However, wherelarger quantities of t h e acid must be added, t h e acid concentration can be brought down t o 0.70N by addition of ammonium hydroxide. T h e maximum a m o u n t of ammonium hydroxide that can he added m-it'hout affecting the absorbance of the complex \vas found to be 0.3 nil. of approximately 11N pcr 10-m1. total volume; addition of lnrger quantities increased the nbsorb:mcc. Tissue Extract. Possible interferences by tissue constituents were t8wtcd \\-it11 avocado fruit est!.net. Acetone powder of the avocado (avocado fruit tissue acetone-extracted anti pondered) was extracted with 1N p~w!iloricacid a t 70' C. for 20 minutes a n d centrifuged. T h r e e milliliters of the superlintant n'ns hcnted for 40 niiniitcs :It 203" C. with 0.5 nil. of 70% p~wliioric:ickl; phoephorus cortent \Y;S fount1 t o be 6.22 y per 3 d.of siipcmxitnnt. Aliquots of 3.5 y of phosphorus (as RHJ'OI) were atcltld to 3-nlI. :tlicjiwts of the supernntant anti !ihosplioriis contcnt, was dctermincd irig for 30 minutcs at 203" C. The rccovei'J- of thc n[ltlml phosphorus w:is 94.2 to 96.74r,, DISCUSSION
The llrcwnt method is much more
Table
1. Effect of Molybdate Concentration on Absorbance
Molybdate Reagent, 111. 1 1 2 3 4
0 5 0 0 0
Absorbance Complex Blank nith 5 y of against P agninst water bhnk 0 0 0 0 1
409 470
5iO
750 800
0 072 0 280 0 .121 0 ,321 0 320
sensitive than the original of nernhart and Wreath ( 2 ) . Parallel dctcrminntions made by the two niethocls using a Beckman DLT spectropliotoriic~ti~r gxve specific absorptivities of 4.3s Y, 104 sq. em. per gram of phosphorus nt 430 mu and 6.48 X los sq. em. per g r u n of phosphorus at 320 m p : approsiinstely a 15fold incrcnse in sensitivity. I n the concentration range of 1 to 10 y of phosphorus thc reaction folloirs Bccr-I,anibert's la^. ,2 typical determination on sis replicate?, c i c h having .i y of phosphorus in a total volume of 10 nil., had n rmxn ahsorhaiicc of 0.324 -t- 0.001. This gives a precision within 0.3C;;. The accuracy of this method may be judgcd from the results of a typical
Table II. Phosphorus Determination by Bernhart and Wreath and Modified Methods
(Results in micrograms of phosphorus) Bernhart and Wreath 1lodifcd Added F a .Idded Found 25
21.9 50.9 99.2
50 100
2.51 4.93 10.0%
2.5 5.0 10 0
determination (Tublo 11). 130th tcstz w m made using th(a same staii(1:ird solution of KI12P04. This method has iic~,nsucwssfully used for t1etormin:~tion of ! ~iiosphorus in p h n t mitochonc1ri:i ant1 in niicroorganisnis such ns Tetr;ihymcna and Chlorclla. LITERATURE CITED
(I) Allen, E. J. L., nlbchern. J .
34,
(1940). ( 2 ) Bernhart, D. S.,\\-rc:ith, .?s.IL. CIIEAI, 27, 4-10 (I!J%),
:I. It.,
858
RECEIVETI for review Ftrbru:try 23, 1939. t\ccegted .higust 2 i : 1959. Invcstigstion supported i n part 113' a grant from the Cancer Rcsenrcli Funds of the Univcrsity of Cdiforiii:i.
Spot Tests for Phenol Vapors and for Aromatic Compounds Containing Oxygen FRITZ FEIGL and ERWIN JUNGREIS laboratorio do Produccio Mineral, Ministeria da Agricultura, Rio de Janeiro, Brazil Translated by RALPH E. OESPER, University o f Cincinnati, Cincinnati, Ohio
b The Gibbs indophenol reaction for the detection of phenols by means of 2,6-dichloroquinone-4-chloroimine can be used in spot test analyses for the detection of volatile phenols or phenol vapors carried over by steam. This test involves the action of phenol vapors with filter paper impregnated with the reagent, and subsequent exposure to ammonia vapors. With the aid of this procedure it was shown that the pyrolysis of compounds of phenolic nature and also aromatic compounds containing oxygen in open or cyclic side chains splits off phenols. This finding is the basis of a useful preliminary test for such compounds. Examples are given to illustrate the usefulness of the pyrolytic production of phenols cnd their detection in the testing of materials.
c1
T
HE procedures used previously in spot test analyses for the detection of dissolved and undissolved phenols cannot be employed with vapors of phenols. However, in the preliminary and actual tests of materials, it is often of practical importance to distinguish between volatile and nonvolatile phenols in the vapor phase and in some cases t o detect phenols split off during the pyrolysis of solid materials. This laboratory found that the color reaction reported by Gihbs ( 5 ) can be employed for the detection of phenol vapors, in which phenols condense with 2,6-dichloroquinone-4-chloroiniine to yield colorcd indophenols. I n the case of the simplest phenol, phenol (carbolic acid), the condensation may be represented:
/
\
C1
C1
'cl 1-arious workers have applied this indophenol reaction for the colorimetric determination of phenols in aqueous solution with niaintenancc of slight basicity. Boyland and his associates (2) have pointed out the utility of this color reaction for the detection and distinguishing of phenols in aqueous solution. They also shorred that phenols react only in the free para-position. VOL. 31,
NO. 12, DECEMBER 1959
0
2099
However, the present writers have found that the occurrence of the Gibbs reaction requires not only a free paraposition (relative t o the hydroxyl group) , but also the absence of carboxy, sulfo, formyl, nitroso, and nitro groups in the same benzene ring as the hydroxyl (OH) group. Accordingly, the above reagent is not generally applicable t o phenols. On the other hand, the occurrence or the nonoccurrence of the Gibbs color reaction in the wet way can be of diagnostic value with respect to phenolic compounds. For instance, l-naphthol5-sulfonic acid reacts promptly, whereas the isomeric 1-naphthol-3-sulfonic acid does not react. The condensation represented above also occurs if solid phenols and phenol vapors are brought into contact with the solid reagent. The formation of indophenol is readily observed in the case of phenol and 1-naphthol through the production of brown-red products. Because indophenols are acid-base indicators, the color change based on the formation of phenolates can also be made to occur through the action of ammonia vapor on the condmsation product formed initially. These partial reactions may be readily carried out within the techniques of spot test analyses and they make it possible to detect phenols in their vapor phase. DETECTION OF VOLATILE PHENOLS
Procedure. A small amount of t h e test material or t h e evaporation residue from a drop of the test solution is heated in a micro test tube in a glycerol b a t h preheated t o 160' C. T h e mouth of the test tube is covered with a disk of filter paper t h a t has been impregnated with a saturated benzene solution of 2,6 - dichloroquinone 4 chloroimine. After several minutes, the paper is held over hmmonia vapors. A blue stain appears in the presence of volatile phenols.
-
The procedure revealed 0.3 y of phenol, 0.5 y of thymol, and 0.5 y of 1naphthol. It is possible t o distinguish between 1- and 2-naphthol, as the former (melting point 96" C.) has so much vapor tension that the indophenol reaction occurs within 1 to 2 minutes when the sample is heated to 120" to 130' C. with the reagent paper held in contact with the vapor. I n contrast, 2-naphthol (melting point 122" C.) gives no indophenol reaction under these conditions. DETECTION OF AROMATIC COMPOUNDS CONTAINING OXYGEN
Phenol is one of the products when salicylic acid is subjected to dry heating ( 1 ) ; therefore, the pyrolysis of other phenolic compounds would be expected to yield volatile phenols through loss of certain moieties, hydrolysis, and oxidation. Trials with ap-
2106
e
ANALYTICAL CHEMISTRY
proximately 100 compounds, including some with rather complicated structures, showed that practically all of them, including phenol esters and phenol ethers, give a positive response to the indophenol reaction. The exceptions were several azo dyes containing phenolic O H groups and also aromatic polynitro compounds. Surprisingly, the pyrolytic splitting off of volatile phenols is not restricted to compounds which are higher phenols or phenol derivatives. The same result was observed with arylarsonic acids, quinones, and with aromatic compounds containing oxygen atoms in opcn or cyclic side chains. The findings observed thus far indicate that the pyrolytic generation of phenols is so general that their detection through the indophenol reaction can serve as a good preliminary test for nonvolatile oxygcnbearing aromatic compounds. The attainable limits of detection frequently are in the range of 100 to 200 y which is not as low as most of the detection limits given by other spot tests. This comparative lack of sensitivity is due to the fact that the pyrolytic production of phenol is only one of various reactions occurring during the thermal decomposition of these compounds, and also because the procedure prescribed here inevitably results in the loss of part of the phenol through its partial combustion. The disadvantage is compensated for by the advantage of being able to detect for the first time the presence of aromatic osygrn-bearing compounds in such simple fashion. The reliability of these preliminary tests was determined by observing the behavior of the gaseous pyrolysis products from about 60 aliphatic compounds of the most varied kinds when tested with 2,6-dichloroquinone-4-chloroimine paper. A blue color was produced on immediate contact with the reagent paper only in the case of the cleavage products obtained from uric acid, Other purine derivatives did not exhibit an analogous behavior. The procedure is not reliable if aliphatic or aromatic nitro compounds are present. When such compounds are pyrolyzed, they yield nitrous acid which destroys the reagent, However, the presence of nitro compounds is easily detected by a separate test, as they yield nitrous acid when subjected to dry heating and this product is easily revealed by the Griess reaction. If this preliminary test ( 3 ) is positive, a second portion of the material under examination should be warmed with zinc and acetic acid to reduce the nitro groups into amino groups. After this operation, the reaction mixture should be taken to dryness and the evaporation residue then taken through the procedure for determining the presence or absence of phenols in the pyrolysis products. Simple
aromatic nitro compounds such as nitrobenzene do yield phenol when pyrolyzed. Procedure. T h e test is made in a micro test tube fixed in a piece of asbestos board. About 0.5 t o 1 mg. of the specimen is used. The mouth of the test tube is covered with a disk of filter paper t h a t has been moistened with a saturated benzene solution of 2,6 - dichloroquinone - 4 - chloroimine. The bottom of the test tube is heated gently a t first and then more strongly. This quasi dry distillation usually results in a dense gray or broirn vapor. It is essential that this fog, R hich gradually rises from the heated material actually come into contact ith the reagent paper. Several minutes' heating are required in most cases. The cooled paper is then exposed to animonia vapors. If the response is positive, a blue spot appears. I t slonly fades but the color can be restored by renewed exposure to ammonia. The vapors from aromatic aniines give blue condensation prodwts as soon as they come into contact with the reagent paper. This result does not interfere with the test for phenols, as the color from amines is discharged by ammonia vapors in contrast to the blue color of the phenols, which appears only in ammoniacal surroundings. The procedure was tried initially on the following phenols and their derivatives, including phenyl esters and ethers. Without exception, there was evidence of the splitting off of a phenol. The compounds tested were: 1- and 2naphthol, resorcinol, pyrocatechol, oand p-phenylphenol, 1,2,4-benzenetriol, naphthalenediol, 2,7-naphthalenediol, tetrahydroxyanthraquinone, (2, 7) ( 2 , 6), (1, 4), (1, 5 ) naphtholsulfonic acids, ellagic acid, 4-amino-3-methylpheno1, 1amino-2-naphtholsulfonic acid, 7-sulfo-8hydroxyquinoline, methyloxpquinoline, sulfosalicylic acid, tetrachlorohydroquinone, 2 - hydroxy - 5 - chlorobenzpaldehyde, 2,4-dihydroxyacetophenoneJ hydroxybenzaldehyde, salicylaldoxime, pentachlorophenol, 2,4-dihydroxybenzaldehyde, 4-hydrouybenzophenone, orcinol, gallic acid, phenolphthalein, fluorescein, phenetole, veratrole, 4propenylveratrole, safrole, isosafrole. anethole, 4 - phenoxybutyric acid, piperonal, rotenone, umbelliferone ethyl ether, narceine, emetine, m-phenetidine, 4 - methoxy - o - formylbenzcnesulfonic acid, anisic acid, narcotine, and acetylsalicylic acid. Phenols also resulted when the following compounds were subjected to dry heating: benzoic, phthalic, naphthoic, mandelic, chloromandelic, o-nitrobenzoic, phenylanthranilic, phenylarsonic, and m-nitrophenylarsonic acids; acetanilide, benzanilide, iV - 1 - naphthylacetamide, p-chloro- and 3,4-dichlorobenzaldehyde, quinone, anthraquinone, and phenanthrenequinone. Especially interesting is the producI
tion of phenols through the pyrolysis of the following aromatic compounds which contain oxygen in open or closed chains: phenoxyacetic acid, benzil, benzophenone, acetophenone, pheiioxathiin, 9-xanthenol, phenylalanine, hippuric acid, phenylacetic acid, henzilmonoxime, ninhydrin, anthrone, diphenylmethylcarbinol, and 7,8-benzoflavone. USE OF PYROLYTIC SPLITTING OFF OF PHENOLS I N TESTING OF MATERIALS
Differentiation of Formaldehyde Plastics. Formaldehyde can be easily
detected by t h e Eegriwe chromotropic acid reaction in plastics or resins prepared b y t h e condensation of formaldehyde with urea, melamine, or phenols (4). A further differentiation is possible through the detection of pyrolytically split-off phenols by the indophenol reaction discussed above, as this test is characteristic for formaldehyde-phenol resins. such as Bakelite. Detection
of Terylene and Dacron.
Among t h e many synthetic fibers pro-
duced t h u s far, only Terylene a n d Dacron, which are polyesters of glycol and terephthalic acid (S), contain aromatic oxygen-containing components. Both of these fibers yield phenol when pyrolyzed and the latter is easily detected in the gas phase by the indophenol reaction. A fraction of a milligrain of specimen is adequate for this test. This finding is taken from a series of studies dealing with the application of spot tests to the investigation of synthetic fibers. Testing of Dyes Containing Phenol Groups. T h e detection of phenol components is of interest in t h e analysis of dyes. S o rapid procedure has been available until now. Accordingly, milligram amounts of such dyes were subjected t o d r y heating and t h e gaseous pyrolysis products were tested b y t h e indophenol reaction. A positive response was obtained from purely phenolic dyes such as alizarin, purpurin, hematoxylin, morin, curcumin, resorcylic acid, carminic acid, and aurintricarboxylic acid. -4s little as 50 to 100 y of any of these
dyestuffs exhibits a pyrolytic splitting off of phenol. On the other hand, azo dyes contaiiiing phenolic hydroxyl groups give a varied pattern when pyrolyzed. With some, the phenol reaction is distinctly weaker than that given by the purely phenolic dyes just noted, and Jyitli othcm no formation of phenol was o1)servod. Therefore, the pyrolysis of such azo dyes apparently follows a coiirsc d i f f ( ~ ent from that taken by purcly phenolic dyes. LITERATURE CITED
( I ) Beilstein, C., "Handbuch der Organische Chemie," Vol. VI, p. 112, 1923. ( 2 ) Boyland, E., Manson, E. D., Solomon, J. B., Kiltshire, G. K , Biochem. J . 53, 420 (1053). (3) Feigl, F., Amaral, J. R.,.If ikrochzm. Acta 1958, 337. (4) Feigl, F., Hainberger, L., Chemist Analyst 44, 47 (1955). (5) Gibbs. H. D.. J . Bio!. Chcm. 72,
RECEIVEDfor review May 14, 1058. Accepted September 23, 1959.
Spot Tests for Phenol Esters and Phenol Ethers FRITZ FEIGL and ERWIN JUNGREIS laboratorio do Produg6o Mineral, Ministerio do Agricultura, Rio de laneiro, Brazil Translafed by
RALPH E. OESPER, University of Cincinnati, Cincinnafi, Ohio
b Spot tests for phenol esters of carboxylic acids and for phenol alkyl ethers can b e based on the fact that these types of compounds yield phenols when subjected a t 150" C. to pyrolytic saponification and dealkylation, respectively. Oxalic acid dihydrate serves as the water donor in the saponification; the dealkylation is accomplished by the action of alkali iodide plus oxalic acid dihydrate. The resulting phenol volatilizes with the steam, and the vapor then gives the indophenol color reaction with 2,6dichloroquinone - 4 chloroimine. Triphenyl phosphate can b e detected through pyroommonolysis to phenol b y heating to 250" C. with guanidine carbonate. The detection limits are within the bounds of microanalysis.
-
I
previous paper (S), it was shown that phenols can be detected in the gas phase by the Gibbs indophenol reaction. Accordingly, it was expected that tests for phenol esters and phenol alkyl ethers could be worked out if phenol vapors could be made to result N A
from the saponification or dealkylation of these esters and ethers. No tests are available for these classes of phenol derivatives at present. This objective can be reached through relatively simple procedures in which use is made of the reactivity of solid materials and topochemical reactions. Hydrolytic cleavage occurs when phenol esters are saponified : R(Ar)COOAr
+ Hz0 -+R (Ar)COOH
+ ArOH
As was reported previously (1, d ) , the hydrolytic cleavage of organic compounds can be accomplished not only by the usual wet method (with participation in all cases of hydrogen and hydroxyl ions), but also by dry heating in the presence of organic or inorganic compounds which give off water at elevated temperatures. I n fact, such thermal treatment sometimes accomplishes hydrolyses which proceed only slowly if at all by the wet method. I n such pyrohydrolyses the active agent is the superheated steam derived from the hydrate (or water donor) at the elevated temperature and is released
in direct contact with the organic compound, Phenol esters of carboxylic acids are among the compounds SUSceptible to pyrohydrolysis. The dihydrate of oxalic acid can serve as the water donor; it starts to lose water at 102" C. and the dehydration continues in the molten oxalic acid up to 160" C. I n this sintering or fusion the released water of crystallization brings about not only the hydrolysis of phenol esters but also provides water vapor which facilitates the volatilization of the phenols. Phenol esters of phosphoric acid (and probably of other noncarboxylic acids) do not undergo pyrohydrolysis when heated with hydrated oxalic acid. I n contrast, heating with guanidine carbonate to 150" C. leads to the liberation of phenol, because under this condition guanidine carbonate loses ammonia which brings about the pyroammonolysis : OP(OC6Hs)a
+ 3NH3 + O P (NH2)J
+ 3CaHaOH
Analogous reactions take place likewise with phenol esters of carboxylic VOL. 31, NO. 12, DECEMBER 1959
2101
