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

Use thin-layer chromatography to uncover the range of pigments the play a duty in photosynthesis and give pipeline their colour.

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Looking out over a lush green valley or forest, it is fascinating to view the variety of different shades. Leaves selection from light to dark and even speckled. The color are identified by the presence of various pigments, many of which space responsible for one of the most interesting and also important metabolic reactions in living organisms: photosynthesis.

Photosynthetic colors are located in the chloroplasts of the leaf. They record energy from the visible irradiate spectrum, i beg your pardon they usage to synthesise carbohydrates from not natural matter. There space many types of photosynthetic pigments, however the two main teams are chlorophylls and carotenoids (which room further break-up into 2 classes: carotenes and also xanthophylls). Each form absorbs a different wavelength, therefore that together they capture much more light.

Chlorophylls room the pigments mostly responsible for photosynthesis. Castle absorb red and blue light, and reflect green light, i m sorry is what offers leaves their green colour. Carotenoids, ~ above the various other hand, reflect yellow, orange and also red – the colour of leaves throughout autumn. During this time of year, chlorophyll breaks down so the carotenoid pigments end up being visible.

Carotenoids help with photosynthesis by taking in wavelengths that light that chlorophylls cannot absorb. They transfer power to chlorophyll molecules and also assist to safeguard the leaf from excess light – lock absorb surplus light energy and also dissipate the as warmth to avoid it native damaging the leaf.

Other non-photosynthetic pigments, such together anthocyanins or other flavonoids, recognize the color of flowers, so their absorption spectra vary. The function of this pigments is to entice insects or birds for pollination.

Absorption spectrum because that photosynthetic pigmentsNicola Graf

Separating leaf pigments using thin-layer chromatography

This short article presents a simple laboratory experiment to understand leaf pigments. Students usage thin-layer color layer analyzer to different the assorted pigments that are current in two various leaf extracts. They determine each pigment and determine whether the 2 extracts have any pigments in common. The experiment is suitable for students age 11–16 and takes 1–2 hrs to complete.

Note that we offered leaves from Epipremnum aureum (commonly known as devil’s ivy) and also Ficus benjamina (commonly well-known as weeping fig), yet any types could be supplied for the sheet extracts. Girlfriend might additionally like to lug out the experiment using a brightly coloured flower, such together those in the Petunia genus, and additionally a yellow or orange leaf.

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

For the thin-layer chromatography, we usage a an unified mobile step of hexane, acetone and also trichloromethane (3:1:1) as it provides the finest separation result. However, the requires part of the activity to be brought out within a fume hood by the teacher. This mobile phase separates the pigments many clearly, however you might adapt the activity to usage mobile phases that hexane or ethanol alone, i beg your pardon the college student can lug out themselves. Both hexane and also ethanol effectively separate the pigments, however the difference between each pigment is no as clear as as soon as the an unified solvent is used.


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

A lab coat, gloves and also eye protection have to be worn. The solvents supplied in this experiment space flammable, therefore they have to not be used near flames. The linked solvent (hexane, acetone and also trichloromethane) need to only be offered inside a fume hood due to the volatility, smell and health risks associated with it.


The following steps should be lugged out by the students:

Place your first leaf sample in the mortar. Pipette 1 ml that acetone right into the mortar and use the pestle to grind the sample till the sheet is damaged down.Transfer the mixture to a well of the spotting tile utilizing the pipette.Wash the mortar and pestle, and repeat measures 1–2 using the 2nd leaf sample. Use a brand-new pipette to include 1 ml the acetone and use this pipette to transfer the mixture come a new well that the spotting tile.Take the chromatography plate and also draw a horizontal heat 1.5 cm from the bottom making use of a pencil. Take care not come touch the plate through your fingers.Using your an initial pipette (take care not to mix up which pipettes were provided for each sheet sample), draw up few of your first leaf sample. Apply a single, tiny drop come the pencil heat on the left hand side of the color layer analyzer plate. Make certain to leaving enough space to right the 2nd sample ~ above the appropriate hand side.Wait a couple of seconds till it dries, and apply a 2nd drop ~ above the exact same spot. Proceed until friend have added around 10 drops.Using your second pipette, repeat measures 5 and also 6 for the 2nd leaf sample by including it come the right hand next of the plate.Allow the bowl to dried completely.

The following steps have to be brought out by the teacher:

Inside the fume hood, combine the solvents in the following proportions: hexane, acetone and also trichloromethane, 3:1:1.Add the an unified solvent come the beaker. You should include only a shallow layer of solvent, so that the pencil line on the chromatography plate will certainly not it is in submerged.Place the chromatography plate vertically into the beaker, v the pencil heat at the bottom, and also cover the beaker with a clock glass. Students have the right to watch as the solvent moves up the plate and also the colors separate.Wait until the solvent has actually travelled roughly 6 cm from the starting point (this will certainly take around 15–30 minutes) prior to removing the plate native the beaker, leaving it within the fume hood.Use a pencil to conveniently mark the furthest allude reached through the solvent. Enable the plate come dry fully before removing that from the fume hood.

The following steps must be carried 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. Print out a copy that the picture for your notes.Using the color layer analyzer photo, try to occupational out how countless pigments are existing in each sheet extract.Now look in ~ the chemical frameworks of various pigments (see figure 1). Can you determine which colors is i beg your pardon (see the explanation ar for much more guidance)? create down your answers.Measure the distances travelled by the solvent and the pigments, and calculate the retardation variable (Rf) using the adhering to 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 your answers correct?

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


The different pigments in a sheet extract room separated based upon their affinities for the stationary phase (the silica top top the thin-layer chromatography plate – a polar substance) and the mobile phase (the solvent – a nonpolar substance). Compounds v a high affinity because that the solvent (i.e. Nonpolar compounds) will move much further than compounds v a high affinity for silica (i.e. Polar compounds).

In our instance (see figure 2), both sheet extracts had four pigments. Colours 4 moved a shorter distance than pigment 1, indicating the pigment 4 is more polar and pigment 1 is less polar. By looking at the chemical frameworks of various pigments and the polar and also nonpolar groups, students can shot to determine the pigments in each of the sheet extracts.

They will require to know that, of the functional groups present in the pigments in figure 1, alcohol groups are the most polar, ester and ether groups the least polar, and aldehyde and also ketone groups are in between. Indigenous this, we can deduce that carotenes are the least polar colors (no polar groups), and xanthophylls are the many polar (two alcohol groups, one at each end of the molecule). Therefore, colours 1 and 2 are most likely to be carotenes, and pigment 4 is likely to be a xanthophyll. Colors 3 is most likely to be chlorophyll, because it is much more polar than carotenes however less polar than xanthophylls. You deserve to observe the characteristic environment-friendly colour from chlorophyll top top the chromatogram.

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

Now look at the Rf values, which variety between 0 and 1, through 0 being a pigment that does not relocate at all, and 1 describe a pigment that moves the same distance as the solvent. The Rf worth varies depending on the solvent used, but the basic order of the colours (from the highest possible to the lowest Rf value) usually remains the same, because the nonpolar link move further than the polar compounds. Rf worths for assorted pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in table 1.

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


After the experiment, you can ask your students few of the complying with questions to gauge their expertise of plant pigments and also thin-layer chromatography.

Look at absorb spectra for various plant pigments. I m sorry pigments absorb the most light native the red finish of the spectrum? What colour room they?If chlorophyll is the most necessary photosynthetic pigment, i m sorry colours of the clearly shows spectrum are most useful to a plant because that photosynthesis?Seaweeds are regularly yellow-brown in colour. Do you think light from the red finish or the blue end of the spectrum penetrates water best?What species of plants have non-green leaves? How might you discover out what pigments lock contained?Where room photosynthetic pigments situated within a leaf?Why is it beneficial for tree to save several various photosynthetic pigments?Why is it essential to use a nonpolar solvent (such together hexane, acetone and trichloromethane) and also not a polar solvent (such as water) to investigate tree pigments utilizing thin-layer chromatography?Why need to you avoid emotional the thin-layer color layer analyzer plate?Why should the key be fully dry before putting it into the beaker?Why do some pigments have a larger Rf value than others?


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

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

Together, they presented this task at the 2018 hand-operated Science conference in Barcelona, and they generally organise and participate in educational activities to aid bridge the gap in between university and an additional school students.


Combining the outdoor aspect of nature through the identification of various chemical structures produces a perfect applied science lesson. The analysis of the various pigments in leaves has a clear visual result that can then be regarded the chemical frameworks of the various photosynthetic pigments.

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

The task is most suitable for students age 14–16 as component of a science club or expansion activity. In enhancement to the main method, the authors provide suggestions because that using different solvents to enable students to carry out the experiment entirely independently. With further detail, the activity could likewise be helpful for students age 16–19.

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Many new terms space introduced, for this reason the post provides an excellent chance to an obstacle students come understand concepts such together mobile and stationary phases, polarity that molecules and how biological is fundamentally based on chemical structure blocks.