Experiment 8: Separation and Rf value determination of pigments in plant leaves
2007 Al-Bio Marking Scheme
Experiment 8: Separation and Rf value determination of pigments in plant leaves
Area B1 (max. 15 marks)
Aim: ( max. )
- to separate and identify the pigments in given leaves samples ( )
- by means of paper chromatograph ( )
- and to determine the Rf values of the separated pigments respectively (
)
Principle: (max. )
Pigments in plants
- in green plants, there is not only one single pigment
- different pigments have different molecular structure
- absorb different wavelength in the light spectrum
-
primary pigment, chlorophyll a, is a green pigment which act as the electron donor during the light
reaction in photosynthesis by emitting electrons after absorption of sufficient energy
other pigments, may be in colours other than green, act as accessory pigments to collect photons of
various wavelength in the spectrum and transmitte the energy to the primary pigment
since the pigments are just mixed inside the plant cells, they can be separated by physical method.
Pigments extraction
- grinding leaves in mortar
 destruction of cell to release cell contents including the pigments
-
extraction solvent
dissolve and extract the pigments out from the destructed cells
(½ )
(½ )
Paper chromatography
- depends upon the differential movement of each pigments through the chromatography paper under the
influence of the developing solvent (½ )
- pigments are separated into spots along the chromatography paper (½ )
- since different pigments have different solubilities in the developing solvent and / or (½ )
- different adhesion on the chromatography paper (½ )
 each has a constant relative position to the solvent front on the paper (i.e. a constant Rf value)
List of apparatus
(0 / -½ )
As those mentioned in teachers’ instructions except the Zebina leaves was replaced by the Spring Onion
leaves.
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Experiment 8: Separation and Rf value determination of pigments in plant leaves
Procedures
-non-reported speech (-1/2)
Steps (max. 3)
Preparation:
- Clean the leaves with tap water
before extraction
2007 Al-Bio Marking Scheme
Quantities and Units (max. 2)
-
Blot dry and cut the leaves
-
Cut the chromatography paper
strip into suitable size
-
width (cm) x length ( cm )
-
Mark the line of origin on the
paper strip with pencil
-
distance from the end of paper strip
(cm)
-
Pour the developing solvent into
boiling tube
-
volume of solvent (cm3 )
-
stopper the tube and stand for
equilibration
-
time to reach equilibrium (min.)
Extraction
- Grind the leaves with mortar and
pestle / test tube and glass rod
-
Standardisation (max. 0)
-
With extraction solvent added little by little
volume of solvent in each addition
(cm3 )
-
Development of pigment
- spot the concentrated extract onto
the center of origin on the paper
strip with a capillary tube
-
repeat spotting whenever the
previous spot was dry until a
concentrated spot is obtained
How many times repeated
-
with a pin, attach the spotted paper
strip to the bottom of the stopper of
the boiling tube
-
suspend the prepared paper strip and immerse the lower end into the
developing solvent in boiling tube
with the origin and concentrated
pigment spot just above the solvent
the length of strip immersed in the
solvent (cm)
-
develop the chromatogram
time duration of the development
(min.) / the minimum length of the
chromatogram (cm)
-
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Experiment 8: Separation and Rf value determination of pigments in plant leaves
-
remove the strip immediately after
development and mark the solvent
front with a pencil
-
outline each spot on the strip with
pencil after air-dry of the strip
2007 Al-Bio Marking Scheme
Measurement
measure the distance of each pigment
spot and the solvent front from the
origin
Precautions (@½ , max. 2)
Steps
Explaination
- Pipette should be erect and be read Prevent reading errors
at the same level as eyes
-
-
the size of the spot should be as
the smaller and the more concentrated spot
small as possible / not greater than of origin, the better speration and
3mm in diameter
important for identification and calculation
of Rf values
-
all used organic solvents should be Prevent contamination of environment via
discarded into waste bottle
sewage discharge
-
the edges of paper strip must not
touch the sides of the tube
The solvent may run faster at the edges of
paper strip where in touch with the tube
wall
-
additional extract solvent was
added during extraction
-
to prevent too viscous and easier for
spotting
-
little by little
-
since too much solvent would give a
too dilute extract and this creates
difficulty in spotting
-
the origin should not be too close to (the longer the chromatogram runs, the
the bottom of the stopper
better the separation of the sample)
-
do not immerse the concentrated
pigment
Control (0 / -½
- nil
Avoid to dissolve the pigment into the the
developing solvent instead of running
upward along the chromatograph.
)
Others (max. 1/2)
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Experiment 8: Separation and Rf value determination of pigments in plant leaves
2007 Al-Bio Marking Scheme
Area B2 (max. 10 marks)
-
attached chromatogram (or diagram of chromatogram) with clear spots outline, mark of solvent front and
origin ( with proper title ) (max. 2)
-
Data table:
-- proper title (max. 1)
-- proper headings (spots label, pigment migrated distance, Rf value, colour description) with S.I. unit (if
applicable) (max. 4)
-- complete table lines (1/2)
-
-
Distance of sovlent front from the origin with S.I. unit (max.1/2)
Calculation:
-- formula for Rf (max. 1)
-- showing how the data is substituted into formula to obtain the Rf values (max. 1)
Any other observation / findings (max. 1)
Area B3 (max. 10 marks)
- data description and interpretation (max. 5)
-- the number of pigment obtained
-- compare / comment on the colour of the different pigments
-- compare on the Rf value of the different pigments / comment the results : e.g. agree with your
prediction in principle?)
-- any observable relationship between the Rf values / between the Rf and the colour / … (only
coincidence or any background knowledge to support the possible relationship)
-- comment on the other observation or findings
-
other significant discussion (max. 4)
-- limitation and sources of error with improvement suggestion
-- hypothesis drawn from the disscussion, assumption or uncertainty with follow up suggestion
-- others
-
conclusion (max. 1)
the number and the Rf values of the separated pigments
* with reference to the above guidelines, reports were marked by overall impression.
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