Lab Report: Isolation of Plant Pigments
The presence of different pigment molecules in the leaves of plants is due to the presence of carotenoids and chlorophyll. These pigments are of different colors such as red, green, yellow and orange. Separation of these different colors involves the application of three different processes namely solvent extraction, chromatography and spectrophotometry. Solvent extraction is used to extract these pigments from the leaves, chromatography applied to separate the extracted pigments. Finally, spectrophotometry is used to identify the different colors separated by chromatography. These three different processes follow each other in succession, and must be done with accuracy and precision. The most common plant pigment is chlorophyll that is green in color. On the other hand, carotenoids because of their oxygen component may be orange, red or yellow in color. Chlorophyll absorbs light at 450 nm and 650-700 nm in the blue and red wavelength regions. In the paper chromatogram, there will be different regions representing the different pigments. Beta carotene appears as yellow-orange, pheophytin as grey, chlorophyll a as blue-green, chlorophyll b as green and xanthophylls as yellow. There are also other pigments called the anthrocyanins that appear reddish in color.
All these pigments absorb light at different wavelengths and resolute on the paper chromatogram at different speeds. As a result, they will occupy different regions on the paper chromatogram based on their resolution. A look at the results of the paper chromatography reveals a chromatogram with five different regions occupied by different pigments. We observe an orange pigment, yellow pigment, yellow-green pigment, blue-green and reddish pigments in that order. These are the pigments that are clearly visible on the chromatogram. These pigments appear in this order from the solvent front. Remember the solvent used in this process must not dissolve any of these pigments. There are other pigments in blurred and appear grey in color. They appear at the bottom of the rest of the pigments.
Looking at the table that provides the results of the distance of each pigment from the solvent front, we can assign each of these pigments a specific pigment number line. In this case, the orange pigment is position 1, yellow pigment is position 2, yellow-green pigment is position 3, blue-green pigment is position 4 and reddish pigment is position 5. Based on the above results, we can say that the orange pigment is the lightest of all and moves quickly on the chromatography paper. On the other hand, the reddish pigment is the heaviest among the visible pigments and moves slowly on the chromatogram. After identifying these regions on the chromatogram, we can correctly associate them with their correct pigments from theory. As a result, the orange pigment represents the carotenes, the yellow pigment represents the xanthophylls, the yellow-green region represents chlorophyll a, the blue-green region represents chlorophyll b and the reddish region represents the anthrocyanins. The blurred grey region represents the pheophytin pigments.
The above results must be supported by another set of results that will confirm the accuracy of identification. As a result, the pigments on the chromatogram are scrapped off and put in different test tubes where we measure the transmittance and absorbance of light. Transmittance of light refers to those wavelengths of light that are not used by plants pigments. As a result, they pass through the pigments without any interference. On the other hand, absorbance of light refers to those wavelengths of light that do not pass through these pigments; hence, used by the pigments. All plant pigments have different absorbance and transmittance of light wavelength. The absorbance of these pigments ranges from 440 nm to 660 nm, where they absorb most light energy. This is basically between the blue and red regions of the electromagnetic spectrum.
An examination of the absorbance and transmittance results provided indicates that maximum absorption is between the 400 nm and 600 nm wavelengths. However, the maximum absorption for every pigment varies depending on the wavelength. For example, the 1st sample show maximum absorption at 500 nm, sample 2 shows maximum absorption at 400 nm, sample 3 shows maximum absorption at 400 nm, sample 5 shows maximum absorption at 600 nm and sample 4 shows maximum absorption at 400 nm.
Using these different absorption rates, we can correctly match the absorbance to the correct pigments. For instance, chlorophyll a and b absorb maximally at 400 nm and 600 nm. Using this information, we can say with confidence that sample 3 that shows maximum absorption at 600 nm is chlorophyll. Beta carotene absorbs maximally at 400 nm than any other pigment. Therefore, based on the results provided, sample 7 absorbs maximally at 400 nm giving an absorbance of 0.24. These are the carotenes. This is followed by xanthophylls that absorb maximally at 400 nm. Therefore, sample 6 represents the xanthophylls. The anthrocyanins absorb minimally at 400 nm compared to other pigments; hence, represented by sample 5. Using this information, we have correctly justified the information revealed by the chromatogram.
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