The Experiment
The main aim of the experiment is to separate the spinach pigments using difference in polarity. It is also aimed at separating aromatic using column adsorption chromatograph. Most of the plant leaves contain different pigments that are colored. The pigments are grouped into two groups. That is Chlorophylls and Carotenoids. There are various compounds that have been used in the study. That is chlorophyll a, Chlorophyll b, beta carotene; azobenzene and 2-nitraoiline.The compounds listed above are colored because of transfer of light. The ultraviolet light has acted on them and made them colored. The ultra violet light is made up of a shorter wavelength.
The wave length is visible and has a lot of light. The ultraviolet light is divided into various groups based on the wavelength. That is UV, UVB and UVC. An electron is always excited when an ultraviolet light is absorbed by molecules. The electron always orbits at a higher energy. The wavelength of light absorbed by the molecule depends on the energy difference that is found between the excited state and ground state. If the difference between the ground state and excited state is small, the wavelength of light is always long.
The wavelength of light absorbed increases with the increase in resonance and conjugation. If the wavelength is long enough, one is able to see color in the visible regions. Thus, the compounds above are colored because the wavelength of light they have absorbed is long enough for one to see color (David, 2001).Most experiments have used a hydrous magnesium sulfate. The magnesium sulfate contains magnesium, sulfur and oxygen. The anhydrous magnesium sulfate is always used as a drying agent. The anhydrous magnesium sulfate absorbs water from the atmosphere. The anhydrous magnesium sulfate was used to dry the fresh spinach.
The magnesium was added in the spinach mixture to eliminate moisture and ensure one is able to get a dry powder (David, 2001).The movement of the substance and the solvent front is shown by the distance traveled by the substance divided by the distance traveled by the liquid. The compounds above have different retention factors. The retention factor for chlorophyll a is 0.7 and the retention factor for chlorophyll b is 0.24.The retention factor for carotenes and Xanthophylls is 0.15 and 0.12.
The compounds used in the experiment have different retention factor because they differ in terms of solubility. They also differ in terms of adsorption strength. Some of the compounds in the spinach leaves tend to be carried farther up the plate used in the study than other substances. Hence, the reason chlorophyll a has the highest retention factor followed by chlorophyll b. The compounds also differ in terms of color because of the difference in strength of ultraviolet light (David, 2001).
The spots have different color under ultraviolet light because of different factors. Some of the alumina powders and silica powders used in the experiment have fluorescent materials. After placing the alumina powder under Ultraviolet light, the areas having fluorescent appear colored while the areas that do not have fluorescent appear clearly visible. The fluorescent areas have different colors. Hence, the reaction between the fluorescent areas and the light produces spots having different colors and colorless areas.
The separation is effective if the bands move in horizontal front, but not jagged front. This is because horizontal bands permit rapid movement of solvents than jagged front. This makes it easy to separate the solvents in spinach leaves. Jagged fronts might interfere with the separation of the substance because they have some bents and this affects the whole process. The results produced are also not reliable (Miller, 2005)..There is evidence for two compounds in the spinach leave. This is shown by the separation of first benzene and 2-netzolanlen.The first compound was first was orange.
The second compound was compound yellow. The hexane was used in the experiment. The hexane carries epithet that is used to eliminate non polar benzene and ethyl acetate was used to eliminate the yellow compound. There was no presence of other compounds apart from the two compounds listed above (Miller, 2005).The separation for colorless compounds can be monitored by monitoring the separation of the compounds from the mixture in column chromatography.
One should monitor the separation of the substances through the Column. Different substances have different interaction levels and they are carried through the mobile phase different. Some of compounds are attracted faster while others are not. This makes it easy to monitor colorless substance when carrying out an experiment. A mixture will always be separated into various compounds depending on the number of substance. After the separation process one will be able to get a clear substance. The results of the study are shown in the table below (Miller, 2005).
| Appearance of compounds under Ultraviolet light | Retention factor | color | Type of compound |
| More intense than chlorophyll b | 0.7 | green | Chlorophyll a |
| green | 0.24 | green | Chlorophyll b |
| Clearly visible | 0.15 | orange | carotene |
| Almost 2 spots | 0.12 | yellow | xanthophylls |
Reference
Miller,J.M.(2005).Chromatography: concepts and contrasts. John Wiley and Sons
David,G.L.(2001).Analytical Chemistry. Universities Press
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