PHENOL TESTS.*

II. NITROUS ACID TESTS. THE MILLON AND SIMILAR TESTS. SPECTROPHOTOMETRIC INVESTIGATIONS.

BY H. D. GIBBS.

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

(Received for publication, November 17, 1926.)

CONTENTS.

Introduction ................... Literature ..................... Experimental. .................

Phenol ..................... Absorption spectra ......... Para-cresol.. ............... Tyrosine ...................

Summary ...................... Bibliography ...................

......... ......... ......... .........

.........

.........

.........

.......

.......

.......

....... . . . . ....... ....... .......

....... ....... ....... ....... . . .

....... ....... .......

PAGE

445 448 450 450 455 456 457 457 458

INTRODUCTION.

A general classification of phenol tests, with a bibliography, was given in the first paper of this series (Gibbs, 1926). This article treats of a number of phenol reagents, consisting of ni- trous acid, nitrites, alkyl nitrites, dilute nitric acid, and any of the above in presence of salts of mercury. These reagents give red solutions which, in some cases, are delicate, but not char- acteristic tests for the species. By various modifications and procedures attempts have been made to develop these tests for distinguishing between certain phenols. In some cases fair success has been attained. By comparison of the colors with a known standard, approximate quantitative accuracy has been developed for certain phenols.

For purposes of classification, these tests may be divided into two groups according to the reagents employed.

*Published by permission of the Surgeon General, United States Public Health Service.

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446 Phenol Tests. II

1. Nitrous acid and nitric acid reagents. 2. Nitrous acid and nitric acid reagents containing salts of

metals. In the latter are the Millon, Hoffmann, Plugge, Lintner, and

similar reagents. Although Millon’s test is’the most delicate and the most popular

of all of the tests and has been made the subject of numerous researches, I have found in the literature no definite proof of the reactions involved. There will be presented in this paper experi- mental evidence of the two stage nature of the reactions. The first stage is essentially the formation of nitrosophenols, and subsequent to t.his is the formation of more highly colored products. This process is catalyzed by the mercury salts and Vaubel (1900) describes a mercury derivative which he isolated and analyzed.

The difference in the behavior of these reagents with phenols, and the discrepancies concerning the delicacy of the tests as performed by different investigators and recorded in the literature, are evidently due to the different proportions of nitric acid, nitrous acid, and mercury salts. These differences affect the speed of the reaction and secondary condensations, and probably do not occasion any fundamental difference in the primary reaction. The most delicate test is obtained when these three ingredient’s are present in the proper proportion, a condition which seems to be fulfilled best by the freshly prepared Millon reagent. Even in the case of this reagent the method of preparation is an important factor.

When the Millon reagent reacts with phenol, p-nitrosophenol is formed, together with a compound which is quite red in acid solution, a distinction from p-nitrosophenol. The nature of this compound has not been investigated by me but the evidence points to its being a derivative of p-nitrosophenol (see Vaubel, 1900).

This view seems reasonable when it is remembered that the nitrosophenols are quite reactive, unstable in the air, capable of many condensations, and that evidence supports both the nitroso- and the quinone oxime formulas. These desmotropic forms are in equilibrium with each other, act as acid-base indi- cators, and give colors to the solutions. Moreover, I have found that dilute solutions of p-nitrosophenol react with solutions of

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mercurous nitrate forming red or brown precipitates which I have not investigated. (See also the work of Ingold and Piggott (1924)

-N-O I I

on the effect of t,emperature on the equilibrium 2N:O G 0 - N - in their studies of nitrosomesitylene in benzene solution).

Of the many colored nitroso compounds (quinone oximes) six commercial products are described by Rowe (1924) in the Colour Index and all are made by the action of nitrous acid on (1) resorcinol, (2) /3-naphthol, (3) a-naphthol, (4) 2,7-dihydroxy- naphthalene, (5) /3-naphthol-6-sulfonic acid, and (6) oc-naphthol- 4-sulfonic acid. It is evident that the nitrous acid may react in a position ortho to the hydroxyl group and that an unsubstituted position para to the hydroxyl is not always necessary. I have indicated this in an incomplete study of the action of nitrous acid upon a very pure sample of p-cresol. Moreover, more than one equivalent of nitrous acid may react, as in the case of resorcinol.

In regard to the effect of the mercury in those tests employing compounds of this metal in connection with nitrous acid, it is only necessary to note the great reactivity of this element with organic compounds, the host of mercury organic derivatives, and the many cases where mercury is employed as a catalyst in or- ganic synthesis, to appreciate the complications that may be introduced by this element as employed in the Millon, Hoffmann, Plugge, Lintner, and other reagents upon various phenols. The effect of mercury is not limited to the formation of mercury organic compounds but may be directional in governing the course of a reaction in substitution. In the presence of condensing agents complicated products well may result (see Whitmore, 1921).

It is interest’ing to recall that nitrosobenzene was first prepared by the action of nitrosyl bromide upon mercury diphenyl, (von Baeyer, 1874), and it cannot be prepared by the action of nitrous acid on benzene; that p-nitrosophenol reacts with nitrous acid and with hydroxylamine to form p-diazophenol, 0: C&H,: Nz, which is susceptible to various couplings forming dyes; and that nitrosobenzenes are capable of polymerization like aldol.

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448 Phenol Tests. II

LITERATURE.

A brief review of the most important literature bearing on these tests follows.

1. Nitrous Acid and Nitric Acid Reagents.-Griessmayer (1871) em- ployed potassium nitrite and sulfuric acid in solution and nitrous acid in nitric as reagents. The latter gives a blood-red color with ellagic acid which is stated to be characteristic. (See also Perkin and Nierenstein (1905).) Eykman (1882) described an ethyl nitrite test for phenols claiming a delicacy for phenol of 1 part in 2,000,OOO. The solution of the phenol was treated with a few drops of an alcoholic solution of ethyl nitrite and then mixed with an equal volume of sulfuric acid, thus producing the red color. Huerre (1922) obtained color reactions with diluted nitric acid and various phenols. Some phenols gave no color, and certain phenol ethers formed nitro derivatives which crystallized out of the solutions. Ware (1925) made use of a dilute solution of nitrous acid and of nitric acid for the detection of certain plant phenols. Ekkert (1925) developed tests for nitrous and nitric acids. He employed a strong hydrochloric acid solution of resorcinol as the reagent and could detect 0.01 mg. of sodium nitrite and 0.05 mg. of potassium nitrate by the color reaction.

9. Nitrous and Nitric Acid Reagents Containing Salts of Metals.-The well known reagents containing mercury are those devised by Millon, Hoff- mann, Plugge, and Lintner. Millon (1849, 1850) proposed a solution of mercury in nitric acid as a protein reagent. It has acquired great vogue, especially in the detection of phenolic compounds. Hoffmann (1853) put forth a reagent for tyrosine consisting of a nitric acid solution of mer- curic oxide. Meyer (1864) showed that there must also be present a trace of nitrous acid, a condition present in Hoffmann’s work but not recognized by him. Plugge (1872) found that a reagent consisting of a solution of mercurous nitrate which contained traces of nitrous acid would detect 1 part of phenol in 200,000. The color with phenol and some other phenols is de- veloped on boiling the solution. AlmCn (1877) compared various tests for delicacy and found the Millon reaction with phenol most delicate, 1 part in 2,000,OOO.

In the mercury-nitrous acid class of reagents Hoffmann’s test was found to be next in delicacy and Plugge’s test least delicate. The great variation in the delicacy of tests with different phenols is brought out in this early work. For example the ferric chloride test will show 1 part of phenol in 3000, and 1 part of salicylic acid in 100,000 while the Millon test has about the same delicacy of 1 part per million for both phenols.

Nasse (1879) believed the action of Millon’s reagent to be a nitration, but 20 years later (1901), in light of further information, he changed his view. Hirschsohn (1881) found the delicacy of the Millon test to be 1 part of thymol in 16,000 parts. He also employed among other tests the nitric acid reaction for thymol and phenol. With thymol, 1 part in 32,000,

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a pale yellow color is obtained. Nickel (1890) experimented with other salts of mercury, such as mercuric chloride, in the nitrous acid reagent, and he also records some experiments with zinc chloride and zinc sulfate. A number of substances, principally glucosides, were found to give blue or violet color reactions with a reagent consisting of potassium nitrite and zinc sulfate. In commenting on Nasse’s view that Millon’s reagent pro- ,duced a nitration, Nickel gave it as his opinion that nitrosophenols were formed which then further transformed into red dyes.

Lintner (1900), in his work upon mercuric salicylate, employed the Millon test in a modified form. He states that the action of Millon’s reagent is due to mercuric nitrate, nitric and nitrous acids, and that mer- curous nitrate plays no rBle in the reaction. The best test for salicylic acid is obtained by first producing the mercuric salicylate by boiling the salicylic acid solution with a few drops of a 10 per cent solution of acid mercuric nitrate, then adding 2 or 3 drops of dilute sulfuric acid and finally sodium nitrite solution, dropwise. The intense red color appears im- mediately. He believed this to be due to a nitroso derivative. The limit of delicacy is about 1 part of salicylic acid in 500,000. Tests for proteins may be made in the same way.

Vaubel (1900), without definite proof of the first stage of the reaction, describes the action of the Millon reagent as occurring in two stages:

(4

NO

/ CeHsOH + HNOz = GH4 + Hz0

\ OH

NO

04 /

CsH4 + 4HgNOs + 3N0 + 3CsHbOH =

\ OH

On heating this mercury derivative with sodium hydroxide solution, it loses its mercury and a compound, apparently of the formula

= (0.CsH4.0Na)z NaON

= (GH4.ONa)z

is formed. This compound is soluble in water and the free acid is pre- cipitated on acidification. He also states that the Millon test is not given by diortho and dimeta substituted compounds.

Nasse (1901) states that the Millon reagent should contain no nitric acid

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Phenol Tests. II

and that it is best prepared by mixing mercuric acetate solution with a few drops of a 1 per cent solution of an alkali nitrite, and employing the same either with or without the addition of acetic acid. It seems to the writer that some nitric acid will be present under these conditions.

Bach (1911) employed the Millon test for phenols in waste waters and found the delicacy to be less than 1 part phenol in 200,000 parts. Elvove (1917) pointed out that the delicacy of the Millon test depends upon the mode of preparing the reagent and the manner of its use, and gives details for the preparation of an efficient reagent. By using comparatively large amounts of the reagent it is possible to detect and to estimate quantitatively phenol and the cresols without the usual procedure of heatingthereaction mixture. He found the delicacy of the test to be I part of phenol in 600,000, and he also observed that a sample of cresol containing some phenol could be differentiated from a sample of pure cresol by its behavior with the Millon reagent. Chapin (1920) noted the qualitative observations of EI- vove and developed a quantitative method for determining phenol in the presence of the three cresols. He claimed a fair accuracy for the method. He also presented a mathematical analysis of his calorimetric procedure and tabulated the colors obtained with a number of phenols, both mono- and polyhydric.

EXPERIMENTAL.

In order to investigate the hypothesis that the primary reaction between phenols and the nitrous acid and nitric acid reagents results in the formation of a nitrosophenol, a study of the develop- ment of color at room temperature was made in the following solutions.

Phenol.

Mixture l.-50 cc. of phenol solution, 2: 1000 in water, were treated with 10 cc. of water containing 10 drops of concentrated nitric acid of the best laboratory reagent quality.

Mixture W.-Mixture 1 plus 1 cc. of a concentrated solution of mercurous nitrate to which just sufficient nitric acid had been added to prevent turbidity.

Mixture 5.-50 cc. of phenol solution, 2:lOOO in water, .were treated with 10 cc. of water containing 2 drops of a concentrated solution of sodium nitrite and 1 drop of concentrated hydrochloric acid.

Mixture ,$.-Mixture 3 to which was added 1 cc. of the con- centrated solution of mercurous nitrate as in No. 2.

Mixture 6.- 50 cc. of phenol solution, 2:1000, plus 10 cc. of Millon’s reagent.

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Mixture 6’.-50 cc. of phenol solution, 2:1000, plus 10 drops of Millon’s rea,gent.

The Millon reagent employed in these tests was prepared according to Elvove (1917) as follows: 50 cc. of concentrated nitric acid, densit,y 1.405 at 25”, were added to 68 gm. of mercury in a 250 cc. beaker. The resulting solution, measuring about 40 cc., was diluted with 92 cc. of water. To this were added 2.76 cc. of nitric acid and mixed until the precipitate, which often formed, was complet,ely dissolved.

The red color developed most rapidly in Mixture 5. The color development in Mixtures 2,4, and 6, all containing mercury, was quite rapid and satisfactory to about the same degree in a given time. By adjusting the proportions of mercury compound used, the color development may be made approximately equal in the various mixtures. A crystal of mercurous’nitrate dropped into the nitric or nitrous acid tests accelerates the formation of the red color in the same way as the solution of mercurous nitrate. Some experiments employing mercuric chloride in place of the nitrite indicate that this compound of mercury possesses the catalytic power to a much lesser degree.

The production of color in Mixtures 1 and 3 was at first a yellow shade and was most rapid in No. 3, the nitrite solution. By increasing the concentration of the nitric acid in Mixture 1 the ‘color development was greatly accelerated. On long standing the colors in Mixtures 1 and 3 became red but not quite the same shade as the solutions containing mercury.

On precipitating the mercury from any of the solutions by the addition of ammonia or sodium hydroxide and filtering there was obtained a clear red solution. By adjustment of the concentra- tion and the pH value of these solutions they can be brought to match the color of the solutions obtained by the nitric acid test, No. 1, and the nitrous acid test, No. 3, when the latter solutions are similarly adjusted for concentration and pH. Neither of these tests, Nos. 1 and 3, employed mercury compounds. The pH of the various mixtures was adjusted by diluting a measured volume with buffers of the Clark and Lubs series (Clark, 1925). At the higher pH ranges the colors were reddish brown, and in the acid ranges yellow.

The test for nitrosophenol was performed on all the mixtures

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452 Phenol Tests. II

as follows: In the case of Mixtures 1 and 3, the addition of concen- trated sulfuric acid produced the condensation of nitrosophenol and phenol to indophenol, the Liebermann reaction.

In the case of the solutions containing mercury (Mixtures 2,4,5, and 6) the result was obtained on the red solutions after precipi- tating the mercury with sodium hydroxide. In some cases it was found that the test could not be obtained until after a little phenol solution, 2: 1000, had been added to the red solution to be tested. This is evidently due to the fact that all of the phenol originally present, had reacted with the nitrous acid reagent.

In order to obtain some idea of the effect of pH on the rate of the reaction between phenol and nitrous acid, 5 cc. portions of phenol solution (2: 1000 = 0.0213 M) were mixed with equal quantities of sodium nitrite solution (0.0213 M) and to this were added 10 cc. of buffers pH 1.2, 2, 3, 4, 5, 6, 7, 8, 9, and 10. These mixtures were allowed to stand 2 days at room temperature. The color gradually developed in the acid ranges, first yellow then slowly deepening to red, no color showing at pH 7, 8, 9, and 10. The best color development seems to be in the range pH 4 to 5. On heating the solutions the rate of color formation was very much accelerated.

The production of the characteristic color test with phenol and dilute nitric acid was proven to be due to nitrous acid, which was either present in the nitric acid or developed by the reducing action of the phenol, by the following tests.

Solutions of three different concentrations of phenol in water were treated with dilute nitric acid and to identical comparison solutions there were added small quantities of urea for the purpose of destroying any nitrous acid that might be present.

In every case the color development was normal in a few hours except where the urea had been added. In the latter experiments no color developed and some gas was evolved from the solutions. On making the test solutions alkaline at the end of the experiment, no color was developed in the solutions containing urea, while the color in the solutions containing no urea was greatly enhanced.

In order to compare with p-nitrosophenol the properties of the colored compounds obtained in the tests described above, a sample of p-nitrosophenol was prepared by the action of nitrous acid on phenol and purified as follows. The product was put into

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H. D. Gibbs 453

hot alcohol containing decolorizing carbon, shaken, and quickly filtered into cold water containing a very little acetic or hydro- chloric acid. On cooling with ice there was obtained a good crop of crystals. After repeating the process several times, a beautiful crop of almost colorless crystals was obtained. They were rapidly separated by filtration with as little exposure to the’air as possible. Without’ further drying, a portion of these crystals was dissolved in warm water, which had been previously boiled to expel air, the solution cooled to room temperature, and filtered. Analysis for nitrogen gave the following results.’

Nitrogen by Kjeldahl. I. 25 cc. solution = 0.00658 gm. N.

II. 25 ” “ = 0.00658 “ “ Calculated, p-nitrosophenol equals a concentration of 2.316 gm. per 1000 cc. or 0.0188 molar.

The dissociation constant of this preparation of p-nitrosophenol, determined by the method of Salm (1906) is 6.6 in terms of PK..

The solutions employed were: for the acid solution 0.5 cc. of p-nitrosophenol solution plus 10 cc. of buffer pH 3; for the alkaline solution 0.5 cc. of p-nitrosophenol solution plus 10 cc. of 0.2 N

sodium hydroxide. Superpositions of these were matched against 1 cc. of solution plus 9.5 cc. of buffer pH 6.6 and 10 cc. of water.

The dissociation constant was also determined by the spectro- photometric method (Holmes, 1924). Two 2 cc. portions of a solution of pure p-nitrosophenol were diluted with 20 cc. of 0.2 N sodium hydroxide and with 20 cc. of buffer pH 2, and the trans- mittancy measured in 10 cm. tubes at wave-length 560 rnh as de- scribed under the heading “Absorption spectra.”

Solution.

0.2 N sodium hydroxide. . 0.&5 - log T 0.968

pH 2.......................................... 1.00 0.0

Since the solution in 0.2 N alkali gave the maximum color ob- tained in alkaline solutions, it is assumed that all of the compound is here present in the colored form and in the solution at pH 2 the colored form is entirely absent. Therefore at the value 0.968 t 2 = 0.484, the two forms are.present in equal quantities.

1 I have Mr. C. G. Remsburg of this laboratory to thank for the analyses.

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454 Phenol Tests. II

FIG. 1.

PH 4, 5, 6, and the cc --log tran

Spectrophotometric absorption curves of p-nitrosophenol at 7, 8 and 0.2 N sodium hydroxide at concentration of 0.0019 M,

)lor produced by Millon’s reagent acting on phenol. Ordinates, smittancy; abscissae, wave-lengths in rn~.

A solution of 2 cc. of the p-nitrosophenol in 20 cc. of buffer pH 6.4 gave the value T = 0.33, -log T = 0.482. This value of 6.4 for pK, is less than that obtained by the method of Salm and I

480 500 520 540 540 580 660 620 640 660 680

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H. D. Gibbs 455

believe it to be more accurate, because of the difficulty of matching color when Salm’s method is applied to such weakly colored solu- tions. It is possible that the rather rapid fading of the solution in 0.2 N sodium hydroxide may have reduced the pK, value somewhat.

A comparison of the above value was made with that of the color obtained in the Millon test as follows: 50 cc. of phenol solution, 2:1000, were treated with 25 cc. of Millon’s reagent. A brilliant deep red color developed in about. 0.5 hour. This solution was treated with a 10 per cent solution of sodium hydrox- ide until no more precipitate formed. It was then filtered, and the red filtrate approximately neutralized with 0.1 N sulfuric a,cid solution. This clear red-brown solution showed substantially the same dissociation value, pK, 6.6, as that found in the case of the solution of pure p-nitrosophenol and also the same spectro- photometric absorption curve when adjusted for concentration and pH values (see Fig. 1).

although p-nitrosophenol is present and probably the first compound formed, it is not the only colored derivative produced by the action of Millon’s reagent as shown by the following investigation of the mercury-sodium hydroxide precipitate from the above experiment.

This precipitate was washed with water until the filtrat,e ap peared practically colorless, and then it was treated on the filter with dilute nitric acid. The filtrate appeared deep red and did not lose color on further acidification with nitric acid. The absorption spectrum of this acid solution in the visible region of the spectrum, very closely approximates the curves for p-nitro- sophenol in the alkaline ranges. The curve has the same slope, shows no tendency to develop bands in this region of the spectrum, and the intense absorption of the acid solution is quite a dis- tinction from p-nitrosophenol.

No further investigation of this color was attempted to deter- mine its constitution or how it is carried down with the precipita- tion of the mercury by sodium hydroxide.

Absorption Spectra.

The absorption spectra of the pure solution of p-nitrosophenol, previously described, were measured in the visible region at

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456 Phenol Tests. II

various pH levels by means of a Keuffel and Esser color anaIyzer. 2 cc. quantities of the solution were diluted with 20 cc. quantities of buffers pH 4, 5, 6, 7, 8 and 0.2 N sodium hydroxide, making solutions 0.0019 molar, which were placed in 10 cm. tubes. The curves are plotted in Fi.g. 1.

There is also plotted on this chart the absorption curve of a phenol solution treated with a few drops of Millon’s reagent. Owing to the rapidity of the color development the curve is difficult to obtain accurately. The readings were commenced at 700 rnp and proceeded toward the short wave-lengths and since the ab- sorption in the blue was increasing rapidly the curve is too steep at the upper end. Disregarding this discrepancy the conformity with the p-nitrosophenol type of curve is satisfactory.

Para-Cresol.

The reactivity of phenols that have the position para to the hydroxyl group unsubstituted is well recognized and in these cases the formation of various derivatives through reactions with the para-hydrogen is easily understood. In cases where the para position is substituted, especially by a methyl group as in the case of para-cresol, certain types of reactions may proceed with difficulty or not at all (compare Pummerer et al., 1922, 1925, mentioned under ferricyanide reagents in Paper I). Since the Millon color test is in general so delicate and might be attributed to impurities of ortho- or meta-cresol contaminating para-cresol, it was decided to prepare this cresol in a pure state to determine its reactivity not only with Millon’s reagent but also with ni- trous acid and some other compounds.

The purification by a new procedure will be described in a later paper.

Millon Test.-Para-cresol gives the Millon test in concentration of 1: 1000 very deep red and 1: 100,000 pink.

Nitrous Acid Test.-At concentrations of 1: 2000 p-cresol gives a positive coloration with nitrous acid. In performing the test equal volumes of solutions of p-cresol 1: 1000 and sodium nitrite of the same molar concentration were mixed and acidified with hydrochloric acid.

In order to establish further the reactivity of p-cresol with nitrous acid 0.1 mol (10.8 gm.) was put into water, 0.1 mol

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H. D. Gibbs 457

sodium hydroxide (4.0 gm.) dissolved in water, added and then 0.1 mol sodium nitrite (7.0 gm.) dissolved in water was added. The total volume was 250 cc.

This solution was surrounded by an ice bath, chilled to between 0” and 7”, and a solution of 25 gm. of sulfuric acid in 100 cc. of water slowly dropped in while the solution was thoroughly agitated.

At the low temperatures there was very little evidence of a reaction between the p-cresol and nitrous acid, the former separat- ing as the solution becomes acid. After all of the sulfuric acid had been added the solution was slowly warmed to room tempera- ture and then there was evidence of a reaction through the for- mation of a red precipitate. On distilling with steam very little of the p-cresol was recovered, the solution becoming red and much resinous material forming. The products of the reaction were not investigated since the study seemed hardly pertinent to this work.

Tyrosine.

OH

1

0 I

CH, . CH . COOH !

NH,

Dilute solutions of tyrosine, at room temperature, treated with dilute nitric or nitrous acid, slowly develop a color first yellow and gradually deepening. The addition of mercurous nitrate to the solutions accelerates the color formation. Millon’s reagent develops the red color in the same way. The color formation in the case of tyrosine is much slower than with phenol and the behavior of the color solutions is similar in changing the pH. No study has been made of the colored compound formed but it is evidently due to the action of nitrous acid.

SUMMARY.

The phenol reagents, nitrous acid, dilute nitric acid, and Mil- Ion’s, have been studied, and the literature reviewed.

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458 Phenol Tests. II

The reaction with phenol produces p-nitrosophenol which may condense to produce other color compounds. The presence of mercury compounds accelerates the color formation.

The pK, value of pure p-nitrosophenol was determined by the method of Salm to be 6.6 and by the spectrophotometric method 6.4. The latter is believed to be more accurate. The dissociation constant for a color compound formed by the Millon reaction was found to be 6.6.

The absorption curves in the visible region are recorded for p-nitrosophenol at pH 4, 5, 6, 7, 8 and 0.2 N sodium hydroxide and for the color produced by the Millon reagent.

Para-cresol and tyrosine react with Millon’s reagent and with nitrous acid. The substitution of groups in the para position to the hydroxyl in general does not seem to interfere with the nitrous acid tests.

BIBLIOGRAPHY.

In the preceding paper of this series there is found a bibliography of phenol tests (Gibbs, 1926). Almen, A., Die relative Empfindlichkeit der Carbol- und Salicylsaurereac-

tionen, Arch. Pharm., 1877, x, 44. Bach, H., Kolorimetrische Bestimmung von Phenolen in Abwassern, 2.

anal. Chem., 1911, 1, 736. von Baeyer, A., Nitrosobenzol und Nitrosonaphthalin, Ber. them. Ges.,

1874, vii, 1638. Chapin, R. M., A new method for the determination of phenol in the

presence of certain other phenols, J. lnd. and Eng. Chem., 1920, xii, 771.

Clark, W. M., The determination of hydrogen ions, Baltimore, 2nd edition, 1925.

Ekkert, L., Noch eine Farbenreaktion der salpetrigen Saure und der Salpetersaure, Pharm. Zentralhalle, 1925, lxvi, 733.

Elvove, E., A calorimetric method for the estimation of the cresol or phenol preservative in serums, Bull. Hyg. Lab., U. S. P. H., 1917, cx, 25.

Eykman, J. F., Nitrous ether as a sensitive reagent for carbolic acid, New Remedies, 1882, xi, 340.

Gibbs, H. D., Phenol tests. 1. A classification of the tests and a review of the literature, C/rem. Rev., 1926, iii, 291.

Griessmayer, V., Ueber das Verhalten von Sttirke und Dextrin gegen Jod und Gerbsaure, Ann. Chem., 1871, clx, 40.

Hirschsohn, E., Comparative experiments on the behavior of thymol and carbolic acid towards certain reagents, Pharm. J., 1881, xii, 21.

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Hoffmann, R., Reaction auf Leucin und Tyrosin, Ann. Chem. u. Pharm., 1853, lxxxvii, 123.

Holmes, W. C., The spectrophotometric determination of hydrogen-ion concentrations and of the apparent dissociation constants of indicators. I. The methods, J. Am. Chem. Sot., 1924, xlvi, 627.

Huerre, R., &uelques reactions de l’acide azotique sur les phenols et les d&hers de la pyrocatechine et de l’homopyrocat&hine, Bull. SC. pharmacol., 1922, xxix, 180.

Ingold, C. K., and Piggott, H. A., The additive formation of four-membered rings. IV. The influence of temperature on the tendency toward self- addition of the nitroso-group, J. Chem. Xoc., 1924, cxxv, 168.

Lintner, C. J., Ueber MercurisalicylsLure und die Millon’sche Reaction, Z. angew. Chem., 1900, 707.

Meyer, L., Ueber die Hoffmann’sche Reaction auf Tyrosin, Ann. Chem., 1864, cxxxii, 156.

Millon, E., Sur un reactif propre aux compo&s proteiques, Compt. rend. Acad., 1849, xxviii, 40.

Millon, E., Sur un rCactif propre aux compos& proteiques, Ann. chim. et phys., 1850, xxix, 507.

Nasse, O., Ueber die aromatische Gruppe im Eiweissmoleciil, Ber. naturjorsch. Ges. Halle, 1879, 23.

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TESTS. SPECTROPHOTOMETRICTESTS. THE MILLON AND SIMILAR PHENOL TESTS: II. NITROUS ACID

1927, 71:445-459.J. Biol. Chem. 

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