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

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

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Looking the end over a lush green valley or forest, the is fascinating to watch the variety of various shades. Leaves range from irradiate to dark and even speckled. The color are identified by the existence of various pigments, countless of which space responsible for among the most interesting and also important metabolic reaction in living organisms: photosynthesis.

Photosynthetic colors are situated in the chloroplast of the leaf. They catch energy indigenous the visible irradiate spectrum, which they usage to synthesise carbohydrate from inorganic matter. There room many species of photosynthesis pigments, yet the 2 main teams are chlorophylls and also carotenoids (which are further separation into 2 classes: carotenes and also xanthophylls). Each type absorbs a different wavelength, so that with each other they capture an ext light.

Chlorophylls room the pigments generally responsible for photosynthesis. They absorb red and also blue light, and reflect green light, i beg your pardon is what provides leaves their eco-friendly colour. Carotenoids, ~ above the other hand, reflect yellow, orange and red – the color of leaves throughout autumn. Throughout this time that year, chlorophyll breaks under so the carotinoid pigments become visible.

Carotenoids help with photosynthesis by absorbing wavelengths that light the chlorophylls can not absorb. They transfer power to chlorophyll molecules and also also assist to safeguard the leaf from excess light – castle absorb excess light energy and dissipate that as warmth to protect against it indigenous damaging the leaf.

Other non-photosynthetic pigments, such together anthocyanins or various other flavonoids, identify the colour of flowers, so your absorption spectra vary. The duty of this pigments is to attract insects or birds because that pollination.

Absorption spectrum for photosynthetic pigmentsNicola Graf

Separating sheet pigments making use of thin-layer chromatography

This write-up presents a an easy laboratory experiment to know leaf pigments. Students use thin-layer chromatography to different the various pigments the are existing in two different leaf extracts. They determine each pigment and determine whether the two extracts have any kind of pigments in common. The experiment is an ideal for students aged 11–16 and takes 1–2 hours to complete.

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

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

For the thin-layer chromatography, we use a combined mobile step of hexane, acetone and trichloromethane (3:1:1) together it gives the ideal separation result. However, the requires component of the activity to be lugged out inside a fume hood by the teacher. This mobile step separates the pigments many clearly, however you could adapt the activity to usage mobile phases of hexane or ethanol alone, i beg your pardon the college student can bring out themselves. Both hexane and ethanol successfully separate the pigments, however the distinction between each pigment is no as clean as when the linked solvent is used.


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

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


The complying with steps need to be carried out by the students:

Place your very first leaf sample in the mortar. Pipette 1 ml that acetone into the mortar and also use the pestle to grind the sample until the leaf 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 repeat actions 1–2 using the 2nd leaf sample. Use a new pipette to include 1 ml of acetone and also use this pipette to transfer the mixture come a new well of 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 v your fingers.Using your very first pipette (take treatment not to mix increase which pipettes were provided for each sheet sample), draw up some of your very first leaf sample. Use a single, little drop come the pencil line on the left hand next of the color layer analyzer plate. Make sure to leave enough room to to the right the 2nd sample ~ above the right hand side.Wait a couple of seconds until it dries, and apply a 2nd drop ~ above the exact same spot. Proceed until girlfriend have included around 10 drops.Using your second pipette, repeat procedures 5 and also 6 for the second leaf sample by including it to the ideal hand side of the plate.Allow the plate to dry completely.

The adhering to steps have to be brought 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 an unified solvent come the beaker. Friend should include only a shallow layer of solvent, so that the pencil heat on the color layer analyzer plate will not be submerged.Place the color layer analyzer plate vertically right into the beaker, with the pencil heat at the bottom, and also cover the beaker with a clock glass. Students deserve to watch as the solvent moves up the plate and also the colors separate.Wait until the solvent has travelled roughly 6 centimeter from the beginning point (this will certainly take roughly 15–30 minutes) prior to removing the plate native the beaker, leaving it within the fume hood.Use a pencil to easily mark the furthest point reached by the solvent. Allow the plate come dry totally before removing that from the fume hood.

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

Photograph the chromatogram as shortly as that is dry. The colours will certainly fade in ~ a few hours. Publish out a copy that the photograph for your notes.Using the color layer analyzer photo, try to occupational out how plenty of pigments are existing in each leaf extract.Now look in ~ the chemical frameworks of different pigments (see figure 1). Can you recognize which colours is i beg your pardon (see the explanation ar for an ext guidance)? write down her answers.Measure the ranges travelled by the solvent and the pigments, and also calculate the retardation variable (Rf) using the complying with equation:Rf = (distance took trip by pigment) / (distance travelled by solvent) 

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

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


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

In our instance (see number 2), both leaf extracts contained four pigments. Colors 4 relocated a shorter distance 보다 pigment 1, indicating that pigment 4 is much more polar and also pigment 1 is much less polar. By looking at the chemical structures of different pigments and also the polar and also nonpolar groups, college student can try to recognize the pigments in each of the sheet extracts.

They will need to know that, the the practical groups existing in the colours in figure 1, alcohol groups are the most polar, ester and ether teams the the very least polar, and aldehyde and also ketone groups are in between. Indigenous this, we deserve to deduce the carotenes room the least polar pigments (no polar groups), and also xanthophylls are the most polar (two alcohol groups, one at each finish of the molecule). Therefore, colours 1 and 2 are most likely to it is in carotenes, and also pigment 4 is likely to it is in a xanthophyll. Colors 3 is most likely to it is in chlorophyll, since it is more polar than carotenes however less polar 보다 xanthophylls. You deserve to observe the characteristic eco-friendly colour from chlorophyll ~ above the chromatogram.

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

Now look in ~ the Rf values, which variety between 0 and 1, with 0 being a pigment the does not move at all, and also 1 denote a pigment the moves the same distance together the solvent. The Rf value varies relying on the solvent used, but the general order the the colours (from the greatest to the shortest Rf value) usually remains the same, since the nonpolar compound move more than the polar compounds. Rf values for miscellaneous pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are presented in table 1.

Table 1: Rf worths for a range of tree pigments, calculated from a chromatogram utilizing 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 some of the adhering to questions to gauge their expertise of tree pigments and also 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 space they?If chlorophyll is the most crucial photosynthetic pigment, i beg your pardon colours that the clearly shows spectrum are most helpful to a plant for photosynthesis?Seaweeds are frequently yellow-brown in colour. Perform you think light from the red end or the blue end of the spectrum penetrates water best?What species of plants have actually non-green leaves? How could you discover out what pigments lock contained?Where room photosynthetic pigments situated within a leaf?Why is it advantageous for plants to save several various photosynthetic pigments?Why is it crucial to use a nonpolar solvent (such together hexane, acetone and trichloromethane) and also not a polar solvent (such as water) come investigate tree pigments utilizing thin-layer chromatography?Why have to you avoid emotional the thin-layer chromatography plate?Why need to the bowl be totally dry before putting it right into the beaker?Why perform some pigments have actually a bigger Rf value than others?


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Josep Tarragó-Celada is a phd student in biochemistry at the faculty of biological in the Universitat de Barcelona, Spain. His work focuses 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 activity at the 2018 manual Science conference in Barcelona, and also they frequently organise and also participate in educational tasks to aid bridge the gap between university and an additional school students.


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

This practical activity affords college student the possibility to move past basic record chromatography come the more facility technique that thin-layer chromatography. This cross-curricular job will engage students who enjoy biology-based object such as photosynthesis as well as students who reap the problem-solving aspect of analytical approaches in chemistry.

The activity is most suitable for students age 14–16 as component of a science club or extension activity. In enhancement to the main method, the authors administer suggestions because that using various solvents to allow students to carry out the experiment entirely independently. With additional detail, the task could likewise be advantageous for students aged 16–19.

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