Exercise X 



PHOTOSYNTHESIS 53 



Optional experiments 



(1) In the above experiment the hght inten- 

 sity limits the rate of photosynthesis. If several 

 independent steps are involved in an overall 

 reaction, the rate at which the process goes is 

 determined by the step which has the lowest 

 rate, i.e., the limiting step. Determine at what 

 concentration the bicarbonate becomes limiting 

 at maximum light intensity. 



(2) Shake a few pieces of leaf for 3 to 4 

 minutes in a test tube with petroleum ether. 

 Pour off the petroleum ether into another test 

 tube, replace with methanol, and shake the 

 leaves as before in this solvent, heating mean- 

 while under the hot-water tap. Pour off the 

 methanol extract into a clean test tube, and 

 compare with the petroleum ether extract. 

 Which contains more pigment? Which is 



therefore the better solvent for leaf pigments? 

 Now mix both together, shake once vigorously, 

 and let the two layers separate. Which contains 

 more pigment? How do you explain these 

 observations? 



(3) Determine in a spectrophotometer the 

 absorption spectra of the mixture of chloro- 

 plast pigments and of the separate pigments 

 isolated on your chromatograms. By dividing 

 this job among several students, you may be 

 able to do all of it. To obtain enough of the 

 isolated pigments it will probably be necessary 

 to combine several chromatographed samples in 

 as small a volume of solvent (acetone or ethanol) 

 as the spectrophotometer will handle. The mix- 

 ture of pigments and the chlorophylls should be 

 measured between 380 and 720 mix; the caro- 

 tenoids between 380 and 550 m/x. The instructor 

 will show you how to use the spectrophotometer. 



EQUIPMENT 



Per student 



6 large test tubes 

 2 small test tubes 

 quart jar 



Per 2 students 



volumeter 



electric light source 



glass tumbler 



Per 30 students 



95% ethanol 



100 ml sucrose (0.5 M) 



500 ml 2,6-dichlorophenolindophenol (2.5 



M) 



500 ml phosphate buffer (0.1 M, pH 6.5) 



2 liters of 1% sodium bicarbonate 



1 liter absolute methanol 



X 10- 



0.5 liter petroleum ether 



2 liters acetone 



50 filter paper sheets, 4.5" X 4.5" 



fresh spinach leaves 



crushed ice 



100 fresh Elodea stems 



fine capillaries for applying pigments to filter paper 



roll of aluminum foil 



Per laboratory 



6 hand spectroscopes 



spectrophotometer (the Bausch and Lomb "Spec- 



tronic 20" instrument will perform adequately) 



cheese cloth \ 



Waring blendor > for instructor before lab 



centrifuge j 



0.5 M sucrose 



ultraviolet lamp and safety glasses 



