PHENOL TESTS.*

III. THE INDOPHENOL TEST.

BY H. D. GIBBS.

(From the Division of Chemistry, Hygienic Laboratory, United States Public Health Service, Washington.)

(Received for publication, January 17, 1927.)

CONTENTS.

Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Qualitative test.. . . . . . . . . . . . . . . . . . . . . . . Quantitative method.. . . . . . . . . . . . . . . . . . . . . . . Quinonechloroimides.. . . . . . . . . . . . . . . . . . . . . .

a. Preparation..................................... b. Solubility of 2,6-dibromoquinonechloroimide.. . .

Bbsorption spectra.. . . . . . Effect of pH upon the rate of indophenol formation. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

. . .

. . . . . .

. . . . .

PAGE

649 652 653 653

. 653 654 657

. 659 662

INTRODUCTION.

Among the many methods of identifying phenolic compounds the test producing the beautiful, intense, blue solutions of the indophenol salts is probably the oldest. It dates back to the work of Robiquet (1835), J. Dumas (1838), R. Kane (1841), and other early workers, who were concerned with the orcein color substances, and who obtained the blue color with cresol, resorcinol, and other phenols without correct knowledge of the reactions involved, or of the composition of the color compounds. Lex (1870) and Weselsky (1871, 1872) obtained colors with nitrous acid and Kopp (1873) obtained the same color with fuming sulfuric acid, a test that is now known as the Liebermann test for phenols, for Liebermann (1874, 1875) showed that Kopp’s fuming sulfuric acid contained nitrous acid.

Prior to 1874, complicated formulae were proposed for the simple indo- phenols, and very little was known concerning their structure. The studies of von Baeyer and Caro (1874) resulted in the first approximation to the

* Published by permission of the Surgeon General, United States Pub- lic Health Service.

649

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Phenol Tests. III

true formula. The first commercially useful dyes of the class were pre- pared by Koechlin and Witt (1881, 1882) and patented by them in many countries.

The indophenols and their closely related derivatives, the indamines, have been made by a large variety of methods’ the most important of which may be classified by four types of con- densations of two unlike molecules.

I. Oxidation.-By simultaneous oxidation of an amine and a phenol in the sense of the expression

HO.CGH4NHa f C6HsOH ? O:G,Ha:N.C&H~.OH

an indophenol is produced. 2. Dehydration.-The elimination of a molecule of water from

a nitrosophenol and a phenol produces an indophenol.

HzSO~ HO.CsH.NO + GHr,OH - O:CsH4:N.C&Hd.OH

3. Deamonozation. -Certain amines and aminophenols may be condensed with the elimination of a molecule of ammonia forming a leuco derivative according to the reaction

4. Deacidation.-The coupling of 2 molecules with the elimina- tion of an acid may produce an indophenol. The most important reactions of this type are t,hose occurring between quinonechloro- imides and phenols in the sense of the equation

G:CeHn:NCl + CoH,OH = O:C6Ha:N.C$H4.0H + HCl

Hirsch (1880) first described the condensation of quinone- chloroimide and phenol and this method has been found to be the most satisfactory for the laboratory production of a great many derivatives of indophenol. The development of this type of reaction as a very delicate qualitative, and a very accurate quantitative, method for the estimation of phenol forms the substance of this paper. The procedure may be applied to many

1 This subject will be more fully treated in a later paper on the synthesis of indophenols and indamines.

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H. D. Gibbs 651

other phenols but only the quantitative data concerning phenol are presented.

It has been found that the dihalogen substituted quinone- chloroimides, 2,6-dichloroquinonechloroimide and 2,6-dibromo- quinonechloroimide give the most delicate tests of any of t,he quinonechloroimides tried and the indophenols formed from them are, in general, the most stable. In addition to these two, there have been investigated quinonechloroimide, o-cresolquinone- chloroimide, and m-cresolquinonechloroimide.

The test employing the 2,6-dibromoquinonechloroimide, has a delicacy of at least 1 part of phenol in 20,000,OOO. Further investigations may greatly extend its delicacy and usefulness. The indophenol formation has been followed quant’it’atively by means of the spectrophotometer and a number of absorption curves are charted.

The fact that the best results are obtained in buffered solutions should not mitigate against the use of this test for it is becoming more and more essential for a well equipped laboratory to be provided with a series of accurately standardized buffering solu- tions. Palitzsch’s (1915,1916) borax buffer solution, having a pH value of 9.24, may be employed. It is easily prepared by dis- solving 19.108 gm. of sodium borate NazB407.1H20 in 1 liter of water.

The quinonechloroimides do not react with all phenols. It is generally believed that the primary requisite is that the posi- tion para to the hydroxyl must be unsubstituted (see Gibbs, 1926, 1927). This may be taken as a general rule although ex- ceptions may be discovered when a larger number of phenols has been studied. Even with the para position free some sub- stituted phenols have failed to react, due to the influence of the adjacent substituted groups.

A series of 2,6-dibromophenol indophenols has been investi- gated in this laboratory by Cohen, Gibbs, and Clark (1924) and a series of 2,6-dichlorophenol indophenols by Gibbs, Cohen, and Cannan (1925), and it has been found that these compounds form a series of easily made, stable indophenols. Since they were made from 2,6-dichloro- and 2,6-dibromoquinonechloroimide they were named, in the papers above referred to, the 2,Bdichloro-

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652 Phenol Tests. III

and 2,6-dibromoindophenols, the numbering of the rings being as follows :

In a paper on the indophenols and indamines now in prepara- tion the question of nomenclature will be fully discussed.

Qualitative Test.

Since the quinonechloroimides are only slightly soluble in water (see section on solubility) it is fortunate that only small concentrations are required for the test as applied to phenols. The quinonechloroimide reagent may be employed as an aqueous suspension or may be filtered to a clear solution. In the latter case a larger volume is required.

A mass of the solid 2,6-dibromoquinonechloroimide (or the dichloro derivative) about the size of a pea is shaken with 10 cc. of water in a test-tube to make a cloudy, yellow suspension. The larger particles sink rapidly and by drawing a portion from the middle of the suspension, into a pipette, the few drops of the reagent required for the test are readily delivered from the pipette into the solution to be tested. This procedure assures an excess of the imide which will increase the speed of the formation of the indophenol.

The quinonechloroimide decomposes slowly in alkaline buffers giving rise to discoloration of the solutions, which colors by no means should be mistaken for an indophenol test by one ex- perienced with the reaction. The solutions to be tested should be very dilute, not stronger than 1 or 2 parts of the phenol in 1000 parts of solution, should be brought to an alkalinity ranging between pH 8 to 10, about pH 9.4 is preferable. 2 or 3 drops of the test solution, carrying some of the quinonechloroimide in suspension, are added to 10 to 50 cc. portions of the solution to be tested. In the presence of reacting phenols the blue color of the indophenol develops, in the more concentrated solutions intense blue almost instantly, and in very dilute solutions, 1 part in 20,000,OOO or more; a pale blue may require an hour or

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H. D. Gibbs

more for full development. Different indophenols require different pH for best formation but all are formed in the alkaline region. The best results are obtained by working in buffered solutions in color comparison tubes, and observing the color formation in a layer of solution 10 to 20 cm. in depth.

This test may be made approximately quantitative by com- parison with known standards.

Quantitative Method.

The quantitative determination is best made by measurements of the color formation by means of the spectrophotometer. For phenol the readings are made at 610 mp, the peak of the absorp- tion band for t,his indophenol. When employing the Keuffel and Esser color analyzer 10 cm. observation tubes may be employed. By means of rough preliminary experiments, the solution to be tested should be brought to about a concentration of 5 X 1O-6 M with a buffering solution of about pH 9.4.

To 20 cc. of this solution in a test,-tube, 2 or 3 drops of the 2,6-dibromoquinonechloroimide suspension, described in the qual- itative procedure, are added, and the 10 cm. tube filled with this mixture.

The color formation may then be observed at time intervals measured in minutes until the maximum of absorption is shown. This requires from 10 to 20 minutes.

Where T is transmittancy, - log T equals 1 for an indophenol concentration of 5 X lop6 M solution, observed through a 10 cm. layer. See “Absorption Spectra” described later in this paper.

Quinonechloroimides.

a. Preparation.-The two quinonechloroimides found to be most useful in this work are: (1) 2,6-dibromoquinonechloroimide and (2) 2,6-dichloroquinonechloroimide. They are readily prepared as follows :

A cold, hydrochloric acid solution of the corresponding amine, (1) 2,6-dibromo-p-aminophenol, (2) 2,6-dichloro-p-aminophenol, is slowly poured into a cold solution of sodium hypochlorite. Both solutions should contain much crushed ice and should be stirred constantly during the reaction. The addition of the

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Phenol Tests. III

aminophenol solution should be stopped before a permanent blue or dark brown color appears and while free chlorine is st,ill present.

The quinonechloroimides separate as canary-yellow precipitates which are collected on a Buchner filter, washed with a little water, and air-dried. When dry they are quite stable.

The solution of sodium hypochlorite is most easily prepared according to the method of Raschig (1907) as follows:

630 gm. of sodium hydroxide are dissolved in a little water, cooled, and poured onto sufficient crushed ice so that the total weight is 10 kilos. Chlorine gas is now run into the mixture in a rapid stream until the weight increases 730 gm. At this time much crushed ice should still be present. The solution of sodium hypochlorite is now ready for use. This quantity is sufficient for about 2.5 mols of the aminophenol.

b. Solubility of 2,6-Dibromoquinonechloroimide.-(With E. El- VOX.) It has been found that the concentration of an aqueous solution of 2,6-dibromoquinonechloroimide can be determined most accurately by spectrophotometric measurement of the in- dophenol formed by the reaction with phenol in alkaline solutions. This reaction has been proved to be quantitative under certain conditions. A determination requires less than 20 minutes, the time depending upon the buffer solution employed. The solu- bility has been found to be 0.0002 M at 20”.

The saturated solution is prepared by shaking for about 10 minutes an excess of the compound, that has been powdered and sifted through a fine seive, about 100 mesh, with water and filtering the suspension several times through a folded filter until the filtrate runs clear.

Care should be taken, however, to avoid too much powdering of the sample or grinding the powder together with the water in the mortar for too long a time, since it appears that under such conditions there is obtained what appears to be a supersaturated solution or a colloidal solution.

It has been found that 2,6-dibromoquinonechloroimide crystals undergo a slight decomposition on standing. This decomposition is more marked if the crystals are moist or in a moist atmosphere. Solutions change more rapidly, becoming deeply colored, a reaction which is accelerated by light. If kept in the dark the

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H. D. Gibbs 655

solutions are serviceable for a number of hours. A nitrogenous decomposition product is formed in the crystals which is more soluble than the 2,6-dibromoquinonechloroimide. The prelim- inary attempts to check the spectrophotometric determinations of solubility by means of determinations of the nitrogen in the solutions and also the oxidizing power, was always found to give results very much too high, the former especially so. By re- peatedly extracting the 2,6-dibromoquinonechloroimide with separate portions of water the greater part of this impurity was removed. The estimation of the amount of imide from the nitrogen determination and from the titration with thiosulfate of the liberated iodine then checked approximately in magni- tude with the spectrophotometric determinations of indophenol formation. A sample of very carefully purified 2,6-dibromo- quinonechloroimide that had been standing for several months in the laboratory in glass stoppered bottles, was found to contain an amount of this soluble impurity equivalent to about 0.06 per cent of the imide as calculated from the nitrogen determinations and compared with the imide as found by the spectrophotom- eter. The decomposition product does not interfere seriously with the accuracy of the indophenol determination although it is better to employ a second extraction in preparing the solutions for accurate work.

The three methods employed for determining the amount of 2,Bdibromoquinonechloroimide in solution are described as follows :

Reduction Method.-2,6-Dibromoquinonechloroimide liberates iodine from a solution of potassium iodide, and the titration of the iodine with standard thiosulfate solution gives a rough measure of the amount of the imide.

To 500 cc. of a saturated solution of the 2,6-dibromoquinone- chloroimide there are added 100 cc. of a 5 N solution of hydrogen chloride, and then 5 cc. of a 20 per cent solution of potassium iodide. After thoroughly mixing, the liberated iodine is titrated with 0.1 N sodium thiosulfate.

1 mol of the imide should require 4 equivalents of hydrogen for complete reduction. This procedure has not been studied sufficiently to recommend it as an accurate method.

Nitrogen Determination.-It has been found most convenient

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656 Phenol Tests. III

to determine the nitrogen in the solution after the thiosulfate titration of the iodine as described in the previous method. The Kjeldahl method was employed, always using 500 cc. port,ions of Dhe 2,6-dibromoquinonechloroimide solution, and all determina- tions were made in duplicate.

Spectrophotometric Method.-This method of estimation of the 2,6-dibromoquinonechloroimide depends upon the spectro- photometric determination of the maximum blue color of t’he indophenol formed when a measured quantity of the imide solu- tion reacts upon phenol in an alkaline solution.

The reaction proceeded very smoothly in a buffered solution of pH 9.0 to 9.5. Buffer of pH 9.4 was usually employed and the concentration of the phenol was at least 10 times that of the imide. The following procedure was found most satisfactory.

About 40 cc. of buffer solution pH 9.4 were put into a 50 cc. measuring flask, 5 cc. of a phenol solution of about 4 X 1O-4 molality added, and then 1 cc. of the Z,B-dibromoquinonechloro- imide solution was delivered into it from a standardized pipette, and then the flask was filled to the mark with the buffer solution. Thorough mixing was then assured by pouring the contents of the measuring flask into a 100 cc. Erlenmeyer flask from which spectroscopic tubes, 10 cm. in length, were filled. These tubes were put in place in the spectrophotometer and readings taken at intervals, at wave length 610 rnp, the peak of the absorption band for this indophenol. The maximum color usually develops in about 18 minutes. The reaction has a high temperature coefficient and may take longer if the temperature is below 20”.

In order to illustrate the points brought out in the previous discussion a series of determinat’ions is described as follo\vs and recorded in Table I.

40 gm. of an excellent sample of 2,6-dibromoquinonechloroimide were extracted consecutively with 3 portions of water of 1200 cc. each, the temperature of which was approximately ZO”, and filtered to clear solutions.

Duplicate 500 cc. samples of each extraction were taken for the iodine titration and the nitrogen determination and 1 cc. portions were taken for the spectrophotometric measurements.

The spectrophotometric method is by far the simplest, quickest, and most accurate. The other methods merely show the magni-

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Phenol Tests. III

These products have been described in the prior work of this laboratory and the analyses showed

Purity. per cent

2,6-Dichloroindophenol .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72.9 2,6-Dibromoindophenol .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85.5 Methylene blue, Sample F . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76.6

FIG. 1. Spectrophotometric absorption curves of the sodium salts of: I, 2,6-dibromoindophenol 0.00001 M; II, 2,6-dichloroindophenol 0.00001 M; III, 2,6-dibromoindophenol 0.000005 M; IV, 2,6-dichloroindophenol 0.000005 M; V, methylene blue chloride 0.000002 M. The ordinates are measured as -log T.

The only impurities in the indophenols are sodium chloride and water and the methylene blue is approximately pure except for water. The latter was most highly purified as described by Clark, Cohen, and Gibbs (1925).

The absorption curves are plotted from data obtained from the study of unbuffered solutions. These were made by dis- solving a molar weight of the compound in mg. without correcting for the purity, in 1000 cc. of water and further diluting this to

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H. D. Gibbs 659

the desired concentration as stated in the chart. The absorption of the solutions was measured in 10 cm. tubes.

It is to be noted that the absorption of equal molar solutions of the dichloro and the dibromo compounds is almost equal while the peaks of the curves occur at different wave lengths; namely, 600 rnp for the chloro and 610 mp for the bromo derivatives. Equimolecular solutions are quite different in quantity of material

chloro 289 1 present being in the ratio of - = -

bromo 379 = 1.31’ While the absorption of methylene blue is in quite a different

region of the spectrum it is noticeable that the coloring power, as measured by the peak of the absorption band, is much greater, the E values being in about the proportion of 0.7 for the indo- phenol to 2.5 for methylene blue.

It is readily seen from the absorption curves that methylene blue is a green-blue while the indophenols are a much purer blue.

Too much reliance is not to be placed upon the statements regarding the relative persistence of the peaks of the indophenols when measured in unbuffered solutions since these compounds act as acid-base indicators and are sensitive to the carbon dioxide of the atmosphere.

An accurate determination of the peak of the absorption band of 2,6-dibromoindophenol at 610 rnp places the value of -log T at 1 for solutions 5 X 1O-6 M in buffer pH 9 when measured in 10 cm. tubes. The deviations from this value, due to pH in the range from pH 8.5 to 10, are very slight and do not affect the value materially. The standard solution, 5 X 10-5~, was pre- pared by dissolving the proper quantity of the indophenol, of 85.5 per cent purity previously mentioned, in water. 5 CC.

portions of this solution were put into a 50 cc. flask which was filled to the mark with buffer pH 9, thus making a solution 5 X 10eE M from which the 10 cm. tubes were filled for the spectrophoto- metric observations.

Effect of pH upon the Rate of Indophenol Formation.

Very early in the investigation it was noted that the pH of the solution greatly affected the rate of the formation of the blue color. The reaction velocity increases with the increase in

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660 Phenol Tests. III

alkalinity. A comparison of the rates at pH 8.5, 9, 9.5, and 10 was made by treating a 5 X lO-'j~ phenol solution in these buffers with an excess of 2,6-dibromoquinonechloroimide, as described under the analytical procedure, and observing the indophenol formation as measured in the spectrophotometer at 610 rnp at time intervals.

2 gm. of pure crystals of phenol were put into water with 50 cc. of 0.2 N sodium hydroxide (about one-half the quantity to form sodium phenolate) and the solution was made to 2 liters. It was clear and colorless. 10 cc. of this solution were diluted to 1 liter, thus making a solution which contained 1 part of phenol in lOO,OOO, or 0.0001064 M. This was used for a stock solution and diluted to varying degrees in buffers for tests. When 1 cc. of this solution was put into 20 cc. of buffer there resulted a solution of the concentration of 5 X lo-” M or 1 part of phenol in Z,lOO,OOO. This concentration was employed in determining spectrophotometrically the effect of pH upon the speed of the reaction.

To 21 cc. of the buffered solution in a test-tube 2 or 3 drops of a Z,&dibromoquinonechloroimide suspension were added. As soon as added the contents of the test-tube were mixed and the 10 cm. tube, in which the absorption is measured, was filled and placed in the spectroscope and readings taken at once.

The first readings were low, due to the slight turbidity of the solution, but soon the solution cleared and read 100 per cent transmittancy. Then the color began to develop and the absorp- tion, due to the indophenol formation, was read at the wave- length 610 mp.

The time required for the clearing of the solution and the be- ginning of the indophenol blue formation, as observed by the spectrophotometer, was quite different in the different buffers, being 16 minutes at pH 8.5, 7 minutes at pH 9, 3 minutes at pH 9.5, and 2 minutes at pH 10. The solubility of the quinone- chloroimide and also the rate of decomposition increases with the pH; that is, with the increasing alkalinity. Discoloration of the solution slowly takes place at the higher alkalinities.

In several instances, near the end of the reaction, the full curves were plotted and they coincide very nearly with the curves for the purified compounds plotted in Fig. 1.

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H. D. Gibbs 661

TABLE II.

Spectrophotometric Data of Formation of Indophenol from 2,6-Dibromoguin- onechloroimide and Phenol in Solutions of Concentration of 1 Part in

2,100,OOO or 0.000005 M at Various Degrees of Alkalinity.

These results are plotted in Fig. 2. T = kansmittancy.

PH 8.5 pH9 I pH 9.5 I pH 10

Time -16 min.

0 10

22 0.85 0.07Of 65 0.60 0.221;

102 135

176 220

~-

0.43 0.366: 0.32 0.4942 --

0.27 0.568( 0.22 0.6571 --

0.185 0.732t 0.16 0.795!

252 285

1685 After.

T - log ?

--

1 .oo O.OOO(

0.93 0.031i

--

0.12 0.9202 is time the

rate of fading is so great that the maximum color is not developed.

1 5

i 7

i

3

3 5

3

Time -7

min.

0 3

6 11

16 21

26 31

34 49

58 71

101 121

0.990.0044 0 0.99 0.0044 0 0.99 0.910.0410 1 0.92 0.0362 2 0.85

---____

0.860.0655 3 0.85 0.0706 4 0.70 0.720.1427 5 0.79 0.1024 7 0.54 ~__~__~ --

0.600.2218 7 0.67 0.1739 10 0.42 0.500.3010 9 0.63 0.2007 12 0.32 __--- ---

0.440.3565 11 0.60 0.2217 14 0.29 0.380.4202 13 0.55 0.2596 17 0.24 --- ----

0.350.4559 15 0.47 0.3279 22 0.15 0.180.7447 17 0.40 0.3979 27 0.13 ~--__- ~-

0.160.7939 19 0.36 0.4437 32 0.11: 0.110.9586 21 0.31 0.5086 40 0.09 __- -__- --

0.101.0000 23 0.28 0.5528 47 0.08E 0.091.04.58 25 0.25 0.6021 -~~-- --

27 0.23 0.6383 29 0.21 0.6778

~--___-~-

31 0.19 0.7212 36 0.16 0.7959

41 0.13 0.8861 46 0.12 0.9208

---A- --

51 0.11 0.9586 61 0.1050.9788

__~ -- -____

86 0.09 1.0458

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662 Phenol Tests. III

The data are tabulated in Table II and plotted in Fig. 2. The zero point is taken at the beginning of the indophenol formation where the transmittancy = 1. The inhibition period mentioned above is omitted from the time.

It is to be noted that the complete indophenol formation at pH 9, 9.5, and 10, from the total amount of phenol present, is shown by the curves.

IO ii0 30 40 50 60 70 80 90 1 Tiine h Ml’nures

FIG. 2. Graphic representation of the data recorded in Table II, showing the formation of 2,6-dibromoindophenol at various degrees of alkalinity as measured by the spectrophotometer readings at 610mp, the peak of the absorption band of this indophenol. The ordinates are measured as -log T.

A quantitative study of the reaction velocity together with a study of the underlying causes in the change in speed due to pH and a mathematical treatment of the same forms the subject of the next paper of this series.

SUMMARY.

A test for phenols, depending upon the quinonechloroimide reaction producing indophenols, is described. Qualitative data for phenols are given and it is shown that the test is made quanti-

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H. D. Gibbs 663

tatively accurate by following the color formation by means of the spectrophotometer. The test is delicate, both qualitatively and quantitatively, to 1 part of phenol in 20 million by ordinary manipulative skill. The effect of the pH of the solution on the velocity of indophenol formation is shown.

Almost all phenols having the position para to the hydroxyl group unsubstituted, and also some other derivatives such as the amines, will form indophenols (or indamines) and, therefore, the method will be useful for differentiating between phenols only on the basis of the absorption spectra. This phase of the test is not treated in this paper.

A method for the preparation of the reagent is given and the solubility of the best reagent, 2,6-dibromoquinonechloroimide, has been determined with considerable accuracy.

BIBLIOGRAPHY.

von Baeyer, A., and Caro, H., Ueber die Einwirkung der salpetrigen S&me auf Dimethylaniline und tiber Nitrosophenol, Ber. them. Ges., 1874, vii, 963; Ueber die Einwirkung der salpetrigen Slure auf Dimethyl- aniline, ibid., 809.

Clark, W. M., Cohen, B., and Gibbs, H. D., Studies on oxidation-reduction. VIII. Methylene blue, Pub. Health Rep. U. S. P. H., 1925, xl, 1131.

Cohen, B., Gibbs, H. D., and Clark, W. M., Studies on oxidation-reduction. V. Electrode potentials of simple indophenols, each in equilibrium with its reduction product, Pub. Health Rep. U. S. P. H., 1924, xxxix, 381.

Dumas, J., Ueber das Orcin, Ann. Chem., 1838, xxvii, 145. Gibbs, H. D., Phenol tests. 1. A classification of the tests and a review of

the literature., Chem. Rev., 1926, iii, 291; Phenol tests. II. Nitrous acid tests. The Millon and similar tests. Spectrophotometric in- vestigations, J. Biol. Chem., 1926, lxxi, 455; Para cresol. A new method of separating p-cresol from its isomers and a study of the boiling point, J. Am. Chem. Sot., 1927, xlix, in press.

Gibbs, H. D., Cohen, B., and Cannan, R. K., Studies on oxidation-reduc- tion. VIII. A study of dichloro substitution products of phenol in- dophenol, Pub. Health Rep. U. S. P. H., 1925, xl, 649.

Hirsch, A., Ueber das Chinonchlorimide und ahnliche Substanzen, Ber. them. Ges., 1880, xii, 1903.

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H. D. GibbsINDOPHENOL TEST

PHENOL TESTS: III. THE

1927, 72:649-664.J. Biol. Chem. 

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