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

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

You are watching: Chlorophyll a or b more polar


Looking the end over a lush eco-friendly valley or forest, that is fascinating to see the range of various shades. Leaves range from light to dark and also even speckled. The colour are determined by the presence of different pigments, numerous of which room responsible for among the many interesting and also important metabolic reactions in living organisms: photosynthesis.

Photosynthetic colors are located in the chloroplast of the leaf. They catch energy from the visible light spectrum, i m sorry they use to synthesise carbohydrate from not natural matter. There room many types of photosynthetic pigments, but the 2 main groups are chlorophylls and also carotenoids (which space further separation into 2 classes: carotenes and xanthophylls). Each kind absorbs a various wavelength, for this reason that together they capture more light.

Chlorophylls room the pigments mainly responsible because that photosynthesis. Lock absorb red and also blue light, and also reflect green light, i beg your pardon is what gives leaves their eco-friendly colour. Carotenoids, on the various other hand, reflect yellow, orange and red – the color of leaves during autumn. During this time of year, chlorophyll breaks down so the carotenoid pigments become visible.

Carotenoids assist with photosynthesis by absorbing wavelengths that light the chlorophylls can not absorb. Castle transfer power to chlorophyll molecules and also also assist to safeguard the sheet from excess light – they absorb surplus light energy and dissipate it as warmth to avoid it indigenous damaging the leaf.

Other non-photosynthetic pigments, such as anthocyanins or other flavonoids, identify the colour of flowers, so their absorption spectra vary. The role of these pigments is to lure insects or birds because that pollination.

Absorption spectrum because that photosynthetic pigmentsNicola Graf

Separating sheet pigments using thin-layer chromatography

This post presents a an easy laboratory experiment to know leaf pigments. Students use thin-layer chromatography to separate the assorted pigments the are current in two different leaf extracts. They identify each pigment and determine whether the two extracts have any type of pigments in common. The experiment is an ideal for students age 11–16 and takes 1–2 hours to complete.

Note that we provided leaves from Epipremnum aureum (commonly recognized as devil’s ivy) and Ficus benjamina (commonly known as weeping fig), but any types could be used for the leaf extracts. You might also like to carry out the experiment making use of a brightly coloured flower, such as those in the Petunia genus, and also 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 known as weeping figJM Garg/Wikimedia Commons, CC through 3.0

For the thin-layer chromatography, we usage a merged mobile phase of hexane, acetone and trichloromethane (3:1:1) together it provides the ideal separation result. However, the requires part of the activity to be brought out inside a fume hood through the teacher. This mobile step separates the pigments most clearly, yet you might adapt the activity to use mobile phases that hexane or ethanol alone, which the college student can bring out themselves. Both hexane and ethanol efficiently separate the pigments, yet the difference between each pigment is not as clear as once the merged solvent is used.


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

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


The complying with steps have to be lugged out by the students:

Place your an initial leaf sample in the mortar. Pipette 1 ml the 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 making use of the pipette.Wash the mortar and pestle, and also repeat steps 1–2 making use of the second leaf sample. Use a new pipette to add 1 ml the acetone and also use this pipette to transport the mixture to a new well the the spotting tile.Take the chromatography plate and draw a horizontal heat 1.5 cm from the bottom making use of a pencil. Take treatment not come touch the plate with your fingers.Using your first pipette (take treatment not to mix increase which pipettes were supplied for each leaf sample), attract up few of your first leaf sample. Apply a single, tiny drop to the pencil line on the left hand side of the chromatography plate. Make sure to leaving enough an are to fit the 2nd sample ~ above the right hand side.Wait a couple of seconds until it dries, and also apply a second drop ~ above the exact same spot. Proceed until girlfriend have included around 10 drops.Using your second pipette, repeat measures 5 and 6 for the 2nd leaf sample by adding it to the best hand next of the plate.Allow the key to dried completely.

The following steps must 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 merged solvent come the beaker. You should add only a shallow layer of solvent, so that the pencil heat on the color layer analyzer plate will certainly not be submerged.Place the chromatography plate vertically into the beaker, v the pencil line at the bottom, and also cover the beaker through a clock glass. Students deserve to watch together the solvent move up the plate and the colours separate.Wait till the solvent has travelled roughly 6 cm from the beginning point (this will take roughly 15–30 minutes) prior to removing the plate indigenous the beaker, leave it within the fume hood.Use a pencil to easily mark the furthest suggest reached by the solvent. Enable the plate to dry fully before removing the from the fume hood.

The complying with steps must be lugged out through the students:

Photograph the chromatogram as quickly as it is dry. The colours will fade in ~ a couple of hours. Print out a copy of the picture for her notes.Using the chromatogram photo, try to job-related out how plenty of pigments are current in each sheet extract.Now look at the chemical frameworks of various pigments (see number 1). Deserve to you determine which colours is i beg your pardon (see the explanation ar for more guidance)? compose down her answers.Measure the distances travelled by the solvent and also the pigments, and calculate the retardation element (Rf) utilizing the following equation:Rf = (distance travelled by pigment) / (distance took trip by solvent) 

Record your outcomes in a table. To compare these come the values 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 groups circled in red. (Click to enlarge)Nicola Graf


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

In our example (see figure 2), both sheet extracts consisted of four pigments. Colours 4 relocated a much shorter distance 보다 pigment 1, indicating the pigment 4 is much more polar and also pigment 1 is less polar. By looking at the chemical frameworks of different pigments and also the polar and nonpolar groups, student can shot to identify the pigments in each of the sheet extracts.

They will require to understand that, the the practical groups present in the colors in number 1, alcohol teams are the most polar, ester and also ether groups the least polar, and also aldehyde and ketone groups are in between. Native this, we deserve to deduce that carotenes are the least polar colors (no polar groups), and xanthophylls space the most polar (two alcohol groups, one in ~ each finish of the molecule). Therefore, colors 1 and also 2 are likely to it is in carotenes, and pigment 4 is likely to it is in a xanthophyll. Pigment 3 is likely to be chlorophyll, due to the fact that it is an ext polar 보다 carotenes but less polar 보다 xanthophylls. You can observe the characteristic eco-friendly colour indigenous chlorophyll top top the chromatogram.

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

Now look at the Rf values, which range between 0 and also 1, v 0 being a pigment the does not move at all, and 1 denote a pigment the moves the same distance together the solvent. The Rf worth varies relying on the solvent used, however the basic order of the pigments (from the greatest to the lowest Rf value) usually remains the same, because the nonpolar link move more than the polar compounds. Rf values for various pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are displayed in table 1.

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


After the experiment, you deserve to ask her 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 many light native the red end of the spectrum? What colour are they?If chlorophyll is the most essential photosynthetic pigment, i beg your pardon colours of the visible spectrum are most helpful to a plant for photosynthesis?Seaweeds are often yellow-brown in colour. Perform you think irradiate from the red end or the blue end of the spectrum penetrates water best?What species of plants have actually non-green leaves? How might you discover out what pigments lock contained?Where space photosynthetic pigments located within a leaf?Why is it advantageous for tree to contain several different photosynthetic pigments?Why is it essential to use a nonpolar solvent (such together hexane, acetone and trichloromethane) and not a polar solvent (such together water) come investigate tree pigments using thin-layer chromatography?Why should you avoid touching the thin-layer chromatography plate?Why need to the bowl be completely dry before putting it into the beaker?Why do some pigments have actually a larger Rf worth 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 of 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 activity at the 2018 hand-operated Science conference in Barcelona, and also they generally organise and also participate in educational tasks to aid bridge the gap in between university and secondary school students.


Combining the outdoor element of nature with the identification of different chemical frameworks produces a perfect used 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 possibility to move past basic record chromatography to the more complex technique the thin-layer chromatography. This cross-curricular job will connect students who enjoy biology-based subject such as photosynthesis and students who enjoy the problem-solving element of analytical approaches in chemistry.

The task is most perfect for students age 14–16 as part of a science club or expansion activity. In enhancement to the main method, the authors carry out suggestions because that using various solvents to allow students to carry out the experiment totally independently. With more detail, the task could additionally be valuable for students aged 16–19.

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Many brand-new terms are introduced, so the write-up provides terrific chance to difficulty students come understand concepts such together mobile and stationary phases, polarity that molecules and also how biology is fundamentally based upon chemical structure blocks.