Simple Sensitive Test for Com pounds Co nta ining the Cyc Io penta diene CH2 Gro uping Application to Air Pollution EUGENE SAWICKI, THOMAS W. STANLEY, and JAMES NOE Air Pollution Engineering Research, Roberf A. Tuff Sanifary Engineering Cenfer, Public Healfh Service, U. S. Deparfment of Health, Education, and Welfare, Cincinnati 26, Ohio
b The reaction of 1,2-dinitrobenzene,
I 7
1,4-dinitrobenzene, and
1,4-dinitIonaphthalene in alkaline solution with compounds containing the cyclopentadiene CH2 group results in a stable blue to green color with long wave length maxima ranging from 600 to 750 mp. The wave length maxima and identification limits obtained from the reaction of 1,2dinitrobenzene with over 100 fluorene derivatives have been tabulated. In a neutral aromatic fraction this test appears to be specific for the cyclopentadiene CH2 grouping. The presence of fluorene and benzo[ a ] - and/or benzo[b]fluorene in air-borne pariiculates has been confirmed with the help of the test procedure.
A
specific, and sensitive test for fluorene and its derivatives would be of value in the analytical investigation of complex organic mixtures. Fluorene and some of its derivatives give brilliant color reactions with piperonal chloride in trifluoroacetic acid solution (4, 6 ) . However, this reagent will react with any aromatic compound which has its basic center a t an aromatic carbon atom and is more basic than benzene. Recently an elegant thermochromic test for fluorene and some of its derivatives was described (6). However, fluorenones or inner-ring p-quinones will interfere with the test. Many fluorene derivatives substituted with strongly electronegative or electropositive groups gave negative results. The thermochromic procedure was also found to be difficult to apply to a quantitative analysis for fluorenic compounds. A search of the literature has disclosed that cyclopentadiene, indene, and fluorene and some of its derivatives give a blue to green color on reaction with 1,4-dinitrobenzene in alkaline solution ( 2 ) . In our investigations many polynitroaromatic compounds gave a color reaction with fluorenic compounds in alkaline solution. Of these, 1,2816
SIMPLE,
e
ANALYTICAL CHEMISTRY
I
L__Lp-p-, --J -
~
p
~
600
Figure 1. spectra
500
700
Lmu Visible absorpiion
--------. . . . .. . ---
I
--
dinitrobenzene appeared to give the best results. The mechanism of the color reaction conceivably could involve the reduction of the dinitro derivative in alkaline solution to a colored dianion: O
'NO2
&
2,
Q*ho:
Visible absorption spectra
Colorimetric procedure with 1,2-dinitrobenzene reagent for Fluorene 2-Acetamidofluorene 2-Nitrofluorene
------.-. . . ..
O " C 2
-
700
600
i,mp
Figure 2.
Colorimetric procedure for cyclopenta[d,e,f]phenanthrene with 1,2-Dinitrobenzene 1,4-Dinitrobenzene 1,4-DinitronaphthoIene
+
--
-
Zh$
UO,
Evidence has been given that this type of anion is formed by the reaction of an organic reductant with p-dinitrobenzene (1). On the other hand Meisenheimer (3) postulated that the colored product had a structure such as
From the data in Figure 1 and Tables I and 11, it would appear that the mechanism is somewhat more complicated. EXPERIMENTAL
Reagents and Equipment. 1,2- and 1,4-dinitrobenzene and 1,4-dinitronaphthalene were obtained from the Aldrich Chemical Co., Milwaukee, W e . , as were the polynuclear hydrocarbons. The fluorene derivatives n-ere prepared by standard literature procedures. Details or references are given in previous publications. A Cary Model 11 recording spectrophotometer was used for determination of the wave length maxima. Spot Test Procedure. To 1 drop (0.02 ml.) of the dimethylformamide test solution mas added 1 drop of 0.25% 1,2-dinitrobenzene in dimethyl-
~
forniamide, follon-ed by 1 drop of 10% aqueous tetraethylammonium hydroxide. ii blue t o green color indicated t h e presence of a compound containing t h e cyclopentadiene CH2 grouping (Table I). Colorimetric Procedure. One milliliter of t h e dimethylformamide test solution mas pipetted into a 10-ml. volumetric flask; 0.2 ml. of a 0.25y0 dimethylformamide solution of 1,2dinitrobenzene was added, followed b y 0.2 ml. of 10% aqueous tetraethylammonium hydroxide. T h e solution was then diluted t o t h e mark with dimethylformamide. The absorption spectrum was determined from 500 to 800 mp against a blank containing everything except the test compound. DISCUSSION
A large number of fluorene derivatives gave positive results in both the spot test and colorimetric procedures (Table I). Carcinogens, such as 2-nitrofluorene, 2-acetamidofluorene, 2trifluoroacetamidofluorene, 2-benzoylaminofluorene, and 2,7-diacetylaminofluorene gave positive results. Substitution in the 1- and 9-positions of fluorene seemed to h a r e an adverse effect on the test. Honever, 9-carbosyfluorene gave positive results, probably because the compound decomposes t o fluorene in alkaline solution ( 7 ) . Hydroxy derivatives, such as 2-, 3-, and 9hydroxyfluorene, gave negative results. On the other hand, 2.5- and 2,7-dinitrofluorene gave red colors in the test. Several other compounds containing the cyclopentadiene C H p grouping were tested with positive results (Table 11). The wide variation in identification limit is probably directly related to the percentage yield of blue to green dye(s) in the test. A miscellaneous group of compounds was also tested, either because a benzyl group was present or the compound could conceivably be found with a fluorenic hydrocarbon in the aromatic fraction of air-borne particulates. All these compounds gave a negative test. They included acenaphthene, anthracene, anthrone, bene jalanthracene, henzo [alpyrene, benzo [elpyrene, benzyl methyl ketone, chrysene, dibenzofuran, dibenzothiophene, 9.10-dihydroanthracene, 9,lO-dihydrophenanthrene, diphenylmethane, fluoranthene, fluorennaphone, lJ2,3,6,7,8-hexahydropyrene, thalene, perylene, phenanthrene, phenanthraquinone, pyrene. triphenylene, and xanthene. Some aliphatic ketones that give a color reaction in the test but are easily separated through volatilieation or chromatography present no serious problem. Several polynitro derivatives produced a blue to green color with fluorene when substituted for 1,2-dinitrobenzene in the test procedure. Some of these were l,4-dinitrobenzene, 1,2-dinitro-3,4-
Table I.
W a v e Length Maxima and Identification Limits of Fluorene Derivatives in the 1,2-Dinitrobenzene Color Test Amax,
Compound F5 -
I-Diacetylamino F 2-Methyl F 2-Ethyl F a-Phenylthio-2-benzyl F h--(2-F-thioacetyl) morpholine 2-Fluor0 F 2-Chloro F 2-Bromo F 2-Methoxy F 2-Methylthio F 2-Ethylthio F 2-Acetylthio F 2-Diacetylamino F 2-A4cetamidoF 2-Difluoroacetamido F 2-Trifluoroacetamido F 2-Perfluorobut yryl-
amino F 2-Trichloroacetamido F 2-Benzoylamino F 2-( 2-Carboxybenzoylamino) F 2-( 3-Toluoylamino) F 2-( 2-Toluoylamino) F 2-(4-8nisoylamino) F 2-Isonicotinoylamino F 2-( 2-Furoylamino) F 2-Methoxycarbonylamino F 2-ethoxy carbonylamino F 2-( 2-Fluoroethoxy-
carbonylamino) F 2-( 2-Chloroethoxycarbonylamino) F 2-( 2-Hydroxyethoxycarbonylamino) F 2-Ethylthiocarbonylamino F Ethyl 2-F-ureylene 2-Mesylamino F A'-Methyl 2-mesylamino F 2-(4-Tosylamino) F iV-Methyl 2-(4-tosylamino) F h7-Ethyl 2-(4-t0sylamino) F S-n-Propyl 2-( 4-tosylamino) F 2-( 2,4-Dimethylbenzenesulfonylamino) F 2-( 3-Kitrobenzenesulfonylamino) F 2-Benzalamino F 2-( 4-AIethylbenzalamino) F a F. Fluorene.
MP 695 585 695 695
Ident. Limit, 0.1 3.0 0.2 2.0
710
1.o
700 700
2.0 0.2
705
0.2 0.2
705
690 698 698 705 675 680 717
0.5 1 .o 2.0
0.3 1.3 1.o 7.0
675
14.0
665
2.0
675 695
8.0
695
0.8 0.5 0.8 8.0 7.3
700
1.5
665 650 650 585
14.0
700
1. o
700
0.4
730
0.4
700
0.5
695
0.5
670 665 710
3.4 0.5 3.0
700
0.3
700
0.8
700
0.8
705
0.3
660
9 .o
675 705
9.0 0.3
700
0.7
Compound 2-( 4-Isopropylbenzalamino) F 2-( 2,PDihydroxybenzalamino) F 2-Piperonalamino F 2-(4-Acetamidobenzalamino) F 2-( 4-Dimethylaminobenzalamino) F 2-Cinnamalamino F 2-( 1-Kaphthalamino) F
2-Sulf0 F ,Y,.Y-Di-n-butyl 2-1:sulfonamide N-n-But yl 2-F-sulfonamide 2-Rlesyl F 2-Acetyl F 2-Benzoyl F 2,2'-di F ketone 2-Sitro F 4-Methoxycarbonylamino F Ethyl 4-F-ureylene 4-Renzalamino F 4-Carboxy F 4-Carbamoyl F ?V-Isopropy1-4-cai-bamoyl F 9-Carboxy F 2-Amino-3-nitro F 2-Acetamido-3-nitro F 2-Mesylamino-3-nitro F
705
2.0
2-(4-Tosylamino)-3nitro F 2-Acetamido-7-methyl F 2-Acetamido-7-ethyl F 2-Acetamido-7-bromo F 2-Acetamido-7-iodo F 2-hcetamido-7-sulfo F 2,7-Diacetylamino F 2,7-Dibenzalamino F 2-Benzalamino-7-ethylF 2-Acetyl-7-hydroxyl F 2-Acetyl-7-amino F 2,7-Diacetyl F 2-Yitro-7-methyl F 2-Nitro-7-ethvl F P-Nitro-7-meihoxy F 2-Kitro-7-trifluoroacetamido F 2-Nitro-7-amino F 2-Kitro-7-sulfo F 2-Xitro-7-acetyl F
Xrn,Y,
;Ilp
Ident . Limit, -i
700
670
1.6 1.5
7 00
0.8
700
1. G
700 705
0.8 1. 5
705
0.8
TOO
0.6
710
0.9
705 705 705
0.3
715
0.7
590 615 715 -675 710
0.3
0.6 0.3 4.3 0.5
710
0.3
715 725
695 750 740
0.3
0.4
0.5 0.5
0.2 2.
0.3
725
0.3
725
0.5
675 065
14.0 1.3
700 700
0.8
665 620 710 710
630 700 710
725 725 720
0.4 2.a
1. 4
0.4 0.3 2.3 0.3 0.5
0.3 0.3 1 .o
735
0.2
720 730 725
0.6 0.7
1.8
Table II. W a v e Length Maxima and Identification Limits of Miscellaneous Compounds Containing the Cyclopentadiene CH2 Grouping in the 1,2-Dinitrobenrene Test
Compound Cyclopentadiene Indene 3-Hydroxyindenea 4H-Cyclopenta [d,e,f]phenanthrene 1-Indanone.
WJ 570
Ident. Limit, Y
10
'05 705
2.0
Amax,
Compound llH-Benzo[a]fluorene 11H-Beneo [b]fluorene 7H-Benzo[c]fluorene 7-Ethyl-9H-fluoreno [ 2,3414 1,2,5]selenadiazole
>I$ 725 725 740 -775
VOL. 39, NQ. 7, JUNE 1960
Ident. Limit, Y
6 0 4 0 2 0
0 3
817-
dimethylbenzene, 1,2-djnitro-3,4-dichlorobenzene, Ar,X-dimethyl-3,4-dinitroaniline, 2,2’-dinitrobiphenyl, 2,4’dinitrobiphenyl, 1,5-dinitronaphthalene, and 1,4-dinitronaphthalene. Of these compounds 1,2-dinitrobenzene, 1,4-dinitrobenzene, and 1,4-dinitronaphthalene were, by far, the best. The type of spectra obtained by the reaction of these three dinitro derivatives with 4Hcyclopenta [d,e,f]phenanthrene is shown in Figure 1. Considering the sensitivity arid the stability of the final colored products and the ready availability of the reagent, 1,2-dinitrobenzene was chosen for a more thorough study. The spectra of the reaction products obtained in the reaction of this reagent with fluorene, 2-nitrofluorene, and 2acetamidofluorene are s h o r n in Figure 2. One standard colorimetric procedure was used for all the reagents and all the test substances. The procedure
appears capable of application to the quantitative analysis of compounds containing the cyclopentadiene CH2 grouping. However, to obtain optimum results in the colorimetric analysis for any one particular compound, the conditions of the test would probably hare to be varied. Application. When t h e benzene extract of urban air-borne particulates is chromatographed through alumina with pentane, a fraction is obtained between the pyrene and chrysene fractions, whose ultraviolet absorption spectrum shorn points of resemblance t o t h e analogous spectra of benzo[a]and benzo[b]fluorene. The test applied to this fraction or to the benzofluorenes gave a grecn coior.,,,A, 730 mp. This result confirms the prcsence of a benzofluorenc in urban air particulates. The combined chromatographic fractions obtaincd after the
aliphatic fraction and before the pyrene fraction also gave a green color, Amax 695 mpj in the test. Apparently fluorene is also present in urban air particulates. LITERATURE CITED
( I ) Kuhn, R., Weygand, F., Ber. 69, 1969
11936). (2j Levy, K.$ Campbell, X., J . Chem. Sac. 1939, 1442.
(3) AIeisenheirner, J., Ber. 36, 4174 (1903). (4) Sanicki, E., Chemzst-Analyst 47, 9 11988). ( 5 ) SaAcki, E., Elbert, W., Ibtcl., 48, 68 (1959). (6) Sawicki, E., Miller, R., Stanley, T., Hauser, T., ~ ~ N A LCHEM. . 30, 1130 (19%). ( 7 ) Rislicenus, IT.,Ruthing, -A,, Be?. 46, 2770 (1913). RECEIVED for review Sovember 3, 1959. Accepted January 25, 1960.
Spectrophotometric Determination of Carboxyl in Oxidized Starch HERBERT C. CHEUNG, BENJAMIN CARROLL, and C. EDWIN WEILL Chemistry Departmenf, Rutgers, The State University, Newark, N. J. ,Methylene blue i s used in a spectrophotometric procedure to determine the carboxyl content of oxidized starches produced b y the hypochlorite oxidation process. The basis for the method i s the apparent constancy of the binding affinity o f methylene blue and carboxylate ion a t infinite dilution of the dye. Prior removal of inorganic cations usually present in carboxylated starches is not essential. For the special case of samples with exceedingly high carboxyl content, a simpler and more direct colorimetric procedure may be used.
T
are several methods for the determination of the carboxyl content of oxidized starch. The two methods generally used are direct titration, which is applicable only to substances having relatively high carboxyl content, and titration of the acid liberated b y cation exchange, using a reagent such as calcium acetate (3, 6). The latter method yields accurate results, if the carboxyl groups are initially in the free acid form, and the sample is free from inorganic cations. Recently Hofreiter, R701ff, and Mehltretter ( 2 ) studied the determination of carboxyl content in dicarboxylated cornstarches by an ion HERE
8 18
ANALYTICAL CHEMISTRY
exchange procedure. This paper reports analytical results using the cationic dye, methylene blue, spectrophotometrically to determine the degree of carboxylation in starch. This method is more convenient and rapid than the conventional titration method, because it is based on one or two colorimetric readings as compared to a complete titration. For substances with lorn carboxyl content, the direct titration method is unreliable. I n previous attempts t o determine the carboxyl content of a carbohydrate using a cationic dye, the sample was kept in the solid form and vias usually washed or equilibrated n ith the dye solution. The uptake of the dye b y the solid was subsequently determined. A one to one correspondence is assumed for the dye-carboxylate ratio. This method was used by Davidson (1) to determine the carboxyl content of cotton cellulose. Attempts to use it for carboxylated starch yielded erratic results (a), although Schoch and Maywald (9) obtained qualitative information. The latter investigators used a wide variety of dyes to identify starch in granular form (9). I n the case of granular carboxylated starch not all the carboxyl groups are available for binding. There is also the possibility of nonspecific adsorp-
tion of the van der TTaals type. I n the method suggested in this paper, the carboxylated starch is dissolved and the uptake of the dye is determined directly in the solution. This is possible because the adsorption is accompanied by a spectral change. If the specific adsorption is extrapolated to infinite dilution, the ratio of the extrapolated values to the carboxyl content of the sample is a constant. EXPERIMENTAL
Buffers were prepared directly from reagent grade sodium hydroxide and monopotassium phosphate. -4histological grade of niethylene blue (Fisher Scientific Co.) 17-as used without further purification. Correction as made, however, for dye purity as given b y the supplier. T h e carboxyl contents of starches prepared by t h e hypochlorite oxidation process (3) Jvere determined by potentiometric titration, using the glass electrode. The starch samples were used without prior removal of the inorganic cations that were present. Apparatus. A Beckman Model G pH meter was used. A Beckman DU spectrophotometer equipped with cells of 1-cm. p a t h was employed for all absorbance measurements. Procedure. T h e starch solution was prepared in the usual manner bj7 first making a slurry, then pouring Materials.
