Question 17

How do cation exchangers work?

Answer 17

• Get polymer beads with a negative charge
• add protein/ pass through the beads
• proteins move through the column

Question 18

In IEX cation exchanges, how do proteins with a more negative net charge move?

Answer 18

Move faster and elite earlier

Question 19

Give the steps in cation exchangers

Answer 19

-proteins bind to an ion exchanger
-wash column with buffer to elute proteins based on how big an affinity thy have
-

Question 20

What do cation exchangers bind to in IEX?

Answer 20

Proteins with positive charges

Question 21

What do anion exchangers bind to in IEX?

Answer 21

Proteins with negative charges

Question 22

What does a high salt elution buffer do?

Answer 22

Increases mobility of remaining bands so they can be eluted

Question 23

What are other names for gel filtration chromatography?

Answer 23

Size exclusion chromatography or molecular sieve chromatography

Question 24

What is gel filtration chromatography used for?

Answer 24

Widely used to purify biological molecules in complex samples like blood

Question 25

What is separation based on in gel filtration chromatography?

Answer 25

Size and shape

Question 26

What does the column consist of in gel filtration chromatography?

Answer 26

Porous beads of dextran or agarose: Sephandex and sephanese are commonly used commercial preps

Question 27

What happens if the proteins can’t fit inside the beads in gel filtration chromatography?

Answer 27

They just pass over and are eluted first

Question 28

What is the elution volume?

Answer 28

The volume of a solvent required to elute a given solute from the column

Question 29

When is gel filtration chromatography usually used?

Answer 29

Near the end of the purification process

Question 30

What is affinity chromatography?

Answer 30

Relatively simple and effective molecular technique which isolated antibodies, antigens, hormones or other proteins by taking advantage of their binding affinity for their respective ligand

Question 31

What does affinity chromatography require?

Answer 31

- Beads matrix - solution containing substance to be isolated - ligand that binds specifically to the target proteins - a wash to elute non-bound substances - final wash with elution buffer containing a competitive ligand to elute the target protein

Question 32

What is affinity material made up of?

Answer 32

Inert support + spacer arms + ligand

Question 33

What must the first wash in affinity chromatography be?

Answer 33

Sufficient solute concentration, temp and pH to elute all unbound impurities

Question 34

What should the second wash in affinity chromatography contain?

Answer 34

Competitive ligand

Question 35

What is the second wash in affinity chromatography used for?

Answer 35

Remove competitive ligand by dialysis after its been eluted

Question 36

What does high performance liquid chromatography allow for?

Answer 36

The used of smaller profile solution for the column material, thus increasing the surface area

Question 37

What does a high surface area allow in high performance liquid chromatography?

Answer 37

Much better separation of samples based on hydrophobicity

Question 38

What is the role of the pump in high performance liquid chromatography?

Answer 38

Force the mobile phase through the column at a specific rule expressed in millilitres per minute

Question 39

What does the injector do in high performance liquid chromatography?

Answer 39

Introduces the sample into the flow stream of the mobile phase

Question 40

What is a typical sample volume of a high performance liquid chromatography?

Answer 40

5-20 micro litres

Question 41

In a chromatogram what does the area underneath the peak signals represent?

Answer 41

How much of the thing there is

Question 42

What are the advantages of high performance liquid chromatography?

Answer 42

``` Speed High resolution Sensitivity Reproducibility Accuracy Automation ```

Question 43

What are the disadvantages of high performance liquid chromatography?

Answer 43

Cost Complexity Coelution

Question 44

What is coelution

Answer 44

Sometimes there are two molecules each time with the same affinity for the mobile phase, so they elute at the same time?

Question 45

What are the two steps in 2D electrophoresis?

Answer 45

Isoelectric focussing | SDS-PAGE

Question 46

What does SDS-PAGE stand for?

Answer 46

Sodium dodecyl sulphate- polyacrylamide gel electrophoresis

Question 47

How does SDS-PAGE work?

Answer 47

Separates proteins by molecular weight so its based on the size of the proteins

Question 48

What does a western blot use?

Answer 48

Electrophoresis for protein seperation

Question 49

Where is the sample placed in elecrophoresis?

Answer 49

Onto the negative end of the polyacrylomide gel

Question 50

What happens after blotting in electrophoresis?

Answer 50

The membrane is blocked in order to prevent and unwanted membrane-blotter interaction to visualise the protein of interest