This protocol outlines a method for thin-layer chromatography. Thin layer chromatography is an experimental technique used to separate, identify and quantify the different chemical substances contained within a sample. In plant biology, these are usually pigments, and this protocol may be used for leaf, fruit or floral tissue.
Chromatography is the separation of substances – usually pigments (Onozaki, Mato et al. 1999) – from a sample, via their movement from a mobile to a stationary chemical phase. Thin-layer chromatography (hereafter TLC) involves a liquid mobile phase and a solid stationary phase, but other chromatographic methods also use gases and liquids under high pressure. TLC is particularly effective for carotenes, xanthophylls and chlorophylls (Chaffai, Tekitek et al. 2006), which are pigments present in all plants. Chlorophylls are involved in the electron-transfer reactions of photosynthesis; carotenoids – encompassing carotenes and xanthophylls – also take part in light capture and are scavengers for reactive oxygen species produced during photosynthesis (Raven, Evert et al. 1999). TLC is a simple procedure which can easily be undertaken by students, since it does not require complicated machinery like some other chromatographic methods. In TLC, the sample is dissolved in a liquid eluent, which together comprise the mobile phase that moves via capillary action over a glass plate coated in silica or alumina. Pigments are adsorbed onto the silica or alumina (stationary phase) at polar sites; depending on the polarity of the mobile phase the more polar the pigment, the faster and stronger it will adsorb, and the lower down on plate a pigment spot will occur (see also Figure 1). The distances travelled along the plate are used to calculate a characteristic -retention factor’ (Rf) for each pigment, from which they can then be identified. Also, an ultraviolet light source can be used to differentiate pigments which are UV-active from those which aren’t. In addition, the pigment spots can be removed from the plate and characterized via spectrophotometric analysis. The emission spectrum will differ for each pigment, depending on the excitation wavelength used.
Units, terms, definitions
The mobile phase refers to the entire body of liquid that travels over the TLC plate, both the solvent and the sample. The eluent is the solvent liquid alone. The stationary phase denotes the alumina or silica powder packed onto the TLC plate. The retention factor (Rf) is a ratio, a measure of proportion of two things with identical units, thus the Rf is a unitless measure.
Procedure (for carotenoids and chlorophyll)
- To prepare your samples, shock freeze in liquid nitrogen and pulverize it using a mortar and pestle.
- Vortex 100μg of each sample with 1000μL of 80% acetone.
- Incubate under light for 10 minutes at 37∘C.
- Centrifuge at 7000g for 2 minutes. Use the supernatant for this experiment.
- Mark a straight line 1cm from the bottom of the TLC plate with a pencil.
- Use a pipette or capillary tube to spot a small amount (approx 10μL) of each sample onto the line, being careful to record which spot corresponds to which sample. Each plate should contain no more than 3-4 spots. (See Figure 1)
- Place the plate upright in the treatment chamber, ensuring that the line with the sample spots is on the end closest to, but still above, the level of the mobile solvent.
- Close the lid and allow the plate to develop until the solvent front has almost reached the top of the plate. This may occur quickly, so be sure to watch carefully.
- Remove the plate from the chamber; mark the solvent front with a pencil. Circle each defined spot or patch of colour.
- If desired, view the plate under a UV lamp and note which pigments are visible under ultraviolet radiation.
- To calculate the Rf for each pigment, divide the distance it travelled along the plate by the total distance of the solvent front.
- Using a razor blade, remove each pigment spot separately from the TLC plate for further spectrophotometric characterization.
- To each pigment sample, add 80% acetone (500μL) by mixing and incubate for 10 minutes under a light with periodic shaking.
- To remove any remaining silica, centrifuge the pigment samples for 2 minutes at 7000g.
- Measure the emission spectra of the pigment-containing supernatant using a spectrophotometer, at excitation wavelengths of every 20nm from 400nm-800nm.
- Plotting absorbance vs. wavelength for each pigment will yield a characteristic emission spectrum.
Figure 1: A silica plate spotted with three samples, showing pencil mark across the bottom. The first and third samples were pigments taken from spinach leaves, largely chlorophylls, while the middle sample was extracted from paprika and the pigments present are carotenoids.
Notes and troubleshooting tips
For a pigment which travelled 19mm along a silica plate, where the solvent front travelled 44mm, the retention factor would be calculated as follows:
Rf = 19mm/44mm
(2008, November 30, 2008). “Pigments and absorption spectra.” from http://phototroph.blogspot.com/2006/11/pigments-and-absorption-spectra.html.
Chaffai, R., A. Tekitek, et al. (2006). “Thin Layer Chromatography Analysis of Organic Acids in Maize.” American Journal of Plant Physiology 1(1): 65-75.
Ellison, M. and T. Everingham (2011). CHEM1101: Laboratory Manual. Research School of Chemistry, the Australian National University
El-Shaer, N. S., J. M. Badr, et al. (2007). “Determination of lawsone in henna powders by high performance thin layer chromatography.” Journal of Separation Science 30(18): 3311-3315.
Estavillo, G., U. Mathesius, et al. (2011). Plant Detectives Project: Identification of Arabidopsis Mutants Linking Genetics to Form and Function Laboratory Manual. School of Botany and Zoology, the Australian National University
Lynn, D. and S. Schanderl (1967). “Separation of chlorophylls and related plant pigments by thin lyaer chromatography.” Journal of Chromatography 26: 442-448.
Onozaki, T., M. Mato, et al. (1999). “Differences in Flower Colour and Pigment Composition Among White Carnation Cultivars.” Scientia Horticulterae 82: 103-111.
Raven, P., R. Evert, et al. (1999). Biology of Plants, W.H. Freeman and Company/Worth Publishers. Tirimanna, A. (1981). “Study of the Carotenoid Pigments of Bix orellana L. Seeds by Thin Layer Chromatography.” Mikrochimica Acta 2: 11-16.
University of Colorado, B., Chemistry and Biochemistry Department. (2011). “Thin Layer Chromatography.” from http://orgchem.colorado.edu/hndbksupport/TLC/TLC.html.
Watson, P. (1966). “Investigation of pigments from Russula spp. using thin layer chromatography.” Transactions British Mycological Society 49(1): 11-17.
Health, safety & hazardous waste disposal considerations
Use extreme caution when handling liquid nitrogen. Always wear gloves, ensure a safe workspace, and use no more than the required quantities.