The Natural Organic Colouring Matters
By
Arthur George Perkin, F.R.S., F.R.S.E., F.I.C., professor of colour chemistry and dyeing in the University of Leeds
and
Arthur Ernest Everest, D.Sc., Ph.D., F.I.C., of the Wilton Research Laboratories; Late head of the Department of Coal-tar Colour Chemistry; Technical College, Huddersfield
Longmans, Green and Co.
39 Paternoster Row, London
Fourth Avenue & 30th Street, New York
Bombay, Calcutta, and Madras
1918
Kaikki kuvat (kemialliset kaavat) puuttuvat // None of the illustrations (of chemical formulas) included.
According to the early researches of Chevreul (Ann. Chim. Phys., 1808, 66, 8, and 1808, 68, 284) and of Geradin and Preisser (J. Pharm. Chim., 1840, 26, 344) the colouring principle of indigotin present in indigo-yielding plants was considered to consist of indigo white, and this theory remained uncontradicted until Schunck (Phil. Mag., 1855, [iv.], 17, 74, and 1858, 15, 127) isolated from the hath tinctoria (woad), Polygonum tinctorium, and Indigofera tinctoria (Schunck and Roemer, Ber., 1879, 12, 2311) a glucoside, which was named indican.
To prepare this substance from woad the leaves were extracted with cold alcohol, the solution treated with a little water, and concentrated at the ordinary temperature by blowing air over it. The waxy matter which thus separated was removed by filtration, and the filtrate shaken up with freshly precipitated cupric hydroxide. The mixture was filtered, the liquid freed from dissolved copper by means of sulphuretted hydrogen, and then evaporated at the ordinary temperature. The residue was extracted with cold alcohol, the extract treated with ether to precipitate certain impurities, and the solution evaporated.
Thus obtained it consisted of a yellow or yellowish-brown syrup, which was of an exceedingly unstable nature, and could not be dried without decomposition. With alcoholic lead acetate it gave a yellow precipitate, whereas in aqueous solution it could only be precipitated by means of basic lead acetate. Analyses of the lead compound indicated that indican possessed the formula C26H31NO17. Schunck found that this compound was a glucoside, and that by the action of dilute acids, alkalis and of a ferment present in the plant, it was readily hydrolysed with the formation of indigotin, and a sugar indiglucin
2C26H31O17N + 4H2O = C16H10O2N2 + 6C6H10O6
For the production of indigotin the presence of air or other suitable oxidising agent was however necessary, and it appeared, therefore, that during the reaction the indigotin at first formed was reduced to indigo white.
Later, however, Schunck and Roemer showed (loc. cit.) that indican, when hydrolysed in the absence of air, gave a product which, on subsequent treatment with oxidising agents, did not yield indigotin. Schunck further obtained by the action of cold dilute acids on his indican a brown powder, from which he isolated six distinct substances, viz. indihumin, indifuscin, and indiretin, soluble in warm sodium hydroxide solution, and α- and β-indifulvins and indirubin, insoluble in alkalis.
When aqueous solutions of the indican were boiled or heated for some time a decomposition ensued, and the product, on treatment with acid, gave indiglucin, but no indigotin, this being replaced by indiretin and indihumin, brown amorphous substances. The latter closely resembled, and was probably identical with, indigo brown. By the action of alkalis or alkaline earths at the ordinary temperature, indican was converted into a new glucoside, indicanin, C20H23NO12, which on treatment with acid gave indiglucin and indirubin.
Oxyindicanin, a brown gummy substance, insoluble in alcohol, was isolated during the preparation of indican, and yielded, under the influence of acids, indiglucin and a brown substance similar to indifuscin.
In 1896 Surg. Lt.-Col. G. S. A. Ranking (Jour. Asiatic Society of Bengal, Ixv., ii., No. 1), as the result of a careful examination of the fermented leaf extract, pointed out for the first time that the soluble compound present and which by air oxidation gives indigotin, cannot be indigo white, but is evidently indoxyl. He also suggested that as CO2 is largely evolved during the leaf fermentation it is not improbable that indoxylic acid, may also be present.
Marchlewski and Radcliffe (J. Soc. Chem. Ind., 1898, 17, 434), evidently unaware of Ranking's paper, and indeed this seems to have been lost sight of by later workers, in a theoretical discussion of the subject, suggested that indican, C14H17O6N, was a glucoside of indoxyl, the hydrolysis of which could be represented by the following equation: C14H17O6N + H2 = C8H7ON + C6H12O6
As a result of the communication of Marchlewski and Radcliffe, Hazewinkel, the director of the experimental station for indigo, Klaten, Java (Proc. K. Akad. Wetensch., Amsterdam, 1900, 2, 512), gave an account of a research, concluded in 1898, which he had hitherto considered to be to the interest of the Java planters to keep secret. In this important paper he shows that indican is an indoxyl glucoside, and that the sugar obtained from it is dextrose.
The elaborate researches of Beyerinck, van Romburgh, and other Dutch chemists proved that the indican present in the various Indigofera and in the Polygonum tinctorium was far more stable than Schunck supposed, and the experiments of these authors eventually led to the isolation of this glucoside, in a crystalline condition from the Indigofera leptostachya and Polygonum tinctorium by Hoogewerff and ter Meulen (Proc. K. Akad. Wetensch., Amsterdam, 1900, 2, 520).
The leaves were immersed in two and a half times their weight of boiling water, boiled for a few minutes, and further systematically exhausted. Without any sensible decomposition the decoction could be evaporated in vacuo if care was taken to keep the reaction alkaline. The dry residue was extracted with methyl alcohol, and to the solution ether was added as long as a precipitate was formed. This was removed, the clear liquid evaporated, the residue completely dried in vacuo, and then dissolved in water. The filtered and concentrated solution deposited on cooling well-defined crystals of indican. This process may be modified by treating the decoction of the leaves with baryta water before concentration, by which means a large proportion of the impurities are precipitated. Seventeen kilos of the leaves of Polygonum tinctorium yielded 5 grams of pure indican.
Thus obtained indican, Cl4H17O6N, crystallises from water in spear-shaped crystals, which contain 3 molecules of water of crystallisation. Heated in a test tube, or on platinum foil, purple-coloured fumes are given off, but this does not take place in an atmosphere of carbon dioxide. By passing a current of air through a hot solution of indican in dilute hydrochloric acid containing a little ferric chloride, 91 per cent, of the indican was converted into indigotin according to the equation
2C14H17NO6 + O2 = C15H10N2O2 + 2C6H12O6 There was no difference between the indican prepared from the I. leptostachya and that obtained from the P. tinctorium.
In a paper by Beyerinck (Proc. K. Akad. Wetensch., Amsterdam, 1900, 3, 102), "On the Formation of Indigo from Woad," this chemist discusses Schunck's well-known work on the same subject, and points out that the indigo yielding substance contained in this plant is not, as Schunck regarded it, identical with the indican present in the Polygonum tinctorium. The colouring principle of woad Beyerinck names isatan, and shows that this compound, unlike indican, is decomposed in feebly alkaline solutions, whereas indican is stable even in concentrated alkaline liquids. In presence of acids both isatan and indican are hydrolysed, but indican with greater difficulty. Isatase, the specific enzyme of woad, does not act on indican, and isatan, on the other hand, is unaffected both by the indigo enzyme or by common bacteria.
Schunck (Chem. News, 1900, 82, 176) considered that the crystalline indican of Hoogewerff and ter Meulen was not the substance obtained by him, and should not be considered as a pure variety of it, but was rather derived from it, by extracting the plant with a hot solvent and the use ot chemicals. He preferred to name his compound a-indican and theirs b- indican.
Bergtheil (Chem. Soc. Trans., 1904, 85, 877), who experimented with the I. sumatrana and I. arrecta, did not find it possible to prepare indican from the leaves of these plants in the manner described by Hoogewerff and ter Meulen.
It was, however, shown by Perkin and Bloxam (Chem. Soc. Trans., 1907, 91, 1715) that crystalline indican can be isolated from both of these plants by such a method, and is in reality the source of the natural indigo which is derived from them.
In a further communication ter Meulen (Rec. trav. chim., 1905, 29, 444) describes a modification of the method previously given for the isolation of indican from the Polygonum tinctorium, which consists in treating a cold solution cf the partially purified substance with sulphuric acid, by which means certain impurities are precipitated. The acid is then removed with barium carbonate. The main object of the investigation was, however, the determination of the sugar that this glucoside yields when hydrolysed by its specific enzyme, and this proved to be dextrose, as already indicated by Hazewinkel (loc. cit.)
As a result of the study of the behaviour of indican with solvents, Perkin and Bloxam (loc. cit.) devised a very simple process for the isolation of this glucoside, by the aid of which large quantities of the pure substance could be readily prepared.
The leaves and stems of the I. sumatrana (1000 grams) were treated with 4 litres of cold acetone, the mixture being occasionally shaken during seven days, and the green-coloured extract was evaporated on the steam-bath to a very small bulk. To the residue light petroleum was added, causing the deposition of a brown viscous precipitate of crude indican, and this was repeatedly agitated with small quantities of light petroleum. The product on treatment with water gave a pale yellow liquid, containing resinous matter in suspension, and the latter was removed by shaking with ether. The clear aqueous solution, treated with 10 c.c. N/2 sodium carbonate, on gradual evaporation in vacuo deposited crystals, and eventually a semi-solid mass was obtained. It was collected, drained, and allowed to dry at the ordinary temperature. When exhaustively extracted, 1000 grams of leaf gave 31,66 grams of indican, and by a continuous system of working more than 500 grams of crystalline indican were prepared. The preparation of this glucoside from the leaves of I. arrecta is more troublesome, owing partly to the presence of kaempferitrin (loc. cit.), but more especially of a colourless sugar-like compound, C6H12O5; melting-point 186-187°; possibly a modification of quercitol. The fact that indican can be so readily isolated without the aid of heat, and merely with the use of acetone, light petroleum and ether, is not in harmony with the contention of Schunck (loc. cit.) that the crystalline glucoside is an alteration product of his amorphous substance, and consequently the terms a- and β-indican suggested by him should disappear.
Indican crystallised from water, C14H17O6N, 3H2O, melts at 57-58°, but in the anhydrous condition as obtained by the addition of boiling benzene to its hot alcoholic solution, at 176-178°. Owing to its somewhat ready solubility in water it can be more economically purified by the latter process, and, according to Perkin and Thomas (Chem. Soc. Trans., 1909, 95, 793), crystallisation from absolute alcohol gives excellent results.
It has been shown by Baeyer (Ber., 1881, 14, 1745) that indoxyl readily condenses with aldehydes and ketones to form the sor called indogenides, and Hazewinkel (loc. cit.) partly identified this substance by means of its condensation products with isatin, benzaldehyde, and pyruvic acid, relying, however, on their qualitative reactions, as he did not prepare these compounds in a pure enough condition for analysis. Almost simultaneously Beyerinck (Proc. K. Akad. Wetensch., Amsterdam, 1899, 2, 120) prepared indirubin by hydrolysing crude indican in the presence of isatin.
Perkin and Bloxam (loc. cit.} and Gaunt, Thomas, and Bloxam (J. Soc. Chem. Ind., 1907, 26, 1174), who experimented with the pure substance, found that when indican dissolved in water is added to a boiling solution of isatin, acidified with a little hydrochloric acid, and the operation is carried out in an atmosphere of hydrogen or carbon dioxide, the yield of indirubin is quantitative according to the following equations:
C14H17O6N + H2O = C8H7NO + C6H12O6
C8H7NO + C8H5NO2 = C16H10N2O2 + H2O
This "isatin" method, for details of which see page 513, affords a ready means, not only for the analysis of the crystalline glucoside, but also for the estimation of the amount which is present in aqueous infusions of the leaf (v. infra).
More recently Perkin and Thomas (Chem. Soc. Trans., 1909, 95> 795)> wno studied in a similar way the condensation of indoxyl derived from indican with p-nitrobenzaldehyde, found that the /-nitrobenzaldehydeindogenide is deposited in quantitative amount, and that this reaction could also be employed for the analysis, both of crystalline indican and of that present in the leaf extract. The reaction takes place with extreme readiness, for with indican solution at a dilution of 1 in 1000, the above compound quickly separates, and even at 1 in 10,000 the condensation can be observed to take place. Piperonal and indican in the presence of dilute acid yield the analogous compound C16H11O3N, orange-coloured needles, melting-point 223-224°; but this process, under analytical conditions, gave only approximately satisfactory results. As a side issue, p-hydroxybenzaldehydeindogenide, C15H11O2N, orange-red needles, melting-point 267-269°, and dihydroxybenzaldehydeindogenide, C15H11O3N, orange-red needles, melting-point 264-265°, were prepared from indican. The latter compound derived from protocatechuic aldehyde dissolves in concentrated sodium hydroxide, with a bluish-violet coloration, and dyes with mordanted woollen cloth welldefined shades.
On the other hand, when indican is hydrolysed with acid in the presence of an oxidising agent it does not appear possible to obtain a quantitative yield of indigotin. Hazewinkel (loc. cit.) states, in regard to this point, that acid oxidising agents convert indican into indigo, and this in turn is oxidised by an excess of the reagent, By the use of ferric chloride and hydrochloric acid, Hoogewerff and ter Meulen obtained from the pure glucoside only 91 per cent, of the theoretical quantity of colouring matter, which appeared to contain indirubin, and was of doubtful purity. Gaunt, Thomas, and Bloxam (loc. cit.), who examined the behaviour of ammonium persulphate, a reagent suggested by Rawson for the analysis of the plant extract (Report on the Cultivation and Manufacture of Indigo, Mozzufferpore, 1904; cf. also Bloxam and Leake, Dalsingh Serai Report, 1904), found that the process was far from quantitative with pure indican, and that the yield of colouring matter averaged but 82 per cent, of the theoretical. Perkin and Thomas (loc. cit.) studied the effect of the hydrolysis of solutions of indican with acid during the aspiration of air through the liquid, under varying conditions of temperature and concentration. The most satisfactory yield of pure colouring matter (93,5 per cent.) was produced when air was passed during eight hours through a solution of 0,5 gram of the glucoside in 850 c.c. of water acidified with 15 c.c. of 33 per cent, hydrochloric acid, and the temperature maintained at 60°.
When, however, the operation was carried out at 70° less colouring matter was obtained (87,6 per cent.), and, curiously enough, replacement of the hydrochloric acid by an equivalent amount of sulphuric acid gave, under similar conditions, a much lower result. The deficiency in the yields given by these air-oxidation processes was due to the fact that a portion of the indoxyl had been converted into substances other than indigotin, and it was observed that whereas in the case of hydrochloric acid the filtrate possessed a pale yellow colour, that containing sulphuric acid had a browner and darker tint. Indirubin was also present in these indigo preparations.
* According to Perkin (Chem. Soc. Trans., 1916, 109, 211) this operation can be so conducted that the total amount of nitrogen present in this product is approximately equivalent to that possessed by the indican originally employed.Whereas Schunck (loc. cit.) had described the production of various brown substances by the action of dilute acids on his indican, and Schunck and Roemer (loc. cit.) had obtained a brown-yellow compound by means of hydrochloric acid in absence of air, the behaviour of the pure crystalline glucoside in this respect was studied by Perkin and Bloxam. When 100 c.c. of a 4 per cent, solution of indican was treated with 3 c.c. of sulphuric acid, and digested at a boiling temperature, the liquid, at first yellow, became brown, a brown resinous substance, together with a little indigotin, quickly separated, and the presence of indole was observed. The product of the reaction was almost identical in weight with that required by the amount of indoxyl which the glucoside would yield on hydrolysis, and consisted chiefly of a dark reddish-brown powder (a), sparingly soluble in alcohol, together with a small quantity of a similar, though more readily soluble substance (b).* Analyses of (a), which is termed indoxyl brown, gave C = 68,10; H = 4,10; N = 9,34, figures almost identical with those found by the same authors for the main constituent of indigo brown, and though these two products differed from one another in certain minor respects, there could be no doubt that they were closely allied. The more readily soluble substance (b) also closely resembled the indoxyl brown, and gave on analysis N =9,65 percent. Indican, when treated with cold hydrochloric acid in the absence of air for ninety hours, gave indoxyl brown and a soluble brown substance similar to that described above (Perkin and Thomas). The acid filtrates from the indoxyl brown preparations contained dextrose, and this was identified by means of its osazone, and also by the preparation of its acetyl derivative.
The indigo enzyme discovered by Schunck (loc. cit.) has been elaborately investigated by the Dutch chemists. Beyerinck (Proc. K. Akad. Wetensch., Amsterdam, 1899, 1, 120) extracts the finely divided leaves of the plant, first with cold 96 per cent, alcohol, and subsequently with more dilute alcohol, which removes chlorophyll, indican, wax, etc., and leaves a snow-white highly active powder. From such preparations the enzyme itself could only be imperfectly removed, for in water it is almost insoluble, very sparingly so in glycerol, and rather more readily in 10 per cent, solutions of sodium and calcium chlorides respectively. The residue which remains after extraction in this way is not perceptibly less active than before treatment. A minute study of these leaf preparations was carried out by Beyerinck in regard to their behaviour with partly purified indican solutions, and he indicates the effect of temperature on the intensity of the hydrolysis by means of curves. Among numerous points of interest it was observed that ammonia quickly destroys the enzyme, and also that emulsin slowly hydrolyses indican, although the intensity of its action was only one-twentieth of that of Indigofera enzyme preparations.
Hazewinkel (ibid., 1900, 2, 513), who also investigated the subject in 1898, arrived independently at Beyerinck's conclusions. Finding that emulsin acted on indican solutions he called the indican enzyme indimulsin, and considered that a 10 per cent, solution of sodium chloride is the best medium for dissolving it. A very interesting point which he mentions is that during fermentation no indican passes from the leaf into the surrounding liquid.
In the paper of van Romburgh (ibid., 1899, 2, 344) allusion again is made to the insoluble character of the enzyme, and to the activity of emulsin with solutions of indican. Finally, Beyerinck (ibid., 1900, 3, 101) demonstrated that the ferment present in woad, Isatis tinctoria, is not capable of hydrolysing indican, though it reacts with isatan, the peculiar indigotin yielding principle of this plant. Bergtheil (Chem. Soc. Trans., 1904, 85, 877), whose paper covers ground already traversed by Hazewinkel, Beyerinck, and van Romburgh, considers that the difficulty which occurs in extracting the enzyme is due to the presence of tannin in the leaves (cf. Brown and Morris, Chem. Soc. Trans., 1893, 63, 604). By pounding the leaves with hide powder the tannin becomes fixed, and a very active solution of the enzyme can be obtained.
Ter Meulen (Rec. trav. chim., 1905, 24, 444) is, however, in agreement with the other Dutch work referred to above, as is evident from his statement "L'enzyme de 1'indigo est insoluble dans 1'eau". According to Gaunt, Thomas, and Bloxam (loc. cit.), Bergtheil's product is not a true solution, as the enzyme is entirely removed from it by means of a Berkfeld filter. Thomas, Perkin, and Bloxam (Chem. Soc. Trans., 1909, 95, 829), again, point out that there is no certainty of the presence of tannin in the leaves of the I. sumatrana and I. arrecta, and that any tannin matter if originally present would be eliminated during the repeated extraction of the material with alcohol. As the result of their experiments the insolubility of the enzyme was confirmed.
A study of the hydrolysis of pure indican by means of the enzyme and subsequent oxidation of the indoxyl solution with air under varied conditions has been made by Thomas, Perkin, and Bloxam (loc. cit.). The fermentation was carried out in an atmosphere of purified hydrogen, and the temperature and dilution of the solution in both this and the subsequent oxidation process were so arranged as to fairly approximate the ordinary factory routine. For full details of apparatus and the analytical precautions adopted the original paper must be consulted.
The results of this investigation show that the hydrolytic action of the enzyme proceeds somewhat rapidly, and that by employing 2 grams of the enzyme and 1 gram of indican under the conditions of dilution stated, the reaction was complete after 2 hours' digestion at 50°. The solution, though free from indican, contains, however, less than the theoretical amount of indoxyl (93 per cent.). This is due to the fact that some quantity of the indoxyl (4 per cent.) is occluded by the enzyme powder, and it was found that by increasing the quantity of this latter a correspondingly greater loss occurs. The residual deficiency (3 per cent, approx.) arises from the instability of indoxyl itself, which even in an atmosphere of hydrogen at 50° is slowly converted into a product which is incapable of giving indigotin on oxidation. This property, which is referred to as the "decay" of indoxyl, is much more evident when the digestion with the ferment is prolonged for several hours, and the experiments of these authors indicate that by such a treatment for 30 hours, at least 20 per cent, of the indoxyl undergoes this transformation. On the other hand, at 15°, in an atmosphere of hydrogen, the indoxyl solution is comparatively stable, and on standing for 24 hours, experienced a loss of only 3 per cent.
According to Beyerinck (loc. cit.} great attention should be paid to the degree of the acidity of indican solutions which are undergoing fermentation, and this is corroborated by Thomas, Perkin, and Bloxam. Thus, by the presence of a trace of sulphuric acid, during the fermentation, the decay of the indoxyl is practically inhibited, and, moreover, by the addition of a further quantity of the acid at the close of the operation, the occlusion of the indoxyl by the enzyme powder is also prevented. As a result of this procedure the solution contained 99,5 per cent, of the theoretical quantity of indoxyl.
When a dilute aqueous solution of indoxyl is oxidised by air the reaction is more complex than has usually been considered the case, and a quantitative yield of indigotin is not produced. Thomas, Perkin, and Bloxam have, for instance, found that the indoxyl solutions produced by the enzyme hydrolysis of indican, when treated with air at 60°, gave only 88 per cent, of the theoretical quantity of indigolin, admixed with a little indirubin. It thus appears evident that in addition to the oxidation of indoxyl to indigotin some secondary reaction occurs, but of the chemical nature of this change there is as yet no certain evidence. The isolation from the indigo thus produced of traces of substances resembling indoxyl brown or indigo brown indicates the effect, at least in part, of a condensation similar in character to that which is involved in the production of the former product. Moreover, the nitrate from the indigo, which is prepared in this manner, was invariably of a dull yellow colour, and yielded, by extraction with ether, a small quantity of a yellowish-brown resin.
This secondary change of indoxyl is facilitated by the presence of potassium acetate in the liquid during the oxidation, for by this means the yield of indigotin was decreased to 81 per cent., and the filtrate obtained from it possessed a rich dichromate colour. It has long been known that the oxidation of indoxyl solutions, in so far as the crude fermented factory liquid is concerned, is facilitated by the presence of ammonia or lime water, and the subject has been discussed by Rawson and by Beyerinck (loc. at.}. According to Thomas, Perkin, and Bloxam, the employment of a small quantity of either of these reagents during the oxidation of the indoxyl derived from pure indican was beneficial, and an increase of about 5 per cent, in the yield of indigo thus took place. On the other hand, the addition of only a trace of these compounds is advisable, because should an excess be present the amount of indigo produced is rather decreased than increased thereby.
But whilst both ammonia and lime water in suitable amount partially inhibit the secondary change of the indoxyl referred to above, a third factor, well known to manufacturers, comes into play, which is represented by the production of notable amounts of indirubin. For the formation of this colouring matter isatin is necessary, and it is likely that, in the presence of a large amount of the above reagents, an excessive production of this substance occurs, and occasions the decreased yield of indigo which, under these circumstances, has been shown to take place. It has, in fact, been pointed out by Perkin (Chem. Soc. Proc., 1907, 23, 30) that traces of isatin exist in Java indigos, which are rich in indirubin.
Curiously enough the presence of a trace of hydrochloric acid during the oxidation acts in the same manner as ammonia, though to a less extent, in increasing the yield of colouring matter, but in this case the reaction proceeds much less rapidly. The employment of pure oxygen with neutral solutions of indoxyl gives 3—4 per cent, less colouring matter than is obtained when air alone is employed, whereas in presence of ammonia the yield is but little affected. The addition of Chile saltpetre to the fermentation vat has been a custom of Indian planters for some time, and Rawson (Report on the Cultivation and Manufacture of Indigo, 2nd ed., 1907) states that although no increase of colouring matter is thus produced in the oxidation vat, the precipitate settles better. The laboratory experiments of Thomas, Bloxam, and Perkin with pure indican corroborate this statement. Finally, there is but little difference in the yield of colouring matter experienced when the solution of indoxyl is oxidised by air at either 30 or 60°, although, if anything, the advantage is in the case of the higher temperature.
As a result, therefore, of the employment of acid during the enzyme hydrolysis of indican, and oxidation of the resulting indoxyl solution under feebly alkaline conditions, the best yields of colouring matter have been obtained. On the other hand, it has not been found possible either with synthetical indoxyl or indoxyl derived from indican to obtain a quantitative yield of pure indigotin or of an admixture of this colouring matter with indirubin. In regard to the bearing of this work on the commercial process, Thomas, Perkin, and Bloxam suggest that the effect of the addition of a small quantity of sulphuric or oxalic acid to the fermentation vat should be studied. They consider, however, that the most satisfactory laboratory results on the preparation of indigotin from the plant extract, or from pure indican, are given when the solution is hydrolysed by hydrochloric acid, with simultaneous oxidation by air. The cost of hot water extraction of the plant is, however, considered by Rawson to be prohibitive.
Bacterial Fermentation.
Though in the manufacture of indigo, hydrolysis of the indican is mainly due to the action of its specific enzyme indimulsin, it is well known that the bacteria which are present exert a similar although minor effect.
In 1887 Alvarez (Compt. rend., 115, 286) isolated from an extract of the indigo plant, an organism Bacillus indigogenus, which was capable of producing this fermentation. Beyerinck (loc. cit.) who studied the matter in considerable detail, points out that a similar effect is produced by infecting indigo plant infusions with garden soil, and that in this case the common gas-producing bacteria perform the chief part. Alvarez, he suggests, went too far in insisting on the existence of a specific bacterium in indigo fermentation. On the other hand, Bergtheil (loc. cit.) considers that at least one organism capable of producing indigo fermentation is invariably found in large quantities in an infusion of the plant. This corresponds very closely with the description given by Alvarez of his Bacillus indigogenus, and is no doubt identical with it.
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