Separation of Plant Pigments Using Thin Layer Chromatography


Thin layer chromatography (TLC) is one of the analytical methods that can be used in qualitatively identification of various components with a particular substance. A mixture material can separate, and its fractions determined. The working principle of thin layer chromatography is that the substance dissolves in a solvent and the solution in relation to their respective molecular masses carries different fractions. The solvent acts as a mobile phase while the paper is the stationary phase (Carey, 2013, pp. 51-56).

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TLC is one of the safest analytical methods. In this experiment, TLC is used to identify different pigments in spinach leaves. The leaves are in three categories of fresh, frozen and tinned. The scientific name of spinach is Spinaciaoleracea. The pigments in spinach are organic oils known as triglyceride (Gordon, 2011, pp. 111-113).


  1. Transfer the sample of ‘Fresh’ spinach (0.5gm) and one bottle of the sand mixture (1gm sand + 0.5gm anhydrous Sodium Sulfate) into the mortar and grind with the pestle to obtain a fine dry powder. The Anhydrous sodium sulfate is a dehydrating agent and will remove the water from the leaves.
  2. Put the powder approximately 2.0 grams into a test tube and mix with 2 ml of acetone solution. Tightly close the test tube and shake well for one minute. Confirm that the solution is homogenous. Please ask a technician if you are unsure about how to use the Parafilm.
  3. The mixture was allowed to stand for ten minutes the distilled, and the distillate poured into a micro-centrifuge tube.
  4. The procedure above was repeated using samples of frozen spinach (‘Froz’) spinach. Repeat yet again with a sample of tinned (‘Tin’) spinach.
  5. Repeat one more time using either green or red pepper.
  6. Centrifuge for 1 minute at 10,000rpm to spin any remaining solid to the bottom of the tube. Ask a technician to help with this!
  7. Obtain a TLC plate and make four dots equal distance apart with a pencil (do not use ink) on the coated side (white) about one centimeter from the edge of the strip.
  8. Capillary tube was dipped in a leaf extract to fill by the capillary action. The extract was the applied on the dots and allowed to dry. In order to make a concentrated dot, the application of the extract on the dots was repeated three times while allowing them to dry.
  9. The TLC paper with the dry dots was then dipped in the solvent. The dots were left about one centimeter from the solvent.
  10. Chromatography was allowed to develop.
  11. The TLC paper was removed, and the solvent front was quickly marked with a pencil.
  12. The paper was allowed to dry.
  13. The pigment marks on the paper were marked, and the measurement taken and recorded.


The picture of complete chromatography
Fig. 1: The picture of complete chromatography
Distance traveled (mm)
Solvent A Solvent B
Fresh Frozen Tinned Pigment Fresh Frozen Tinned
60.0 60.0 60.0 Solvent front 57 57 57
58.0 57.0 57.0 β-Carotene 43 43 43
29.0 35.0 Pheophytin a 39 42
28.0 34.0 Pheophytin b 38 40
28.5 27.0 33.0 Chlorophyll a 39 36 38
26.0 25.0 31.0 Chlorophyll b 36 32 36
21.0 21.0 25.0 lutein 30 24 28


The rate of pigment movement up the plate is reported as a Rf value that is described as the relation of the distance moved by the spot to the distance moved by the solvent. The Rf values are calculated using specific formula (Carey, 2013, pp. 51-56).


The literature gives Rf values of 0.61 and 0.49 for pheophytin a and pheophytin b.

Rfvalue of ß-carotene = 58.0/60 = 0.97
Rfvalue of pheophytin a = 42/60 = 0.70
Rfvalue of pheophytin b = 40/60 = 0.67
Rfvalue of chlorophyll a = 39/60 = 0.65
Rfvalue of chlorophyll b = 36/60 = 0.60
Rfvalue of lutein = 30/60 = 0.50

Pictures of different pigments separated on TLC paper

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The picture above shows the marks on paper of different pigments. The arrows on the picture show the names
Fig. 2: The picture above shows the marks on paper of different pigments. The arrows on the picture show the names

The principal chemical components of spinach are ß-carotene and pheophytin a. it is important to understand how storage affects the chemical component of spinach to maintain nutritional value. In terms of elucidating the components of spinach using thin layer chromatography the principal chemical components ß-carotene and pheophytin a are significant (Carey, 2013, pp. 51-56).

From results of solvent A and B, the quantities of all pigments are more or less the same except lutein that shows a significant increase in volume in solvent B.

The highly non-polar component was ß-carotene because it had the longest distance moved by the solute while lutein was the most polar component because its solute distance moved was the shortest (Gordon, 2011, pp. 111-113).

Some limitations of TLC is that certain impurities cannot be identified when they are insoluble in the solvent, or they have the same molecular masses as the identified components like the case of isomers. The stationary and the mobile phases of any chromatography must be of different polarity. Meaning when the mobile phase is polar, the stationary phase must be non-polar and vice versa. Separation of mixtures in chromatography relies on the difference of polarity between the two phases. (Gordon, 2011, pp. 111-113).

TLC does not give structural properties of the molecules but only proves the presence of different molecules. Another analytical technique like spectroscopy provides more information on the molecules. Some of the spectroscopic methods that can be used in structural elucidation are nuclear magnetic radiation, UV-Visible spectroscopy and mass spectroscopy (Gordon, 2011, pp. 111-113).

For faster separation of thermally sensitive compounds like plant pigments, High-performance liquid chromatography (HPLC) can be used. Gas chromatography is faster but because it uses heat for volatilization of compounds, it will denature the plant pigments (Gordon, 2011, pp. 111-113).

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HPLC find application in the large-scale separation of plant products for example in the separation of pyrethroids from pyrethrum in the manufacture of environmentally friendly insecticides and manufacture of medicinal products from plants (Gordon, 2011, pp. 111-113).


Spinach contains different organic pigments in varied quantities that are affected by storage. The experiment was of great benefit for food scientists and other stakeholders in the food industry. Conditions that affect nutritional values of foodstuffs must be known to maintain the supply of high-quality food products of great nutritional benefit to the consumers. ß-carotene is the main component of spinach. TLC is effective in the separation of thermal sensitive components, as it does not involve the use of heat.


Carey, M. (2013). Pharmacy. Chemistry of natural products, New Yok: Johns Hopkins University Press, p. 51-56.

Gordon, B. (2011). Natural Compounds. An approach to biomolecules and synthetic chemistry. America: Oxford University Press, p. 111-113.

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