in a chromatography experiment chlorophyll pigments are separated using paper

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

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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

Paper Chromatography of Plant Pigments

Learning Objectives

After completing the lab, the student will be able to:

• Extract pigments from plant material.
• Separate pigments by paper chromatography.
• Measure R f (retention factor) values for pigments.

Activity 2: Pre-Assessment

• The leaves of some plants change color in fall. Green foliage appears to turn to hues of yellow and brown. Does the yellow color appear because carotenoids replace the green chlorophylls? Explain your reasoning.
• Examine the molecular structures of photosynthetic pigments in Figure 10.1. Photosynthetic pigments are hydrophobic molecules located in thylakoid membranes. Will these pigments dissolve in water?

Activity 2: Paper Chromatography of Plant Pigments

Paper chromatography is an analytical method that separates compounds based on their solubility in a solvent.

The solvent is used to separate a mixture of molecules that have been applied to filter paper. The paper, made of cellulose, represents the stationary or immobile phase. The separation mixture moves up the paper by capillary action. It is called the mobile phase. The results of chromatography are recorded in a chromatogram. Here, the chromatogram is the piece of filter paper with the separated pigment that you will examine at the end of your experiment (see Figure 10.4).

We separate the compounds based on how quickly they move across the paper. Compounds that are soluble in the solvent mixture will be more concentrated in the mobile phase and move faster up the paper. Polar compounds will bind to the cellulose in the paper and trail behind the solvent front. As a result, the different compounds will separate according to their solubility in the mixture of organic solvents we use for chromatography.

This video demonstrates the principles and examples of chromatography. You will experiment with only paper chromatography in this lab; however, you will see that you are already familiar with some uses of thin layer chromatography.

Safety Precautions

• Work under a hood or in a well-ventilated space and avoid breathing solvents.
• Do not have any open flames when working with flammable solvents.
• Wear aprons and eye protection.
• Do not pour any organic solvent down the drain.
• Dispose of solvents per local regulations.
• Use forceps to handle chromatography paper that has been immersed in solvent and wash your hands after completing this activity.

For this activity, you will need the following:

• Plant material: intact leaves of spinach and Coleus (one leaf of each plant per pair of students)
• Filter or chromatography paper
• Ruler (one per group)
• Colored pencils
• Beakers (400 mL) (Mason jars are an acceptable substitute)
• Aluminum foil
• Petroleum ether: acetone: water in a 3:1:1 proportion
• If no hood or well-ventilated place is available, the mixture can be substituted with 95 percent isopropyl alcohol. Note that, if isopropyl alcohol is used, the pigment bands will smear. You may not be able to separate and identify the chlorophylls or carotene from xanthophyll.

For this activity, you will work in pairs .

Structured Inquiry

Step 1: Hypothesize/Predict: Discuss with your lab partner what color pigments will likely be present in the spinach leaves. Write your predictions in your lab notebook and draw a diagram of how you think the pigments will separate out on the chromatography paper.

Step 2: Student-led Planning: Read step 3 below. Discuss with your lab partner the setup of the experiment. Then agree upon the dimensions of the filter/chromatography paper that you will use. To allow good separation, the paper should not touch the walls of the container. The paper must fit inside the container while being long enough for maximum separation. Write all your calculations in your lab notebook.

Step 3: Follow the steps below to set up your filter paper and perform the chromatography experiment.

• Prepare the chromatogram by cutting a piece of filter paper. Transfer pigments from spinach leaves as in Activity 1. A heavy application line will yield stronger colors when the pigments separate, making it easier to read results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.
• Form a cylinder with the filter paper without overlapping the edges (to avoid edge effects). The sample should face the outside of the cylinder. Secure the top and bottom of the cylinder with staples.
• Pour enough separation mixture to provide a mobile phase while staying below the origin line on the chromatogram. The exact volume is not critical if the origin, the start line where you applied the solvent, is above the solvent. See Figure 10.4.

• Label the beaker with a piece of tape with your initials and your partner’s initials.
• Lower the paper into the container with the band from the extraction in the lower section. The paper must touch the solvent, but not reach the band of pigment you applied. Why must the band be above the solvent line? Write your answer in your notebook.
• Cover the container tightly with a piece of aluminum foil.
• Track the rising of the solvent front. Can you see a separation of colors on the paper?
• When the solvent front is within 1 cm of the upper edge of the paper, remove the cylinder from the beaker using forceps. Trace the solvent front with a pencil before it evaporates and disappears! Draw the colored bands seen on your chromatography paper in your lab notebook immediately. The colors will fade upon drying. If no colored pencils are available, record the colors of the lines.
• Let the paper dry in a well-ventilated area before making measurements because the wet paper is fragile and may break when handled. This is also a precaution to avoid breathing fumes from the chromatogram.
• Discard solvent mixture per your instructor’s directions. Do not pour down the drain.

Step 4: Critical Analysis: Open the dried cylinder by removing the staples. Measure the distance from the first pencil line to the solvent front, as shown in Figure 10.5. This is the distance traveled by the solvent front. Measure the distance from the pencil line to the middle point of each color band and the original pencil line. Record your results in your notebook in a table modeled after Table 10.1. The retention factor (R f ) is the ratio of the distance traveled by a colored band to the distance traveled by the solvent front. Calculate R f values for each pigment using the following equation:

R f=Distance traveled by colored band/Distance traveled by solvent front

Step 5: After determining the color of the band, tentatively identify each band. Did your results support your hypothesis about the color of each band? Discuss which aspects of the experiments may have yielded inconclusive results. How could you improve the experiment?

Guided Inquiry

Step 1: Hypothesize/Predict: What type of pigments are present in Coleus leaves and where are the different colors located? Can you make a hypothesis based on the coloration of the variegated leaves? Write your hypothesis down in your lab notebook. Would there be a difference if you performed chromatography on pigment composition from different colored regions of the leaves?

Step 2: Student-led Planning: Cut the chromatography/filter paper to the dimensions needed. Apply pigments from different parts of the Coleus leaves following the procedure described under Activity 1, keeping in mind that a darker line will yield stronger colors when the pigments are separated, which will make it easier to read the results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.

Step 3: When the solvent front reaches 1 cm from the top of the filter paper, stop the procedure. Draw the pigment bands you see on the filter paper in your lab notebook. Clearly indicate the color you observed for each band.

Step 4: Let the cylinder dry and measure the distance the front traveled from the origin and the distances traveled by each of the pigments. If the bands broadened during separation, take measurements to the middle of each band.

Step 5: Critical Analysis: Calculate R f for each of the bands and record them in a table in your notebook. Compare the R f you obtained with those of other groups. Are the R f values similar? What may have altered R f values?

Assessments

• Carotenoids and chlorophylls are hydrophobic molecules that dissolve in organic solvents. Where would you find these molecules in the cell? What would happen if you ran the chromatography in this lab with water as the solvent?
• All chlorophyll molecules contain a complexed magnesium ion. Your houseplant is developing yellow leaves. What may cause this, and how can you restore your plant’s health?
• Seeds that grow under dim light are said to be etiolated, which describes their pale and spindly appearance. They soon waste away after exhausting their food reserves. Can you explain this observation?

Lab Manual for Biology Part I Copyright © 2022 by LOUIS: The Louisiana Library Network is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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• Biology Article
• Separation Of Plant Pigments Through Paper Chromatography

How to Separate Plant Pigments Using Paper Chromatography

To distinguish and study the various pigments present in plants through the process of paper chromatography.

Plants carry out the process of photosynthesis, during which light energy from the sun is converted into chemical energy (food). The capturing of light energy is carried out by molecules known as pigments, which are present within the plant cells.

Explore more:   Plant cells .

What are Pigments?

Pigments are chemical compounds, which are able to reflect only a particular range of wavelengths of visible light. Leaves of plants primarily contain different types of pigments within their tissues. The four different types of pigments are listed below in a tabular column along with their colours.

In order to view and distinguish the primary four plant pigments, a simple technique known as chromatography can be used.

Read more:  Pigments

What is Chromatography?

It is a technique that is used to distinguish between different molecules. This differentiation is based on these attributes-shape, size, charge, mass, adsorption and solubility.

Types of chromatography:

• Column chromatography
• Paper chromatography
• Partition chromatography
• Thin-layer chromatography

Mechanism of Paper Chromatography

In this technique, the interaction between three components is involved – solid phase, separation of a mixture and a solvent.

• At first, the mixture is spotted onto the paper and is dried.
• The solvent is made to flow through the capillary attraction.
• While the solvent moves through the paper, the various components of the mixture differentiate into varied coloured spots.
• Later the paper is allowed to dry and the position of various compounds is viewed.
• The substance, which is the most soluble moves further on the paper as compared to the other substances that are less soluble.

Material Required

• Chromatography chamber
• Spinach leaves
• Mortar and pestle
• Ether acetone solvent
• Capillary tube
• Filter paper strips
• Watch glass
• In this experiment, spinach leaves are used to separate different pigments.
• Pick a few fresh and green leaves of spinach and wash it.
• Cut out small pieces of spinach using scissors. Add them to the mortar.
• Accurately measure 5ml acetone using a measuring cylinder and add it into the mortar.
• With the help of mortar and pestle, grind the spinach leaves into a smooth paste.
• Shift the prepared paste of spinach into the watch glass with the help of a spatula.
• Place a filter paper strip with a tapering notch towards one ending of the strip.
• Horizontally trace a line with a scale and a pencil that is 2 to 3 cm apart from the notch’s tip.
• Using a capillary tube, add 1 drop of the extract of the pigment in the midsection of the line.
• Let the drop dry. Repeat the same process of adding a drop and allowing it to dry for 4-5 times.
• In the chromatographic chamber, pour the ether acetone solvent.
• Make sure to folded and stapled an end side of the paper.
• Suspend the strip in the chamber.
• The loading spot remains about 1 cm above the level of the solvent.
• Let the chamber remain uninterrupted for a while.
• We can notice that the solvent passes along the paper scattering various pigments of the blend to different distances.
• Once the solvent reaches 3/4 th  of the strip, carefully take the strip off.
• Allow the strip to dry.

Observation

The dried paper strip displays four different bands. Discrete pigments can be distinguished with the help of colours.

• The Carotene pigment is observed at the topmost as an orange-yellow band of pigments distinctively.
• Just below this band, a yellowish band appears which indicates the pigment xanthophyll.
• The third band appearing dark green indicates chlorophyll-a pigment.
• The yellowish-green band present at the bottom is the chlorophyll b pigment.

Precautions

• The leaves that are selected should be green and fresh spinach leaves
• From the tip of the notch, the loading spot needs to be 2 to 3 cm apart
• While suspending the filter paper strips in the chamber, one need to ensure that the loading spot needs to be set up above 1 cm from the level of the solvent.

Viva Questions

Q.1. What Rf value or Retention factor?

A.1. The Retention factor or Rf value applies to chromatography to make the technique scientific. It is defined as the distance travelled by the compound divided by the distance travelled by the solvent.

Rf value = Distance travelled by the compound / Distance travelled by the solvent.

Q.2. What is Phycobilin?

A.2. Phycobilins are light-capturing bilins found in chloroplast organelles, cyanobacteria and in a few algae.

Q.3. What is the significance of pigment in photosynthesis?

A.3. It helps in the absorption of energy from light. The free electrons in the pigments present in their chemical structure transfer their energy to other molecules during photosynthesis when they turn into high energy electrons, thereby liberating energy they captured from light. This released energy is then used up by other molecules for the formation of sugars and related nutrients with the use of water and carbon dioxide.

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Leaf chromatography

In association with Nuffield Foundation

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Try this class practical using paper chromatography to separate and investigate the pigments in a leaf

Most leaves are green due to chlorophyll. This substance is important in photosynthesis (the process by which plants make their food). In this experiment, students investigate the different pigments present in a leaf, from chlorophyll to carotenes, using paper chromatography.

The experiment takes about 30 minutes and can be carried out in groups of two or three students.

• Eye protection
• Pestle and mortar
• Chromatography paper
• Beaker, 100 cm 3
• Small capillary tube (see note 1)
• Cut-up leaves, or leaves and scissors (see note 2)
• Propanone (HIGHLY FLAMMABLE, IRRITANT), supplied in a small bottle fitted with a teat pipette (see note 3)

Equipment notes

• The capillary tubing can be ‘home-made’ from lengths of ordinary glass tubing (diameter: 3–4 mm) using a Bunsen burner fitted with a flame-spreading (‘fish-tail’) jet.
• A variety of leaves can be used. Best results are obtained from trees or bushes with dark green leaves, eg holly.
• Preferably use teat pipettes that do not allow squirting, eg those fitted to dropper bottles of universal indicator.

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Propanone, CH 3 COCH 3 (l), (HIGHLY FLAMMABLE, IRRITANT) – see CLEAPSS Hazcard HC085A .  The vapour of propanone is HIGHLY FLAMMABLE. Do not have any source of ignition nearby.
• Finely cut up some leaves and fill a mortar to about 2 cm depth.
• Add a pinch of sand and about six drops of propanone from the teat pipette.
• Grind the mixture with a pestle for at least three minutes.
• On a strip of chromatography paper, draw a pencil line 3 cm from the bottom.
• Use a fine glass tube to put liquid from the leaf extract onto the centre of the line. Keep the spot as small as possible.
• Allow the spot to dry, then add another spot on top. Add five more drops of solution, letting each one dry before putting on the next. The idea is to build up a very concentrated small spot on the paper.
• Attach the paper to the pencil using sellotape so that when placed in the beaker, the paper is just clear of its base.
• Place no more than about 10 cm 3 of propanone in the beaker and hang the paper so it dips in the propanone. Ensure the propanone level is below the spot.

Source: Royal Society of Chemistry

The equipment required for using paper chromatography to separate the different pigments in leaves

• Avoid moving the beaker in any way once the chromatography has started.
• Leave the experiment until the propanone has soaked near to the top, and then remove the paper from the beaker.
• Mark how high the propanone gets on the paper with a pencil and let the chromatogram dry.

Teaching notes

This experiment works very well providing care is taken over preparing the spot on the chromatography paper. It should be as small and as concentrated as possible. Encourage students to be patient and to wait until each application is dry before adding the next.

At least three spots should be obtained, and one of these should be yellow due to carotenes.

The extent to which any particular component moves up the paper is dependent not only on its solubility in propanone but also on its attraction for the cellulose in the chromatography paper. The yellow carotene spot (with a higher RF value) tends to move up the paper the furthest.

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Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• Practical experiments
• Chromatography

Specification

• 2. Develop and use models to describe the nature of matter; demonstrate how they provide a simple way to to account for the conservation of mass, changes of state, physical change, chemical change, mixtures, and their separation.
• Chromatography as a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.
• 6 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• Chromatography involves a stationary phase and a mobile phase. Separation depends on the distribution of substances between the phases.
• The ratio of the distance moved by a compound (centre of spot from origin) to the distance moved by the solvent can be expressed as its Rf value: Rf = (distance moved by substance / distance moved by solvent)
• Mixtures can be separated by physical processes such as filtration, crystallisation, simple distillation, fractional distillation and chromatography. These physical processes do not involve chemical reactions and no new substances are made.
• Recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases.
• Interpret chromatograms, including measuring Rf values.
• Suggest chromatographic methods for distinguishing pure from impure substances.
• 12 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• 2.11 Investigate the composition of inks using simple distillation and paper chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates…
• C2.1g describe the techniques of paper and thin layer chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates o…
• C5.1.4 recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases
• 3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• C3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• 1.9.5 investigate practically how mixtures can be separated using filtration, crystallisation, paper chromatography, simple distillation or fractional distillation (including using fractional distillation in the laboratory to separate miscible liquids…
• 1.9.7 interpret a paper chromatogram including calculating Rf values;
• carry out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms;

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Separation of Photosynthetic Pigments with Chromatography ( Edexcel International A Level Biology )

Revision note.

Separation of Photosynthetic Pigments with Chromatography

• Chloroplasts contain several different   photosynthetic pigments   within  photosystems embedded in their thylakoid membranes
• The colour of a pigment is due to the wavelengths of light reflected by that pigment, e.g. chlorophylls absorb light at the red and blue ends of the visible spectrum and reflect light in the green part of the spectrum, so appear as green pigments
• Chromatography can be used to   separate and identify chloroplast pigments   that have been extracted from a leaf

Chromatography

• Different components   within a mixture travel through materials at   different speeds due to their size or charge
• This causes different components to   separate
• An R f value can be calculated for each component of the mixture on the basis of its rate of movement
• The mixture of pigments is passed through paper made of cellulose
• The mixture of pigments is passed through a thin layer of an adsorbent, e.g. silica gel
• The pigments travel faster than through paper, so they separate more distinctly
• Leaf sample
• Dropping pipette
• Pestle and mortar
• Filter paper or TLC paper
• Capillary tube
• Beaker or boiling tube
• Chromatography solvent
• Do not use a pen as the ink will separate into pigments within the experiment and obscure the results
• It is important to choose a healthy leaf that has been in direct sunlight so you can be sure it contains many active photosystems
• Acetone is an organic solvent and therefore fats, such as the phospholipid membranes in plant cells, dissolve in it
• Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid membranes to release the pigments
• Extract some of the pigment using a capillary tube and spot it onto the dot in the centre of the pencil line you have drawn
• The solvent will move up the paper
• The pigment mixture will be   dissolved   in the   solvent  and carried with the solvent as it moves
• Leave the paper suspended in the solvent until the solvent has almost reached the top of the paper
• The solvent may continue moving after the paper is removed from it, so it is important to draw a pencil line immediately
• The pigments should have separated out and there should be different spots on the paper at different heights above the pencil line; these are the separate pigments
• Calculate the R f value for each pigment spot

R f   value = distance travelled by pigment ÷ distance travelled by the solvent

• Always measure to the centre of each spot of pigment
• Chromatography can be used to   separate and identify chloroplast pigments   that have been extracted from a leaf as each pigment will have a unique R f   value
• Larger, less soluble molecules will travel more slowly and therefore have a   smaller R f   value
• Smaller, more soluble molecules will travel faster and therefore have a   larger R f   value
• Carotenoids   have the   highest R f   values, usually close to 1
• Chlorophyll   b   has a   much lower R f   value
• Chlorophyll   a   has an R f   value somewhere   between   those of carotenoids and chlorophyll   b

Chromatography can be used to separate photosynthetic pigments, which can then be identified by their R f   values. 

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Kitchen Science Experiments to Try at Home

• Electricity
• Kitchen Science Experiments

Chlorophyll Chromatography

Part of the show the science of farming.

Ingredients

Instructions

Cut a piece of filter paper about 1cm wide and a bit longer than your jar is tall.

Place your leaf over the top of the strip and roll the coin across the strip about 2cm from the bottom.

Now add a little acetone (nail varnish remover) to the bottom of your jar, half a cm is plenty.

Suspend your strip of paper so the bottom end is sticking a few mm into the acetone. you can hold it up by folding the top end of the strip over forming a hook which will rest over the lip of the jar.

Wait a few minutes, with any luck you should see an interesting effect.

Try the experiment with a strongly coloured leaf like a purple one.

- Chromatography is pretty  much a black art, and is very dependent on the exact type of paper you are using and the solvent you are using, so if it doesn't work try a different type of paper.

- To get more pigment into the paper and produce a better result you can add some leaf juice, let the paper dry out, and then add some more - repeat as many times as you have the patience for.

- Don't let the paper touch the side of the jar, or all the solvent will move up through the gap between the paper and the glass bypassing your sample.

- Doing the experiment inside a jar seems to be important - probably because it gets filled with acetone vapour and so reduces the evaporation rate of the acetone.

- The experiment only seems to work well on filter paper of one sort or another - probably because it is designed not to stick to things where as most other forms of paper are designed to stick to inks, or what you spilt on the table.

You should find that the acetone slowly rises up the paper, and when it reaches the green mark it takes some of the colour with it, and seperating out the colours.

You should find that may purple leaves still contain green pigments - otherwise they couldn't photosynthesize.

Explanation

This is a technique called chromatography, it works because when you put the paper into the solvent, the solvent is drawn up into the paper by surface tension. This means that there is a flow of liquid over the paper. Some substances spend more time in the solvent and others spend more time on the paper, so they move at different speeds, and get separated out.

This is a very powerful technique which is widely used by chemists to separate out mixtures, both analytically to find out what is in the mixture and to separate out different products of a reaction. The speed of each substance is very dependent on the solvent and the substrate (in this case filter paper) so it is a case of finding the right combination of the two to separate out the substances you want to separate.

Why was there more than one colour of green?

This particlular set  of substrate and solvent has been optimised to separate out different forms of chlorophyll - the pigment which gives plants their colour and is sued in photosynthesis (apparently the ideal combination is dry acetone moving over icing sugar, but dry acetone is hard to get hold of at home). There are in fact 4 types of chlorophyll each having a different absorption spectra. There are two main types found in plants, both which look green but one bluer and the other yellower, so you see two greens. This means that the plant can absorb a greater range of colours of light, and wastes less of the spectrum.

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Separation of Plant Pigments by Paper Chromatography

Plant pigments – an introduction.

A compound that absorbs light is called a pigment. Chlorophylls a and b are primary photosynthetic pigments that absorb light for photosynthesis . The accessory pigments carotenoids and xanthophyll absorb light and pass it to chlorophyll a. Even though chlorophyll is the primary pigment , the other pigments are essential to the plant's ability to produce colour and engage in photosynthesis because they absorb each light differently and effectively across the electromagnetic spectrum.

Paper Chromatography

Chromatography, which means "colour writing," is a Greek term that is formed from the words "chromo" and "graph". Chromatography enables the separation of the constituent parts of a given mixture, enabling scientists to observe and produce findings and theories. 

Paper chromatography is a method for classifying dissolved substances according to how soluble they are in a given solvent, such as chlorophyll, carotene, and xanthophyll. Paper chromatography can be used to separate the colours in plant cells. The stationary element in chromatography paper permits the reaction between the solute and solvent to take place and produce results.

Leaf Chromatography

The separation of leaf colours using chromatography is known as leaf chromatography. Leaf chromatography is an experiment that is conducted to determine the colour of the photosynthetic pigments.

The experiment is conducted to learn about the pigments in the leaf, and it is mostly done by using paper and thin-layer chromatography. Let’s discuss some brief points of leaf chromatography.

There is a procedure by which this experiment is conducted in labs.

Sample leaves should be crushed into small pieces and put in a mortar for pestle grinding. Add solvent and keep using the pestle to crush.

Then, carefully draw a pencil line 1 cm from the bottom of the chromatography paper, spot a little amount of leaf extract repeatedly onto the centre of the line, and let each spot dry. 

Make sure the paper dips into the solvent but the spot of leaf extract doesn't by suspending it using a pin attached to a bung within a test tube with a 1 cm depth of solvent. 

The solvent is allowed to run up the paper until it is close to the bung, at which point the paper is removed. The solvent's location is marked, and the paper is allowed to dry. 

The final chromatography paper is known as a chromatogram, and it may be photographed to determine the exact position of each pigment. Next, determine the Rf value for each pigment spot on the chromatogram.

The retention factor is pronounced Rf. The retention factor is calculated by dividing the component's travel distance by the solvent's travel distance.

The colour dissolves as the alcohol goes through the filter paper. Some pigments in the leaf travel more quickly than others because of their properties. 

The pigment's movement rate is measured by the Rf (retention factor) value. Rf value = distance transported by pigment from origin to centre of pigment spot/distance from the origin to the solvent front. By applying this formula, you can determine the Rf value.

The pigments in the plant's leaf are separated by paper chromatography, i.e., separation chromatography. It is the same as a leaf chromatography experiment. The process of paper chromatography is also the same as the leaf chromatography experiment. 

Separation of Chlorophyll Pigments by Paper Chromatography

The chlorophyll molecule is present in the leaf and can be separated by using paper chromatography. The paper chromatography separates the pigments in the leaf based on the distance travelled by pigment molecules on the paper in a nonpolar solvent.

Separation of Plant Pigments by Paper Chromatography Diagram

The Experimental Setup of Paper Chromatography

Chromatogram Report

The final chromatography paper is known as a chromatogram, and it may be photographed to determine the exact position of each pigment. The pattern of pigment spots on the chromatography paper at the conclusion of the experiment is called a chromatogram. Along with the alcohol, the pigments also migrate along the strips of paper.

Chromatography Conclusion

Carotene is identified as having the lowest molecular weight by its yellow to orange tint near the top of the paper. In the pigment separation of chlorophyll, chlorophyll may be distinguished by its blue or dark green hue. When chlorophyll pigments are separated, the colour yellow-light green identifies chlorophyll B. In the chromatography solvent, xanthophyll is more soluble since it has gone up the paper. This describes the conclusion of paper chromatography.

Conclusion 

The pigments are light-absorbing molecules and are separated by using paper chromatography techniques in the lab. The pigments move on the paper based on their solubility in the solvent. Along with the alcohol, pigments also migrate along the strips of paper. Some pigments in the leaf travel more quickly than others because of their properties.