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

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

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Looking out over a lush green valley or forest, the is fascinating to watch the range of various shades. Leaves range from irradiate to dark and also even speckled. The colour are determined by the visibility of different pigments, plenty of of which are responsible for among the many interesting and important metabolic reactions in living organisms: photosynthesis.

Photosynthetic colors are located in the chloroplast of the leaf. They capture energy native the visible irradiate spectrum, i m sorry they usage to synthesise carbohydrate from inorganic matter. There are many varieties of photosynthetic pigments, but the two main groups are chlorophylls and also carotenoids (which room further separation into 2 classes: carotenes and also xanthophylls). Each form absorbs a different wavelength, so that with each other they capture an ext light.

Chlorophylls space the pigments mostly responsible because that photosynthesis. Lock absorb red and also blue light, and reflect green light, which is what offers leaves their eco-friendly colour. Carotenoids, on the various other hand, reflect yellow, orange and also red – the colour of leaves during autumn. Throughout this time of year, chlorophyll breaks under so the carotenoid pigments come to be visible.

Carotenoids assist with photosynthesis by soaking up wavelengths the light the chlorophylls can not absorb. Castle transfer power to chlorophyll molecules and also also assist to safeguard the leaf from excess irradiate – they absorb surplus light energy and dissipate it as warm to protect against it native damaging the leaf.

Other non-photosynthetic pigments, such as anthocyanins or other flavonoids, identify the colour of flowers, so your absorption spectra vary. The function of this pigments is to entice insects or birds for pollination.

Absorption spectrum for photosynthetic pigmentsNicola Graf

Separating sheet pigments using thin-layer chromatography

This write-up presents a basic laboratory experiment to know leaf pigments. Students use thin-layer color layer analyzer to different the various pigments that are present in two different leaf extracts. They recognize each pigment and also determine even if it is the two extracts have any type of pigments in common. The experiment is suitable for students aged 11–16 and takes 1–2 hrs to complete.

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

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

For the thin-layer chromatography, we use a merged mobile phase of hexane, acetone and trichloromethane (3:1:1) as it gives the finest separation result. However, the requires part of the activity to be carried out inside a fume hood through the teacher. This mobile phase separates the pigments many clearly, yet you can adapt the activity to usage mobile phases that hexane or ethanol alone, i m sorry the college student can lug out themselves. Both hexane and ethanol effectively separate the pigments, yet the distinction between each pigment is no as clear as when the linked solvent is used.


Leaf samples (e.g. E. Aureum and F. Benjamina), reduced into piece measuring about 2 centimeter x 2 cmThin-layer chromatography plates (10 cm x 5 cm) pre-coated with silica gelOrganic solvent comprised of:3 parts hexane, C6H141 part 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 leaf sample)Pencil

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


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

Place your an initial 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 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 procedures 1–2 using the second leaf sample. Usage a brand-new pipette to add 1 ml that acetone and use this pipette to deliver the mixture come 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 utilizing a pencil. Take treatment not to touch the plate through your fingers.Using your first pipette (take care not come mix up which pipettes were provided for each sheet sample), draw up few of your very first leaf sample. Use a single, tiny drop come the pencil heat on the left hand side of the color layer analyzer plate. Make certain to leaving enough room to right the second sample on the appropriate hand side.Wait a couple of seconds till it dries, and also apply a 2nd drop on the very same spot. Proceed until you have included around 10 drops.Using your second pipette, repeat procedures 5 and also 6 because that the second leaf sample by including it to the right hand side of the plate.Allow the bowl to dry completely.

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

Inside the fume hood, combine the solvents in the complying with proportions: hexane, acetone and trichloromethane, 3:1:1.Add the combined solvent to the beaker. You should include only a shallow class of solvent, so the the pencil line on the chromatography plate will certainly not it is in submerged.Place the color layer analyzer plate vertically right into the beaker, v the pencil heat at the bottom, and cover the beaker v a watch glass. Students deserve to watch together the solvent move up the plate and the pigments separate.Wait till the solvent has travelled approximately 6 cm from the starting point (this will certainly take around 15–30 minutes) prior to removing the plate from the beaker, leave it inside the fume hood.Use a pencil to easily mark the furthest suggest reached by the solvent. Permit the plate come dry totally before removing the from the fume hood.

The adhering to steps should be lugged out through the students:

Photograph the color layer analyzer as soon as it is dry. The colours will fade within a few hours. Publish out a copy the the photo for her notes.Using the color layer analyzer photo, shot to job-related out how plenty of pigments are present in each sheet extract.Now look at the chemical frameworks of various pigments (see number 1). Have the right to you identify which colors is which (see the explanation section for more guidance)? write down your answers.Measure the ranges travelled through the solvent and also the pigments, and calculate the retardation element (Rf) using the adhering to equation:Rf = (distance travelled by pigment) / (distance took trip by solvent) 

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

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


The different pigments in a leaf extract room separated based on their affinities for the stationary step (the silica ~ above the thin-layer color layer analyzer plate – a polar substance) and also the mobile phase (the solvent – a nonpolar substance). Compounds through a high affinity for the solvent (i.e. Nonpolar compounds) will relocate much additional 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. Colors 4 relocated a much shorter distance 보다 pigment 1, indicating that pigment 4 is an ext polar and pigment 1 is less polar. By looking in ~ the chemical structures of different pigments and also the polar and also nonpolar groups, college student can shot to identify the colors in each of the leaf extracts.

They will need to recognize that, the the useful groups current in the colours in number 1, alcohol teams are the many polar, ester and ether groups the least polar, and also aldehyde and also ketone groups are in between. Indigenous this, we deserve to deduce the carotenes room the the very least polar colors (no polar groups), and xanthophylls room the many polar (two alcohol groups, one at each finish of the molecule). Therefore, colors 1 and 2 are most likely to it is in carotenes, and also pigment 4 is most likely to be a xanthophyll. Pigment 3 is most likely to be chlorophyll, since it is much more polar than carotenes but less polar than xanthophylls. You have the right to observe the characteristic eco-friendly colour indigenous chlorophyll top top the chromatogram.

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

Now look in ~ the Rf values, which selection between 0 and 1, v 0 being a pigment that does not move at all, and 1 indicating a pigment the moves the very same distance together the solvent. The Rf value varies relying on the solvent used, but the general order of the colours (from the highest possible to the lowest Rf value) usually remains the same, because the nonpolar compound move further than the polar compounds. Rf values for assorted pigments (using hexane, acetone and also trichloromethane (3:1:1) because that the solvent) are displayed in table 1.

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


After the experiment, you have the right to ask your students several of the complying with questions to gauge their understanding of tree pigments and also thin-layer chromatography.

Look at absorb spectra for miscellaneous plant pigments. I beg your pardon pigments absorb the most light from the red end of the spectrum? What colour room they?If chlorophyll is the most essential photosynthetic pigment, i beg your pardon colours of the clearly shows spectrum space most valuable to a plant for photosynthesis?Seaweeds are frequently yellow-brown in colour. Do you think light from the red end or the blue finish of the spectrum penetrates water best?What varieties of plants have non-green leaves? How can you find out what pigments they contained?Where are photosynthetic pigments situated within a leaf?Why is it advantageous for tree to save on computer several various photosynthetic pigments?Why is it important to usage a nonpolar solvent (such as hexane, acetone and trichloromethane) and also not a polar solvent (such as water) to investigate tree pigments utilizing thin-layer chromatography?Why have to you avoid touching the thin-layer color layer analyzer plate?Why should the bowl be fully dry before putting it right into the beaker?Why do some pigments have a larger Rf worth than others?


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Josep Tarragó-Celada is a doctor student in biochemistry in ~ the faculty of biological 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 department of biochemistry and also molecular medicine at the Universitat de Barcelona.

Together, they presented this activity at the 2018 hand-operated Science conference in Barcelona, and also they commonly organise and also participate in educational activities to assist bridge the gap in between university and second school students.


Combining the outdoor element of nature with the to know of various chemical structures produces a perfect applied science lesson. The evaluation of the various pigments in leaves has a clear visual result that deserve to then be related to the chemical frameworks of the different photosynthetic pigments.

This practical task affords student the opportunity to move beyond basic document chromatography to the more facility technique of thin-layer chromatography. This cross-curricular task will connect students who enjoy biology-based subject such together photosynthesis and students who reap the problem-solving aspect of analytical approaches in chemistry.

The task is most an ideal for students aged 14–16 as part of a scientific research club or expansion activity. In addition to the main method, the authors administer suggestions for using different solvents to allow 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 new terms space introduced, for this reason the article provides fantastic chance to challenge students come understand ideas such together mobile and also stationary phases, polarity of molecules and how biology is fundamentally based upon chemical structure blocks.