Lab 6- manual-Extraction of photosynthetic pigments from plants

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Purdue University *

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Chemistry

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Jan 9, 2024

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Lab 6: Extraction of photosynthetic pigments from plants. To extract proteins, carbohydrates, other metabolites, or pigments in a cell, one must use various techniques to break open a cell. The lysis of cells depends on the organism used and the substances to be extracted. Bacteria are commonly lysed using a French press, as shown below, which breaks cells by pressurizing the cell suspension in a closed chamber (e.g., 7-10,000 psi) and suddenly releasing the pressure. The release of pressure creates a liquid shear capable of lysing the cells. Bacteria can also be lysed by sonication, which focuses sound waves to create a liquid shear and cavitation. Tissue culture cells or cell suspensions can often be lysed by a hand-held or motor-driven homogenizer, as will be shown in the lab. Solid or more fibrous tissue requires other approaches. Small samples can be extracted using a mortar and pestle. These are often frozen in liquid N 2 and then powdered before adding an extraction buffer. Larger samples may require the use of a blender. A polytron homogenizer is a device with counter-rotating blades that can be useful for very fibrous tissue. In this lab, you will be extracting chlorophyll from spinach leaves. Along with chlorophyll and b, spinach leaves have other pigments, such as carotenoids, anthocyanins, and xanthophylls. Since the amount of chlorophylls is the highest and anthocyanin and xanthophyll are the lowest, we see the light reflected from the chlorophylls; hence, the leaves appear green. Each pigment absorbs and reflects light at a specific wavelength, as shown in the diagram below. For this lab we will only focus on chlorophylls. Relative absorbance of photosynthetic pigments as a function of the wavelength of light. Exercise 6.1 Extraction of Chlorophylls.
Materials: 1. Spinach leaves (on TA/TI benches) 2. Mortar pestle 3. 1000 µl tips and pipettes on each bench 4. Centrifuge on each bench 5. Parafilm -cut pieces. 6. Transfer pipettes 7. Cuvettes 4 on each bench, plus a few extra at TA tables. Procedure: 1. You will work as a group of two students for this lab. 2. Label a 1.5 ml microfuge tube as E and draw a line at the 1 ml mark on the tube with a sharpie. 3. Obtain a healthy-looking spinach leaf from the TA bench. 4. Excise the leaf, remove prominent veins as feasible, and weigh 0.5g of the spinach leaf pieces. Add the weighed leaf pieces directly to the mortar. * think about how much is the fresh weight of the spinach tissue you are using. 5. Then add 4 ml of 95% Ethanol and proceed to grind. Grind vigorously until the leaf is thoroughly homogenized (like “pea syrup” without many particles, ~ 2 mins). Transfer 1 ml of the extract to the microfuge tube from # 2 using a plastic transfer pipette. 6. Working with the other group of two students, centrifuge your E tubes with extract together. 7. Make sure the sample tubes have equal volumes. Place the balanced tubes opposite to each other in a centrifuge. The rotor MUST be balanced before spinning to avoid damage to the centrifuge and possible injury. Do not forget to put the lid on. 8. Spin for 4 min at maximum speed (approximately 10,000 × g . While waiting on the centrifugation, wash your mortar and pestle, spray it with Ethanol, and put it back on the bench. 9. Transfer all the supernatant to a new clean tube and label it SN. Record the volume. Keep the tube with the pellet aside. The pellet may be very small and clear, so it might be hard to see. 10. Keep the tube with the pellet aside. Pellet may be very small and clear so it might be hard to see. : Add 1 ml of 95% ethanol to the pellet and vortex vigorously for ~ 30 s. Place the tube in the drawer for 5 min, then centrifuge for 5 mins at maximum speed.
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