Color Reactions of Amine Antioxidants H. P. BURCHFIELD AND J. N. JUDY Naugatuck Chemical Division, U.S . Rubber Co., Naugatuck, Conn. Several color reactions of the amine antioxidants are useful for the detection and identification of the most important classes of these materials used commercially. The principal tests are based on coupling and oxidation reactions in organic solution in the presence of anhydrous stannic chloride. They may he applied to isolated materials or to solvent extracts of vulcanized rubber samples.
A
S ASALYTICAL problem frequently encountered by the
obtained from diphenylamine is optically identical with that obtained by the oxidation of diphenylbenzidine. I t is of interest to note that N-nitrosodiphenylamine mill react in the absence of amyl nitrite.
rubber chemist is to identify or confirm the presence of the amine antioxidants either in rubber samples or as chemical compounds. The task may be the relatively simple one of establishing whether a rubber batch has been mixed correctly, or it may involve more extensive testing to determine the type of stabilizer used in a vulcanized compound of unknown source. To identify the isolated materials, standard procedures, such as the determination of melting point and ultimate analysis, are frequently used. Hawever, it is helpful to have available methods by which a preliminary idea of the nature of the sample can be obtained. End6 ( 2 ) has described tests for the detection of the arylamines by the colors formed on oxidation in the presence of sulfuric acid. In the case of the diarylamines, it is probable that a benzidine rearrangement occurs, folloiyed by a partial oxidation of the intermediate product. The mechanism of this reaction has been investigated in detail by Kehrmann (4). While it is useful in some applications, the colors formed are difficult to reproduce, and the test suffers from a technical disadvantage in that it must be carried out in the presence of aqueous sulfuric acid. This paper describes several new color reactions suitable for the detection of a wide variety of antioxidants, n.hich can be carried out in the organic solvent used to extract the rubber sample. In addition to the obvious advantages inherent in the use of a medium in which the reactants are easily soluble, a much wider variety of reaction conditions can be employed. This allows for a sharper distinction between the various classes of antioxidants. Several of the tests described are variations of familiar procedures, such as the coupling of diazonium salts with amines and the indamine reaction with p-phenylenediamine in the presence of ferric chloride (IO). However, the reactions of greatest interest are several new coupling and oxidation procedures which are carried out in the presence of anhydrous stannic chloride. This reagent is colorless and soluble in most organic solvents. It can increase its covalence to 6 and thus acts as a powerful electron acceptor, which leads to the ready formation of intermediate complexes that can rearrange or react further to give the desired products. The colors formed are reproducible and relatively stable. In a number of specific cases they have been made the basis for quantitative methods of analysis. As the composition of many commercial antioxidants is indefinite, they are generally referred to by their trade names. A list of the materials discussed is given in Table I.
Table I.
Approximate Compositions of An tioxidants
AgeRite Resin AgeRite Resin D AgeRite Syrup
\
AgeRite White Aminox Aranox Betanox
Commercial
Aldol 1-naphthylamine Acetone-aniline condensation products containing 2,2,4-trimethyl-1,2-dihydro. quinoline and polymers LV,N’-bis(2-naphthyl)-p-phenylenediamine
Diphenylamine-acetone condensationproduct p-(p-Tolylsulfonylamido)-diphenylamine
BLE
1
Flectol A Flectol B Flectol H Neosone . Neosone D, AgeRite Powder Santoflex B
Phenyl-2-naphthylamine-acetone condensation product Diphenylamine-acetone condensation product Acetone-aniline Condensation products Phenyl-1-naphthylamine Phenyl-2-naphthylamine Acetone - w - aminodiahenvl condensation
Stabilite Stabilite Alba
St ali te VGB
The complex formed is an intermediate Oxidation product, and the color fades on the addition of a large excess of reagent. When the reaction is carried out in glacial acetic acid, in which the intermediate product is more stable, it can be used for the quantitative estimation of diphenylamine and related materials. I t is necessary to maintain the amyl nitrite concentration a t 0.0005 molar or less to avoid excessive fading. The transmission is measured a t 590 nip a t a concentration of 2.5 X mole per liter of diphenylamine. The color formation follows the BeerLambert law except a t concentration ranges below 1.0 X 10-b mole per liter where fading occurs. -4s the N-acetyl derivatives do not react, it is necessary to avoid long standing or refluxing in acetic acid solution. In making both the qualitative and quantitative tests, the stannic chloride must be added before the amyl nitrite. In a qualitative test intense colors ranging from blue-violet to blue-green are obtained from the diaryl amines, while the products formed from the naphthyl aryl amines are green. This permits the detection of these materials in many types of rubber compounds. Typical antioxidants which give strong positive tests are phenyL2-naphthylamine, phenyl-1-naphthylamine, AgeRite Stalite, and N,N’-diphenyl-p-phenylenediamine. Stabilite and Stabilite Alba form brilliant red precipitates. Thus the test serves to detect both the diaryl amines and the arylsubstituted ethylenediamines and to distinguish them from one another. Acetone-aniline antioxidants and aldehyde-amint accelerators give yellow to orange colors, which are not sufficient11 intense or characteristic to serve as criteria of identity. 11. Stannic Chloride-Benzotrichloride Reaction. A very intense color test semispecific for the diarylamine-ketone conden-
DESCRIPTION OF METHODS
I. Stannic Chloride-Amyl Nitrite Reaction.
Diary1 amines and naphthyl aryl amines unsubstituted in a t least one of the para positions are readily oxidized to intensely colored products by nitrous acid in the presence of anhydrous stannic chloride. Amyl nitrite is used as the source of acid and benzene as the solvent. The reaction is similar to the condensation and subsequent oxidation of diphenylamine in strong sulfuric acid; however, here the reaction proceeds readily in 0.1 M stannic chloride solution. The mechanism is demonstrated by the fact that the product
786
187
OCTOBER 1941 sation products is obtained by reacting the antioxidant with benzotrichloride in the presence of anhydrous stannic chloride. Ethylene dichloride is used as the solvent. The reaction is believed to proceed through the formation of carbonium ions by the interaction of the reagent and the starfnic chloride, which results in coupling a t the para position of the amine to form highly colored arylmethane chlorostannates. Stannic chloride is particularly efficient in this reaction because of its ability to abstract chlorine from the reagent and product to form the stable SnCle-anion ( 3 ) . The reactive constituents in the antioxidants are the alkyl-substituted acridans produced in the condensation reaction :
none dianil by benzoyl peroxide (9). The intense orange-yellow color produced in this reption is useful for the detection of the aryl-substituted p-phenylenediamines and several other easily oxidized materials, as most of the antioxidants react slowly, if at all. I n some cases the color is deepened to red-violet or blue by the addition of anhydrous stannic chloride. This is a valuable aid, as it allows the operator to distinguish between a number of additional types, and produces a color more easily recognized in the extract from a rubber sample. The reactions obtained on several materials of this class on solutions containing 0.05 gram per liter are tabulated below, The Mulliken system of color notation is used ( 7 ) :
H .* Antioxidant .V,S’-diphenyl-p-phenylenediamine AgeRite White Aranox
R
BzzOz
OY-S 041 OY-T2
BzzO*
+ such
RV-S B-N V-T2
R
The selectivity of the reaction is evidently dependent on the basic properties of the amines. Those which can donate the unshared electron pair on the nitrogen to the tin to form stable coordination complexes are deactivated a t the ortho and para positions, while those of low basicity such as triphenylamine and 5,5dimethylacridan react readily. This is to be expected in view of the difference in reactivity between aryl amines and aryl amine hydrochlorides. The use of ethylene dichloride as a solvent partially satisfies the electron-accepting powers of the tin and greatly increases the rate a t n hich the reaction can take place when compared to its speed in benzene. Traces of polar solvents which form stable complexes with stannic chloride completely inhibit the reaction. When carried out under strictly anhydrous conditions the colors are sufficiently stable and reproducible for accurate photometric estimation. Caution must be used in the choice of the solvent, for somp types of commercially available ethylene dichloride contain unsaturated impurities which modify the results of the test. BLE, Aniinox, and other diphenylamine-ketone condensation products normally produce violet colorations, while Betanox forms a green, and triphenylamine a red product. hlost of the other aryl amines react slowly to form pale yellow to orange colorations which are unimportant analytically. In the presence of the contaminated solvent the reactions of Betanox and triphenylamine are unaltered; however, the products obtained from materials of the BLE type are a deep blue violet, while phenyl-1-naphthylamine which normally does not react a t all yields an intense scarlet a*oloration. This is the most specific reaction described. I t can be used for the detection of the diaryl amine-ketone condensation products either alone or in the prespnce of accelerators and other antioxidants. 111. Coupling Reaction. A familiar reaction, and one which has probably found use in many rubber laboratories, is the coupling of diazonium salts with amines to form azo dyes. As all aromatic amines react, the distinction is one of degree rather than kind.. The reagent chosen for this work because of its stability and activity in coupling reactions is p-nitrobenzenediazonium chloride. Many other compounds will serve equally well, if the reactions with amines of known composition are observed and classified. The reagent is added to an acetone solution of the amine, and the color develops immediately. Phenyl-l-naphthylamine gives a violet coloration, while phenyl-2-naphthylamine and acetone-aniline condensation products such as Flectol B and AgeRite Resin D yield red solutions. Most other antioxidants give colors ranging from orange to pale yellow. While many of them can be distinguished by direct comparison, the differences in color are slight, which precludes the use of the test in many applications. IV. Benzoyl Peroxide Oxidation. I n benzene solution S,N ’diphenyl-p-phenylenediamine is oxidized quantitatively to qui-
V. Indamine Reaction. The rvaction of aniline with pphenylenediamine in aqueous ferric chloride solution to form a green indamine dye has been widely used in the analysis of both primary amines and diamines ( I O ) :
HC1,H2N - - ( I I Z ) - S = ~ NH.HCl+
3FeC12
+ 2HC1
If an acetone solution of an amine antioxidant is diluted with two volumes of water and p-phenylenediamine and ferric chloride are added, a characteristic color is produced. The reaction takes place with diaryl amine and aryl naphthylamines as well as with primary amines. In some cases an oxidation product is formed in the presence of ferric chloride alone. Many of the antioxidants react to give brilliant green colors similar to that obtained from aniline. However, phenyl-lnaphthylamine produces only faint yellow color, while phenyl-2naphthylamine forms a bright red. Thus diaryl amines may be detected in the presence of the former and a characteristic color reaction is available for the latter. A4cetone-aniline condensation products, which give tests difficult to distinguish from those of phenyl-2-naphthylamine in some previous reactions, form green products similar in appearance to those of the primary amines. These materials frequently produce intense blue-green colors on the addition of ferric chloride alone. Aldehyde-amine antioxidants give reactions similar to those of the primary amines. The applicability of the test is limited, as a relatively high concentration of antioxidant is required to produce a color sufficiently intense for analytical purposes. If the sample does not contain large amounts of asphaltic softeners or extenders, this disadvantage may be partly overcome by extracting a large sample and reducing the volume of solvent on a steam bath. VI. Stannic Chloride-Bromine Test. Stannic chloride and bromine react with aniline-acetone condensation products in ethylene dichloride solution to give orange-red insoluble addition complexes, the formation of which is specific for materials of this class. Excess bromine is removed by the addition of an unsaturated compound such as allyl chloride. The test is not sufficiently sensitive to be applied to the analysis of rubber samples. S,N’-diphenyl-p-phenylenediamine and other easily oxidized materials produce dark green-blue solutions which interfere. Antioxidants which give positive tests include Flectols A, B, and H, AgeRite Resin D, and AgeRite Syrup, all of which are described as acetone-aniline condensation products containing 2,2,4-trimcthyl-1,2-dihydroquinoline and polymers.
7aa
V O L U M E 19, NO. 10
V. Indamine Reaction. Place 5 ml. of the acetone solution of antioxidant in a test tube and add 5 drops of Test I I1 I11 IV V p-phenylenediamine solution. Add 10 Phenyl-1-naphthylamine G-SI yo-n VR-N ... Y-n ml. of water and 3 drops of ferric chloPhenyl-2-naphthylamine G-S1 cy-n R-N ... R-N AgeRite Stalite BG-S1 y-n ride solution. The color requires several v- N BLE BG-SI minutes to develop. The optimum conBetanox GY-N G-S1 A',"-diphenyl-p-phenylenecentration of antioxidant is about 0.25 diamine BV-S2 y-n yo-n OY-N-RV-N OR-N gram per liter. AgeRite White B-91 y-n RO-N 0-S1-B-N 0-tl VI. Stannic Chloride-Bromine Test. Flectol A oy-n y-n ... BG-N R-N Stabilite Red prey-n To 5 ml. of an ethylene dichloride soluRO-N *.. VR-SI cipitate tion of the antioxidant add 1 ml. of Santoflex B oy-n y-n yo-n RV-N RV-T2 VGB stannic chloride solution. Mix and add oy-tl gy-tl yo-n ... G-S1 3 drops of bromine solution. After 30 5 BG-S1 blue-green shade one, RV-S red-violet normal, etc. seconds add 2 drops of allyl chloride to remove the excess bromine. The formation of a red precipitate indicates the presence of an acetone-aniline condenREAGENTS sation product. Many easily oxidized amines produce green t o blue-green colors under these conditions and obscure the test, The Stannic Chloride Solution. Dissolve 14.7 ml. of fuming stanoptimum concentration of antioxidant is 0.25 gram per liter. nic chloride in anhydrous analytical reagent grade benzene and The test cannot be applied to the analysis of rubber samples. make the volume up to 250 ml. Amyl Xitrite Solution. Dilute 5 ml. of amyl nitrite to 100 ml. with benzene. DISCUSSION p-Nitroaniline Solution. Dissolve 1.0 gram of p-nitroaniline in B mixture of 25 ml. of concentrated hydrochloric acid and 25 ml. The purpose of this paper is to present a detailed description of .of water and make the volume up to 100 ml. with water. a number of specific reactions with only broad general indications Sodium Nitrite Solution. Dissolve 2.5 grams of sodium nitrite of how they can be used. This is in part necessary because of the in water and make the volume up to 100 ml. Benzoyl Peroxide Solution. Dissolve 5.0 grams of benzoyl large number of amine antioxidants now available and the many peroxide in a mixture of 10 ml. of glacial acetic acid and 40 ml. of more which may be mkketed in the future. To include informabenzene. Prepare fresh daily. tion on all the materials in use would be impractical; furthermore, Ferric Chloride Solution. Dissolve 10 grams of ferric chloride many commercial products are mixtures of several chemical types. hexahydrate in a mixture of 10 ml. of concentrated hydrochloric acid and 90 ml. of water. On these, the color tests may be intermediate, or in some cases p-Phenylenediamine Reagent. Dissolve 0.1 gram of recrystalgive evidence of the presence of several components. lized p-phenylenediamine in 10 ml. of acetone. Prepare fresh The principal values of these reactions are (1) to establish daily. whether or not an amine antioxidant is present in a given stock, Bromine Solution. Dilute 5 ml. of bromine to 100 ml. with carbon tetrachloride. and (2) to determine the broad general class to which it belongs. Benzotrichloride, allyl chloride, ethylene dichloride, benzene, Minor variations in structure or composition cannot be detected and acetone. unless they influence the reactivity of the material toward a given reagent. None of the tests described will distinguish SAMPLE PREPARATION Flectol A from Flectol B, or BLE from Aminox. The most we can say is that the former materials are .acetone-aniline condenWeigh out 0.05 gram of the antioxidant and dilute to 20 ml. with the solvent specified in the procedure. Dilute 1.0 ml. to sation ptoducts, while the latter belong t o the diary1 amine50 ml. for tests I, 11, 111, or IV. This solution will contain apketone group. proximately 0.05 gram per liter. Prepare a solution containing A convenient approach to a systematic identification of the 0.25 gram per liter by diluting 1.0 ml. of the original solution to isolated compounds is to examine the reactions of mhterials ex10 ml. Use this for tests V and VI. The following procedures have been found satisfactory for pected to be of interest and classify the colors produced in the testing tread stocks containing 1.0 part of antioxidant on 100 various tests by comparison with standards such as those proparts of rubber. Prepare the sample by breaking it down on a vided by Mulliken ( 7 ) . A summary of the reactions obtained on rubber mill. Place 0.5 gram in a test tube and add 5 ml. of the some typical commercial products a t the concentrations specified specified solvent, Heat gently in water bath for 4 to 5 minutes. Cool to room temperature and proceed with test I, 11, 111, or in the analytical procedures is shown in Table 11. IV. Under some circumstances test V may be used by refluxing The notation is an abbreviated form of the one described by 5.0 grams of sample with 20 ml. of solvent for 30 minutes and Mulliken. The reactions which can be used to detect or charactesting the extract. The sample should not contain intensely terize the antioxidants are printed in capitals while those which colored materials. yield faint colors or are not sufficiently specific are printed in lower case letters. In choosing the reactions and classifying the PROCEDURE tests emphasis was placed on those yielding strong red, blue, and I. Stannic Chloride-Amyl Nitrite Reaction. To 5 ml. of a green colorations which can be perceived a t high dilution. The benzene solution of the antioxidant containing approximately orange and yellow colors are not so useful, as they are less char0.05 gram per liter, add 1 ml. of stannic chloride solution and 3 drops of amyl nitrite solution. Note the color which develops. acteristic and more difficult to detect in solvent extracts. 11. Stannic Chloride-Benzotrichloride Reaction. To 5 ml. of The use of the Mulliken notation is of limited value for the an ethylene dichloride solution of the antioxidant containing detection of antioxidants in rubber, as the concentration is not approximately 0.05 glram per liter, add 1 ml. of stannic chloride solution and 2 drops of benzotrichloride. The color develops in known and the depth of the color may vary considerably from the a few seconds. standard. Judgment, therefore, must be based on hue alone. 111. Cou ling Reaction. Add 5 ml. of sodium nitrite solution While this is usually reproducible, it may sometimes be altered by t o 25 ml. o! p-nitroaniline solution and permit the mixture to the presence of other materials in the extract. Thus, a strong stand for 15 minutes. Add 3 drops of reagent to an acetone solution of the antioxidant containing 0.05 gram per liter. The yellow background may cause a blue to appear green-blue. color develops immediately. The reagent should be prepared The tests described were evaluated on a series of tread stocks fresh daily. containing 5 parts of softener and 1 part of antioxidant, and were IV. Benzoyl Peroxide Oxidation. To 5 ml. of a benzene solution of the antioxidant containing 0.05 gram per liter add 3 drops found to give results in agreement with those obtained on soh- . of benzoyl peroxide solution. Record the color which develops tions of pure materials. Samples may occasionally be encoun1minute. Add 1 ml. of stannic chloride solution and record after ....~ tered, however, where this is not the case. In such instances it is the modified color. Table 11.
-
~
Classification of Reaction Colors According to Mulliken"
-
189
OCTOBER 1947 frequently possible to concentrate or isolate the material sought by auxiliary physical methods such as steam-distillation ( I ) , chromatographic adsorption, or selective solubilities of the amines or their hydrochlorides in various solvents. The materials most likely t o interfere are softeners and extenders which produce dark colored extricts. Dithiocarbamate and thiazole accelerators do not react. Diphenylguanidine and aldehyde-amine accelerators give colors similar to those of the antioxidants in test V but do not interfere in other tests. Many of the phenolic antioxidants couple with p-nitrobenzene diazonium chloride in test I11 to yield pale yellow reaction products. Aged samples in which the antioxidant has deteriorated may fail to give satisfactory results. This is to be expected, howevcr, as the reactions described are characteristic of the amines but not of their oxidation or deromposition products. There are many color tests other than those described above which can be used to characterize the antioxidants. Primary amines such as aniline can be diazotized and coupled with 2naphthol, or reacted with p-dimethylaminobenzaldehyde to form yellow Schiff’s bsscs (8). Diphenylamine can be detected by a modification of t kit, Zerelyitinoff reaction in which benzoyl chloride is added to the product obtained on treating the amine with methyl magnesium iodide in the presence of anisole (6). Aldol I-naphthylamine gives a red-violet color in the presence of cupric chloride ( b ) . Further investigations in this laboratory on reactions carried out in the presence of anhydrous stannic chloride indicate that phenyl-2-naphthylamine can be oxidized by tertbutylhydroperoxide to an orange-red complex useful for its quantitative determination, while the tetraaryl hydrazines produced by lead peroxide oxidation of the diarylarnines yield the highly colored tetraaryl hydrazinium ehlorostannates (11).
These, and other methods may be of value in some applications. However, the six procedures described above appear to be the most useful of those investigated. The colors produced are brilliant and reproducible and sufficiently selective to allow the operator to distinguish among a large number of antioxidants. ACKNOWLEDGMENT
The authors wish to thank Virginia S. Martin for her aid in carrying out this work. LITERATURE CITED
(1) Craig, D., IND. ENG.CHEM.,ANAL.ED.,9,56 (1937). (2) EndB, H., J . SOC.Chem. Ind., J a p a n , 38 (suppl. binding), 618 (1935). (3) Hammett, L. P., “Physical Organic Chemistry,” 1st ed., pp. 53-4, New York, 51cGraw-Hill Book Co., 1940. (4) Kehrmann, F., and Roy, G., Ber., 55, 156 (1922). (5) Kirchhoff, F., K a u t s c h u k , 7, 7 (1931). (6) Meade, E. M., J . Chem. SOC., 1939,1808. (7) Mulliken, S. P., “Method for Identification of Pure Organic Compounds by a Systematic Analytical Procedure Based on Physical Properties and Chemical Reactions,” 1st ed., pp. 230-4, New York, John Wiley & Sons, 1904. (8) Schoorl, N., P h a r m . Weekblad, 77, 1381 (1940). (9) Sidgwick, N. V., and Taylor, T. W. J., “Organic Chemistry of Nitrogen,” new ed., p. 98, Oxford, Clarendon Press, 1937. (10) Snell, F. D., and Snell. C. T., “Colorimetric Methods of Analysis,” Vol. 2, pp. 411-12, New York, D. Van Nostrand Co., 1937. (11) Weitz, E., andSchwechten, H. W., BeT., 60, 1203 (1927). RECEIVED January 21, 1947.
Fluorometric Method for Estimating Small Amounts of Chlorophyll a RICHARD H. GOODWIN Department of Botany, Connecticut College, New London, Conn. In purified preparations of chlorophyll a and b in acetone, chlorophyll a fluoresces more t h a n chlorophyll b, when excited by t h e monochromatic ultraviolet, violet, and blue bands of t h e mercury arc spectrum. There is about a tenfold difference in fluorescent acti\ity of t h e two chlorophyll c0.mponents when irradiated with \iolet light (40t7 A.); a threefold difference when irradiated with blue light (4358 Chlorophylls a and b fluoresce independently of one another i n mixtures. The composition of a mixture of pure chlorophylls a and b can be estimated by determining its fluorescence when irradiated by violet and by blue light, since the fluorescence ratio, violet/blue, is characteristic for each percentage mixture of a and b. The presence of carotenoids does not interfere appreciably with the fluores-
k).
T
HE present paper reports an investigation of fluorometric methods for the quantitative measurement of chlorophyll. Kavanagh (8), in describing a new photoelectric fluorometer, suggested its use in the determination of the chlorophylls. His data indicate that concentratations as low as 1 microgram per liter could be determined, if a weaker quinine standard (0.25 mg. of quinine sulfate per liter) were used. The fluorometric method has a certain number of advantages: (1) It is very sensitive. Much smaller quantities of chlorophyll
cence of very low concentrations of chlorophyll a unless thk carotenoid concentration becomes greater t h a n t h a t of t h e chlorophyll. Chlorophyll a can be estimated i n a mixture of a and b by determining the fluorescence of t h e mixture when excited by violet light and by assuming t h a t all the fluorescence is due to chlorophyll a. Under these conditions, errors in t h e estimate due to t h e presence of b will be constant for any given percentage and will be less t h a n 670 for any mixture containing less t h a n 40% chlorophyll b. Crude acetone extracts of less t h a n 1 mg. of green tissue can be assayed for chlorophyll directly and rapidly by the fluorescent method. .4bsolute values of less than 2 micrograms of chlorophyll a can be obtained with a standard deviation between a series of similar samples of less t h a n 10%.
can be determined in this way than by the usual spectrophotometric and colorimetric methods using standard equipment (1, 4, 11), although special colorimeters (7) and spectrophotometers (13)have been devised which allow measurements of comparable sensitivity to be made. (2) Purification of extracts may be omitted under certain conditions, since the red chlorophyll fluorescence can be measured a t such great dilutions and other pigments present have little or no effect, and since chlorophylls are the only substances, naturally occurring in most planta in appre-
