APPLICATION OF PAPER CHROMATOGRAPHY 1657 



and a second fractionation made with another solvent, moving under right 

 angle to the first one, thus producing a two-dimensional pattern. The re- 

 sulting spots, usually invisible to the eye, can be "developed" by color 

 reagents (such as ninhydrin for amino acids or molybdate for phosphoric 

 acid); radioactive spots are easily detected by "radioautography" — for 

 this purpose, the filter paper is pressed against an x-ray film, and the latter 

 developed. 



Individual spots can be identified from the known solubilities of the 

 compounds, whose presence appears likely, and the identification can be 

 confirmed by coincidence checks with known compounds. If a spot con- 

 tains sufficient material it can be cut out, eluted and investigated by the 

 usual chemical methods. 



This technique was first applied by Stepka, Benson and Calvin (1948) 

 to the C*-tagged amino acids from Chlorella and Scenedesmus. 



After 30-sec. photosynthesis in the presence of C*02, the material from Scenedesmus 

 showed activity predominantly in aspartic acid, somewhat less in alanine. Radioauto- 

 graphs revealed some activity also in asparagine, serine, alanine and phenylalanine. 

 In similar material from Chlorella, there were about equal amounts of active aspartic 

 acid and alanine. 



Calvin and Benson (1948), and Benson, Bassham, Calvin, Goodale, 

 Haas and Stepka (1949) extended the chromatographic method to tagged 

 products other than the amino acids. 



Figure 36.13 shows the location on the paper chromatogram of a num- 

 ber of compounds which may be of interest in connection with the work on 

 photosynthesis. Water-saturated phenol is the one (basic) solvent; a 

 mixture of butanol with propionic acid and water the second (acidic) sol- 

 vent. Cationic substances are moved toward the left, anionic toward the 

 top of the figure. Lipids and lipophilic compounds are moved into the 

 upper left corner, farthest from the origin ; while sugars, phosphate esters, 

 and other neutral, hydrophilic compounds stay near the original spot, in 

 the right bottom corner. A mixture containing acetic (instead of pro- 

 pionic) acid was used when it was desirable to move phosphate esters more 

 effectively. 



In these experiments, algae (or leaves) were killed by dropping them, 

 after exposure to C*02, into hot 80% aqueous ethanol (instead of acid, as 

 before). The extract was concentrated to 2 cc. and 0.01-0.2 cc. were ap- 

 plied to a 1.5-cm. circle in the corner of the sheet. Amounts varying be- 

 tween a few microgram (acids) and 1-2 mg. (sugars) could be separated in 

 this w^ay. 



Radiograms were made from cells exposed to C*02 for between 5 and 

 90 sec. in light, after having been engaged in steady photosynthesis in 1-4% 



