A S A L Y T I CAL E DI T I O S
114
1-01, 2 , s o . 1
Colorimetric Determination of Acriflavine and Neutral Acriflavine',' Edward Lee Howes DEPARTXEST
OF
SURGERY, YALE vNIVERSITY, S C H O O L
CRIFLAT'ISE (3, 6-dianiino-iiiethylacridiiiiuin chloride monohydrochloride) or neutral acriflavine (3,6-diamino-A--methylacridinium chloride) may be determined gravimetrically by weighing the precipitated silver chloride after the addition of dilute sulfuric acid and silver nitrate t o the dye. The total weight is calculated on the percentage of chloride in the total molecular \\-eight. The acid acriflavine also may be determined by titration with 0.1 N sodium hydroxide. These methods are subject to considerable error and are only available for large amounts. Qualitatively acriflavine and neutral acriflavine are easily determined, even in small quantities, by the marked fluorescence imparted t o a n aqueous solution. Acriflavine hydrochloride may be differentiated from the base by the effervescence i t gives with an equal volume of a saturated solution of sodium bicarbonate. On the addition of coiicentrated fuming nitric acid (commercial) t o a strong solution of acriflavine, there develops a deep purple color. This colorimetric method is based on this reaction.
A
Experimentation
Table I-Intensity ( u ) a n d Duration ( b ) "of Reactions of Standard Solutions of Acriflavine a n d Neutral Acriflavine with Various Dilutions of Nitric Acid ACRIFLAVINE
a
"02
0 . 2 5 CC.
Concd. 20 10
0 2+ 3+
10
9
3+
8
Concd. 20 10 5 Duration
0
0.5cc.
0 3c
3+ 42+
0 0
0 2
6 7
10
0 23-
s
4+
3 7
0 1+ 32+ 3+ 4+ is given in minutes. 0 0
8
14
3+
2 5 12 8
0
3 15 8
3
23+
J
2-
1T
2-
4-3-
8
1 h
15 b
NEUTRALACRIFLAVINE Color Dilution 1-100 Deep purple 1-500 Characteristic 1-1000 Characteristic 1-1500 Characteristic 1-2000 Characteristic 1-2500 Faint 1-3000 Very faint Range: 1-500 t o 1-2000
The color reaction obtained with 1 cc. of 10 per cent nitric acid and 1 cc. of the standard acriflavine solution or 2 cc. of the neutral acriflavine was selected as having the best initial intensity and the longest duration. The nitric acid is not diluted accurately, but it must be made fresh from fuming nitric acid (commercial]. ChemiReceived November 6 , 1929. of this investigation has been defrayed by Davis and Geck, Inc. 1
* T h e expense
Table 111-Volume of Standard Solutions T h a t \Vi11 Give Satisfactory Color with 1 cc. of 10 Per Cent Nitric Acid ACRIFLAVINE * Intensity a t start Duration
VoLLMc
cc
NEUTRAL ACRIFLAVISE Intensity a t start Duration
Jlznutes
0 5 1 2
2 T 3 f 4+ Red, n o t purple
Table IV-Comparison
8 15 18
2+ 3 7
4r
Minutes -3 8 12 10
of Colorimetric Readings after Standing Various Periods of Time
(Solutions mixed simultaneously, same dilutions) R ~ W D I NOF C TIMEOF .4V. READIKGO F SOLK. 1 STAKDINC SIMILAR SOLNI. Minutes 25 3 24 8 "0 5 19 9 15 8 14 8 25 12 23 4
The coinparison therefore should be completed within 10 minutes after the reaction is started. Technic
I+
Table 11-Color Reactions Obtained with 1 CC. of 10 Per Cent Nitric Acid a n d 1 CC. of Acriflavine a n d Neutral Acriflavine of Various Dilutions ACRIFLAVINE Color Dilution 1-100 Brick red Purple b u t not characteristic 1-5000 Characteristic 1-6000 Characteristic 1-10,000 Characteristic 1-11,000 1-12,000 Characteristic 1-13,000 Too faint Range: 1-6000 t o 1-12,000
cally pure nitric acid will not give the desired color reaction. As the commercial acid is a variable product and the amount of fuming even more so, according to the freshness of solution, it is necessary to experiment with the dilution of the nitric acid until a maximum color of longest duration is obtained. This dilution has been found to vary from 1 to 20 per cent depending entirely on the fuming properties of the acid. However, once this factor is determined, the reactions with the diluted nitric in two standard solutions started a t the same time are practically identical and of sufficient duration t o obtain accurate colorimetric readings. Table I V gives the colorimetric comparison of the reactions of many standard solutions after definite time intervals.
2 cc.
1 cc.
0 8 20 12
lfEDICIKE, Now HAVEN,COSN.
3
Two standard solutions of neutral acriflavine and aci iflavine, each containing 10 mg. of the dye dissolved in 100 ce. of water, were tested against various amounts of nitric acid in varying dilutions. Tables I t o I11 gire the results of these experiments.
Dilution of
OF
One cubic centimeter of 10 per cent nitric acid (fresh) is added to 1 cc. of standard acriflavine solution ( 2 cc. of neutral base) and to 1 cc. of the unknown solution in colorimeter cups. These are stirred vigorously until the color reaction is complete. If the colors do not match roughly, it is necessary to dilute the unknown further. Strong solutions of the dye give a Burgundy red with nitric acid instead of purple. The volumes are then made u p to 10 cc. each and read in the colorimeter. The standard gives the strongest color a t 20, but may be used a t 15 or 25. I n those cases where the unknown solution is weaker than the standard, it is best to adjust the colorimeter until the unknown gives its best hue and to adjust the standard to it. Table V gives the results of determinations x i t h known solutions of acriflavine. Table V-Determinations XO.
with Known Solutions of Acriflavine READING Gram 0.00054 0.00061 0.002 0 0016 0.00099 0.00045 0.013 (strong s o h )
KNOWN
Gram 0.0005 0.00O61 0.002
0,0015 0.0001 0.0005 0.0100 (calcn. X 100)
The error is less than 10 per cent and the method is accurate to the third decimal place. The writer is greatly indebted to Treat B. Johnson, of the Department of Chemistry, Yale University, for his helpful suggestions.
