Chromatography is a technique that is widely used for the separation of mixtures of compounds. Owing to the convenience and effectiveness of the process, it is carried out for the separation of a particular component from the mixture of unknown components. Chromatographic separation is mainly based on the differential affinities of the components of mixture towards the stationary and mobile phase leading to the separation of the components. Because of the factors that affect separation; each component of the mixture moves differently in the chromatographic system, some stay longer and move slowly through the system while others pass rapidly into the mobile phase and leave the system faster.
Several types of chromatographic techniques are complex processes that require a variety of organic solvents, expensive equipment and prolonged periods of time. Out of all this, Thin Layer Chromatography comes out to be the most effective and superior to all chromatographic techniques when it comes to the separation of amino acids and nucleotides. Excellent sharpness of separation, high sensitivity, and speed are some of the greatest advantages of TLC that makes it a first choice for the separation of photosynthetic pigments. Thin Layer Chromatography is one of the easiest and cheapest techniques, commonly used to carry out the separation of natural components. Here, less polar mobile phase is used with the more polar stationary phase and this is referred to as normal phase chromatography.
This method of chromatography is a commonly used technique to separate non-volatile mixtures. It is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel or aluminium oxide (alumina). This layer of adsorbent is known as the stationary phase. In thin-layer chromatography, a small spot of the mixture to be separated is applied near one end of the TLC slide and then dipped into the mobile phase allowing it to run through the slide by capillary action, further leading to separation of the components of the mixture. The mobile phase should be different from the stationary phase for better elution. For eg; polar stationary phase such as silica gel is used with a non-polar mobile phase. Sometimes, a mixture of solvents is also used for fine-tuning the bulk properties of the mobile phase that leads to effective separation of components from the mixture.
Photosynthetic pigments are the components in question here that are found in plants, algae, and bacteria, these photosynthetic pigments absorb light energy at different wavelengths of the visible spectrum and convert it to chemical energy. This energy is transferred to and stored inside the components like glucose and starch. Majorly, there are three types of photosynthetic pigments that are involved in the absorption of light. These are Chlorophyll, Carotenoids, and Phycobilins that are distributed among algae, bacteria and higher plants. As all these pigments are associated with the process of photosynthesis, they absorb only specific wavelengths of visible light, while reflecting others that give a particular colour to the containing plant and organism. The most important of these pigments are chlorophylls. Chlorophylls are commonly present in all plants, primarily in cyanophytes and several bacteria. Chlorophyll a and chlorophyll b are the most important types of chlorophyll and both of them differ slightly in their structure, hence slightly differing in their distribution as well. Chlorophyll is found in all photosynthetic plants except bacteria and absorbs radiation at wavelengths along the two ends of the visible spectrum i.e; red and blue. Chlorophyll b is the pigment found in algae and higher plants.
It is through these photosynthetic pigments that the plants generate various essential sugars for their cellular functions. Extraction of these pigments gives us insight into their characteristic features which further helps to understand their mechanism by which they function owing to their different molecular structure. Study shows that accessory pigments such as pheophytin a, xanthophyll and carotenoids are present alongside the primary pigment and they are found to increase the absorption spectrum of the photosystem while protecting the primary pigment from oxidation in presence of light.
As the photosynthetic pigments are of profound importance for the whole process of photosynthesis, it becomes of paramount importance to study them in detail. We have learned a lot about them through carrying necessary experiments involving their extraction and separation but a lot remains to be known yet. It is through these experiments that we now know the presence of accessory pigments causing primary pigments to absorb light at a wide range of wavelengths also protecting them against the dangers of photo-oxidation by running photoprotective cycles. Thin Layer Chromatography of plant pigments allows the effective separation of each pigment without destroying any of its properties and making it available for carrying out further analysis of its characteristic features.
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