(3) (4)
nitronium ion is formed by reducing the effective concentrations of both the dichromate and lead ions. Likewise, the lead combined with bromide is not accessible for precipitating the chromate, and the bromide so combined may or may not he available for oxidation. The bromide probably is oxidized by direct combination in a n acid-base reaction with nitronium ion yielding NOzBr as an intermediate, thereby ruling out PbBr+ ion as directly oxidizable. Thus, it is necessary to calculate the free bromide, lead, and dichromate ions present in Equations 3 and 4 to determine the actual dependence of nitronium ion on the concentrations of materials in solution. This was done, using the equilibrium constants 40 for Equation 3 (1) and 30 for Equation 4
(5)
($1.
but the order in bromide ion, based on past experience, could be either first or second. I n aqueous solutions, there is a strong tendency for halogen atom intermediate to be stabilized by the presence of the species XZ-, a free-radical ion (3, 4). The necessity for such stabilization in the activated complex might be expected to be reduced by the high temperature of fused salt solvents; however, a t least part of the reaction might go by the second order (in bromide ion) path. Thus, i t is necessary to establish the order in bromide. K h e n dichromate, bromide. and lead ions are mixed together in a solvent, the following equilibria occur :
+ +
Pb+2 Cr20;2 Pb+* Br-
e PbCrQOT ( I ) PbBr+ ( 2 )
+ + 2 CrO4-a Pb+* + Cr04-2e PbCrOl 4 (I, 8 ) Crz07-9 IiO3- *KOz+
(6)
The formation of soluble lead dichromate affects the equilibrium in which the
Elimination of nitronium ion from the rate expression and the equilibrium expressions for Equations 5 and 6 leads to :
The value of the constant kKs/K: was evaluated for all conditions with n = 1 and a satisfactory constant was obtained (Table I). Apparently there is no second order effect in bromide, and Equation 7, n = 1, satisfactorily represents the data, provided all concentrations are corrected for complex ion formation. LITERATURE CITED
(1) \ , Duke. F. R.. Iverson. ?\I. L.. Chem. Boc. 81.’5061 (1958). ’
J. Am.
Iverson, M. L., J . Phys. :1958). V., Rrav, W. C., J . Am. #6). W. C., Ibid., ’
.,,
62. 3357’(1940). ’ ( 5 ) -$Vesth&er,’ F. H., Chem. Rev. 45,
419 (1949). RECEIVED for reviev October 17, 1958. Accepted March 5, 1959. Division of Analytical Chemistry, 11th Annual Summer Symposium, ACS, Schenectady, N. Y., June 1958.
Sensitive Colorimetric Method for Determination of Fructose LYLE E. HESSLER Textile Research laboratories, Cofton Research Committee o f Texas, Texas Technological College, Lubbock, Tex.
,A rapid, direct method is needed for the determination of fructose in plant material in the presence of aldopentoses and aldohexoses. Several aromatic amines, notably p-anisidine and 3,3-dimethoxybenzidine, gave accurate, rwroducible results when used with 85% pbospboric acid for determining fructose in the presence of glucose. The method gave low COefficients of variation on known quantities of fructos-. Good reproducible results were found in determining fructose in the cotton boll in a range of 9 to 236 mg. of fructose per gram of dry weight material. The method will find application in many biological systems.
A
s
of an extensive study of cellulose synthesis in the development of cotton bolls, a n accurate method for the determination of fructose was required. The colorimetric determination of this natural sugar in biological systems has ah-ays been difficult because of interference from aldose sugars. Methods of sugar analysis are very often empirical and time-consuming. PART
1234
ANALYTICAL CHEMISTRY
Adjustments, compensations, and factors are necessary to obtain a fair degree of accuracy in determining the constituents of a sugar mixture. The colorimetric determination of sugars and related substances has recently been reviewed rather extensively (8). Several anthrone methods (1,2,6) for the determination of fructose in the presence of glucose, have appeared, based on different rates of color formation for the two sugars, a t different temperatures. The methods are rather limited in their application, because of the large number of substances that interfere with the anthrone color reaction. Chromatographic separation of sugars has intensified the search for colorimetric reagents. I n most cases these reagents have been developed for sprays and are not specific. Mukherjee and Srivastava (4) used p-anisidine phosphate in ethyl alcohol as a spray reagent in chromatographic separations. They found that fructose gave a lemon yellow color, on paper, after heat development. Glucose and other aldoses also gave some color with the reagent. A study has now been made of anisidine and dimethoxybenzidine, and a number of other aromatic amines as
reagents for the colorimetric determination of fructose in the presence of aldoses. EXPERIMENTAL
REAGENTS.p-Anisidine and 3,3-dimethoxybenzidine (Eastman Kodak Co.) were used. The p-anisidine was of higher purity than the technical grade 3,3-dimethoxybenzidine, which may account for less color in the former reagent. PHOSPHORIC ACID, reagent grade, 85%. SUGARS. The following monosaccharides were used: fructose (Eastman Organic Chemicals), dextrose (reagent grade), mannose, arabinose, rhamnose, and xylose (Nutritional Biochemical Corp.) . APPARATUS. A Bausch & Lomb Spectronic 20 colorimeter was used for the colorimetry. IIWESTIGATION OF REAGENTS. Fructose gave a greenish yellon. color with p-anisidine in 85y0 phosphoric acid, after heating, with little interference from aldose sugars. Dimethoxgbenzidine behaved similarly. The transmittance at various n-ave lengths \yas very much the same for both reagents. The most suitable m-ave length was around 450 mg. Standard curves
020 n in
1
I
I
I
I
0.05 0.10 0.5 0.20 0.25 FRUCTOSE MG.
0.05 0.10 0.15
Figure 2.
020 0.25
Determination of reagent concentration
FRUCTOSE MG.
Figure 1. Calibration curve for fructose by means of p-anisidine and of dimethoxybenzidine reagents
(Figure 1) were straight-line relationships between absorbance and concentration of fructose for both reagents. I n phosphoric acid, dimethoxybenzidine had a slight color which did not interfere with the determination. This color diminished on standing. Anisidine had almost no color. The optimum concentration of reagent was determined by experimentation (Figure 2). Both anisidine and dimethoxybenzidine gave the best results with 0.5% reagent in 85% phosphoric acid. T h e length of time for maximum color development was 30 minutes (Figure 3). Determinations with known concentrations of fructose showed little interference of glucose with either reagent. Mannose, arabinose, xylose, and rhamnose developed a slight color by this method (Table I). T h e determiuation of sugar involves extraction, evaporation of alcohol, and clarification in aqueous solution. Interference from the accompanying salts was investigated. Sodium chloride, phosphate, and acetate up to 25 mg., in the determination, did not interfere with the color development with panisidine reagent. Procedure. Fructose or t h e unknown containing not more t h a n 0.3 mg. or less t h a n 0.05 mg. of fructose was measured into a large borosilicate glass test t u b e (25 x 200 mm.), allowing t h e volume of solution not t o evceed 2 ml. If t h e fructose solution is less t h a n 2 ml. in volume, i t is necessary t o increase t h e volume t o 2 ml. with distilled Trater. T o t h e sugar solution were added 4 ml. of reagent (0.5q10 anisidine in 85% phosphoric acid). A water blank was made up a t the same time. The solutions were gently heated t o boiling, over a gas burner, and cooled to room tem-
Figure 3. Rate of color development for glucose and fructose as determined by p-anisidine and dimethoxybenzidine reagents
P---e--o
*Ic-o---o---
0
z
FRUCTOSE
a
0
--- ANlSlDlNE
a
D
M
0.10 p
--
T
-0-
-
e----I
To obtain information on the reproducibility of the method, six determinaTable 1.
~
E
-GLUCOSE 6- -
4 0 5 0 TIMEMNUITS
IO
perature. After 30 minutes of cooling, the greenish Yellow color reached a maximum and the spectrometric analysis Was made at a Tvave length Of 450 mp.
I
H
2030
a
60
tions v, eie inade on approxiniately 0.1 mg. of fructose (Table 11). A standard deviation of 0.00258 found, with a coefficient of variation of 2.69% for the anisidine reagent and a standard deviation of 0.00152 with a coefficient of variation of 1.52% for the diniethoxybenzidinr reagent.
Interference from Aldose Sugars in Determination of Fructose
Aldose Sugar
Sugars
Taken, Mg.
Rhamnose Xylose Arabinose Mannose Glucose
0.2 0.2 0.2 0.2 0 2
Rhamnose Xylose Arabinose hlannose Glucose
0.2 0.2 0.2 0.2
0.2
Fructose Found Absorbance Mg, % Anisidiiie Rengent 0.020 0.007 0.35 0.020 0.007 0.35 0.003 0 15 0.008 0,001 0.20 0.010 0.004 0 001 0.05 DimethoxybenEidine Reagent 0.028 0,009 0.45 0,030 0,009 0.45 0.007 0.35 0.020 0.015
0.015
0.004
0.004
VOL. 31, NO.
7, JULY 1959
0.20 0.20
1235
Precision of Fructose Determination
Table (I.
Reagent .\iii*ictine
Al)sorl)-
Found,
ante
Ng. 0.0'33 0.098
Iletn.
0 220
0.235 0 220
0.235 0.235 0.235 Av. Ilimet hoxybenzidine
0 0 0 0
5 6
0.1000 0.1000 375 0 0975 380 0 1000 385 0 1025 380 0 1000 Av. 0,1000
During the 1957 crop year, s o m ~160 -nmples of cotton bolls of various ages w r e analyzed for fructose. The rew l t s ranged from 9 to 236 nig. of fructose per gram of dry w i g h t . Glucose micentration was, in most cases, greater than the concentration of the fructose. The recovery of known quantities of fructose added to a n unknon n sugar holution extracted from cotton bolls and tletermined with the anisidine reagent is shown in Table 111. The unknon-n cnontains glucose and plant constituents found in the cotton boll. The accurate recovery indicates little interference from plant constituents.
*ample S O
I 2 3
Standard Deviation 10.00258
70
2.67
+0.0015 t0.0015 0,0000
0.0000 -0 0025
~0.00158'2
Fructose, hIg./Gr:tma
Saniplt.
Bolls,
Analyst
S0
Days
1
'3
65 67 69
21
165 75 17
169
28
35 Dry weight.
1236
Analyst
ANALYTICAL CHEMISTRY
-ii
17
Sample Bolls,
Fructose. lIg./
so.
Ph y s
Reagent
Grama
19
'31
127.5
21
"8
23
35
.Inisidine Dirnethosybenzidine Anisidine Dimethouybenzidine Anisidine Dimethoxy benzidine
0 0000 +O 0025 0 0000
127 6 61 '3 58 6
14 8
14 T
Dry weight.
Table IV shows the analysis of cotton boll3 of various degrees of development and changing fructose concentration. Each analyst started with a solution extracted from the cotton boll, and carried the fructose analysis through to completion. The results are well within the experimental error, as shown in Table 11. Table V shows that there is not much difference in the anisidine and dimethoxybenzidine reagent when used to determine fructose. ilnisidine gives a more colorless blank and is perhaps preferred, although either reagent can be relied on to give good results.
DISCUSSION
of
Age of
1.52
Recovery of Fructose as Determined with Anisidine Reagent b y Adding Known Amount to Unknown Fructose, 11Ig Unknoan, Standard, Ml. 0 1 11g /I11 Absorbance In samplr Found 0 0550 0 0550 0 130 20 0 0775 0 0777 0 5 0 185 1 0 0 1275 0 1250 10 0 300 1 0
Table IV. Determination of Fructose in Cotton Bolls by Anisidine Reagent
Table V. Determination of Fructose in Cotton Bolls of Various Ages b y Means of Anisidine and Dimethoxybenzidine Reagents
+0.0015
0.098 0.0965
RESULTS
Table 111.
+0.0015 -0.0035
0.098 0.098
0,380
:3 1
-0,0035
0,093
0.380
1
->
Error, vg.
Coefficient' of Variation,
The reagent hydrolyzes fructose containing compounds such as sucrose and inulin. Both compounds gave a straight-line relationship with p-anisidine, indicating that the reagent nil1 determine fructose in dissaccharides and polysaccharides without previous hydrolysis. I n biological systems if sucrose is present, a correction can be made by determining the amount of
invert sugar. Khere a complete sugar analysis is made, this is usually performed anyway. Ketose sugars which are not common to biological systems, such as sorbose, tagatose, sedoheptulose, and lactulose, will develop color with the fructose reagent. d number of aromatic amines were investigated for specificity in the determination of fructose. Most of these amines were specific for fructose in the presence of aldose sugars, but they lack color intensity for determining small quantities of ketose sugars. I n exploratory tests, phenylhydrazine and benzidine gave indications of the same specificity and good color intensity as anisidine and dimethoxj-benzidine reported here. ACKNOWLEDGMENT
Appreciation is expressed to Harken McAda for assistance in running the tests and preparing the manuscript. LITERATURE CITED
(1) Bonting, S. L., Arch. Biochem. 5 2 , 272 (1954). ( 2 ) Brown, W. L., Young, bI. K., Seraile, L. G., J . Lab. Clin. M e d . 49, 630 (1957). (3) Dubois, M., Gilles, K. A,, Hamilton, J. K., Reber, P. A., Smith, F., ANAL. CHEX 28, 350 (1956). (4) ?Mukhejee, S., Srivastava, H. C., hature 169, 330 (1952). (5) Wise, C. S., Dimler, R. J., Davis, H. A,, Rist, C. E., Ax.4~.CHEJI. 27, 33 (1955). RECEIYEDfor review August 18, 1958. iiccepted February 24, 1959.
