Keywords: Separating mixtures, structural determination, useful techniques, Bonding and polarity, Photosynthesis

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

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Looking the end over a lush environment-friendly valley or forest, the is fascinating to check out the variety of different shades. Leaves variety from irradiate to dark and even speckled. The colour are identified by the visibility of different pigments, plenty of of which are responsible for one of the most interesting and also important metabolic reaction in living organisms: photosynthesis.

Photosynthetic pigments are situated in the chloroplast of the leaf. They record energy native the visible light spectrum, i m sorry they use to synthesise carbohydrates from not natural matter. There room many types of photosynthesis pigments, yet the 2 main groups are chlorophylls and also carotenoids (which space further break-up into 2 classes: carotenes and xanthophylls). Each kind absorbs a various wavelength, so that together they capture more light.

Chlorophylls room the pigments primarily responsible because that photosynthesis. Lock absorb red and also blue light, and reflect green light, i m sorry is what provides leaves their eco-friendly colour. Carotenoids, on the other hand, reflect yellow, orange and red – the colour of leaves throughout autumn. Throughout this time of year, chlorophyll breaks down so the carotenoid pigments come to be visible.

Carotenoids assist with photosynthesis by taking in wavelengths of light that chlorophylls can not absorb. Lock transfer power to chlorophyll molecules and also help to protect the sheet from excess irradiate – lock absorb excess light energy and also dissipate that as heat to avoid it from damaging the leaf.

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

Absorption spectrum because that photosynthetic pigmentsNicola Graf

Separating leaf pigments utilizing thin-layer chromatography

This write-up presents a simple laboratory experiment to understand leaf pigments. Students usage thin-layer chromatography to different the miscellaneous pigments that are present in two different leaf extracts. They identify each pigment and also determine even if it is the two extracts have any type of pigments in common. The experiment is perfect for students age 11–16 and takes 1–2 hours to complete.

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

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

For the thin-layer chromatography, we use a merged mobile step of hexane, acetone and trichloromethane (3:1:1) as it gives the finest separation result. However, it requires component of the task to be lugged out within a fume hood by the teacher. This mobile step separates the pigments most clearly, but you can adapt the activity to usage mobile phases the hexane or ethanol alone, i m sorry the students can carry out themselves. Both hexane and ethanol effectively separate the pigments, but the distinction between every pigment is not as clean as as soon as the an unified solvent is used.


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

A lab coat, gloves and also eye protection need to be worn. The solvents provided in this experiment space flammable, for this reason they should not be used near flames. The merged solvent (hexane, acetone and trichloromethane) have to only be used inside a fume hood as result of the volatility, smell and also health risks linked with it.


The complying with steps need to be brought out through the students:

Place your very first leaf sample in the mortar. Pipette 1 ml that acetone into the mortar and use the pestle to grind the sample until the sheet is damaged down.Transfer the mixture to a well of the spotting tile using the pipette.Wash the mortar and also pestle, and also repeat procedures 1–2 utilizing the 2nd leaf sample. Usage a brand-new pipette to include 1 ml of acetone and also use this pipette to carry the mixture come a brand-new well the the spotting tile.Take the chromatography plate and draw a horizontal line 1.5 cm from the bottom using a pencil. Take treatment not to touch the plate through your fingers.Using your an initial pipette (take care not to mix up which pipettes were used for each leaf sample), attract up several of your an initial leaf sample. Apply a single, little drop to the pencil heat on the left hand side of the color layer analyzer plate. Make certain to leave enough room to to the right the 2nd sample ~ above the best hand side.Wait a couple of seconds till it dries, and also apply a 2nd drop on the exact same spot. Continue until girlfriend have added around 10 drops.Using your 2nd pipette, repeat procedures 5 and 6 for the 2nd leaf sample by adding it to the best hand next of the plate.Allow the bowl to dried completely.

The adhering to steps must be carried out by the teacher:

Inside the fume hood, integrate the solvents in the adhering to proportions: hexane, acetone and also trichloromethane, 3:1:1.Add the linked solvent come the beaker. Girlfriend should include only a shallow great of solvent, so the the pencil line on the chromatography plate will certainly not be submerged.Place the color layer analyzer plate vertically right into the beaker, with the pencil heat at the bottom, and cover the beaker v a watch glass. Students can watch as the solvent move up the plate and also the pigments separate.Wait till the solvent has actually travelled approximately 6 cm from the beginning point (this will certainly take approximately 15–30 minutes) prior to removing the plate indigenous the beaker, leave it within the fume hood.Use a pencil to quickly mark the furthest point reached by the solvent. Permit the plate to dry fully before removing it from the fume hood.

The adhering to steps have to be brought out by the students:

Photograph the color layer analyzer as shortly as it is dry. The colours will certainly fade in ~ a couple of hours. Publish out a copy the the photo for her notes.Using the color layer analyzer photo, try to work out how many pigments are present in each leaf extract.Now look at the chemical structures of different pigments (see figure 1). Have the right to you identify which pigment is i beg your pardon (see the explanation section for more guidance)? compose down your answers.Measure the ranges travelled by the solvent and the pigments, and calculate the retardation variable (Rf) utilizing the following equation:Rf = (distance travelled by pigment) / (distance took trip by solvent) 

Record your results in a table. To compare these to the worths in table 1: were her answers correct?

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


The different pigments in a leaf extract are separated based on their affinities because that the stationary step (the silica ~ above the thin-layer chromatography plate – a polar substance) and also the mobile step (the solvent – a nonpolar substance). Compounds v a high affinity for the solvent (i.e. Nonpolar compounds) will move much additional than compounds through a high affinity because that silica (i.e. Polar compounds).

In our example (see figure 2), both sheet extracts had four pigments. Pigment 4 moved a shorter distance than pigment 1, indicating the pigment 4 is more polar and also pigment 1 is less polar. By looking in ~ the chemical structures of different pigments and also the polar and nonpolar groups, college student can try to determine the pigments in each of the leaf extracts.

They will require to recognize that, that the sensible groups current in the pigments in figure 1, alcohol groups are the most polar, ester and ether teams the least polar, and also aldehyde and ketone groups are in between. Native this, we deserve to deduce the carotenes are the the very least polar colours (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 likely to be carotenes, and pigment 4 is likely to it is in a xanthophyll. Colours 3 is likely to be chlorophyll, since it is more polar 보다 carotenes but less polar than xanthophylls. You have the right to observe the characteristic environment-friendly colour native chlorophyll top top the chromatogram.

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

Now look in ~ the Rf values, which selection between 0 and also 1, with 0 being a pigment that does not move at all, and 1 describe a pigment that moves the very same distance together the solvent. The Rf value varies depending on the solvent used, yet the general order that the colours (from the highest possible to the shortest Rf value) usually remains the same, due to the fact that the nonpolar link move additional than the polar compounds. Rf values for miscellaneous pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in table 1.

Table 1: Rf worths for a selection of tree pigments, calculated native a chromatogram using hexane, acetone and trichloromethane (3:1:1) because that the mobile step (Reiss, 1994).PigmentRf value
Chlorophyll a0.59
Chlorophyll b0.42


After the experiment, you have the right to ask her students few of the adhering to questions to gauge their knowledge of tree pigments and also thin-layer chromatography.

Look at absorb spectra for miscellaneous plant pigments. Which pigments absorb the many light native the red finish of the spectrum? What colour room they?If chlorophyll is the most important photosynthetic pigment, i beg your pardon colours the the visible spectrum space most advantageous to a plant for photosynthesis?Seaweeds are regularly yellow-brown in colour. Do you think light from the red finish or the blue finish of the spectrum penetrates water best?What types of plants have actually non-green leaves? How might you find out what pigments they contained?Where space photosynthetic pigments located within a leaf?Why is it advantageous 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 as water) come investigate plant pigments making use of thin-layer chromatography?Why have to you avoid touching the thin-layer color layer analyzer plate?Why should the plate be totally dry before putting it right into the beaker?Why perform some pigments have a bigger Rf value than others?


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

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

Together, they presented this activity at the 2018 hands-on Science conference in Barcelona, and also they generally organise and also participate in educational activities to aid bridge the gap in between university and secondary school students.


Combining the outdoor element of nature v the to know of various chemical frameworks produces a perfect used science lesson. The evaluation of the different pigments in leaves has a clear visual outcome that have the right to then be pertained to the chemical structures of the various photosynthetic pigments.

This practical task affords student the possibility to move beyond basic file chromatography come the more complicated technique that thin-layer chromatography. This cross-curricular job will communicate students who reap biology-based subject such as photosynthesis and also students who gain the problem-solving aspect of analytical approaches in chemistry.

The task is most perfect for students age 14–16 as component of a science club or expansion activity. In addition to the key method, the authors carry out suggestions because that using different solvents to permit students to carry out the experiment completely independently. With more detail, the activity could also be useful for students aged 16–19.

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Many new terms are introduced, for this reason the post provides terrific chance to difficulty students come understand principles such together mobile and also stationary phases, polarity of molecules and how biologic is fundamentally based on chemical building blocks.