DCPIP is a redox dye commonly used as a monitor of the light reactions in photosynthesis because it is an electron acceptor that is blue when oxidized and colourless when reduced. The dye changes color when it is reduced, due to its chemical structure. The nitrogen atom in the center of the molecule is the atom that accepts electrons, and it changes the double N-C bond to a single bond, which forces bonds between carbons in the entire left ring to change.
This microscopic shift in the DCPIP structure causes the macroscopic change in colour, from dark blue to colourless. The amount of DCPIP reduced can be found by measuring the solution's light transmittance with a spectrophotometer. If vitamin C, which is a good reducing agent is present , the blue dye, which turns pink in acid conditions and is reduced to a colourless compound by ascorbic acid. The end point is a pink colour that persists for 10 seconds or more.
This article may be confusing or unclear to readers. Please help us clarify the article. There might be a discussion about this on the talk page. It is not quite so easy to demonstrate the other reactions in photosynthesis. For the reduction of carbon dioxide to carbohydrate there must be a source of electrons.
In the cell, NADP is the electron acceptor which is reduced in the light-dependent reactions, and which provides electrons and hydrogen for the light-independent reactions. In this investigation, DCPIP 2,6-dichlorophenol-indophenol , a blue dye, acts as an electron acceptor and becomes colourless when reduced, allowing any reducing agent produced by the chloroplasts to be detected.
This investigation depends on working quickly and keeping everything cool. Your students will need to understand all the instructions in advance to be sure that they know what they are doing. Cold pestle and mortar or blender or food mixer which has been kept in a freezer compartment for 15—30 minutes if left too long the extract will freeze.
Note 4. Although DCPIP presents minimal hazard apart from staining, it is best to avoid skin contact in case prolonged contact with the dye causes sensitisation. Do not handle electric light bulbs with wet hands. Na 2 HPO 4. Sucrose Dissolve in phosphate buffer solution pH 7. Dissolve 0. Use at room temperature. Note that Potassium chloride is a cofactor for the Hill reaction. Keep solutions and apparatus cold during the extraction procedure, steps 1—8, to preserve enzyme activity.
Carry out the extraction as quickly as possible. Place in a cold mortar or blender containing 20 cm 3 of cold isolation medium. Scale up quantities for blender if necessary. Gather the edges of the muslin, wring thoroughly into the beaker, and add filtrate to the centrifuge tubes. Re-suspend the pellet with about 2 cm 3 of isolation medium, using a glass rod. Squirting in and out of a Pasteur pipette five or six times gives a uniform suspension.
Place tubes 1, 2 and 4 about 12—15 cm from a bright light W. Place tube 3 in darkness. If the extract is so active that it decolourises within seconds of mixing, dilute it with isolation medium and try again. Traditionally the production of oxygen and starch are used as evidence for photosynthesis. The light-dependent reactions produce a reducing agent. The loss of colour in the DCPIP is due to reducing agent produced by light-dependent reactions in the extracted chloroplasts.
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