In order to understand the significance of the discovery of CO2 reduction cycle, we should undetstand the experimental methods followed by Calvin. To trace the various steps of conversion of CO2 after its entry into the plant system until the formation of glucose, Calvin allowed the experimental plants to photosynthesize for various periods of time and analysed the various compounds formed after different periods of photosynthesis.
Calvin chose for his experimentation two green algae – Chlorella pyrenoidosa and Senedesmus obliquus. The plants have the same pigmentation as higher green plants and can be easily cultured and harvested. To make sure that the compounds to be analysed can be easily traced and not mistaken for the compounds already present in the plant, the experimental plants were starved of CO2 and later supplied with a radioactive isotope of CO2 (C14).
Only those compounds in which C14 was incorporated were taken into consideration because they were produced after the experiment began. By varying the time of photosynthesis (1/16 sec to 5 sec) and then killing the plant to stop photosynthesis and analysing the intermediate compounds formed, Calvin and his co-workers were able to map the whole of the CO2 reduction cycle.
Two, very simple but efficient tools namely Chromatography and Radioautography helped Calvin to trace the path of carbon in photosynthesis. Chromatography, in particular paper chromatography is an ideal technique for the separation of compounds present in a small mixture. The extract (mixture) of the plant to be analysed is placed as a spot on a filter paper and dipped in a suitable solvent. The solvent travels on the paper due to capillary action and carries with it the different substances in the extract. The rate of migration of the different substances depends upon their solubility and specific gravity.
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The filter paper (Chromatogram ) should be removed from the solvent before it reaches the edge of the papte and dried. By spraying a suitable reagent (For e.g., ninhydrin for amino acids) the compounds can be detected as several spots widely separated on the paper. The substances may be identified by their relative positions on the Chromatogram or by comparing with a standard Chromatogram. (In a standard Chromatogram compounds whose identity are known are spotted and their position is marked after the solvent run).
Radioautography is a technique to find out, if a particular source consists of a particular type of isotope. Different radioactive isotopes emit different types of radiation. Chiefly there are 3 types. These are: a) Alpha rays, b) Beta rays c) Gamma rays.
When we use a particular isotope we already know what the type of emission is. For instance, C14 emits β rays. When these rays are directed to a photographic film they form characteristic markings and by these one can identify the type of radiation and thereby the isotope.
In the present experiment, Melvin Calvin and associates allowed the algae to Photosynthesise for varying periods (using C14) after which they (plants) were killed in boiling methanol. The plant extract was then spotted on the Chrornatogram to indentify the compounds. In order to make sure that these contain C14, the chromatogram was held against a photographic fill of equal size.
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Only those compounds which had incorporated C14 leave markings on the film and these were taken into consideration (as produced during experimentation). In order to vary the time of photosynthesis flashes of light from 1/16 sec to 5 sec were given to identify the earliest compound formed. In this way Calvin and his associates found out all the intermediaries in the CO2 reduction cycle.