EXPERIMENTAL METHODS. 27 



ground in a meat-chopper. By using a fairly coarse cutter, there was no 

 loss of juice. The manipulation was carried out as rapidly as possible. 

 The ground mass was thoroughly mixed and put into a dish, covered, and 

 placed in a hot-air oven at 98. In this manner the plant material is quickly 

 killed and all danger from enzyme actions, which by the usual means of 

 drying are first considerably accelerated, is thus avoided. On account of 

 the danger of subsequent action of enzymes on the carbohydrates, expression 

 of the juice of a plant is also impractical. After heating thus for about 

 30 minutes, the cover was removed and the material dried for 24 hours at 

 98. It was then ground to a fine homogeneous powder and was ready for 

 analysis. 



THE DETERMINATION OF DRY WEIGHT. 



As has been indicated, in order to gain a clear conception of the carbo- 

 hydrate transformations in a vegetable organism, it is essential to know the 

 water relations. For this reason each sugar analysis was supplemented 

 with a determination of the dry weight. A small amount (about 10 grams) 

 of homogeneous freshly ground material was placed in a tared wide-mouth 

 weighing bottle, weighed, first heated with the cover on as described above, 

 and then dried at 98 and cooled in a desiccator before weighing. This 

 always was done in duplicate or triplicate and the results agreed well with 

 each other. Further heating at 98 or keeping in a desiccator for several 

 days caused no further appreciable loss in weight. Separate experiments 

 showed that in this drying process the disaccharides were not appreciably 

 inverted. 



PREPARATION OF MATERIAL FOR ANALYSIS. 



A separation of the simpler sugars from the condensed polysaccharides 

 was attained by extracting one portion of the dry material with 95 per cent 

 alcohol and by hydrolyzing another portion with dilute acid. For the 

 alcoholic extract 30 to 50 grams of the dry material was mixed with 2 to 5 

 grams of powdered calcium carbonate in order to neutralize the plant acids 

 present and thus prevent inversion of the disaccharides. Although calcium 

 carbonate gives a very slightly alkaline solution, this is not sufficient to cause 

 enolization and consequent rearrangement of hexoses or formation of 

 saccharinic acids. Stronger alkalies are to be avoided, as they cause the 

 reactions mentioned. This is a very serious source of error in much of the 

 work in the literature on the nature of hexoses in leaves, as apparently the 

 ease of mutual rearrangement of, e. g., d-glucose, d-mannose, and d-levulose 

 was entirely disregarded. These transformations have already been dis- 

 cussed in the introductory discussion. 



For the present purpose no attempt was made to distinguish these sugars 

 in the analyses, but they are regarded rather as belonging to one physio- 

 logical group. The calcium salts of most of the plant acids are quite 

 insoluble in alcohol. The mixture of the dry plant material and calcium 

 carbonate was extracted twice, with 10 times its weight of 95 per cent 



