paper chromatography experiment report

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Paper Chromatography Experiment

March 17, 2021 By Emma Vanstone Leave a Comment

This simple felt tip pen paper chromatography experiment is a great way to learn about this particular method of separating mixtures .

WHAT IS CHROMATOGRAPHY?

Chromatography   is a technique used to separate mixtures. Information from a chromatography investigation can also be used to identify different substances.

In chromatography, the mixture is passed through another substance, in this case, filter paper. The different-coloured ink particles travel at different speeds through the filter paper, allowing the constituent colours of the pen ink to be seen.

All types of chromatography have two phases: a mobile phase where the molecules can move and a stationary phase where they can’t move. In the case of paper chromatography, the stationary phase is the filter paper, and the mobile phase is the solvent ( water ).

The more soluble the ink molecules, the further they are carried up the paper.

The video below shows chromatography in action.

You’ll need:

Filter paper or paper towel

Felt tip pens – not washable or permanent

A container – glass, jar or plate

Instructions

Pour a small amount of water onto a plate or into the bottom of a jar.

Find a way to suspend the filter paper over the water so that just the very bottom touches the water. If you do the experiment in a jar, the easiest way to do this is to wrap the top of the filter paper around a pencil, clip it in place, and suspend it over the top of the jar.

Our LEGO holder worked well, too!

Use the felt tip pens to draw a small circle about 1cm from the bottom of the filter paper with each colour pen you want to test.

Suspend the filter paper in the water and watch as the ink moves up the filter paper.

You should end up with something like this! The end result is called a chromatogram.

What happens if you use washable pens?

If the inks are washable, they tend to contain just one type of ink, so there is no separation of colour.

Below, only a couple of the inks have separated compared to the non-washable pens above.

Why does chromatography work?

When the filter paper containing the ink spots is placed in the solvent ( in this case, water ), the dyes travel through the paper.

Different dyes in ink travel through the chromatography filter paper at different speeds. The most soluble colours dissolve and travel further and faster than less soluble dyes, which stick to the paper more.

I’ve created a free instruction sheet and chromatography experiment write up to make the activity even easier.

Extension task

Experiment with different types and colours of pens. Depending on the type of ink used, some will work better than others.

Try chromatography with sweets .

Steamstational also has a great leaf chromatography investigation.

More separation experiments

Clean up water by making your own filter .

Separate water and sand by evaporation .

Make colourful salt crystals by separating salt and water.

Separate liquid mixtures with a bicycle centrifuge .

Last Updated on May 20, 2024 by Emma Vanstone

Safety Notice

Science Sparks ( Wild Sparks Enterprises Ltd ) are not liable for the actions of activity of any person who uses the information in this resource or in any of the suggested further resources. Science Sparks assume no liability with regard to injuries or damage to property that may occur as a result of using the information and carrying out the practical activities contained in this resource or in any of the suggested further resources.

These activities are designed to be carried out by children working with a parent, guardian or other appropriate adult. The adult involved is fully responsible for ensuring that the activities are carried out safely.

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BIOLOGY JUNCTION

Test And Quizzes for Biology, Pre-AP, Or AP Biology For Teachers And Students

Leaf Chromatography Experiment – Easy Paper Chromatography

Leaf chromatography is paper chromatography using leaves. Paper chromatography is a separation technique. When applied to leaves, it separates the pigment molecules mostly according to their size. The main pigment molecule in green leaves is chlorophyll, which performs photosynthesis in the plant. Other pigments also occur, such as carotenoids and anthocyanins. When leaves change color in the fall , the amount and type of pigment molecules changes. Leaf chromatography is a fun science project that lets you see these different pigments.

Leaf Chromatography Materials

You only need a few simple materials for the leaf chromatography project:

• Rubbing alcohol (isopropyl alcohol)
• Coffee filters or thick paper towels
• Small clear jars or glasses with lids (or plastic wrap to cover the jars)
• Shallow pan
• Kitchen utensils

You can use any leaves for this project. A single plant leaf contains several pigment molecules, but for the most colors, use a variety of leaves. Or, collect several of each kind of leaf and compare them to each other. Good choices are colorful autumn leaves or chopped spinach.

Perform Paper Chromatography on Leaves

The key steps are breaking open the cells in leaves and extracting the pigment molecule and then separating the pigment using the alcohol and paper.

• Finely chop 2-3 leaves or several small leaves. If available, use a blender to break open the plant cells. The pigment molecules are in the chloroplasts of the cells, which are organelles encased within the plant cell walls. The more you break up the leave, the more pigment you’ll collect.
• Add enough alcohol to just cover the leaves.
• If you have more samples of leaves, repeat this process.
• Cover the container of leaves and alcohol and set it in a shallow pan filled with enough hot tap water to surround and heat the container. You don’t want water getting into your container of leaves.
• Replace the hot water with fresh water as it cools. Swirl the container of leaves around from time to time to aid the pigment extraction into the alcohol. The extraction is ready when the alcohol is deeply colored. The darker its color, the brighter the resulting chromatogram.
• Cut a long strip of coffee filter or sturdy paper towel for each chromatography jar. Paper with an open mesh (like a paper towel) works quickly, but paper with a denser mesh (like a coffee filter) is slower but gives a better pigment separation.
• Place a strip of paper into jar, with one end in the leaf and alcohol mixture and the other end extending upward and out of the jar.
• The alcohol moves via capillary action and evaporation, pulling the pigment molecules along with it. Ultimately, you get bands of color, each containing different pigments. After 30 to 90 minutes (or whenever you achieve pigment separation), remove the paper strips and let them dry.

How Leaf Chromatography Works

Paper chromatography separates pigments in leaf cells on the basis of three criteria:

• Molecule size

Solubility is a measure of how well a pigment molecule dissolves in the sol vent. In this project, the solvent is alcohol . Crushing the leaves breaks open cells so pigments interact with alcohol. Only molecules that are soluble in alcohol migrate with it up the paper.

Assuming a pigment is soluble, the biggest factor in how far it travels up the paper is particle size. Smaller molecules travel further up the paper than larger molecules. Small molecules fit between fibers in the paper more easily than big ones. So, they take a more direct path through the paper and get further in less time. Large molecules slowly work their way through the paper. In the beginning, not much space separates large and small molecules. The paper needs to be long enough that the different-sized molecules have enough time to separate enough to tell them apart.

Paper consists of cellulose, a polysaccharide found in wood, cotton, and other plants. Cellulose is a polar molecule . Polar molecules stick to cellulose and don’t travel very far in paper chromatography. Nonpolar molecules aren’t attracted to cellulose, so they travel further.

Of course, none of this matters if the solvent doesn’t move through the paper. Alcohol moves through paper via capillary action . The adhesive force between the liquid and the paper is greater than the cohesive force of the solvent molecules. So, the alcohol moves, carrying more alcohol and the pigment molecules along with it.

Interpreting the Chromatogram

• The smallest pigment molecules are the ones that traveled the greatest distance. The largest molecules are the ones that traveled the least distance.
• If you compare chromatograms from different jars, you can identify common pigments in their leaves. All things being equal, the lines made by the pigments should be the same distance from the origin as each other. But, usually conditions are not exactly the same, so you compare colors of lines and whether they traveled a short or long distance.
• Try identifying the pigments responsible for the colors.

There are three broad classes of plant pigments: porphyrins, carotenoids, and flavonoids. The main porphyrins are chlorophyll molecules. There are actually multiple forms of chlorophyll, but you can recognize them because they are green. Carotenoids include carotene (yellow or orange), lycopene (orange or red), and xanthophyll (yellow). Flavonoids include flavone and flavonol (both yellow) and anthocyanin (red, purple, or even blue).

Experiment Ideas

• Collect leaves from a single tree or species of tree as they change color in the fall. Compare chromatograms from different colors of leaves. Are the same pigments always present in the leaves? Some plants produce the same pigments, just in differing amounts. Other plants start producing different pigments as the seasons change.
• Compare the pigments in leaves of different kinds of trees.
• Separate leaves according to color and perform leaf chromatography on the different sets. See if you can tell the color of leaves just by looking at the relative amount of different pigments.
• The solvent you use affects the pigments you see. Repeat the experiment using acetone (nail polish remover) instead of alcohol.
• Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955).  A Manual of Paper Chromatography and Paper Electrophoresis . Elsevier. ISBN 978-1-4832-7680-9.
• Ettre, L.S.; Zlatkis, A. (eds.) (2011). 75 Years of Chromatography: A Historical Dialogue . Elsevier. ISBN 978-0-08-085817-3.
• Gross, J. (1991). Pigments in Vegetables: Chlorophylls and Carotenoids . Van Nostrand Reinhold. ISBN 978-0442006570.
• Haslam, Edwin (2007). “Vegetable tannins – Lessons of a phytochemical lifetime.”  Phytochemistry . 68 (22–24): 2713–21. doi: 10.1016/j.phytochem.2007.09.009
• McMurry, J. (2011). Organic chemistry With Biological Applications (2nd ed.). Belmont, CA: Brooks/Cole. ISBN 9780495391470.

Related Posts

Separation of Plant Pigments by Paper Chromatography

The separation of plant pigments by paper chromatography is an analysis of pigment molecules of the given plant. Chromatography refers to colour writing . This method separates molecules based on size, density and absorption capacity.

Chromatography depends upon absorption and capillarity . The absorbent paper holds the substance by absorption. Capillarity pulls the substance up the absorbent medium at different rates.

Separated pigments show up as coloured streaks . In paper chromatography, the coloured bands separate on the absorbent paper. Chlorophylls, anthocyanins, carotenoids, and betalains are the four plant pigments.

This post discusses the steps of separating plant pigments through paper chromatography. Also, you will get to know the observation table and the calculation of the Rf value.

Content: Separation of Plant Pigments by Paper Chromatography

Paper chromatography, plant pigments, steps of plant pigment separation, observation, calculation.

It is the simplest chromatography method given by Christian Friedrich Schonbein in 1865. Paper chromatography uses filter paper with uniform porosity and high resolution.

The mixtures in compounds have different solubilities . For this reason, they get separated distinctly between the stationary and running phase.

• The mobile phase is a combination of non-polar organic solvents. The solvent runs up the stationary phase via capillary movement.
• The stationary phase is polar inorganic solvent, i.e. water. Here, the absorbent paper supports the stationary phase, i.e. water.

Plant pigments are coloured organic substances derived from plants. Pigments absorb visible radiation between 380 nm (violet) and 760 nm (red).

They give colour to stems, leaves, flowers, and fruits. Also, they regulate processes like photosynthesis, growth, and development.

Plants produce various forms of pigments. Based on origin, function and water solubility, plant pigments are grouped into:

• Chlorophylls (green)
• Carotenoids (yellow, orange-red)
• Anthocyanins (red to blue, depending on pH)
• Betalains (red or yellow)

Chlorophyll : It is a green photosynthetic pigment. Chlorophyll a and b are present within the chloroplasts of plants. Because of the phytol side chain, they are water-repelling . Their structure resembles haemoglobin. But, they contain magnesium as a central metal instead of iron.

Carotenoids : These are yellow to yellow-orange coloured pigments. Also, they are very long water-repelling pigments. Carotenoids are present within the plastids or chromoplasts of plants.

Anthocyanins : These appear as red coloured pigments in vacuoles of plant cells. Anthocyanins are water-soluble pigments. They give pink-red colour to the petals, fruits and leaves.

Betalains : These are tyrosine derived water-soluble pigments in plants. Betacyanins (red-violet) and betaxanthins (yellow-orange) are the two pigments coming in this category. They are present in vacuoles of plant cells.

You can separate all the above pigments using paper chromatography.

Video: Separation of Plant Pigments

Preparation of Concentrated Leaf Extract

• Wash spinach leaves in distilled water.
• Then take out the spinach leaves and allow the moisture to dry out.
• After that, take a scissor and cut the leaves into the mortar.
• Take a little volume of acetone into the mortar. Note : Acetone is used instead of water to mash the leaves because it is less polar than the water. This allows a high resolution of the molecules in the sample between the absorbent paper.
• Then, grind spinach leaves using a pestle until liquid paste forms. Note : The liquid in the crushed leaf paste is the pigment extract.
• After that, take out the mixture into the watch glass or Petri dish.

Load the Leaf Extract onto Absorbent Paper

• Take Whatman filter paper and draw a line above 2 cm from the bottom margin. You can use a pencil and scale to draw a fainted line. Note : A pencil is used because pencil marks are insoluble in the solvent.
• Then, cut the filter paper to make a conical edge from the line drawn towards the margin end. You can use a scissor to cut the Whatman filter paper. Note : The conical end at the bottom of the filter paper results in better separation.
• Put a drop of leaf extract on the centre of a line drawn on the absorbent paper.
• Then, at the same time dry the absorbent paper.
• Repeat the above two steps many times so that the spot becomes concentrated enough.

Setup the Chromatography Chamber

• Take a clean measuring cylinder and add rising solvent (ether acetone) up to 4 ml.
• Bend the strip of paper from the top. Then, using a pushpin attach the paper to the bottom of the cork.
• Adjust the length of the paper. The absorbent paper should not touch the surface of the measuring cylinder.
• After that, allow the solvent to move up the absorbent paper.
• When the solvent front has stopped moving, remove the paper.
• Allow it to dry for a while until the colours completely elute from the paper.
• At last, mark the front edge travelled by each pigment.

Over the dried paper strip, you will see four different bands. Different colour streaks form because of different affinities with the mobile phase (solvent).

• The carotene pigment appears at the top as a yellow-orange band.
• A yellowish band appears below the carotene, which indicates xanthophyll pigment.
• Then a dark green band represents the chlorophyll-a pigment.
• The chlorophyll-b pigment appears at the bottom as a light green band.

Observation Table

1. Light green spot indicates chlorophyll-b pigment.

• Rf value= Distance chlorophyll-b travelled / Distance solvent travelled = 2/10 = 0.2

2. Dark green spot represents chlorophyll-a pigment.

• Rf value= Distance chlorophyll-a travelled / Distance solvent travelled = 3.7/10 = 0.37

3. The yellow band represents xanthophyll pigment.

• Rf value= Distance xanthophyll travelled / Distance solvent travelled = 5.6/10 = 0.56

4. The yellow-orange band indicates carotene pigment.

• Rf value= Distance carotene travelled / Distance solvent travelled = 9/10 = 0.9

Factors affecting the Rf values of a particular analyte are:

• Stationary phase
• The concentration of the stationary phase
• Mobile phase
• The concentration of the mobile phase
• Temperature

The Rf value of compounds in the mixture differs by any changes in the concentration of stationary and mobile phases.

Temperature affects the solvent capillary movement and the analyte’s solubility in the solvent. Rf value is independent of the sample concentration. Its value is always positive .

Related Topics:

• Difference Between Budding and Grafting
• Phototropism in Plants
• Potometer Experiment

1 thought on “Separation of Plant Pigments by Paper Chromatography”

Nice experiment and understanding.

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