Keywords: Separating mixtures, structure determination, valuable techniques, Bonding and polarity, Photosynthesis

Use thin-layer chromatography to uncover the variety of pigments that play a function in photosynthesis and give leaves their colour.

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Looking the end over a lush green valley or forest, it is fascinating to view the array of various shades. Leaves range from irradiate to dark and also even speckled. The colours are identified by the existence of different pigments, many of which space responsible for among the many interesting and also important metabolic reactions in life organisms: photosynthesis.

Photosynthetic colors are located in the chloroplast of the leaf. They capture energy native the visible irradiate spectrum, which they usage to synthesise carbohydrates from inorganic matter. There room many varieties of photosynthetic pigments, however the two main teams are chlorophylls and carotenoids (which space further separation into two classes: carotenes and also xanthophylls). Each type absorbs a different wavelength, for this reason that with each other they capture an ext light.

Chlorophylls space the pigments mostly responsible because that photosynthesis. They absorb red and blue light, and reflect green light, i m sorry is what provides leaves their green colour. Carotenoids, ~ above the various other hand, reflect yellow, orange and red – the colour of leaves throughout autumn. Throughout this time that year, chlorophyll breaks down so the carotinoid pigments become visible.

Carotenoids help with photosynthesis by soaking up wavelengths that light the chlorophylls can not absorb. Castle transfer energy to chlorophyll molecules and also also help to safeguard the leaf from excess irradiate – castle absorb surplus light energy and dissipate the as warm to prevent it native damaging the leaf.

Other non-photosynthetic pigments, such together anthocyanins or other flavonoids, determine the color of flowers, so your absorption spectra vary. The function of these pigments is to tempt insects or birds for pollination.

Absorption spectrum for photosynthetic pigmentsNicola Graf

Separating sheet pigments utilizing thin-layer chromatography

This post presents a simple laboratory experiment to know leaf pigments. Students usage thin-layer chromatography to different the miscellaneous pigments that are existing in two various leaf extracts. They recognize each pigment and also determine even if it is the 2 extracts have any type of pigments in common. The experiment is suitable for students aged 11–16 and also takes 1–2 hours to complete.

Note the we provided leaves from Epipremnum aureum (commonly known as devil’s ivy) and also Ficus benjamina (commonly recognized as weeping fig), but any varieties could be provided for the leaf extracts. Girlfriend might likewise like to lug out the experiment utilizing a brightly coloured flower, such together those in the Petunia genus, and likewise a yellow or orange leaf.

Leaves the Epipremnum aureum, typically known as devil’s ivyJoydeep/Wikimedia Commons, CC BY-SA 3.0
Leaves the Ficus benjamina, commonly known as weeping figJM Garg/Wikimedia Commons, CC by 3.0

For the thin-layer chromatography, we use a linked mobile step of hexane, acetone and also trichloromethane (3:1:1) together it provides the finest separation result. However, the requires component of the activity to be lugged out within a fume hood by the teacher. This mobile phase separates the pigments most clearly, but you could adapt the task to use mobile phases that hexane or ethanol alone, which the students can bring out themselves. Both hexane and also ethanol effectively separate the pigments, but the distinction between each pigment is no as clear as once the linked solvent is used.


Leaf samples (e.g. E. Aureum and F. Benjamina), reduced into pieces measuring around 2 centimeter x 2 cmThin-layer chromatography plates (10 cm x 5 cm) pre-coated through silica gelOrganic solvent consisted of of:3 parts hexane, C6H141 component acetone, (CH3)2CO1 component trichloromethane, CHCl3A beaker and watch glass (or color layer analyzer chamber)Spotting tileMortar and pestle1 ml Pasteur pipettes (one because that each sheet sample)Pencil

A lab coat, gloves and eye protection must be worn. The solvents provided in this experiment space flammable, therefore they need to not it is in used close to flames. The merged solvent (hexane, acetone and also trichloromethane) need to only be supplied inside a fume hood because of the volatility, smell and health risks linked with it.


The following steps need to be lugged out by the students:

Place your an initial leaf sample in the mortar. Pipette 1 ml the acetone into the mortar and also use the pestle come grind the sample till the sheet is broken down.Transfer the mixture to a well of the spotting tile making use of the pipette.Wash the mortar and also pestle, and also repeat measures 1–2 making use of the 2nd leaf sample. Use a new pipette to add 1 ml that acetone and also use this pipette to carry the mixture to a brand-new well that the spotting tile.Take the color layer analyzer plate and draw a horizontal heat 1.5 centimeter from the bottom making use of a pencil. Take care not to touch the plate with your fingers.Using your very first pipette (take care not to mix increase which pipettes were provided for each sheet sample), draw up few of your first leaf sample. Use a single, tiny drop come the pencil heat on the left hand next of the color layer analyzer plate. Make certain to leave enough room to fit the second sample on the right hand side.Wait a few seconds until it dries, and apply a second drop on the same spot. Proceed until friend have added around 10 drops.Using your 2nd pipette, repeat measures 5 and 6 because that the 2nd leaf sample by adding it come the ideal hand side of the plate.Allow the plate to dry completely.

The following steps need to be lugged out by the teacher:

Inside the fume hood, incorporate the solvents in the adhering to proportions: hexane, acetone and trichloromethane, 3:1:1.Add the an unified solvent come the beaker. You should add only a shallow great of solvent, so that the pencil line on the color layer analyzer plate will not be submerged.Place the color layer analyzer plate vertically into the beaker, through the pencil line at the bottom, and also cover the beaker through a clock glass. Students deserve to watch as the solvent move up the plate and also the colors separate.Wait until the solvent has travelled roughly 6 cm from the beginning point (this will take around 15–30 minutes) before removing the plate indigenous the beaker, leaving it within the fume hood.Use a pencil to conveniently mark the furthest suggest reached by the solvent. Allow the plate come dry totally before removing it from the fume hood.

The complying with steps should be carried out by the students:

Photograph the color layer analyzer as soon as it is dry. The colours will fade in ~ a couple of hours. Print out a copy the the photo for your notes.Using the color layer analyzer photo, shot to occupational out how countless pigments are present in each leaf extract.Now look in ~ the chemical frameworks of different pigments (see figure 1). Can you recognize which colours is which (see the explanation ar for more guidance)? write down your answers.Measure the distances travelled through the solvent and also the pigments, and also calculate the retardation aspect (Rf) making use of the adhering to equation:Rf = (distance took trip by pigment) / (distance travelled by solvent) 

Record your results in a table. Compare these come the worths in table 1: were her answers correct?

Figure 1: Chemical frameworks of photosynthetic pigments: chlorophyll a and b, β-carotene, and also violaxanthin (a xanthophyll pigment). Polar teams circled in blue, nonpolar groups circled in red. (Click come enlarge)Nicola Graf


The various pigments in a leaf extract space separated based upon their affinities because that the stationary step (the silica ~ above the thin-layer chromatography plate – a polar substance) and the mobile step (the solvent – a nonpolar substance). Compounds with a high affinity because that the solvent (i.e. Nonpolar compounds) will relocate much more than compounds v a high affinity because that silica (i.e. Polar compounds).

In our instance (see number 2), both sheet extracts contained four pigments. Colours 4 relocated a much shorter distance than pigment 1, indicating that pigment 4 is an ext polar and also pigment 1 is much less polar. By looking in ~ the chemical frameworks of various pigments and also the polar and also nonpolar groups, student can try to identify the pigments in each of the sheet extracts.

They will require to recognize that, the the sensible groups current in the colors in figure 1, alcohol teams are the many polar, ester and also ether groups the the very least polar, and aldehyde and ketone teams are in between. Indigenous this, we deserve to deduce that carotenes space the least polar pigments (no polar groups), and also xanthophylls room the most polar (two alcohol groups, one at each end of the molecule). Therefore, colours 1 and also 2 are most likely to be carotenes, and pigment 4 is most likely to be a xanthophyll. Colours 3 is most likely to be chlorophyll, because it is much more polar than carotenes however less polar than xanthophylls. You have the right to observe the characteristic green colour native chlorophyll on the chromatogram.

Figure 2: Chromatograms and also corresponding Rf values for two leaf samples (E. Aureum and F. Benjamina) using a mobile phase of hexane, acetone and trichloromethaneJosep Tarragó-Celada

Now look in ~ the Rf values, which selection between 0 and also 1, v 0 gift a pigment that does not move at all, and also 1 indicating a pigment the moves the very same distance as the solvent. The Rf worth varies relying on the solvent used, however the general order that the colours (from the highest possible to the shortest Rf value) usually continues to be the same, because the nonpolar compounds move more than the polar compounds. Rf worths for miscellaneous pigments (using hexane, acetone and also trichloromethane (3:1:1) for the solvent) are presented in table 1.

Table 1: Rf values for a range of plant pigments, calculated from a chromatogram utilizing hexane, acetone and trichloromethane (3:1:1) because that the mobile phase (Reiss, 1994).PigmentRf value
Chlorophyll a0.59
Chlorophyll b0.42


After the experiment, you deserve to ask your students some of the complying with questions to gauge their knowledge of tree pigments and thin-layer chromatography.

Look at absorb spectra for miscellaneous plant pigments. Which pigments absorb the most light native the red end of the spectrum? What colour space they?If chlorophyll is the most vital photosynthetic pigment, i m sorry colours of the visible spectrum room most advantageous to a plant because that photosynthesis?Seaweeds are frequently yellow-brown in colour. Carry out you think light from the red end or the blue finish of the spectrum penetrates water best?What species of plants have non-green leaves? How might you uncover out what pigments lock contained?Where are photosynthetic pigments situated within a leaf?Why is it beneficial for tree to save on computer several various photosynthetic pigments?Why is it important to use a nonpolar solvent (such as hexane, acetone and trichloromethane) and also not a polar solvent (such together water) to investigate plant pigments making use of thin-layer chromatography?Why must you avoid touching the thin-layer chromatography plate?Why need to the bowl be fully dry prior to putting it right into the beaker?Why do some pigments have actually a bigger Rf value than others?


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Josep Tarragó-Celada is a phd student in biochemistry at the faculty of biologic in the Universitat de Barcelona, Spain. His work concentrates on the metabolic reprogramming that cancer metastasis.

Josep M Fernández Novell is a professor in the room of biochemistry and molecular biomedicine at the Universitat de Barcelona.

Together, they gift this task at the 2018 manual Science conference in Barcelona, and they commonly organise and also participate in educational tasks to assist bridge the gap between university and second school students.


Combining the outdoor aspect of nature with the identification of various chemical structures produces a perfect used science lesson. The evaluation of the different pigments in leaves has actually a clean visual outcome that can then be concerned the chemical frameworks of the various photosynthetic pigments.

This practical activity affords student the possibility to move past basic file chromatography come the more complex technique the thin-layer chromatography. This cross-curricular task will interact students who reap biology-based topics such together photosynthesis as well as students who reap the problem-solving aspect of analytical techniques in chemistry.

The task is most suitable for students aged 14–16 as component of a scientific research club or extension activity. In enhancement to the main method, the authors provide suggestions for using different solvents to permit students to carry out the experiment totally independently. With more detail, the activity could likewise be advantageous for students aged 16–19.

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Many brand-new terms space introduced, therefore the write-up provides an excellent chance to challenge students come understand ideas such together mobile and also stationary phases, polarity that molecules and how biology is fundamentally based upon chemical structure blocks.