PAG 6 - identification of amino acids in protein using paper chromotography

Question 1

stationary phase

Answer 1

chromatography paper - cellulose

Question 2

mobile phase

Answer 2

solvent - travels up stationary phase & carries bio molecules

Question 3

adsorption

Answer 3

molecules bond to surface of substance

Question 4

outline the steps (1-14)

Answer 4

1. pour 30cm3 solvent into 500cm3 beaker & cover w/ aluminium foil
2. draw pencil line (faint) w/ ruler ~1.5cm from bottom
3. mark 10 dots at intervals of ~2cm – label each with corresponding amino acid (G, A, L, T & unknown is U)
4. using capillary tubes, place each solution to appropriate dot (no larger than 3mm) – allow to dry & repeat
5. roll paper to form cylinder – clip w/ paper clips
6. place in drying oven at approx. 100 degrees for few min until dry
7. lower into beaker of solvent - ensure doesn’t touch sides & solvent below pencil line
8. cover w/ aluminium foil
9. leave for 1-2 hours
10. take paper out, mark where solvent has reached (pencil) & set upside down to dry
11. once solvent evaporated, remove paper clips & hang in fume cupboard
12. spray paper lightly w/ ninhydrin & leave to dry
13. place in oven at 100-110 degrees for ~10 min until spots developed
14. draw around spots & measure distance travelled from origin line as well as distance solvent has travelled

Question 5

calculate Rf values

Answer 5

distance moved by spot / distance moved by solvent front

Question 6

extension q. - why do amino acids move at different rates up chromatography paper

Answer 6

amino acids have different r-groups so they also have different degrees of solubility in water v. non-polar solvent

Question 7

extension q. - which property of amino acids is responsible for this

Answer 7

r-groups

Question 8

extension q. - your Rf values are unlikely to agree exactly with exact published values, why

Answer 8

values will differ slightly due to differences in types of paper used, conc. of solvent, purity of amino acid samples & temperature