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C. Spectrophotometric Measurements of the Hill Reaction 1. Place 15 mL of the 0.05-mg chlorophyll/ mL suspension in a test tube. Wrap the tube with aluminum foil and chill in an ice bucket. This is the source of unheated chloroplasts. 2. Place the remaining 10 mL of the 0.05-mg chlorophyll/mL suspension in a test tube and heat until boiling using an alcohol lamp. Heating inactivates the chloroplasts. Stand the heated mixture for 5 min. to allow the debris to settle.

C. Spectrophotometric Measurements of the Hill Reaction 1. Place 15 mL of the 0.05-mg chlorophyll/ mL suspension in a test tube. Wrap the tube with aluminum foil and chill in an ice bucket. This is the source of unheated chloroplasts. 2. Place the remaining 10 mL of the 0.05-mg chlorophyll/mL suspension in a test tube and heat until boiling using an alcohol lamp. Heating inactivates the chloroplasts. Stand the heated mixture for 5 min. to allow the debris to settle.

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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C. Spectrophotometric Measurements of the Hill Reaction 1. Place 15 mL of the 0.05-mg chlorophyll/ mL suspension in a test tube. Wrap the tube with aluminum foil and chill in an ice bucket. This is the source of unheated chloroplasts. 2. Place the remaining 10 mL of the 0.05-mg chlorophyll/mL suspension in a test tube and heat until boiling using an alcohol lamp. Heating inactivates the chloroplasts. Stand the heated mixture for 5 min. to allow the debris to settle.
Transcribed Image Text:C. Spectrophotometric Measurements of the Hill Reaction 1. Place 15 mL of the 0.05-mg chlorophyll/ mL suspension in a test tube. Wrap the tube with aluminum foil and chill in an ice bucket. This is the source of unheated chloroplasts. 2. Place the remaining 10 mL of the 0.05-mg chlorophyll/mL suspension in a test tube and heat until boiling using an alcohol lamp. Heating inactivates the chloroplasts. Stand the heated mixture for 5 min. to allow the debris to settle.
ne e e a 3. Prepare the contents of each setup listed in Table 5.1 in clean test tubes. Pipet contents in each test tube in the order given. For the "Unheated (Dark)" setup, immediately wrap the test tube with aluminum foil even before the first reading is made. Table 5.1. Contents of tubes for spectrophotometric determination of the Hill reaction. PBS Contents Heated chloroplasts Unheated chloroplasts 0.1% DPIP (to be added immediately before the first reading) Heated 3.5 1.0 Volume Added (mL) Blanks Unheated 3.5 1.0 Heated 2.5 1.0 1.0 Experimental set-ups Unheated (Light) 2.5 1.0 1.0 Unheated (Dark) 2.5 1.0 1.0 4. Your instructor will demonstrate the handling of a cuvette and operating a spectrophotometer. Set the spectrophotometer at 605 nm wavelength. Zero the spectrophotometer using the appropriate blank. Use the "Heated Blank" for "Heated" experimental tube, and use the "Unheated Blank" for both "Unheated (Light)”, and “Unheated (Dark)" experimental tubes. 5. Add 1 mL 0.1% DPIP to experimental tubes "Heated", "Unheated (Light)", and "Unheated (Dark)" prior to the initial readings. DPIP must be added to experimental tubes when about to be placed in spectrophomoter. Cover the mouth of the test tube with a piece of aluminum foil and the thumb and invert the test tube several times to mix its contents. Pour the solution in a clean cuvette. Do not delay reading if DPIP was already mixed. 6. Take the initial readings of the experimental set-ups "Heated", "Unheated (Light)", and "Unheated (Dark)". For the "Unheated (Dark)" setup, immediately wrap the test tube in aluminum foil after taking the initial reading. Measure the absorbance again after 30 min. Uncover only upon reading the absorbance. 7. After the initial readings, place all the test tubes in a beaker placed in an ice bath and illuminate the test tubes with a 100-watt bulb placed 18 inches away. Measure the absorbance of the "Heated" and the "Unheated (Light)" tubes at 5-min. intervals up to 30 min. Mix the contents before reading. 8. Record all the data as Table 5.1 in your laboratory notebook.
Transcribed Image Text:ne e e a 3. Prepare the contents of each setup listed in Table 5.1 in clean test tubes. Pipet contents in each test tube in the order given. For the "Unheated (Dark)" setup, immediately wrap the test tube with aluminum foil even before the first reading is made. Table 5.1. Contents of tubes for spectrophotometric determination of the Hill reaction. PBS Contents Heated chloroplasts Unheated chloroplasts 0.1% DPIP (to be added immediately before the first reading) Heated 3.5 1.0 Volume Added (mL) Blanks Unheated 3.5 1.0 Heated 2.5 1.0 1.0 Experimental set-ups Unheated (Light) 2.5 1.0 1.0 Unheated (Dark) 2.5 1.0 1.0 4. Your instructor will demonstrate the handling of a cuvette and operating a spectrophotometer. Set the spectrophotometer at 605 nm wavelength. Zero the spectrophotometer using the appropriate blank. Use the "Heated Blank" for "Heated" experimental tube, and use the "Unheated Blank" for both "Unheated (Light)”, and “Unheated (Dark)" experimental tubes. 5. Add 1 mL 0.1% DPIP to experimental tubes "Heated", "Unheated (Light)", and "Unheated (Dark)" prior to the initial readings. DPIP must be added to experimental tubes when about to be placed in spectrophomoter. Cover the mouth of the test tube with a piece of aluminum foil and the thumb and invert the test tube several times to mix its contents. Pour the solution in a clean cuvette. Do not delay reading if DPIP was already mixed. 6. Take the initial readings of the experimental set-ups "Heated", "Unheated (Light)", and "Unheated (Dark)". For the "Unheated (Dark)" setup, immediately wrap the test tube in aluminum foil after taking the initial reading. Measure the absorbance again after 30 min. Uncover only upon reading the absorbance. 7. After the initial readings, place all the test tubes in a beaker placed in an ice bath and illuminate the test tubes with a 100-watt bulb placed 18 inches away. Measure the absorbance of the "Heated" and the "Unheated (Light)" tubes at 5-min. intervals up to 30 min. Mix the contents before reading. 8. Record all the data as Table 5.1 in your laboratory notebook.
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