some evidence of salting out. The salting out was completed by the addition of a 50 per cent solution of potassium acetate. A heavy precipitate occurred. The mixture was then brought just to boiling when the precipitate went into solution. The mixture was next cooled and chilled in ice for one hour. A heavy precipitate occurred, a mixture of the tetrasulphonate and potassium sulphate. The precipitate was collected on a Büchner funnel and was washed with a little potassium acetate. It was dissolved in 2,000 c. c. of hot water. The solution was filtered warm and chilled to 0° in ice and salt. A heavy precipitation of large, transparent, red, crystalline plates occurred. These were filtered and washed with 95 per cent ethanol. The tetrasulphonate was further purified by crystallization from 1,000 c. c. of aqueous solution. The final crystals were filtered on hardened filter paper and washed with a little water. Dried for a time by suction and then at 85° for an hour, the tetrasulphonate was ground and bottled as preparation 18. Dried at 120° it was found to contain 8.14 per cent moisture. With correction for moisture, the compound furnished the analyses shown in Table I.

Three preparations of potassium indigo tetrasulphonate were made. Preparation 4 was made on a small scale and was used up in the measurements described in our preliminary paper (Sullivan & Clark, 1921). Preparation 8 was from unpurified indigo. Preparation 18 is considered our best sample.

ANALYSES OF THE INDIGO SULPHONATES.

Moisture was determined by drying the samples at 120-125°, as recommended by Halland (1918). The samples were started at 60° and were gradually brought to 120-125° and dried to a constant weight. The dried samples were very hygroscopic.

Nitrogen was determined by the Kjeldahl method.

Sulphur was determined as BaSO, after fusing with 10-15 parts of a mixture of 7 parts sodium carbonate and 1 part potassium nitrate.

Potassium was determined from the ash as potassium sulphate, and, in a few cases, as potassium platinic chloride.

Ashing was done at a final temperature of 780° in an electrically controlled electric furnace. If not white, the ash, treated with a drop of nitric acid and a drop of concentrated sulphuric acid, was placed in the oven again and the temperature raised gradually to 780°. The white ash was presumed to be K,SO,. The potassium determined by precipitation as potassium platinic chloride agreed with the potassium computed from ash as K,SO. The averages analyses are given in Table I.

of

POSITIONS OF SULPHONIC ACID GROUPS.

The sulphonates of indigo have long been known, for references in the very early literature are to be found. So far as we have ascertained, the first salts were made by Crum in 1823. He prepared the mono- and the disulphonates. Dumas (1836, 1837) also prepared sulphonates. He called the monosulphonic acid "sulphopurpuric acid," and the disulphonic acid he called "sulphindylic acid.” Apparently Juillard (1892) separated the trisulphonate from a mixture, and later Hönig (1899) gave details for its preparation. Vorländer and Schubart (1901) and Schubart (1902) synthesized from intermediates in which the position of the sulphonic acid was known, isomeric disulphonates of indigo:

(I) With the sulphonic group para to the NH; (II) with it meta to the NH.

These disulphonates Vorländer and Schubart designated as the 1.2.5 disulphonate and the 1.2.4 disulphonate. The 1.2.5 disulphonate they found to be identical with indigo carmine. On the basis of Baeyer's formula and the now customary atom numbering

(III) we can call Vorländer and Shubart's 1.2.5 disulphonate the 5.5' disulphonate; their 1.2.4 disulphonate the 6.6' disulphonate. The 5.5' disulphonate, which they found identical with indigo carmine, gives a precipitate with barium chloride and basic lead acetate and is insoluble in excess of these precipitants. The 6.6' acid (Vorländer and Schubart's 1.2.4 acid) gives no precipitate in

dilute solutions with barium chloride, and the precipitate with basic lead acetate is soluble in excess of the precipitant. Spectroscopically, also, the isomers were found to differ.

The potassium disulphonate employed in the present investigation was completely precipitated by barium chloride and with basic lead acetate, so it is probably the 5.5' disulphonate.

Grandmougin (1999, 1921) points out that bromination or sulphonation occurs successively, first in position 5 para to the NH of the indole nucleus, and next in position 7 ortho to the NH.

On the basis of the work of Vorländer, Schubart, and Grandmougin, the positions of the sulphonic groups are as follows: "mono," 5; "di," 5.5"; "tri," 5.5', 7; " tetra" 5.5', 7.7'.

It is possible that some of our preparations may have been mixtures of isomers at the same stage of sulphonation. This could be detected if the characteristic equilibrium potentials of two isomers are distinctly different from one another. Since we have no information on this matter we shall consider the deviations treated in the last section of this paper to be due to contaminations by other sulphonates and not by isomers.

The four sulphonates of indigo have solubilities which increase with the degree of sulphonation. The monosulphonate, while sufficiently soluble in water to give a deep blue solution, precipitates on addition of salts.

With increase in sulphonation, the colors of the dilute solutions shift from an apparently pure blue to a decided reddish blue. Both this shift and a decrease of specific absorption are shown by the absorption curves plotted by Walter C. Holmes (1923) of the Color Laboratory, Bureau of Chemistry, United States Department of Agriculture. Using preparation No. 5 of the "mono," preparation 17 of the "di," preparation 7 of the "tri," and preparation 18 of the "tetra," Holmes (1923) found that, in alcoholic or aqueous solution, sulphonation shifts the absorption band toward the blue, the maximum in ethanol changing from X 6150 for the monosulphonate to A 5980 for the tetrasulphonate, and in aqueous solution from A 6080 for the mono. to A 5900 for the tetrasulphonate. At the same time in either solvent there is developed a greater general absorption in the blue. In alcoholic solution the maxima of the absorption bands are reduced from extinction coefficients 1.63 of the monosulphonate to 0.81 of the "tetra," and the "di" and "tri" lie almost equally spaced between these values. The corresponding data for aqueous solution are not so definite because of the colloidal nature of the monosulphonate. However, the general order is the same.

Dr. Edgar T. Wherry has given us the following descriptions of the crystalline potassium sulphonates:

"Tetra."-Rods and plates with strong pleochroism from brown crosswise to intense blue lengthwise. One refractive index crosswise about 1.50, others indeterminate.

"Tri."-Needles with strong pleochroism from pinkish gray lengthwise to deep blue crosswise. Refractive indices indeterminate. "Di."-Very minute needles, with some pleochroism from deeper to paler blue. Refractive indices indeterminate.

Mono."-Very minute needles or plates, with some pleochroism from deeper to paler violet. Refractive indices indeterminate.

Procedure in Electrode Measurements.

The potential measurements were made with the equipment described in the preceding paper of this series; and, in general, the determination of the characteristics of each set of equilibria followed essentially the course of the study of 1-naphthol-2-sulphonic acid indophenol.

In the latter part of the work the troublesome penetration of oxygen through the rubber tube connected to the apparatus shown in Figure 3 of the previous paper was obviated by attaching the apparatus directly to a copper tube leading from the nitrogen purification chain. After being filled, the apparatus was closed, detached for moving, and gas to compensate for removal of solution was furnished by an attached reservoir of nitrogen stored over mercury. The study of each set of equilibria included three main series of measurements.

(1) First, one or more titration experiments were made in which the oxidant was reduced or the reductant was oxidized at approximately constant pH. This furnished the value of n, the number of electrons concerned in the process. Since n is evidently 2 in all the cases to be cited, we shall not pause to tabulate calculations. The titration experiments also furnish values of the potentials of known mixtures of oxidant and reductant at constant pH in so far as the pH can be controlled or estimated under the circumstances. This control is not always so rigid as in other series of measurements, but in the titrations with ferricyanide we have applied a correction estimated as follows:

The dye solution was prepared by adding 5 c. c. of approximately 0.006 molar leuco-indigo sulphonate to 50 c. c. buffer No. 7. The oxidizing reagent was prepared by making buffer No. 7 approximately 0.004 molar with respect to K,FeCy, and diluting 50 c. c. of this to 55 c. c. It was now assumed that the pH of the buffer was such as to suppress the acid property of the leuco-compound and that the shift in pH which would be produced during the titration would be due to the formation of HK,FeCy, from K,FeCy, and to the altering salt content of the mixture. Therefore an estimate of this effect was made by titrating 50 c. c. buffer No. 7+5 c. c. water with buffer

No. 7+5 c. c. 0.04 NH,SO, +0.0522 gm. K,SO,. There is probably a slight overcorrection, due to the fact that HK,FeCy, is not so completely ionized as H2SO, at the pH of buffer No. 7.

While this procedure still leaves a small degree of uncertainty, it is probable that the correction is of the proper order and that the experimental curve, when corrected, will show only slight deviations from the theoretical type when the compound is pure. If, however, the dye under investigation is a mixture of compounds, each capable of a reversible oxidation-reduction, the deviation of the experimental titration curve from the type should be large. This sort of analysis will be discussed later.

(2) The second series of experiments consisted in the measurement of the electrode potentials of known mixtures of oxidant and reductant in solutions the pH values of which were considered identical with those of the buffer diluted to the same concentration as the buffer-dye mixture. These measurements give the potentials of known mixtures at a few pH values.

In these first two series of measurements, pH is assumed to be constant. As shown in previous papers, the electrode equation for this condition is:

ᎡᎢ [S,]
En=E'。- In
2F [S.]

(1)

(3) The third series of experiments consisted in adding an indefinite, but approximately equimolecular, mixture of oxidant and reductant to different buffer solutions and thus determining the effect of pH change. With this set of data the character of the third term of the equation can be determined (cf. the second paper).

Since many of the buffer solutions had the same composition we may save space by tabulating below (Table II) the composition of those most frequently used. Since slight differences of pH occur by reason of variation in stock material and air contamination, no pH values are included in Table II. The pH values given in subsequent tables were determined in each case on the solution used and within a reasonable period before or after its use.

Just as in the study of 1-naphthol-2-sulphonic acid indophenol (see previous paper), so in the study of the indigo sulphonates by the method of mixture, slight but distinct drifts of potential occurred after adding the mixtures to buffer solutions. As in the former case, so in this the values taken, unless otherwise stated, were those of a period of apparent constancy when the curve of drift with time had reached a "plateau." The magnitude of the drift was about the same as that described for 1-naphthol-2-sulphonic acid indophenol, and to the discussion given in the previous paper we have now nothing to add.