Subunits That Adhere to Paper During Chromatography

Subunits That Adhere to Paper During Chromatography.

Use thin-layer chromatography to discover the multifariousness of pigments that play a function in photosynthesis and requite leaves their colour.


Looking out over a lush green valley or wood, it is fascinating to see the array of dissimilar shades. Leaves range from light to nighttime and even speckled. The colours are adamant past the presence of different pigments, many of which are responsible for one of the most interesting and important metabolic reactions in living organisms: photosynthesis.

Photosynthetic pigments are located in the chloroplasts of the leafage. They capture free energy from the visible lite spectrum, which they use to synthesise carbohydrates from inorganic matter. There are many types of photosynthetic pigments, but the ii chief groups are chlorophylls and carotenoids (which are further separate into 2 classes: carotenes and xanthophylls). Each type absorbs a unlike wavelength, so that together they capture more light.

Chlorophylls are the pigments primarily responsible for photosynthesis. They absorb ruby-red and bluish light, and reflect greenish light, which is what gives leaves their green colour. Carotenoids, on the other manus, reverberate yellow, orange and cherry – the colour of leaves during autumn. During this fourth dimension of year, chlorophyll breaks downwardly then the carotenoid pigments become visible.

Carotenoids assist with photosynthesis past absorbing wavelengths of light that chlorophylls cannot absorb. They transfer energy to chlorophyll molecules and besides help to protect the foliage from excess light – they absorb surplus low-cal energy and misemploy information technology as heat to forbid information technology from dissentious the foliage.

Other non-photosynthetic pigments, such as anthocyanins or other flavonoids, determine the colour of flowers, so their absorption spectra vary. The function of these pigments is to attract insects or birds for pollination.

Absorption spectrum for photosynthetic pigments
Absorption spectrum for photosynthetic pigments

Nicola Graf

Separating leaf pigments using thin-layer chromatography

This article presents a elementary laboratory experiment to sympathize leafage pigments. Students use sparse-layer chromatography to divide the various pigments that are present in two different leaf extracts. They identify each pigment and determine whether the 2 extracts accept any pigments in common. The experiment is suitable for students aged 11–16 and takes 1–2 hours to complete.

Note that we used leaves from
Epipremnum aureum
(normally known as devil’s ivy) and
Ficus benjamina
(unremarkably known every bit weeping fig),
but whatsoever species could be used for the leaf extracts. Y’all might likewise like to behave out the experiment using a brightly coloured flower, such equally those in the
genus, and also a yellow or orangish leaf.

For the sparse-layer chromatography, we employ a combined mobile phase of hexane, acetone and trichloromethane (3:1:i) as information technology provides the all-time separation upshot. Notwithstanding, it requires part of the action to be carried out inside a smoke hood by the teacher. This mobile phase separates the pigments most clearly, but you could adapt the activity to employ mobile phases of hexane or ethanol alone, which the students can carry out themselves. Both hexane and ethanol successfully separate the pigments, only the distinction between each paint is not as clear as when the combined solvent is used.


  • Leaf samples (e.chiliad.
    East. aureum
    F. benjamina), cut into pieces measuring approximately 2 cm x 2 cm
  • Thin-layer chromatography plates (10 cm ten 5 cm) pre-coated with silica gel
  • Organic solvent comprised of:
    • iii parts hexane, C6H14
    • 1 part acetone, (CHthree)2CO
    • 1 role trichloromethane, CHCl3
  • A beaker and watch glass (or chromatography chamber)
  • Spotting tile
  • Mortar and pestle
  • 1 ml Pasteur pipettes (one for each leafage sample)
  • Pencil

Safety note

A lab coat, gloves and heart protection should be worn. The solvents used in this experiment are flammable, and then they must not exist used near flames. The combined solvent (hexane, acetone and trichloromethane) must only be used inside a smoke hood due to the volatility, smell and health risks associated with it.


The post-obit steps should exist carried out past the students:

  1. Identify your first leaf sample in the mortar. Pipette 1 ml of acetone into the mortar and employ the pestle to grind the sample until the leaf is broken down.
  2. Transfer the mixture to a well of the spotting tile using the pipette.
  3. Launder the mortar and pestle, and repeat steps 1–2 using the 2nd leaf sample. Employ a new pipette to add 1 ml of acetone and use this pipette to transfer the mixture to a new well of the spotting tile.
  4. Take the chromatography plate and draw a horizontal line ane.5 cm from the lesser using a pencil. Take care not to touch on the plate with your fingers.
  5. Using your first pipette (take intendance not to mix upward which pipettes were used for each leaf sample), draw upwards some of your first leaf sample. Utilise a single, small driblet to the pencil line on the left hand side of the chromatography plate. Make sure to leave enough infinite to fit the second sample on the right manus side.
  6. Wait a few seconds until information technology dries, and utilise a second drib on the same spot. Go along until you have added effectually ten drops.
  7. Using your 2nd pipette, echo steps v and 6 for the second foliage sample past adding information technology to the right hand side of the plate.
  8. Allow the plate to dry completely.

The post-obit steps must be carried out by the instructor:

  1. Inside the fume hood, combine the solvents in the following proportions: hexane, acetone and trichloromethane, three:ane:i.
  2. Add the combined solvent to the beaker. Y’all should add only a shallow layer of solvent, so that the pencil line on the chromatography plate will not exist submerged.
  3. Identify the chromatography plate vertically into the chalice, with the pencil line at the lesser, and cover the beaker with a spotter glass. Students can sentry as the solvent moves up the plate and the pigments separate.
  4. Expect until the solvent has travelled roughly 6 cm from the starting bespeak (this will take approximately 15–30 minutes) before removing the plate from the chalice, leaving it inside the fume hood.
  5. Utilise a pencil to quickly mark the furthest point reached by the solvent. Allow the plate to dry completely before removing information technology from the fume hood.

The following steps should exist carried out past the students:

  1. Photograph the chromatogram every bit soon as it is dry. The colours will fade within a few hours. Print out a copy of the photo for your notes.
  2. Using the chromatogram photo, try to piece of work out how many pigments are nowadays in each leaf excerpt.
  3. Now look at the chemical structures of different pigments (see figure 1). Tin you determine which pigment is which (see the explanation section for more guidance)? Write down your answers.
  4. Measure the distances travelled past the solvent and the pigments, and summate the retardation gene (Rf) using the following equation:
    Rf = (distance travelled past pigment) / (distance travelled by solvent) 

Record your results in a tabular array. Compare these to the values in table i: were your answers correct?

Chemical structures of photosynthetic pigments
Figure i: Chemical structures of photosynthetic pigments: chlorophyll a and b,
β-carotene, and violaxanthin (a xanthophyll pigment). Polar groups circled in bluish, nonpolar groups circled in carmine. (Click to enlarge)

Nicola Graf


The different pigments in a leaf extract are separated based on their affinities for the stationary phase (the silica on the sparse-layer chromatography plate – a polar substance) and the mobile stage (the solvent – a nonpolar substance). Compounds with a high analogousness for the solvent (i.eastward. nonpolar compounds) will move much farther than compounds with a high analogousness for silica (i.e. polar compounds).

In our example (run into figure 2), both foliage extracts independent four pigments. Pigment iv moved a shorter altitude than pigment 1, indicating that pigment iv is more than polar and pigment i is less polar. Past looking at the chemical structures of dissimilar pigments and the polar and nonpolar groups, students can try to place the pigments in each of the leaf extracts.

They will need to know that, of the functional groups present in the pigments in figure 1, booze groups are the most polar, ester and ether groups the least polar, and aldehyde and ketone groups are in between. From this, nosotros tin deduce that carotenes are the least polar pigments (no polar groups), and xanthophylls are the most polar (two alcohol groups, one at each end of the molecule). Therefore, pigments ane and 2 are probable to exist carotenes, and pigment four is likely to be a xanthophyll. Pigment 3 is likely to exist chlorophyll, since it is more polar than carotenes merely less polar than xanthophylls. You can find the characteristic green color from chlorophyll on the chromatogram.

Chromatograms and corresponding Rf values for two leaf samples
Figure 2: Chromatograms and corresponding Rf values for two leaf samples (E. aureumandF. benjamina) using a mobile phase of hexane, acetone and trichloromethane

Josep Tarragó-Celada

Now expect at the Rf values, which range between 0 and one, with 0 being a pigment that does not move at all, and ane indicating a pigment that moves the same distance every bit the solvent. The Rf value varies depending on the solvent used, but the full general order of the pigments (from the highest to the lowest Rf value) usually remains the aforementioned, because the nonpolar compounds move further than the polar compounds. Rf values for various pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in tabular array 1.

Table 1: Rf values for a multifariousness of plant pigments, calculated from a chromatogram using hexane, acetone and trichloromethane (3:1:1) for the mobile phase (Reiss, 1994).
Pigment Rf value
β-carotene 0.98
Chlorophyll a 0.59
Chlorophyll b 0.42
Anthocyanins 0.32-0.62
Xanthophylls 0.fifteen-0.35


After the experiment, you can ask your students some of the following questions to gauge their understanding of institute pigments and thin-layer chromatography.

  • Look at absorption spectra for various plant pigments. Which pigments absorb the near light from the red end of the spectrum? What colour are they?
  • If chlorophyll is the most important photosynthetic pigment, which colours of the visible spectrum are most useful to a establish for photosynthesis?
  • Seaweeds are often yellow-brown in color. Do y’all think light from the cherry-red terminate or the blue end of the spectrum penetrates water best?
  • What species of plants have not-green leaves? How could y’all find out what pigments they contained?
  • Where are photosynthetic pigments located within a leaf?
  • Why is information technology useful for plants to contain several different photosynthetic pigments?
  • Why is it important to use a nonpolar solvent (such as hexane, acetone and trichloromethane) and not a polar solvent (such every bit water) to investigate constitute pigments using thin-layer chromatography?
  • Why should you avoid touching the thin-layer chromatography plate?
  • Why should the plate exist completely dry earlier putting it into the beaker?
  • Why do some pigments have a larger Rf value than others?


  • Reiss C (1994)
    Experiments in Constitute Physiology. Englewood Cliffs, NJ, The states: Prentice Hall. ISBN: 0137012853


  • For an infographic explaining the chemicals behind the colour of leaves, visit the Compound Involvement website.
  • Read more nearly the chemic structure of different plant pigments past visiting the Harvard Wood website from Harvard University.


Josep Tarragó-Celada is a PhD pupil in biochemistry at the faculty of biological science in the Universitat de Barcelona, Espana. His work focuses on the metabolic reprogramming of cancer metastasis.

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

Together, they presented this activity at the 2018 Hands-on Science briefing in Barcelona, and they frequently organise and participate in educational activities to help span the gap between academy and secondary schoolhouse students.


Combining the outdoor element of nature with the identification of different chemical structures produces a perfect technology lesson. The analysis of the different pigments in leaves has a clear visual outcome that tin can so exist related to the chemic structures of the different photosynthetic pigments.

This practical activity affords students the opportunity to movement beyond basic paper chromatography to the more circuitous technique of sparse-layer chromatography. This cross-curricular task will appoint students who relish biological science-based topics such equally photosynthesis as well every bit students who enjoy the problem-solving aspect of analytical techniques in chemistry.

The activity is virtually suitable for students anile fourteen–sixteen every bit function of a science club or extension activity. In addition to the primary method, the authors provide suggestions for using different solvents to enable students to carry out the experiment entirely independently. With further detail, the activeness could too be useful for students aged 16–19.

Many new terms are introduced, so the article provides an excellent chance to claiming students to understand concepts such as mobile and stationary phases, polarity of molecules and how biological science is fundamentally based on chemical building blocks.

Caroline Evans, caput of chemistry, Wellington Higher, U.k.


Subunits That Adhere to Paper During Chromatography