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10 Cards in this Set
- Front
- Back
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What is chromatography used for? |
To separate the substances in a mixture |
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What is meant by the mobile phase in chromatography? |
Where the molecules can move - e.g. a liquid solvent |
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What is meant by this stationary phase in chromatography? |
Where the molecules can’t move e.g. a piece of chromatography paper, thin layer of solid (silica gel) |
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Describe the basic principle of chromatography that describes what happens in all types? |
• The mobile phase moves through the stationary phase • The components in the mixture spend different amounts of time in the mobile phase and the stationary phase • The components that spend longer in the mobile phase travel further • The time spent in the different phases is what separates out the components of the mixture |
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What is meant by an Rf value? |
The distance a substance has moved through the stationary phase in relation to the solvent |
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Leaves of different species of plant have different proportions and mixtures of pigments (photosynthetic and other pigments like UV protection pigments). Describe how you can identify the pigments using TLC? (Required practical 7) |
•Grind up several leaves with anhydrous sodium sulfate, add a few drops propanone • transfer liquid to test tube • Add petroleum ether + gently shake (two distinct layers form) • Transfer some of the liquid from the top layer into a second test tube with some anhydrous sodium sulfate • Draw a horizontal pencil line near the bottom of a TLC plate • Build up a concentrated spot of the liquid in the second test tube on the line by applying several drops • Once the plate is completely dry - put the plate into a glass container with some prepared solvent in the bottom - just enough so the point of origin is a little bit above the solvent • Put a lid on the container and leave the plate to develop • As the solvent spreads up the plate the different pigments move with it (at different rates) • When the solvent has nearly reached the top, take the plate out and mark the solvent front (furthest point solvent has reached) with a pencil • Leave plate to dry • The separated pigments will appear as several discrete coloured spots • calculate Rf value for each spot • compare the Rf values to values in a database - to identify the pigments
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The whole point of identifying leaf pigments in RP7 is to see if they’re different in shade-tolerant and shade-intolerant plants. Why might you expect them to have different pigments in different proportions? |
• photosynthetic pigments - shade-tolerant plants adapted to the light conditions, allow the plants to make the best use of the light available • non-photosynthetic pigments - The chloroplasts of shade tolerant plants are adapted for photosynthesis in low levels of light - very sensitive to high light intensity. Produce dark pigments (anthocyanins)which protect their chloroplasts from brief exposure to higher light levels |
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When looking up Rf values to compare to yours in a database, what piece of information is it important to know? |
Need to check that the Rf values were recorded under the same conditions as your experiment |
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What are the two types of chromatography? |
• Paper chromatography • Thin-layer chromatography |
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Describe how are you can investigate the effect of light intensity on activity of dehydrogenase in chloroplasts? (Required practical 8) |
In photosystem 1 during LDR, NADP acts as an electron acceptor (is reduced) - the reaction is catalysed by a dehydrogenase enzyme. The activity of this enzyme can be investigated by adding a redox indicator dye to extracts of chloroplasts. The dye (like NADP) acts as an electron acceptor - gets reduced by the dehydrogenase enzyme. As the dye is reduced there is a colour change. The dye used is DCPIP which goes blue to colourless when it gets reduced. The faster the absorbance decreases, the faster the rate of dehydrogenase activity. • Cut a few leaves into pieces (removing any tough stalks) • Grind up the leaves with chilled isotonic solution • Filter the liquid you make into a beaker through a funnel lined with muslin cloth • Transfer the liquid to centrifuge tubes + centrifuge at high speed for 10 mins - chloroplasts father at bottom in a pellet • Get rid of the liquid from the top of the centrifuge tubes, leaving the pellets • Resuspend the pallets in chilled isotonic solution - this is your chloroplast extract • Store your chloroplast extract on ice for rest of experiment • Set up a colorimeter with red filter + zero it using a cuvette containing distilled water • Set up a test tube rack at a set distance from a lamp • Put a test tube in the rack + Add a set volume of chloroplast extract to the tube + Add a set volume of DCPIP - mix contents • Take a sample of the mixture from the tube + add it to a clean cuvette • Place the cuvette in your colorimeter and record the absorbance - do this every 2 minutes for 10 minutes. • Repeat the last few steps for each distance under investigation • Also do 2 negative control tubes: one containing DCPIP and chilled isotonic solution (no chloroplast extract), the second containing DCPIP and chloroplast extract but tube wrapped in tin foil • plot a graph of absorbance vs time for each distance from the light source • compare your results to determine how light intensity affects the rate of dehydrogenase enzyme
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