Practical 2 - Investigation into the separation of choroplast pigments by chromatography

Question 1

Where are chloroplast pigments located?

Answer 1

On the membranes of the thylakoids and grana

Question 2

What do chloroplast pigments do in summary?

Answer 2

Harvest light in the light-dependent reactions of photosynthesis, and transfer its envy into the light-independent reactions, in the synthesis of complex organic molecules

Question 3

What are the 2 major groups of chloroplast pigments in flowering plants?

Answer 3

The chlorophylls
Carotenoids

Question 4

Types of chlorophyll pigments

Answer 4

Chlorophyll a
Chlorophyll b
Phaeophytin

Question 5

Which type of pigment is the commonest and is found in all photosynthetic organisms that will be studied?

Answer 5

Chlorophyll a

Question 6

Where is chlorophyll b found?

Answer 6

In flowering plants

Question 7

What is phaeophytin?

Answer 7

A type of chlorophyll pigment, which is a break down of chlorophyll a molecule, lacking the central magnesium ion and is seen in flowering plants and in purple sulphur bacteria

Question 8

What are the types of carotenoids pigments?

Answer 8

a- and B- carotene
Lycopene
Xanthophylls

Question 9

What colour are both a- and B- carotene?

Answer 9

Orange

Question 10

What colour is lycopene?

Answer 10

Red

Question 11

Types of xanthophylls

Answer 11

Lutein and zeaxanthin

Question 12

What colour do xanthophylls such as lutes and zeaxanthin appear?

Answer 12

Yellow

Question 13

What is being done in this practical in summary?

Answer 13

Chloroplast pigments will be extracted from the leaves of an angiosperm, separate by chromotography and identified

Question 14

Flowering plant

Answer 14

Angiosperm

Question 15

Angiosperm

Answer 15

Flowering plant

Question 16

What does the distance each pigment travels on the chromatography depend upon?

Answer 16

1.) its solubility in the solvent - more soluble pigments travel further
2.) its absorption by the paper or silica gel - pigments that are absorbed less travel further

Question 17

What happens to more soluble pigments on a chromatogram?

Answer 17

Travel further

Question 18

What happens to a pigment that is absorbed more on a chromatogram?

Answer 18

Absorbed less = travel further

Question 19

Apparatus

Answer 19

Dark green leaves (e.g - stinging nettles, spinach)
Scissors
Sand
Pestle
Mortar
Propanone
Distilled water
2x stopper
Pipette
Vial
Chromatography paper or silica gel chromatography plates
Pencil
Ruler
Capillary tube
Hair drier
2 x boiling tube
Petroleum ether

Question 20

What are the hazardous substances used in this practical?

Answer 20

Propanone
Petroleum ether

Question 21

What do we make sure to do with flammable substances?

Answer 21

Keep in the fuming cupboard with the fan on

Question 22

What do we need to use for the chromatography as we can’t use water?

Answer 22

Chromatography solvent

Question 23

Method for preparing the pigment solution

Answer 23

1.) chop 1g leaf material finely with scissors and place in the motar
2.) add a pinch of sand and 5cm^3 propanone
3.) grind the leaf fragments to a slurry
4.) place slurry in a boiling tube
5.) add 3cm^3 distilled water, shake vigorously and stand for 8 minutes
6.) add 3cm^3 Petroleum ether mix by gentle shaking and allow layers to separate
7.) collect the upper, petroleum ether layer, which contains the chloroplast pigments in a pipette and transfer to a vial

Question 24

Steps for preparing the chromatography paper

Answer 24

1.) draw a pencil line across the chromatography paper or plate approximately 2cm from one end
2.) draw chloroplast pigment solution into a capillary tube and put a small spot in the centre of the pencil line. Ensure that the capillary tube does not pierce or tear the chromatography paper
3.) dry and spot as quickly as possible, preventing its spread
4.) repeat steps 2 and 3 \until there is a small but intense spot of pigment

Question 25

Steps for running the chromatogram

Answer 25

1.) place freshly-made 2:1 propanone:petroleum ether solvent mixture in a boiling tube until it is approximately 5mm deep
2.) without touching the sides of the boiling tube, slide the chromatography paper into the boiling tube so that its end is below the surface of the ethanol but the spot is above, and not touching it
3.) hold the chromatography paper in place with the stopper, folding the paper over the rim of the boiling tube at the top
4.) leave the boiling tube ensuring that it is not moved, until the solvent has climbed up the paper to within 10mm of the top
5.) remove the chromatography paper from the boiling tube and immediately, draw a pencil line across the paper to mark the solvent front
6.0 mark the position of the top of each pigment spot with a pencil

Question 26

How do we identify the pigments from a chromatogram?

Answer 26

1.) use a ruler to measure the distance from the origin to the solvent front
2.) measure the distance from the origin to the top of each pigment spot
3.) Rf for a h pigment
Distance travelled by pigment
—————————————-
Distance travelled by solvent front
4.) published data allow the pigments to be identified, as Rf is constant for each pigment in a given solvent

Question 27

Rf

Answer 27

Distance travelled by pigment
———————————————
Distance travelled by solvent front

Question 28

Where do we measure to when measuring from the origin to the solvent spot and to each pigment spot?

Answer 28

The top every time

Question 29

Why should chromatogram be kept out of sunlight?

Answer 29

They’re photosensitive, so the colours could fade