Unit 2C Natasha Owusu
Unit 2C: Practice Scientific Procedures and Techniques: Separate to Identify
Aim: Undertake Chromatographic Techniques to Identify Components in Mixtures from Extracted
Plant Pigment.
Introduction: What is Chromatography and How Does It Work? Chromatography is a method of
separating mixtures by using a moving solvent on filter paper. A drop of mixture solution is spotted near
one end of the paper and then dried. The end of the paper, nearest the spot, is then dipped into the solvent
without submerging the spot itself. It is used because different components of the mixture travel through
the stationary phase at a different speed causing them to separate from one another.
The distance and speed travelled by a solvent along the chromatogram relies as to how closely bound the
substance is to the Stationary Phase. The Polarisation of the substance also defines how far the Silica Plate/
Chromatography Paper can go. Non-polar components dissolve easily in non-polar solvents. Polar
components dissolve easily in polar solvents. If we want to see a separation of spots or lines, we must pick
a solvent in which the components are only slightly soluble, and the polarity of the solvent is very important
in helping us decide. If a component of the mixture is very soluble in the mobile phase, it moves with the
solvent up the plate. If the components are only slightly soluble, the mobile phase will not be able to move
it for up the plates.
Equipment & Materials:
Large Beaker White Tile Red Leaves/Spinach
Wooden Splint Pegs Green Leaves/Spinach
Paper Clip Mortar and Pestle Propanone (acetone)
Chromatography Paper Sand Petroleum (ethyl) Alcohol
TLC (Silica) Plate Tweezers 70% Petroleum Ether and
30% Propane
Pipette Pencil
Hair Dryer
Cocktail Sticks Ruler
Glass Spotting Tub
Scissors Grass
Preparation of Plant Solution:
1. Obtain a Leaf from the desired plant and grind it using a mortar and pestle. A few grains of sand
can be added to facilitate grinding. Add a few drops of Acetone (Propanone) to extract the pigments
from the leaf.
2. Continue grinding until a dark-coloured paste has formed.
When Preparing the solution, it is important to ensure that the concentration is high, but the amount is low,
allowing the pigment to not be heavily diluted. If it is too dilute, the separation would have been very
, Unit 2C Natasha Owusu
difficult to see and take place.
Method Paper Chromatography & TLC:
1. Obtain a strip of Chromatography paper or a Silica Plate.
2. Using a pencil, draw a line 2cm from the tapered tip. This is the baseline.
3. Using a pipette or a cocktail stick, apply a small dot of the pigment, on the baseline. Let the dot dry
and continue to re-apply, until there is a dark spot of pigment.
4. Pour the required amount of Petroleum Ether or Petroleum ether and Propanone into a large/long
Beaker, ensuring that the solvent is below the baseline. Attach a pencil to the top of the
Chromatography paper or Silica Plate using a paper clip/peg or cover with a lid.
5. Allow the Chromatogram to run until the solvent almost reaches the top of the paper strip, for about
30 minutes.
6. Remove the paper and use a pencil to mark the solvent front.
7. Dry Chromatogram with a hair dryer, the measure the distance of the solvent front and distance of
spot moved. Using this information, find out the Rf Values.
Risk Assessment/ Safety Precautions:
Do not allow solvents to come into contact skin and do not breathe it in.
Solvents should be stored in fume cupboards, to minimize toxicity and flammability risk of Solvent vapors.
Lid should be used in experiments as well.
Results for Paper Chromatography 1: Pencil & Paper Clip:
From these diagrams, you can see the distinct
separations from each of the plant pigment. Pigment A
is a big Green leaf and Pigment B is Red/Green Lettuce.
Pigment A travelled further than B. In both Pigment A
and B, Xanthophyll is present, due to the visible pale-
yellow green, which has been separated out. The rf
value for A is 2.5/7=0.3cm and the rf value for B is 5/7=0.7cm. The results were okay; however, they
could be heavily improved.
Results for Paper Chromatography 2: Lid Used:
From these diagrams, you can see the distinct
separations from each of the plant pigment.
Pigment C is Red/Green Spinach and Pigment
D is Green Grass. Pigment D travelled further
than C. In Pigment C, Xanthophyll is present,
due to the visible pale-yellow green, which has
been separated out and Pigment D has signs of
Chlorophyll b and Xanthophyll, due to the deep green and pale yellow. The rf value for C is 4/8.5=0.4cm
and the rf value for D is 6.5/8.5=0.8cm. This Paper Chromatography technique with the Lid, produced
Unit 2C: Practice Scientific Procedures and Techniques: Separate to Identify
Aim: Undertake Chromatographic Techniques to Identify Components in Mixtures from Extracted
Plant Pigment.
Introduction: What is Chromatography and How Does It Work? Chromatography is a method of
separating mixtures by using a moving solvent on filter paper. A drop of mixture solution is spotted near
one end of the paper and then dried. The end of the paper, nearest the spot, is then dipped into the solvent
without submerging the spot itself. It is used because different components of the mixture travel through
the stationary phase at a different speed causing them to separate from one another.
The distance and speed travelled by a solvent along the chromatogram relies as to how closely bound the
substance is to the Stationary Phase. The Polarisation of the substance also defines how far the Silica Plate/
Chromatography Paper can go. Non-polar components dissolve easily in non-polar solvents. Polar
components dissolve easily in polar solvents. If we want to see a separation of spots or lines, we must pick
a solvent in which the components are only slightly soluble, and the polarity of the solvent is very important
in helping us decide. If a component of the mixture is very soluble in the mobile phase, it moves with the
solvent up the plate. If the components are only slightly soluble, the mobile phase will not be able to move
it for up the plates.
Equipment & Materials:
Large Beaker White Tile Red Leaves/Spinach
Wooden Splint Pegs Green Leaves/Spinach
Paper Clip Mortar and Pestle Propanone (acetone)
Chromatography Paper Sand Petroleum (ethyl) Alcohol
TLC (Silica) Plate Tweezers 70% Petroleum Ether and
30% Propane
Pipette Pencil
Hair Dryer
Cocktail Sticks Ruler
Glass Spotting Tub
Scissors Grass
Preparation of Plant Solution:
1. Obtain a Leaf from the desired plant and grind it using a mortar and pestle. A few grains of sand
can be added to facilitate grinding. Add a few drops of Acetone (Propanone) to extract the pigments
from the leaf.
2. Continue grinding until a dark-coloured paste has formed.
When Preparing the solution, it is important to ensure that the concentration is high, but the amount is low,
allowing the pigment to not be heavily diluted. If it is too dilute, the separation would have been very
, Unit 2C Natasha Owusu
difficult to see and take place.
Method Paper Chromatography & TLC:
1. Obtain a strip of Chromatography paper or a Silica Plate.
2. Using a pencil, draw a line 2cm from the tapered tip. This is the baseline.
3. Using a pipette or a cocktail stick, apply a small dot of the pigment, on the baseline. Let the dot dry
and continue to re-apply, until there is a dark spot of pigment.
4. Pour the required amount of Petroleum Ether or Petroleum ether and Propanone into a large/long
Beaker, ensuring that the solvent is below the baseline. Attach a pencil to the top of the
Chromatography paper or Silica Plate using a paper clip/peg or cover with a lid.
5. Allow the Chromatogram to run until the solvent almost reaches the top of the paper strip, for about
30 minutes.
6. Remove the paper and use a pencil to mark the solvent front.
7. Dry Chromatogram with a hair dryer, the measure the distance of the solvent front and distance of
spot moved. Using this information, find out the Rf Values.
Risk Assessment/ Safety Precautions:
Do not allow solvents to come into contact skin and do not breathe it in.
Solvents should be stored in fume cupboards, to minimize toxicity and flammability risk of Solvent vapors.
Lid should be used in experiments as well.
Results for Paper Chromatography 1: Pencil & Paper Clip:
From these diagrams, you can see the distinct
separations from each of the plant pigment. Pigment A
is a big Green leaf and Pigment B is Red/Green Lettuce.
Pigment A travelled further than B. In both Pigment A
and B, Xanthophyll is present, due to the visible pale-
yellow green, which has been separated out. The rf
value for A is 2.5/7=0.3cm and the rf value for B is 5/7=0.7cm. The results were okay; however, they
could be heavily improved.
Results for Paper Chromatography 2: Lid Used:
From these diagrams, you can see the distinct
separations from each of the plant pigment.
Pigment C is Red/Green Spinach and Pigment
D is Green Grass. Pigment D travelled further
than C. In Pigment C, Xanthophyll is present,
due to the visible pale-yellow green, which has
been separated out and Pigment D has signs of
Chlorophyll b and Xanthophyll, due to the deep green and pale yellow. The rf value for C is 4/8.5=0.4cm
and the rf value for D is 6.5/8.5=0.8cm. This Paper Chromatography technique with the Lid, produced
