CP11 Flashcards
IV DV
IV: Type of Leaf
DV: Pigments in Leaf
What is the purpose of chromatography?
To separate different components in a sample
3 things the could effect separation:
- solvent type / solubility of pigment in solvent (1)
- type of chromatography plate / paper (1)
- size of pigment molecules / polarity of pigment molecules (1)
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 using the same
paper and solvent.
Outline the method for chromatography
- Add concentrated spot of mixture to the bottom of stationary phase
- End of chromotgraphy strip (stationary phase) is lowered into the solvent
- Pigments/compounds are carried different distances, according to their solubility
- If compounds are not visible add stain eg, amino acids -ninhydrin
- Stop before sovent front reaches the end of the stationary phase
- Identify compounds by comparing results to known samples
- RF values
State the hazards and precautions in this practical.
Solvents are irritant and flammable.
Keep away from naked flame, wear eye
protection and avoid contact with skin.
Leaf extract may be a biohazard. Wash
hands after use.
Why should the paper be dryed before the next drop?
- the paper should be dryed before the next drop because:
- drying evaporates the solvent (1)
- so that a small concentrated spot of extract can build up (1)
- in order to prevent the extract spreading out (1)
Why do plants contain different pigments
Plants contain different pigments because
- because different pigments absorb light at different wavelengths for photosynthesis (1)
- chlorophyll a and b absorb at lower and higher ends of visible/electromagnetic spectrum (1)
- accessory pigments/carotenoids absorb other wavelengths which enables the plant to use more of the spectrum (1)
EVALUATION
● Pigment spots can fade relatively quickly so Rf values need to be calculated immediately
● The coarseness of chromatography paper can vary which will affect the movement of the solvent & pigment up the paper.
● Maybe better to test known pigment concentrations on the same paper as the plant extracts to give accurate Rf values for specific chromatography paper
- mark baseline in pencil above solvent at start (1)
- ensure the spot dries thoroughly before applying another drop (1)
- mark solvent front before it reaches the top (1)
Explain why some substances do not move up the chromatography paper at all.
Some substances do not move at all because they are insoluble in the mobile phase/solvent
It is likely that you obtained Rf values that were similar, but not identical, to published values or
to the values provided. Explain why this might be the case.
The Rf value of a particular substance should always be the same provided the chromatogram is treated in the same manner in each case.
Different paper, solvent and running conditions
may affect the Rf values.
While the values in this table are for the same solvents, there may be slight differences.
What approach would be preferable to using published Rf values for comparison?
It would be more accurate to use pure extracts of the pigments as reference standards and test them alongside the leaf extract in an identical chromatography procedure then compare Rf
values.
Explain why chloroplasts usually contain several different photosynthetic pigments.
Each of the pigments absorbs and captures energy from light from particular areas of the spectrum.
As a result, far more of the energy from the light falling on the plant can be used than if only one pigment was present.
Photosynthetic pigments are found in plant leaves.
(a) Describe how you could use chromatography to separate these pigments.
(3)
add {extract / pigment} to (start) line (1)
(concentrate spot by) dry and repeat (1)
place paper in solvent eg ethanol (1)
obtain solvent front / place paper so line or spot above solvent / until reaches near top
