« ForrigeFortsett »
of the frog and studied its excretion. They found the excretion to be chiefly by the second segment of the tubules and that the indigo carmine became apparent only after oxidation with hydrogen peroxide. In commenting on the work of Heidenlmin, Nussbaum (1878) cites an experiment showing that the liver may take up and reduce indigo. The absorption was made evident only on oxidizing the reduced indigo, a precaution to be remembered wherever indigo is used as a stain. Indigo carmine (indigo disulphonate), which is now * permissible food color (Pugsley, 1922), has recently been employed by Thomas (1914, 1922) and Thomas and Min Hsu (1922) as a kidneyfunction test. Hatiegan (1922) used it in a test for hepatic hyperpermeability.
It goes without saying that a substance so well known and so common as indigo should have been employed both with and without reason. A review in the American Journal of Medical Science, 20, 483 (1837), cites the use of indigo as a therapeutic agent in epilepsy, hysteria, amenorrhea, St. Vitus's dance, and infantile convulsions. It has been used by the chemist as a test for urinary sugar, for oxygen, for chlorine, for nitrates, for tannin, and as an indicator for caustic alkali.
Popular writers on the romance of synthetic indigo have emphasized those achievements of the chemist which have led to the artificial production of a pleasing color. But it should be noted that the researches which led to the final synthesis revealed the nucleus of indigo, indole. This group is now found in an essential nutrient, tryptophane, and in an essential hormone, thyroxin. In the metabolism of tryptophane the indole group persists, appearing as indolethylamine, a stimulant of smooth muscle, as indole, a bacterial product, and as indican, an end product found in mammalian urine. In plants the occurrence of the indigo-yielding glucosidc in the genus Indigofera and of free indole in certain odorous flowers is but an indication of the wide distribution of the indole group, a distribution curiously brought to general attention through the discovery that the "royal purple of the ancients," the product of a snail, is brominated indigo. Wherever found, one method of detecting the indole group is to convert it to indigo, and thus the study of the system indigo ^ indigo-white is a first, although minor, step toward an understanding of processes of oxidation or reduction by which the indole group in nature is carried through a wide variety of transformations.
In many of the investigations briefly mentioned above, indigo has been used under circumstances or for definite purposes which will hardly bear critical examination. Of these instances we shall later refer to one and shall now mention another, a brief discussion of which will reveal both the nature and the limitations of our own contribution.
The coloration of indigo-white has been used as a test for free oxygen, and, conversely, the decoloration of indigo blue has been used as an end-point indication of oxygen exhaustion. We have already emphasized, in the first article of this series, the fact that many reversible oxidation-reductions can be accomplished by a variety of reactions and can be formulated by a variety of schemes. We shall show in this paper that whatever the manner in which a given state of the system indigo sulphonate ^ leuco compound is established, that state may be expressed in units of universal applicability in essential accord with the formulations of our second paper. The significance may be illustrated by means of an analogy.
Everyone is familiar with the fact that a certain state of colorchange or virage of phenolphthalein indicates only that its aqueous solution has a certain definite hydrion concentration. If now phenolphthalein is to be used as an end-point indicator in the titration of an acid by a base, the acid and base chosen must be such that equivalent quantities mixed in the given concentration furnish a hydrion concentration within the range of color change of phenolphthalein. Otherwise, or if there be present material tending to buffer the solution at or above the zone of hydrion concentration within which phenolphthalein changes color, this indicator wdl be quite useless for end-point work. The failure to recognize this has led to gross mistakes both in theory and in practice. The first and fundamental task in the development of a rational theory of acidimetric titration was the establishment of the acid-base equilibrium-constant or at least the pll zone of color change of each indicator.
The reader will at once perceive the analogy. Indigo, according to the circumstances of its use, may or may not be valuable as an end-point indicator of free oxygen. Most certainly it has sometimes been employed in solutions poised 3 by material active between the true end point of oxygen removal and the potential at which indigo is reduced; but for the present we need not discuss this. We are now concerned in establishing universally applicable data of the oxidation-reduction intensities of different states of the systems. This is a first and fundamental step. Without this information it is easy to misjudge results; and were it worth while, instances of mistakes could be cited from the chemical and medical literature. The establishment of the reduction potentials of each system should make possible the use of that system in the colorimetric estimation of reduction intensity. The possible applications of this fundamental knowledge are numerous.
»See the first article of this scries.
In the previous pages wc have used for brevity the expression "indigo ^ indigo-white" where wc have intended one or another of the following five systems:
1. Indigo ^ indigo-white.
2. Monosulphonate of indigo ^ monosulphonate of indigo
3. Disulphonate of indigo ^ disulphonate of indigo-white.
4. Trisulphonate of indigo ^ trisulphonatc of indigo-white.
5. Tetrasulphonate of indigo ^ tetrasulphonate of indigo
white. These will be discussed separately, and it will be found that henceforth they must be sharply distinguished.
PREPARATION OF THE SULPHONATES OF INDIGO.4
Commercial samples of synthetic and natural (Bengal) indigo which we tested were found to contain impurities as described by Perkins and Bloxam (1907) and Perkins (1907). Purification by sublimation, first used by Crum (1823) was employed by Dumas (1834) in his study of the constitution of indigo and by von Sommaruga (1879) in determining the molecular weight. Bloxam (1905) especially recommends it, but, since we were to depend largely upon recrvstallization of the sulphonates, a preliminary purification of indigo was carried out by the possibly less satisfactory method of extraction. The finely powdered, crude indigo was extracted successively with hot ethanol, cold acetic acid, and, again, with hot ethanol, in both of which solvents indigo red and indigo brown are somewhat soluble. The final ethanol extract was no longer reddish, but had a blue tinge. The material was then dried, reground, and washed with ten times its weight of 10 per cent hydrochloric acid solution—first hot and then cold—to extract the so-called indigo gluten and mineral material. It was finally washed with hot water, dried first at 60° C, and finally at 110° C, reground, and bottled. By nitrogen analysis a synthetic indigo was found to have been raised from 94 to 98 per cent purity by this method. By the same criterion, Bloxam's (1905) purest preparation was about 98 per cent indigo. Such was the stock from which most of our sulphonates were prepared.
Bloxam (1906) has detailed the preparation of the sulphonates, ind we have followed his descriptions, in the main.
The sulphonates are formed by the action of sulphuric acid. Dependent upon temperature, time of action, and the amount and strength of sulphuric acid, there arc formed successively the mono, di-, tri-, and tetra-sulphonic acids. Room temperature and 10 to 20 parts of ordinary sulphuric acid to 1 of indigo arc conditions yielding the monosulphonic acid. Warming under the above conditions leads to the disulphonic acid, indigo carmine. Moderate warming with sulphuric acid containing 15 per cent excess sulphur trioxide gives the trisulphonic acid; while the tetra sulphonic acid is obtained at high temperatures and with fuming sulphuric acid containing 20 per cent excess sulphur trioxide.
'Many authors have distinguished the chief constituent of crude indigo by the Urm "indigotin" or "indigoiine." However, this term has been loosely used or made synonymous with "indigo'' by other*wareful writers. That constituent of crude Indigo which is regarded as the important constituent Wisjcnemical individual has been referred to as indigo from the beginning of chemistry. We shall retain
As the degree of sulphonation increases, the practically insoluble indigo is converted into products of progressively higher solubility. Consequently, in the crystallization of the salts the tendency is for the contaminant to be one or more of the lower sulphonates. The aim in sulphonation should be, therefore, to carry the reaction as completely as possible to the stage desired without oversulphonation.
Potassium indigo monosulphonate was prepared as follows: One part indigo and 20 parts concentrated sulphuric acid were thoroughly mixed and allowed to stand one hour at room temperature. The mixture was frequently stirred and the progress of the reaction tested by transferring a drop of the mixture into water. When a violet solution in hot water was obtained, the whole mass was mixed with enough water to dilute the sulphonic acid approximately ten times. The diluted mixture was allowed to stand several hours and the mono sulphoiuc acid settled out. The precipitate was filtered with suction on a Biichner funnel and was washed with a little water. The filtrate, which may have contained some disulphonic acid, was discarded.
The reddish precipitate was dissolved in boiling water (approximately 300 cubic centimeters of water for each gram of original indigo used) and filtered. To the hot filtrate, powdered potassium carbonate was added, a little at a time, with stirring, until a precipitate began to occur. Then about 100 cubic centimeters of a 50 per cent solution of potassium acetate were added and tho whole was cooled first in water and then in ice to 5°. A precipitate of potassium indigo monosulphonate formed. This salt was not definitely crystalline. It was purified by dissolving in boiling water and precipitating with the addition of a few cubic centimeters of the potassium acetate solution while chilling in ice and salt. This process was repeated. Then followed two crystallizations from water without addition of potassium acetate. Finally the sulphonate was filtered on hardened paper and washed with a little water and with ethanol to remove traoes of potassium acetate. The compound was heated in an oven at 60° for two hours and at 80° for one hour, to free it from ethanol. It was then heated at 100° for a short time until it separated from the hardened filter paper.
Two sets of the potassium monosulphonate were made; preparation 1 was on a small preliminary scale; preparation 5 on a large scale. The analyses of the samples are given in Table I.
Since preparation 5 was made from an unpurified synthetic indigo, it was thought possible that it might contain some indigo red monosulphonate. Accordingly it was ground with ethanol in which indigo red sulphonates are soluble. Preparation 5 was slightly soluble in 95 per cent ethanol but with a blue color. After treatment with ethanol the insoluble part was recrystallized from water and labeled 5 A.
Table I.—Analyses of indigo sulphonates.
Potassium indigo disulphonate was prepared as follows: One part indigo and 20 parts concentrated sulphuric acid were heated on a, water bath one and one-half to two hours with frequent stirring. The product was poured into water, 200 parts, and the mixture stirred and filtered hot. After cooling to about 15° to allow any monosulphonic acid present to settle out, the solution was filtered again. Practically no insoluble matter was present. The filtered solution was then treated with solid potassium carbonate, a little at a time, with stirring, until the potassium salt of the dye showed evidence of precipitating out. About 100 c. c. of a filtered 50 per cent solution of potassium acetate were then added, and the mixture was brought to boiling and filtered hot. Cooling first in water and then to 5° in ice for an hour gave a precipitate of blue needles, more or less contaminated with potassium sulphate. The precipitate was filtered on a Buchner funnel. If the filtrate happened to be a deep blue, a little of the potassium acetate solution was added and the mixture warmed with stirring and chilled as before until more disulphonate began to crystallize out. This second precipitate was filtered on the same Buchner funnel, washed with potassium acetate to wash out sulphate and with ethanol to wash out the acetate. The precipitate was purified by taking up in a minimum amount of boiling water, filtering hot, cooling in ice, and filtering