Separation of plant pigments using thin layer chromatography.
Virtual experiment.

To become familiar with the technique in a real laboratory, you can watch this video

Performing the assay:

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Step 1

Step 2

Step 3

photo

This experiment may be done with two types of samples. Please choose one of them:

Samples (F1 and F2) are two fractions obtained from a plant extract using column chromatography*; the aim is to analyse its composition.
The code for this particular sample set is
Samples (number 1 and 2) are extracts obtained from several plant species.
The code for this particular sample set is

To guide identification, 5 standards are available: solutions of a pure pigment in acetone:

Steps of the experiment

1: Samples and standards are applied on the plate covered with silica gel (stationary phase for the chromatography)

2: The plate is introduced into the tank containing the mobile phase (a mixture of solvents: hexane, acetone and chloroform in 3:1:1 ratio), the tank is covered and we wait for the mobile phase to ascend; it is stopped by switching to step 3.

3: The plate is extracted, we wait for the solvent to evaporate and we proceed to study the results.

Instructions

  1. To move the capillary, hold it with the mouse pointer near its lower end.
  2. Drag the capillary to one of the tubes containing either a sample or a standard, and release it.
  3. Drag the capillary to the plate with stationary phase, positioning its lower end on one of the application targets (a, b, c, d, e, f), at the level of the lateral reference lines.
  4. Drag the capillary to the waste container (a new capillary will later appear at the left-hand side)
  5. Repeat the process with all samples and standards, applying each one on a different position. Write down what you have applied on each of the 7 positions in the plate.
    You may apply samples in any order and position.
  6. Press the ▶ button to go to next step (nº2), where the chromatography will be automatically run.
  7. Before the mobile phase reaches the top of the plate, press the ▶ button to extract the plate and proceed to the next step (nº3).
  8. Observe and interpret the result.
  9. You may press on the photo camera to capture an image that may be copied or saved to disk using the context menu of the web browser (the photo will also have stamped the code of the experiment, date and time).

In your lab notebook: write down the code assigned to your experiment (in this case it is and what you have applied on each position in the plate. Add the photo of the result. Justify the different mobility of each standard relating them to their structure. By comparing the result of samples to that of standards, explain which types of pigment each sample contains.

Samples

* The original extract which was subjected to chromatography came from spinach, chard or microalgae, and was applied on a column filled with activated alumina. Chromatographic fraction number 1 resulted from elution with hexane; fraction number 2 was obtained with a latter elution with acetone.

Author: Angel Herráez. Part of the Biomodel.uah.es website

CC by-nc-sa Offered for use under the terms of the Creative Commons Attribution – NonCommercial – ShareAlike License.

Programmed in HTML5, CSS and JavaScript and foreseeably compatible with all types of web browsers, operating systems and devices. Features are used from the open surce libraries Dragula (by bevacqua, MIT License), DOM to Image (Anatolii Saienko and Paul Bakaus, MIT License), as well as TinyBox.

Based on Rf data from C. Reiss (1994) Experiments in Plant Physiology. Prentice Hall. ISBN 0137012853, cited by Josep Tarragó-Celada and Josep Mª Fernández Novell (2019) Colour, chlorophyll and chromatography. Science in School 47:41-45.