What is the purpose of chromatography?
To separate different components in a sample
State the factors affecting the rate of migration of different pigments
- Solubility
- Mass
- Affinity to the paper
What is the formula of the RF value?
Distance moved by pigment/distance moved by solvent
What is the purpose of finding the RF value of a pigment?
- Experimental RF value can be compared to a standard value in a database to identify the pigment
- The standard value should be measured using the same paper and solvent
Outline the procedure of using chromatography to separate photosynthetic pigments
- Draw a horizontal pencil line 1cm above the bottom of the filter paper
- Add some acetone and use the mortar and pestle to grind up the leaf sample and release the pigments
- Use a capillary tube to transfer the pigment onto the pencil line
- Suspend the paper in the solvent so that the level of liquid does not lie above the pencil line and leave the paper until the solvent has run up the paper near to the top
- Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to
- Calculate the Rf value for each spot
State the hazards and precautions in this practical
- Solvents are irritant and flammable
- Keep away from naked flames, wear eye protection and avoid contact with skin
- Leaf extract may be a biohazard. Wash hands after use
State the null hypothesis
There is no difference in the distance moved by the pigments / any difference in the distance moved by the pigments is due to chance. /
The type of leaf will not affect the distance moved by pigment spot nearest to origin.
You were told to use a pencil to mark the origin. It was important not to use a pen to mark this line.
Ink would dissolve in solvent / ink would be confused with substances on chromatogram / ink will run
Explain why you should measure to the centre of the pigment spot.
-Would standardise readings;
-Allow comparisons to be made;
-Pigment is spread out / moves different distances;
-It is the mean / average distance moved by the pigment
Holly usually has dark green leaves. Some cultivated varieties of holly have yellow leaves. Explain why these cultivated varieties grow more slowly than holly with dark green leaves.
-Yellow leaves have less / no chlorophyll;
-Less / no photosynthesis;
-Products of / named product of photosynthesis needed for growth / synthesis
Leaves change colour in the Autumn. This is partly due to loss of chlorophyll. Describe how you could use chromatography to show that chlorophyll is lost
-Carry out chromatography at different times;
-Find Rf for chlorophyll;
-Look for substance at predicted position / look for green substance
The technique you used to extract pigments from the leaves may not produce good results if it is used with the leaves of xerophytic plants. Use your knowledge of the leaves of xerophytic plants to suggest why.
1 Thick cuticle;
2 Prevents pigments penetrating / prevent pigment being transferred to paper;
OR
1 Thicker / stronger leaves;
2 Lower concentration of pigment / lower amount of pigment / more cells without pigment / more tissue without pigment / harder to crush;
OR
1 Leaves are spines;
2 Have little / no pigment;
You were told not to move the bottle once you had put the TLC paper in it.
Suggest why this is important.
Pigments / solvent would not run straight / would run off side of paper / pigments could wash off paper
Explain why it was necessary to draw the line before the filter paper dried.
Cannot see solvent
You could use Rf values to find out if a pigment on your chromatogram was the same as a pigment on another student’s chromatogram. Explain why it would be better to use the Rf value to do this and not the distances moved by the pigment spots.
1 The solvent / pigment moves different distances;
2 Rf value is distance moved (by spot) divided by distance moved by solvent front / Rf value is a ratio of distance moved (by spot) to distance moved by solvent front/Rf is a proportion;
3 Rf value constant (for the same pigment) / Rf values can be compared;
