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Plant Detectives Manual: a research-led approach for teaching plant science

Activity 4: Qualitative analyses of photosynthetic pigments by thin-layer chromatography (TLC)

4.1) Introduction and objectives

Chromatography is an analytical technique that allows for the separation and identification of a wide range of compounds. The separation of the components in a mixture is a function of their different affinities for a stationary phase, such as a solid or a liquid, and their differential affinity for a moving phase, such as a liquid or gas. When the stationary phase is solid and the moving phase is liquid, the separation of compounds is governed by their tendency to associate with the mobile (usually hydrophobic) liquid phase or to adsorb onto the solid (usually hydrophilic) surface. The solid phase might be paper, starch or silica gel. If the solid is applied in a thin layer to a supporting glass or plastic plate, the method is called thin-layer chromatography (TLC). In this protocol you will use pre-prepared glass TLC plates coated with silica gel. Click here to view the original procedure.

In TLC, the mixture to be separated is first applied as a spot or a line to the solid phase, and then the mobile solvent is allowed to pass through the applied compounds along the immobile phase (Fig. 6B). The compounds will dissolve in and move with the solvent. The distance travelled by a particular compound will depend on its affinity for the hydrophobic (mobile) phase versus its affinity for the hydrophilic (solid) phase, thus assisting with the identification of the compound. The ratio of the distance travelled by a compound to that of the solvent front is known as the Retardation factor (Rf) value. Unknown compounds may be identified by comparing their Rfs to the Rfs of known standards.

 

The different chemical structure of the plant pigment not only confers different absorption properties, but also different affinities for mobile and stationary phases that allows for their separation. Following the protocol below you will separate, identify and compare the pigment composition of your wild type and mutant plants.

 

The main objectives of Activity 4 are to:

  1. separate individual plant photosynthetic pigments from both wild type and mutant plants
  2. compare pigment composition between genotypes
  3. measure the absorbance spectra of pigments to identify their nature.

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Figure 6. Pigment extraction and characterisation by TLC and spectrophotometry

A) Concentrating pigments prior to TLC. Pigments dissolved in the acetone extract can be concentrated by lowering the acetone concentration to less than 70% (by adding water) to facilitate the partitioning of the pigments to the hexane phase. After mixing and spinning the mixture, pigments will accumulate in the more hydrophobic, less dense organic solvent hexane (dark top phase). B) Schematic representation of a TLC run. Note that samples must be loaded on the pencil line. Load as much as possible to obtain darker, more concentrated spots. Pigments (yellow and green dots) will be resolved as the solvent migrates from bottom to top. C) Absorption spectrum of chlorophyll a (Chla) and chlorophyll b (Chlb). Pigments were removed from the silica, extracted with acetone and their absorbance measured at regular intervals in the range 400 to 700 nanometres (nm). Note the distinctive Chla peak at around 660 nm.

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4.2) Materials

  1. heating block (or desk lamp)
  2. capped bottle. This can be a ~250 millilitre beaker covered with a glass Petri dish. A microscope slide staining dish works best if TLC plates are of the proper size
  3. forceps
  4. gloves
  5. goggles (one per person)
  6. chemical hood
  7. micropipettes of 5–50 microlitres (µl), or glass capillaries; check with demonstrator if you have questions about how to use these
  8. pencil
  9. ruler
  10. scanner or camera
  11. TLC plates (three) (i.e., TLC Silica gel 60 F254, Merck Cat#115341)
  12. waste container
  13. razor blades
  14. weighing paper
  15. spectrophotometer or micro plate reader (i.e., BIO-TEK uQuant)
  16. hexane: acetone: chloroform (2:1:1)
  17. hexane

 

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4.3) Procedure

4.3.1) Pigment concentration

In order to load sufficient pigment sample in the TLC plate, it is necessary to concentrate the pigments that were extracted in Activity 3 in an organic phase (hexane).

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Materials

Method

Hexane, vortex, pipettes, tips

  1. Add 80 µl of hexane and 100 µl of water to each of the two samples (one wild type, one mutant) that you aliquotted in Step 8 of Activity 3 in the tubes containing 500 µl of supernatant.2

Vortex

  1. Vortex for ten seconds.

Microcentrifuge, Eppendorf tubes

  1. Centrifuge four tubes for two minutes at 7000 g in a microcentrifuge to separate organic and aqueous phases.

Pipettes, tips, Eppendorf tubes

  1. Transfer the upper phases (hexane) containing the concentrated pigments to new, LABELLED tubes (Fig. 6A). Keep the tube closed at all times as the hexane phase evaporates rapidly. These samples will be loaded onto the TLC plates next.

4.3.2) Thin-layer chromatography (TLC)

 

Materials

Method

Silica plate, pencil, ruler, gloves

  1. Wearing gloves, mark the edge of each pre-prepared TLC plate with a pencil about 1 centimetre (cm) above the bottom, trying not to touch the silica gel surface. TIP: It is critical that the silica plates are dry before loading the sample to avoid smudges leading to poor runs.

Micropipette or capillary tube

  1. With a micropipette or capillary tube spot the wild type sample on the left of the plate and the mutant one on the right of the plate. Try to make the spots evenly spaced on the pencil line.

Heating block, or lamp

  1. Place the plate on the heating block to evaporate residual hexane. TIP: the sample can be also be dried by placing the slide underneath a desk lamp.

 

  1. Continue to load more sample on to the plate by repeatedly touching the silica with the micropipette or capillary containing 5–10 µl of the pigment. Let the spot dry for ten to 20 seconds and apply again. Be careful not to damage the silica gel layer.

 

  1. Load the sample at least five times, but be careful not to overload as the spots may overlap, resulting in cross contamination. Check with your demonstrator. IMPORTANT: record the number of loads so you know the amount of loaded sample.

Forceps, gloves, chemical hood, beaker covered with glass Petri dish, or microscope slide staining dish; hexane: acetone: chloroform (2:1:1)

  1. Carefully, place the plate into the equilibrated chamber containing enough hexane: acetone: chloroform (2:1:1) mixture to cover the bottom and replace the lid. TIP: a beaker covered with a glass Petri dish, or microscope slide staining dish can be used for this step. IMPORTANT: ensure the plate is placed evenly to ensure the solvent comes into contact with the entire bottom of the TLC plate at once, otherwise the samples will not run evenly. Only the bottom of the TLC plate should be in contact with the solvent, NOT the sample. The solvent will be absorbed by the silica gel and will start to move up the plate through the spotted samples. When it reaches the pigment spot, the pigments will dissolve in the solvent and move with it along the silica gel.

Pencil

  1. Once a clear separation of the pigments is achieved (check with demonstrator), remove the plate before the solvent front runs off at the end of the plate and immediately mark the solvent front with a pencil. The solvent will evaporate rapidly, so act quickly. Alternatively, you can use the end of the plate as your reference.

Block heater or lamp, scanner or camera

  1. Allow the plate to air dry on a block heater or under a lamp. At this point, you can scan or photograph the plate.

Waste container

  1. When you are finished, pour the remaining solvent mixture into the waste container provided and leave the bottles in the chemical hood to dry.

4.3.3) Pigment identification

Although the pigments can be recognised by their colours, a more accurate way to characterise them is by measuring an absorption spectrum. In this section, you will remove the pigment bands for Chla and Chlb (or any other pigment of your choice) from the TLC plate and measure their absorbance at different wavelengths. The amount of light absorption by a substance can be graphed as a function of wavelength; this graph is called an absorption spectrum (Fig. 6C). Absorption spectra are unique for individual pigments and should allow you to identify that molecule by comparing with published spectra.

Materials

Method

Razor blades, weighing paper

  1. Remove the pigment bands from the silica plate you want to analyse by scraping the silica gel from the plate with a razor blade onto a square of weighing paper or aluminium foil.

Eppendorf tubes

  1. Combine all of the scrapings for a given band together in one labelled Eppendorf microcentrifuge tube. Each tube will then contain all of a particular pigment present in the total loaded volume of hexane/pigment extract. (Remember that the total volume of the hexane/pigment phase was 80 µl; it is expected that ALL the pigments were partitioned from the acetone phase to the hexane one.)

Pipette and tips, 80% acetone, vortex

  1. Extract the pigments from the silica gel by adding 500 µl of 80% (v/v) acetone, close the tube and mix well by vortexing.

Block heater

  1. Incubate the silica/acetone mix under a light or in a heated bath for ten minutes, shaking periodically (notice the colour of the silica gel. Is the silica gel white or has some pigment been retained?).

Microcentrifuge

  1. Sediment the mixtures for two minutes at 7000 g in a microcentrifuge to remove the silica gel.

Pipette and tips, 80% acetone, 96 well plate

  1. Transfer 200 µl of 80% acetone into the wells as blanks.

Eppendorf tubes, pipette and tips

  1. After pelleting the silica, call your supervisor before proceeding. Recover the cleared supernatant with a pipette and transfer to a properly labelled microcentrifuge tube. Transfer 200 µl of your samples into the wells as duplicates for each sample.

Plate reader

  1. Transfer the plate into the plate reader. Read the blank and sample at 400 nm and every 20 nm until 800 nm.

 

  1. Calculate the ‘Final’ absorbance by subtracting the blank absorbance from the sample absorbance. Use the ‘Final’ absorbance and graph ‘Absorbance’ vs ‘Wavelength’ (nm) (Fig. 6C).

4.4) Expected outcomes

  1. Characterise the pigment profile for both wild type and mutant plants. For this, use visual examination and also measure the Rf for each pigment spot. Rf is the ratio between the total distance travelled by the pigment and the total distance travelled by the mobile phase (solvent front). The starting point for each measurement is the 1 cm mark.
  2. Identify each of the pigments using your knowledge of pigment structure and colour. The pigments visible on the chromatograms are Chla and Chlb, carotene, xanthophylls (possibly more than one), and pheophytin. Based on time and your interest, you could examine all pigments or those that differ between wild type and mutant. Once you have identified the pigments, you can scan or photograph your plate and label each band.
  3. Plot the absorption spectrum for the pigment of your choice.
  4. OPTIONAL: compare the pigment profile among different genotypes and conditions (i.e., light- versus etiolated-grown tissue, if available).

 


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