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

Use thin-layer color layer analyzer to uncover the selection of pigments the play a role in photosynthesis and also give pipeline their colour.

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Looking the end over a lush green valley or forest, it is fascinating to watch the array of different shades. Leaves selection from irradiate to dark and also even speckled. The colour are determined by the presence of various pigments, plenty of of which space responsible for one of the most interesting and also important metabolic reaction in living organisms: photosynthesis.

Photosynthetic colors are located in the chloroplasts of the leaf. They capture energy from the visible light spectrum, i beg your pardon they usage to synthesise carbohydrates from inorganic matter. There space many types of photosynthesis pigments, yet the 2 main groups are chlorophylls and carotenoids (which room further split into two classes: carotenes and xanthophylls). Each type absorbs a various wavelength, so that together they capture more light.

Chlorophylls space the pigments mainly responsible for photosynthesis. They absorb red and blue light, and also reflect eco-friendly light, which is what gives leaves their green colour. Carotenoids, on the other hand, reflect yellow, orange and also red – the colour of leaves throughout autumn. Throughout this time of year, chlorophyll breaks under so the carotinoid pigments end up being visible.

Carotenoids aid with photosynthesis by soaking up wavelengths that light that chlorophylls cannot absorb. Lock transfer power to chlorophyll molecules and also also aid to protect the sheet from excess irradiate – they absorb excess light energy and also dissipate it as warmth to protect against it from damaging the leaf.

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

Absorption spectrum for photosynthetic pigmentsNicola Graf

Separating sheet pigments utilizing thin-layer chromatography

This short article presents a straightforward laboratory experiment to understand leaf pigments. Students usage thin-layer color layer analyzer to separate the miscellaneous pigments that are present in two different leaf extracts. They recognize each pigment and also determine whether the 2 extracts have any kind of pigments in common. The experiment is an ideal for students age 11–16 and also takes 1–2 hours to complete.

Note that we offered leaves indigenous Epipremnum aureum (commonly well-known as devil’s ivy) and also Ficus benjamina (commonly known as weeping fig), however any varieties could be used for the sheet extracts. Friend might likewise like to bring out the experiment using a brightly coloured flower, such as those in the Petunia genus, and additionally a yellow or orange leaf.

Leaves of Epipremnum aureum, commonly known as 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 combined mobile phase of hexane, acetone and trichloromethane (3:1:1) together it gives the ideal separation result. However, that requires component of the task to be brought out inside a fume hood by the teacher. This mobile phase separates the pigments most clearly, however you can adapt the activity to usage mobile phases that hexane or ethanol alone, i m sorry the students can bring out themselves. Both hexane and also ethanol effectively separate the pigments, but the distinction between each pigment is no as clean as when the combined solvent is used.


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

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


The adhering to steps must be lugged out by the students:

Place your an initial leaf sample in the mortar. Pipette 1 ml that acetone right into the mortar and also use the pestle to grind the sample until the sheet is broken down.Transfer the mixture come a well of the spotting tile using the pipette.Wash the mortar and also pestle, and repeat actions 1–2 using the 2nd leaf sample. Usage a brand-new pipette to include 1 ml the acetone and use this pipette to deliver the mixture come a new well the the spotting tile.Take the chromatography plate and also draw a horizontal line 1.5 cm from the bottom utilizing a pencil. Take treatment not come touch the plate v your fingers.Using your first pipette (take treatment not to mix increase which pipettes were supplied for each leaf sample), draw up some of your an initial leaf sample. Use a single, tiny drop come the pencil heat on the left hand next of the chromatography plate. Make sure to leaving enough an are to to the right the second sample ~ above the best hand side.Wait a couple of seconds till it dries, and also apply a second drop ~ above the very same spot. Continue until friend have added around 10 drops.Using your second pipette, repeat procedures 5 and also 6 because that the 2nd leaf sample by including it to the appropriate hand next of the plate.Allow the plate to dried completely.

The following steps must be brought out by the teacher:

Inside the fume hood, integrate the solvents in the complying with proportions: hexane, acetone and also trichloromethane, 3:1:1.Add the combined solvent to the beaker. Friend should add only a shallow class of solvent, so that the pencil line on the color layer analyzer plate will not it is in submerged.Place the chromatography plate vertically into the beaker, with the pencil line 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 until the solvent has actually travelled around 6 centimeter from the beginning point (this will certainly take approximately 15–30 minutes) prior to removing the plate indigenous the beaker, leaving it inside the fume hood.Use a pencil to conveniently mark the furthest allude reached through the solvent. Allow the plate come dry fully before removing the from the fume hood.

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

Photograph the chromatogram as quickly as that is dry. The colours will fade in ~ a couple of hours. Publish out a copy of the photo for her notes.Using the chromatogram photo, try to work-related out how many pigments are current in each sheet extract.Now look at the chemical frameworks of various pigments (see number 1). Can you determine which colors is which (see the explanation ar for an ext guidance)? write down your answers.Measure the ranges travelled by the solvent and the pigments, and calculate the retardation element (Rf) utilizing the complying with equation:Rf = (distance took trip by pigment) / (distance took trip by solvent) 

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

Figure 1: Chemical structures of photosynthesis pigments: chlorophyll a and 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 various pigments in a leaf extract space separated based on their affinities for the stationary step (the silica ~ above the thin-layer chromatography plate – a polar substance) and the mobile phase (the solvent – a nonpolar substance). Compounds with a high affinity because that the solvent (i.e. Nonpolar compounds) will move much more than compounds through a high affinity because that silica (i.e. Polar compounds).

In our example (see figure 2), both sheet extracts consisted of four pigments. Colors 4 moved a shorter distance 보다 pigment 1, indicating the pigment 4 is much more polar and also pigment 1 is much less polar. Through looking in ~ the chemical structures of different pigments and the polar and nonpolar groups, student can shot to determine the colours in every of the sheet extracts.

They will require to understand that, of the useful groups present in the colours in number 1, alcohol groups are the most polar, ester and ether teams the least polar, and also aldehyde and ketone groups are in between. From this, we have the right to deduce that carotenes space the the very least polar colors (no polar groups), and also xanthophylls space the many polar (two alcohol groups, one at each end of the molecule). Therefore, colors 1 and 2 are most likely to it is in carotenes, and pigment 4 is likely to be a xanthophyll. Colors 3 is likely to be chlorophyll, because it is much more polar than carotenes yet less polar 보다 xanthophylls. You deserve to observe the characteristic eco-friendly colour indigenous chlorophyll ~ above the chromatogram.

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

Now look in ~ the Rf values, which range between 0 and 1, through 0 gift a pigment that does not relocate at all, and also 1 denote a pigment that moves the same distance together the solvent. The Rf value varies relying on the solvent used, but the general order that the pigments (from the highest possible to the lowest Rf value) usually remains the same, because the nonpolar compounds move additional than the polar compounds. Rf values for various 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 plant pigments, calculated from 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 can ask your students some of the following questions to gauge their expertise of plant pigments and thin-layer chromatography.

Look at absorption spectra for assorted plant pigments. I beg your pardon 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 the the visible spectrum space most useful to a plant because that photosynthesis?Seaweeds are often yellow-brown in colour. Perform 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 can you uncover out what pigments lock contained?Where room photosynthetic pigments located within a leaf?Why is it useful for plants to save several different photosynthetic pigments?Why is it necessary to use a nonpolar solvent (such as hexane, acetone and trichloromethane) and not a polar solvent (such together water) to investigate plant pigments utilizing thin-layer chromatography?Why should you avoid touching the thin-layer chromatography plate?Why have to the key be completely dry before putting it into the beaker?Why perform some pigments have actually a larger Rf value than others?


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

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


Combining the outdoor aspect of nature v the identification of various chemical frameworks produces a perfect used science lesson. The evaluation of the different pigments in leaves has actually a clean visual result that have the right to then be related to the chemical structures of the different photosynthetic pigments.

This practical task affords students the possibility to move beyond basic file chromatography to the more complicated technique of thin-layer chromatography. This cross-curricular job will engage students who reap biology-based object such together photosynthesis and students who gain the problem-solving facet of analytical techniques in chemistry.

The activity is most perfect for students aged 14–16 as part of a scientific research club or expansion activity. In addition to the key method, the authors administer suggestions because that using various solvents to enable students to bring out the experiment completely independently. With further detail, the activity could also be helpful for students age 16–19.

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Many brand-new terms space introduced, therefore the short article provides an excellent chance to challenge students to understand ideas such together mobile and stationary phases, polarity of molecules and also how biology is fundamentally based on chemical building blocks.