Colorimetric Determinationof 5-Nitro-2-furfuridene
Aminoguanidine
BYSHICHIRO AKIYA and RYOJI SAWAMURA(Received June 15, 1952)

It has been often reported on the color reaction of 5-nitro-2-furfuralderivatives. In this paper, the following two methods are applied tofind whether they are useful for the detection and the colorimetricdetermination of its aminoguanidine derivative: oner) is to detect nitrosoradical which is obtained by reduction of nitroradical and the other 2 )is the same method as 5-nitro-2-furfural and its semicarbazone derivative
whose color is changed into red with caustic alkali.
(I)

Coloration by sodium nitroprusside after reduction in neutrality.
Aqueous solution of 5-nitro-2-furfuridene aminoguanidine (Guanofracin, the commercial name, G. F. is abbreviated in this paper) isfiltered after reduction with zinc dust. · To the filtrate are added
sodium nitroprusside solution and KOH solution, and it turns reddish
brown color. Its detectability is so weak that the limit is in 10 mg%(1/10,000) solution, and the distilled water which does not contain G.F.
turns deep yellow by the same treatment as mentioned above. It istherefore difficult to apply this color reaction to the colorimetric determination for our purpose.
Coloration with caustic alkali.
By the addition of caustic alkali, G.F. aqueous solution turns orangeor red or makes fine red crystal in strong alkalinity, corresponding toG.F. concentration. To the reaction mixture are added dioxane, carbowax, aceton or other various solvents, respectively, in order to increase
the stability and intensity of this coloration, and, as the result, aceton
is the best one for this purpose. Using Beckmann's spectrophotometer,
it is found that no strictness is required in the quantity of alkali to beadded, 1 cc of N-NaOH solution being enough for 10 cc of test solution,and that the absorption maximum is observed approximately at 410 mµ.
Further studies clarified that the following method is the most suitable
one for the determination: the test solution is prepared in a proportionof 8 cc of G.F. solution, 2 cc of aceton and 0.1 cc of lON-NaOH, andthen the absorption spectrum is measured at 410 mµ after kept standingfor 1.5 hours,(II)s. AKIYA and R. SAWAMURA

32

So the optical density of 0.1-2 mg G.F. aquous solution is measured
by Beckmann's spectrophotometer. The result (Fig. 1) shows that theoptical density is measurable at this range of concentration and there
is a proportional relationship between the concentration of G.F. and theoptical density at 410 mµ. (In Fig. 1 the lines show the optical densityof G.F. solution of various concentrations at 410 mµ and the dottedlines show the relationship between the wave length and the opticaldensity.) The result of the measurement of 0.1-100 mg% G.F. aqueous
solution by the Duboscq colorimeter (Fig. 2) shows an approximately proportional relation. (In Fig. 2 the dotted line indicates the values of thesame test solution which are measured by Beckmann's spectrophotometer.
1·100i·ooo.900

I.
I

G.F. watersolution
Aceton

800

I___1
1

Dist. waterlON-NaOH

I

1--II__ii 2cc

l-e--1-- ·cc
12iISample Contrast Sample !Contrast

8cc

20.1

1I 8

I 0.1.. ..

j1

11'1,0.1

I lOcc
I 0.1700IIer i

600

500·400(II)300

20J0'F-

681·0Fig, 1.

1·4'
16Colorimetric Determination of 5-Nitro-2-furfuridene AminoguanidineII40·401

J20~10~llL'-mg % 2Fig.

g,..._o5·33

3
3~5- - - - 10

rnL':m9.% 201002a.

/r"'

l!
3I

0·2

0·5l·O

2·05·010

2050

100-Log mg.%

Fig.

2b.

(b) is summarized from (a).
In case of color reaction using caustic alkali, the above describedrange of concentration (0.1-2 mg%, viz. l/1,000,000-1/50,000) gives avery suitable condition for the determination by spectrophotometer 1 ands. AKIYA and R. SAWAMURA

34

using the Duboscq colorimeter, colorimetric determination can be madeeasily at a wide range of concentration (O.l-100mg%, viz. 1/1,000,0001/1,000).
Table 1.
Wave length

15 min.

45 min.

1 hr.

I 1.5 hr.
I

360 mµ380
400-

-

0.327
0.470

0.360
0.490

0.232
0.373
0.498

410

0.502

0.515

420
430
460-

0.475
0.330

I

2 hr.

3 hr.

--0.243
0.380
0.502

0.500

0.502

0.522

0.515

0.5150.515

0.505
0.468
0.360

0.502
0.461
0.366

0.495

0.488

-0.360I-Table 2.
Wave lengthIIIIIIIV390 mµ400
410
420

0.465
0.522
0.528
0.510

0.470
0.525
0.531
0.510

0.465
0.512
0.530
0.510

0.448
0.495
0.518
0.495

The authors wish their thanks to Toyama Chemical Co. Ltd., forthe supply of reagents and also to Messrs. S. Fukai and Y. Nakaji for
their assistance in experiments. A part of the expenses of this study
owes to the Experimental Research Fund of the Ministry of Education,
to which also they are much obliged.
Experimental PartColoration by sodium nitroprusside after reduction in neutrality.
1. ProcedureTo 3 cc of G.F. aqueous solution in a test tube are added 6-7 dropsof 10% CaCl2 and about an spatuful of zinc dust. The reaction mixtureis heated for a few minutes in a boiling water bath and then filteredthrough filter paper. After the filtrate is cooled, to it are added 6-7
drops of newly prepared 2.5% sodium nitroprusside solution. The mixture is vigorously shaken and then to it are also added 3 drops of 8%
KOH solution, and the precipitate therein is removed by filtration. The
filtrate turns reddish brown color in a few minutes.
(I)Colorimetric Determination of 5-Nitro-2-furfuridene Aminoguanidine

352.

Discussion( 1) The deep yellow, transparent filtrate is obtained by theabove mentioned treatment from both whale blood and distilled water
which do not contain G.F.
( 2) This coloration is conspicuously observed in 100 mg% G.F.
solution but not so in 10 mg% solution.

(II)

Coloration by caustic alkali.
1. Procedure
To 8 cc of G.F. solution are added 2 cc of aceton and the mixture
is vigorously shaken. And then to it is added 0.1 cc of lON-NaOH andshaken vigously again. 1.5 hours later, absorption is measured witha colorimeter. In case of using Beckmann's spectrophotometer, a mixture of 8 cc of water, 2 cc of aceton and 0.1 cc of lON-NaOH or amixture of 10 cc of water and. 0.1 cc of lON-NaOH is used as a control
and the absorption at 410 mµ is measured.
2. Discussion( 1) Absorption spectra of aceton-alkali-aqueous solution
Absorption spectrum of various concentrations in aceton aqueoussolution, to which alkali is added is measured with Beckmann's spectrophotometer between 330 mµ and 450 mµ, and then the values obtained
are almost constant, showing about 0.020 of optical density. The fluctuation of absorption by various quantities of aceton added is negligible.
( 2 ) Effect of the reaction time
The results of the measurement for 15 minutes to 3 hours after
preparation of test solution are shown in Table 1. The values areconstant for 45 minutes to 3 hours after preparation of test solution(The value which is obtained for 1 hour after preparation is a littlelarger but the difference is only 1.3% from other values).
( 3 ) Effect of various proportions of quantity between acetonand G.F. aqueous solution
G. F. solution (in cc)
Ace ton(lON-NaOH
Water(II(IIII))

I
8.5
1.5

0.1

82

III
7.5
2.5

IV
73

0.1

0.1

0.1

II

Control

0.1
10.0

The result (Table 2.) shows that the mixture of proportion II gives
the largest value in optical density. As the values for mixtures ofproportion I and II fluctuate only 0.3-0.6 % of the value of II, there i1:3
no necessity to change proportion mor~ closely.

36s. AKIYA and R. SAWAMURA( 4) In case of 100 mg% G.F. solution, there is no appearance ofred precipitate even after the test solution is kept standing for severalhours.
Summary

For the purpose of the colorimetric determination of aqueous solutionof 5-nitro-2-furfuridene aminoguanidine, the two methods of coloration
with sodium nitroprusside after reduction in neutrality and by caustic
alkali were tried. The former was not suitable because of its dull sensitivity (10 mg% is the limit of detection). The latter was, on thecontrary, applicable between in 0.1 and 100 mg% of G.F. concentration.
Literatures
1) Seiichi Ooguma: Kagaku no Ryoiki (Jr. of Jap. Chemistry) 4, 622 (1950).
2) Seishi Takagi: Jap. J. of Pharmacy and Chemistry 20, 28 (1948).
(Pharmaceutical Department, Faculty of Medicine, Tokyo University)