SEPARATION OF CHLOROPLASTIC MATTER 369 



of volume II. However, pigments represent only a fraction of the total 

 dry matter of the chloroplasts, of the order of 5 or 10%, as we shall see 

 later. Until 1938, not much attention was paid to the nature of the 

 remaining 90-95%. Since then, however, several attempts have been 

 made to carry out a quantitative separation and analysis of chloroplastic 

 matter, by Chibnall (1924, 1939), Menke (1938l■2•^ 1940^'2), Granick 

 (19381-2), Mommaerts (1938, 1940), Neish (1939'-'), Krossing (1940), 

 Bot (1942), Comar (1942), and Galston (1943). 



The first stage in the isolation of chloroplast matter is the grinding 

 of leaves, either in distilled water or in a hypertonic, 0.5 molar sugar 

 solution. The fractionation of the green suspension obtained in this 

 way can be carried out by different methods. The cell wall debris is 

 removed by centrifugation. The remaining suspension contains the 

 water-soluble components (originating in the cell sap, cytoplasm, and 

 chloroplasts), whole or broken nuclei and chloroplasts, forming a more 

 or less stable suspension, and the cytoplasm, in the form of a colloidal 

 solution. Chibnall (1924, 1939) separated the chloroplastic matter 

 (together with a small quantity of nuclear matter) from the cytoplasmic 

 and vacuolar material by filtration through paper pulp. Menke (1938^), 

 Granick (19380, Bot (1942), Comar (1942), and Galston (1943) used 

 fractional centrifugation, in which nuclear, chloroplastic and cytoplasmic 

 matter were precipitated in that order. Instead of utilizing differences 

 in the size and density of the particles, as in the mechanical precipitation 

 and filtration methods, the separation of chloroplastic matter from the 

 cytoplasmic material can also be based on the larger content of the 

 former in hydrophobic (lipoid) compounds, which causes it to be prefer- 

 entially salted out. Menke (1938^) used fractional coagulation by 

 ammonium sulfate, hydrochloric acid, or carbonic acid, while Neish 

 (19390 and Comar (1942) used calcium chloride. 



To check whether the composition of the "chloroplastic matter," 

 separated by fractionation is identical with that of intact chloroplasts, 

 Menke (1938') prepared by centrifugation, a small fraction (containing 

 only 1-2% of the total chloroplastic material), consisting entirely of 

 whole chloroplasts or large chloroplast fragments. He found in this 

 fraction 48% proteins and 37%o "lipides," while the salted-out "chloro- 

 plast fraction" contained 56% protein and 32% "lipides." Menke 

 concluded that as much as 15% of cytoplasmic matter was coprecipitated 

 by ammonium sulfate with the "chloroplastic matter," and applied a 

 corresponding correction to all analyses. 



An uncertain point in some of these fractionations is the fate of the colorless 

 "stroma" of the chloroplasts. If by grinding the leaves one obtains whole chloroplasts, 

 or large fragments of these bodies, then the stroma must follow the grana— certainly 

 in centrifugation and probably in coagulation as well. On the other hand, if the grinding 



