NO. 19 CHLOROPHYLL DETERMINATION JOHNSTON-WEINTRAUB 5 



In determining the percentage transmission of a chlorophyll solu- 

 tion the galvanometer deflections with and without the absorption cell 

 and solution in position are observed alternately. The percentage 

 transmission obtained from the average data is used in reading the 

 concentration of chlorophyll from the calibration curve. 



About 5 to 10 minutes are required to make the transmission deter- 

 minations. With the 5-cm. absorption cell the sensitivity is o.i micro- 

 gram (1/10.000 milligram) of chlorophyll. One square centimeter of 

 leaf is sufficient for duplicate determinations which check within 2 to 

 3 percent. This method is independent of any visual comparisons of 

 intensity or color. 



In a study of the influence of the extraction technique on the results, 

 it has been found that preliminary killing of the leaves by immersion in 

 boiling water, or the addition of calcium carbonate during the grinding 

 does not affect the amount of chlorophyll extracted. 



Furthermore, in these studies made with barley leaves it was found 

 that the presence of light during the short extraction period is negli- 

 gible. The chlorophyll content of the extract remains unaltered during 

 at least 2 weeks storage in the refrigerator (about 4 C). 



SUMMARY 



The method of determining small amounts of chlorophyll herein 

 described is based on the transmission of light in the region of the 

 red absorption band of a solution of chlorophyll in acetone. The 

 transmitted energy is determined by means of a galvanometer and a 

 vacuum thermocouple of extremely high sensitivity. The percentage 

 transmission of the acetone extracts of plant material is then compared 

 with a calibration curve constructed from data obtained with solutions 

 of purified chlorophyll. 



This method eliminates the constant use of standard chlorophyll 

 solutions and is not influenced by the presence of carotenoid pigments 

 in the extract. Furthermore, it is unaffected by minor fluctuations in 

 the light intensity, and errors involved in subjective intensity and 

 color comparisons are avoided. 



