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1

Chromatography can be applied to which elements within chasm

1- charge
2- hydrophobicity
3- Affinity
4- Molecular weight

2

Ion exchange theory works on the basis of?

Overall charge can be determined if we know the sequence

3

Surface charge vs overall protein charge?

may be different

4

What type of interaction is occurring in ion exchange chromatography?

electrostatic

5

Matrix within Ion exchange chromatography contains

ionised groups

6

Anion exchange therefore?

exchanges anions
matrix- pos
proteins-neg

7

Cations charge

cats pur positive

8

Strength of the interaction between the ionised groups and the protein is due to?

number of ionic charges the proteins have
separation due to charge

9

Matrix functional group examples

Anion exchanger
- Diethylamino ethyl (DEAE)
- Quaternary ammonium
Cation exchanger
- Carboxymethyl
- Methyl sulphonate

10

Stronger anions and cations

will interact over a larger range

11

issue with really strong or really weak binders?

will bind too much or not enough

12

Issue with anion and cation exchanges

may not be the appropriate pH

13

Conditions for adsorption?

Low ionic strength buffer
pH to generate opposite charge to ion exchange resin (know pI)

14

pH

protonated (positively charged)

15

pI for a cation exchanger

- high (neg can bind to positive)

16

How to choose buffer pH

one pH unit away from the pI

17

Choose a suitable buffer pH for a cation exchanger when the pI=8.5

Can choose 1 pH unit above or bellow
- 9.5- too high (alkali)
- 7.5 better due to this being a cation exchanger (therefore your ions will be pos charged)

18

Elution method ?

change in ionic strength

19

Highly charged ions will come off?- elution

last

20

What type of gradient is drown in elution

stepwise
gradient

21

What needs to be maintained during elution

pH as this may change the proteins ability to bind

22

Choose gradient such that?

they ate separated

23

Elution by changing the pH

Molecules with the same isoelectric point are focused in narrow bands during the separation.

24

What type of gradient in the collum- Elution by changing the pH. Usually is ?

pH gradient

usually low to high

25

Focussing increases?

resolution

26

when might we want to use the focussing technique to separates proteins?- range ?

isoforms
eg haemoglobin
usually over a range of 1 and a Half pH unit

27

Advantages of ion exchange chromatography?

1- Load large volumes, elute small (concentrating)
2- High capacity, good resolution

28

Disadvantages of ion exchange chromatography?

denaturation at extreme pH
product in high salt
pH gradient technically difficult to produce

29

Seperation due to?- Hydrophobic interaction chromatography

differing surface hydrophobicity

30

Hydrophobic interaction chromatography promoted by?

Salting out