PRACTICAL #11

SEPERATION OF PIGMENTS USING CHROMATOGRAPHY

THEORY:

Photosynthetic organisms do not rely on a single pigment to absorb light, but instead benefit
from the combined action of many
 These pigments include chlorophylls, xanthophyll and carotenes

Chromatography is an experimental technique by which mixtures can be separated

 A mixture is dissolved in a fluid (called the mobile phase) and passed through a static
material (called the stationary phase)
 The different components of the mixture travel at different speeds, causing them to
separate
 A retardation factor can then be calculated (Rf value = distance component travels ÷
distance solvent travels)

Two of the most common techniques for separating photosynthetic pigments are:
 Paper chromatography – uses paper (cellulose) as the stationary bed
 Thin layer chromatography – uses a thin layer of adsorbent (e.g. silica gel) which
runs faster and has better separation
 ● For photosynthesis to transform light energy from the sun into chemical energy in
plants, pigment molecules absorb light to power the chemical reactions.
● Photosynthetic organisms do not rely on a single pigment to absorb light, but
instead benefit from the combined action of many.
● Plant pigments are macromolecules produced by the plant, and these pigments
absorb specific wavelengths of visible light to provide the energy required for
photosynthesis.
● Chlorophyll is necessary for photosynthesis, but accessory pigments collect and
transfer energy to chlorophyll. Although pigments absorb light, the wavelengths
of light that are not absorbed by the plant pigments are reflected back to the
eye. The reflected wavelengths are the colors we see in observing the plant.
(Example: green pigments reflect green light)
● Plants contain different pigments, some of the pigments observed include:
o chlorophylls (greens)
o carotenoids (yellow, orange red)
o anthocyanins (red to blue, depending on pH)
o betalains (red or yellow)

Two of the most common techniques for separating photosynthetic pigments are:

1. Paper chromatography – uses paper (cellulose) as the stationary bed

2. Thin layer chromatography – uses a thin layer of absorbent (e.g. silica gel)
which develops faster and has clearer separation of pigments making it easier to
calculate Rf values. TLC chromatography also uses less solvent.

Overview of the Chromatographic Separation of Photosynthetic Pigments:

YouTube Video of Experiment: https://www.youtube.com/watch?v=6sZBtANyuZ8
·
 ● Chromatography is an experimental technique by which mixtures can be
separated.
● The process of chromatography separates molecules because of the different
solubility’s of the molecules in a selected solvent.
● In chromatography, the paper is marked with a small amount of plant extract
that is allowed to dry, this is repeated several times so that the pigments
becomes stronger in colour due to now being more concentrated.
● It is then placed in a chamber with a specified solvent. The solvent carries the
dissolved pigments as it moves up the paper.
● The pigments are carried at different rates because they are not equally soluble.
● A pigment that is the most soluble will travel the greatest distance and a
pigment that is less soluble will move a shorter distance.
● The distance the pigment travels is unique for that pigment in set conditions and
is used to identify the pigment based on its Rf value.
● A mixture is dissolved in a fluid (called the mobile phase) and passed through a
static material (called the stationary phase).
● The different components of the mixture travel at different speeds, causing them
to separate.
● A retardation factor can then be calculated (Rf value = distance pigment travels
÷ distance solvent travels).

The pigments within the mixture moved at different rates depending on how well they
dissolved in the solvent, therefore different plants and different running solvents will yield
different Rf values.

Aim: To identify plant pigments by separation and isolation of the pigments using thin
layer paper chromatography.

Background Information: Thin layer chromatography is a quick and effective method of

separating pigments within leaves. The pigments run clearly on the chromatogram with
little outward spread, allowing Rf values to be calculated and chlorophylls to be easily
identified.

Safety: Wear eye protection.

Avoid inhaling vapour. Ensure the lab is well-ventilated. Minimise the escape of vapour
from the bottle.
Ensure no naked flames or other sources of ignition. Isopropanol and propanone are
flammable – keep away from naked flame.

Equipment:
-Plant leaves; grass, spinach or geraniums work well
-TLC plate with silica coating
-Universal bottle and lid
-Forceps
- Coin (or equivalent)
-Scalpel, sharp scissors
-Ruler & pencil
- White tile
-Running solvent made up of a 5:2:2 mixture (by volume) of cyclohexane, ethyl
ethanoate and propanone

Instructions:
1. Using sharp scissors cut a strip of TLC plate that will fit into a universal bottle (approx.
8cm x 1cm) and draw a line in pencil 5mm from the bottom edge of the plate.
2. Pour running solvent into the universal bottle so that it is approximately 3mm high (it
must be under the 5mm line of the TLC plate), and replace the lid for the solvent vapour
to saturate the bottle.
3. Place a leaf, or leaves, (grass works well) on the TLC plate to cover the line. Place a
ruler over the leaf so it lines up with the pencil line.
4. Using a coin, press down firmly and roll along the ruler edge to form a green line.
5. Allow the green line to dry. To extract more of the green mixture, repeat steps 3 & 4
several times with a new leaf until a narrow green band forms.
6. Using forceps, place the TLC plate into the universal bottle and replace the lid. Do
not move the bottle while the chromatogram is running.
7. Once the ‘solvent’ has moved 7/8ths of the way up the plate (around 10 minutes),
use forceps to remove it from the bottle. Mark the position of the solvent (before it
evaporates) and leave the plate to dry on a tile.
8. Photograph the result as soon as the solvent has evaporated because the
pigments are light-sensitive and will fade and UPLOAD HERE.
9. To calculate Rf values, mark the location of each distinct pigment
by drawing a dot in the centre of the line.

ASSESSMENT:
Skill – Separate pigments using chromatography to determine Rf values of each
photosynthetic pigment using the equation below:
Rf = distance moved by the pigment ÷ distance moved by the solvent
Results Table (Rf Values):

Colour of Distance Distance Rf Value Name of pigment

band Pigment Solvent
Travels Travels

Past examination question

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