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Plant Chromatography


Paper chromatography is a useful technique in the separation and identification of

different plant pigments. In this technique, the mixture containing the pigments to be separated is

first applied as a spot or a line to the paper about 1.5 cm from the bottom edge of the paper. The

paper is then placed in a container with the tip of the paper touching the solvent. Solvent is

absorbed by the paper and moves up the paper by capillary action. As the solvent crosses the area

containing plant pigment extract, the pigments dissolve in and move with the solvent. The

solvent carries the dissolved pigments as it moves up the paper.

The pigments are carried along at different rates because they are not equally soluble.

Therefore, the less soluble pigments will move slower up the paper than the more soluble

pigments. This is known as developing a chromatogram.

Paper chromatography is useful for identifying unknown compounds - often used in

crime scene investigations to match ink, lipstick, or colored fibers. There are many examples of

chromotography at youtube.com. This set-up shows two different pen inks.


1. To apply chromatography techniques

2. To identify plant pigments by separation and isolation of the pigments using thin layer

paper chromatography.


1. Test tube
2. Leaf extraction
3. Solvent
4. Pestle and mortar
5. Pin
6. Parafilm
7. Stopper tube
Green spinach and red spinach extraction


1. The pin head was used as the dropper, the chloroplast extract was dropped on the

prepared chromatography paper .

2. The extract was dropped at about 1.0 cm from the pointing end of the paper. The drop

were dried with a hair dryer and the process were repeated for 3-4 times until one small

dot of thick pigment available.

3. The paper strip were attched at the cork stopper using a pin. The strip were placed

vertically and straight into the test tube which contained solvent.
4. Let the solvent moves and remove the paper before the solvent front reaches the top of

your chromatography paper.

5. Marked with pencil the last range of the solvent.

the “solvent front” is the position of the liquid solvent on the chromatography paper at any given time. the s

After apply plant chromatography techniques

Type of extraction Observation Compound distance travel

Red spinach 2 compound were observed Green = 2.0cm

Red = 6.4cm

Green spinach 1 compund only Green = 5.6 cm

By using equation of:

Red spinach has two spot

I. Green spot (frist compound)

2.0 cm
=0.28 ¿ xanthophyll one)

II. Red spot (second compound)

6.4 cm
=0.89 ¿ pheophytin)
7.0 cm

It should be that pheophytin pigment appears green but it appears red

Green spinach has one spot only

I. Green spot (first compound)

5.6 cm
=0.82 ¿ pheophytin)
6.8 cm

A "pigment" is simply a molecule that absorbs and reflects light. Recall that white light

actually consists of many colors . I do learned in high school physics as a way to remember the

colors of light that make up the white light of the “visible spectrum”. Different pigments appear

different colors because they have differing abilities to absorb and reflect various colors of light.

(A more thorough discussion of the light-absorbing properties of pigments will be presented in

the Spectrophotometry lab.) The broad array of colors found in plant tissues such as leaves,

flowers, and fruits, can be accounted for by the presence of literally thousands of different kinds

of plant pigments.

How does chromatography paper work? . Think of chromatography as a race and you'll

find it's much simpler than it sounds. Waiting on the starting line, you've got a mixture of

chemicals in some unidentified liquid , just like a load of runners all mixed up and bunched

together. When a race starts, runners soon spread out because they have different abilities. In

exactly the same way, chemicals in something like a moving liquid mixture spread out because

they travel at different speeds over a stationary solid. The light compound will always move
upwards, heavy will stuck at the bottom. The key thing to remember is that chromatography

paper is a surface effect.

Extraction prepared in the laboratory need to take from the fresh sample. We need to use

fresh sample as it contains a lot of pigments in it. Using sample other than that could affected our

result as there contain least of pigment making the experiment becomes more difficult to

determine the compound travelled. When handle the experiment make sure to not submerge the

pigment in the solvent as the pigment may dissolve in the solvent itself. Just the tip of the paper

was dipped into the solvent and let the paper of chromatography do their work by absorbing light

compound upwards. It is crucial to cover the test tube with parafilm or even test tube stopper to

make sure that the surrounding inside the test tube will not mixed up with the environment

outside the test tube. Evaporation will occurs if we did not cover the tube and making the result

accuracy affected. Make sure to mark the solvent travelled and compound travelled at

chromatography paper so that we can calculated the rf value. Throughout rf value that we get,we

manage to determine what type of pigment is it. The fastest pigment moving is carotene orange

coloured pigment. But if you do not get the orange coloured pigment it means that you should

dip the paper in the solvent longer so that the solvent can move upwards for the carotene

pigments. It is just you did not give enough time for the solvent to do their work. Wait patiently.

One more thing, do not dip too long until the solvent reach up the end of paper or worst exceed

from the paper. We cannot calculate the rf value without knowing the solvent traveled distance.

So you really need to be good at prediction.

In this experiment the the rf value for red spinach 0.28 and 0.89 indicates the presence of

xantophyl 1 pigment and pheophytin pingment. The green spinach were only observed with one

compound which is the presence of pheophythin with the rf value of 0.82


I do learned that in nature, color is an important attribute of plants that serves to attract

pollinators to receptive flowers and signal fruit ripeness to seed dispersers. In some instances,

colors may also serve to warn potential predators of poisonous or toxic substances contained in

plant tissues.

Throughout the reading about plant chromatography I found out that, color-producing

pigments have other important roles in plants beyond regulating interactions with animals.

Chlorophyll is a pigment that reflects green light, but absorbs red and blue wavelengths and is

critical for the light reactions of photosynthesis. Flavonoids are an important class of plant

pigments that block ultraviolet (UV) radiation that can damage cell proteins and DNA. Many

flavonoids, including anthocyanins (a subcategory of flavonoids) have a role in the chemical

defense of plants as they are toxic to many herbivores and pathogens – especially insects and


1. Paper Chromatography






2. Paper chromatography