This mobile phase separates the pigments most clearly, but you could adapt the activity to use mobile phases of hexane or ethanol alone, which the students can carry out themselves. Both hexane and ethanol successfully separate the pigments, but the distinction between each pigment is not as clear as when the combined solvent is used.
A lab coat, gloves and eye protection should be worn. The solvents used in this experiment are flammable, so they must not be used near flames. The combined solvent hexane, acetone and trichloromethane must only be used inside a fume hood due to the volatility, smell and health risks associated with it.
Record your results in a table. Compare these to the values in table 1: were your answers correct? The different pigments in a leaf extract are separated based on their affinities for the stationary phase the silica on the thin-layer chromatography plate — a polar substance and the mobile phase the solvent — a nonpolar substance.
Compounds with a high affinity for the solvent i. In our example see figure 2 , both leaf extracts contained four pigments. Pigment 4 moved a shorter distance than pigment 1, indicating that pigment 4 is more polar and pigment 1 is less polar. By looking at the chemical structures of different pigments and the polar and nonpolar groups, students can try to identify 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, alcohol groups are the most polar, ester and ether groups the least polar, and aldehyde and ketone groups are in between.
From this, we can 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 1 and 2 are likely to be carotenes, and pigment 4 is likely to be a xanthophyll.
Pigment 3 is likely to be chlorophyll, since it is more polar than carotenes but less polar than xanthophylls. You can observe the characteristic green colour from chlorophyll on the chromatogram. Now look at the Rf values, which range between 0 and 1, with 0 being a pigment that does not move at all, and 1 indicating a pigment that moves the same distance as the solvent.
The Rf value varies depending on the solvent used, but the general order of the pigments from the highest to the lowest Rf value usually remains the same, because the nonpolar compounds move further than the polar compounds. Rf values for various pigments using hexane, acetone and trichloromethane for the solvent are shown in table 1. After the experiment, you can ask your students some of the following questions to gauge their understanding of plant pigments and thin-layer chromatography.
Visible light, or white light, is made up of the colors of the rainbow. Some of these colors are absorbed "used" by pigments and others are reflected. Pigments appear the color of the reflected light, so the chlorophyll pigments do not use the green portion of the spectrum. The other two pigments are types of carotenoids, which appear yellow, orange, or brown. The top band of pigments in the separation are carotenoids called carotenes, most likely beta-carotene, and appear yellowish-orange.
The second type of carotenoid separated in the experiment are xanthophylls, which appear bright yellowish and are most likely lutein. The "loading line" is the location of the original pigment line painted on the paper. A second experiment using the chloroplast pigment extract obtained using the methods described above can be easily done. Green-colored pigment extract is added into a test tube.
When a light is shone on the extract, pigment molecules absorb energy. In the pen experiment and in other paper chromatography experiments, the process works because of this phenomenon of pigments traveling at different speeds. Usually, if two identical spots on the chromatography paper where the ink or pigments have spread out are at the exact same distance, then that means two pigments were the same in the substance being tested.
However, some limited exceptions exist. Some pigments and compounds will not be visible on their own in chromatography experiments unless you mix them with food coloring or dye. For example, when some amino acids mix together, they will not be visibly separated by using chromatography paper.
However, you can mix food coloring and dye in, which would enable you to use a chromatograph to see the different amino acids once they separate. Alexis Writing has many years of freelance writing experience. She has written for a variety of online destinations, including Peternity. She holds a Bachelor of Arts in communication from the University of Rochester. What Is Contained in a Permanent Marker? Simple Chromatography Experiments. How to Separate the Components of Ink.
Food Coloring Experiments.
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