2.2.14 practical biochem 3: chromotography

Question 1

aim of chromatography

Answer 1

separate mixture into constituents

Question 2

2 components of chromatography

Answer 2

• stationary phase
• mobile phase

Question 3

stationary phase

Answer 3

= chromatography paper or thin-layer chromatography (TLC) plate
- paper made of cellulose
- TLC plate often plastic sheet coated w/ thin layer of silica gel or aluminium hydroxide
- free OH- groups pointing out, in contact w/ mobile phase

Question 4

mobile phase

Answer 4

= solvent for bio molecules
- water (polar molecules) or ethanol (non-polar molecules)
- flows through/across stationary phase, carrying bio molecules with it

Question 5

(if ink) how can the pigments be determined

Answer 5

• each travel at different speeds
• use relative distance travelled to help identify pigments

Question 6

calculate Rf value

Answer 6

measure distance from pencil line to centre of pigment spot & distance from pencil line to solvent front

Question 7

solvent front

Answer 7

distance travelled by solvent

Question 8

3 solutions to identify colourless molecules

Answer 8

1. UV light (TLC plate)
2. ninhydrin - too see amino acids, let plate dry & spray
3. iodine - allow plate to dry & place in enclosed container w/ few iodine crystals

Question 9

what does the speed at which molecules move depend on

Answer 9

• solubility in solvent
• polarity
• size (paper chromatography)

Question 10

how does chromatography work

Answer 10

• exposed -OH groups make surface very polar
• allows surface to form hydrogen bonds with molecules alongside other dipole interactions

Question 11

highly polar solute

Answer 11

tend to stick to surface (absorbed) & move more slowly

Question 12

non-polar solute

Answer 12

travel very quickly

Question 13

what should you do if 2 molecules travel at similar speeds (difficult to separate)

Answer 13

try using different solvent or changing pH

Question 14

how is thin-layer chromatography used (TLC)

Answer 14

• monitor progress of reactions (works quickly)
• urine testing of athletes for illegal drugs
• analysing drugs for purity of components
• analysis of foods to determine presence of contaminants