Biological molecules - Thin Layer Chromatography

Question 1

what is the aim of chromatography

Answer 1

to separate a mixture into its constituents

Question 2

what is the stationary phase

Answer 2

the thin-layer-chromatography plate.

Question 3

describe the stationary phase

Answer 3

often a sheet of plastic coated with a thin layer of silica gel or aluminium hydroxide

Question 4

how does the stationary phase work

Answer 4

there are free -OH groups pointing outwards in contact with the mobile phase

Question 5

what is the mobile phase

Answer 5

the solvent for the biological molecules

Question 6

how does the mobile phase work

Answer 6

it flows through and across the stationary phase, carrying the biological molecules with it

Question 7

what can we use for the mobile phase

Answer 7

water (for polar molecules) or ethanol (for non-polar molecules)

Question 8

describe a basic process for chromatography

Answer 8

• draw a line in pencil on a piece of paper
• spot the solution mixture onto pencil line
• when spots are dry, lower paper into solvent, ensuring line is above the level of solvent
• cover beaker and wait

Question 9

what happens in chromatography

Answer 9

as the solvent travels up the plate, the components of the solution mixture travel with it. by the time the solvent has reached the top of the plate, some are travelling slowly and some quickly, so they are at different positions on the plate

Question 10

what can you use in TLC to reveal colourless molecules

Answer 10

• UV light
• Ninhydrin

Question 11

how does UV light work to reveal molecules

Answer 11

most TLC plates have a chemical that fluoresces under UV. most of the plate will disappear, except places where the spots have travelled

Question 12

how does UV light work to reveal molecules

Answer 12

to see amino acids, allow the plate to dry, then spray with ninhydrin, which binds to amino acids and makes it visible as purple or brown spots

Question 13

what is the formula for Rf value

Answer 13

distance travelled by compound/distance travelled by solvent

Question 14

if you repeat the investigation under the same conditions and the same solvent, what will happen to the Rf values

Answer 14

they will be the same

Question 15

how can amino acids by identified on a chromatogram

Answer 15

the Rf value for each amino acid is constant

Question 16

how does chromatography work

Answer 16

• the speed at which molecules move along the TLC plate depends on their solubility in the solvent (more soluble, faster)
• exposed OH groups make the surface of the TLC plate very polar, allowing it to form hydrogen bonds with molecules
• a highly polar solute will tend to stick to the surface and hence move more slowly, whereas a non-polar solute will travel more quickly

Question 17

if two molecules move at the same speed, it will be difficult to separate them. what could you try

Answer 17

• use a different solvent
• change the pH

Question 18

suggest why gloves should be worn

Answer 18

prevent contamination of stationary phase, for example with bacteria

Question 19

suggest why beaker should be covered

Answer 19

to stop the solvent from evaporating