May 3,1924 Modifications of the Picric Add Method for Sugars 
481 
present. Evidently the sucrose is inverted in a short time and the invert sugar 
produced is exposed to the picric acid for the greater part of the 10-minute period. 
Fructose was not tried alone, but sucrose gave quantitatively the same effect as 
glucose alone. Because of this, and because Okey got similar results, it was as¬ 
sumed that both glucose and fructose were affected in the same way by the picric 
acid. The ratio of the color values with and without the heating in presence of 
picric acid, or in other words the “total” to “reducing sugar” values, was de¬ 
termined, and found to be 1.045. 
This difference in color development may be dealt with in two different ways 
in making analyses by this method: Either two standards may be used, one for 
“total” and one for “reducing” sugars, or one standard can be used for both and 
a factor applied. The latter method was decided upon and the factor 1.045 has 
been used throughout the present work. 
Table I-Effect of heating on an 0.08 per cent glucose solution in picric acid before 
heating with sodium carbonate—comparison made with a standard 0.08 per cent 
glucose set at 30 mm. 
Time of heating, minutes______ 
10 
28.7 
28.6 
28.8 
15 
20 
25 
30 
Colorimeter readings: 
Series T __ 
Series II . __ _ 
Series III__-...- 
28.8 
28.4 
27.9 
28.6 
EFFECT OF DILUTING THE PICRAMATE COLOR 
In analyzing an unknown material, a color too intense for reading in the colori¬ 
meter may be obtained when the solution is diluted in the conventional manner 
to 10 c.c. Therefore it was desired* to know whether the colored solution could 
be diluted to other volumes without destroying the proportionality of intensity. 
Determinations were run on 0.64, 0.32, and 0.16 per cent glucose and the result¬ 
ing colors made to 80, 40, and 20 cc. respectively. These were then compared 
with an 0.08 per cent glucose made to 10 cc. The results are shown in Table II. 
It is apparent that either the high ratio of sugar to picric acid, or the dilution 
itself, disturbs the proportionality rather seriously. Hence dilution cannot be 
used to remedy a color that is too strong for comparison, although this has often 
been recommended {2, 11, 12e). Dehn U) lists this dilution effect among his 
fallacies of colorimetry. 
CLARIFICATION 
The choice of a clarifying agent is of primary importance in any sugar method. 
In trying to choose the best one for the present method some surprising results 
were obtained, which tend to cast doubt on the necessity of clarification in the 
case of many materials. 
Benedict ( 1 ) clarified blood from proteins with picric acid. Folin and Wu ( 6) 
used a tungstic acid preparation for clarification. Host and Hatlehol (7) made 
a comparative study of blood sugar results and found the Folin-Wu method gave 
good results both on tungstate and picric-picrate filtrates; while the Benedict 
method gave high results with picric-picrate filtrates. 
