PERKIN: CONSTITUENTS OF NATURAL INDIQO. PART 11. 435

XLII1.-Constit uents of Ncttuiml Iw.ligo. Part 11. By ARTHUR GEORGE PERKIN.

THE first application of the term l L Tndigo-yellow ’’ to a substance existing in natural ind.igos is due to Bolley and Crinsoz (Jahresb., 1866, 573), who state tha t it is to be found in the Bengal variety, and can be isolated by sublimation, It is described as consisting of golden-yellow needles, sublimiag at 1 30°, sparingly soluble in water, but dissolved by soda lye. A reference to the existence of a yellow colouring matter in the Polygonurn tinctorium, an indigo-yielding plant, by Henry (Gmelin’rr Handbook of Chemistry, 1846, xiii, 50), ie, in- teresting, and he mention3 that this is obtained by treating the dried aqueous extract of the plant with ether.

More recently, Rawaon (J. Xoc. Chem. I d , 1899, 18, 251) observed that Java indigos contained a peculiar compound present usually to the extent of 2 to 3 per cent., but in one special sample as much as about 20 per cent, was found to exist. This substance was soluble in alkalis, with a deep yellow colour ; on heating it partially sublimed, and had the properties of an adjective dye-stuff, the shades given with a chromium mordant being similar to those given by vine-leaves or weld. It could readily be detected by adding to the ground indigo a solution of an alkaline hydroxide or ammonia, Rawson considered it quite possible that this yellow substance was derived from the Jndigofera uwecta, though it might perhaps originate from some other plant accidentally growing with the indigo or purposely added to the vats. Ultimately he ascertained that the former surmise was correct.

Bergtheil (Report of the Indigo Slation, Simiah, Calcutta, 1906) states, “ A very small quantity oF the yellow colouring matter

* A preliminary account of soine of this work h i s been given in Proc. , 1904, 20, 172 and ibid., 1906, 22, 199.

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online / Journal Homepage / Table of Contents for this issue

436 PERKIN: CONSTITUENTS OF NATURAL INDIGO. PARL' 11.

described by Rawson was present in nearly all indigos made a t Pusa,* and has been characteristic of most of the indigos made from it in Bihnr this year (p. 15). Further, there is no doubt that manu- facturing with immature plant tends to produce this yellow matter '' (P. 15).

EXPERIMENTAL.

Some years ago attempts were made to reproduce the indigo-yellow of Bolley and Crinsoz, but it was found that on subliming samples of the best Bengal indigos, containing as was considered a t the time about 75 per cent. of indigotin, no sublimate of this character was obtained, and moreover experiments have failed to detect the presence of such a substance in the indigos themselves. On submitting, however, refined Rengal indigo, stated t o contain 92 per cent. of indigotin, to sublimation with limited access of air, a small quantity of a most interesting yellow compound, C15HS02N2, was produced. A preliminary account of these experiments has been published (Proc., 1906, 22, 19S), but this, a t first suspected t o be Bolley and Crinsoz's indigo-yellow, was not identical with their product, because, not only was it insoluble in alkaline solutions, but it could be heated to 160' without appreciable loss. The appearance of Mr. Rawson's interesting communication again brought the subject to mind, and on application to him he kindly forwarded a small sample of Java indigo containing his yellow compound. This, which weighed approximately 3 grams, could not be distinguished in appearance from an ordinary commercial indigo, but gave with dilute alkalis a deep yellow extract.

To identify, i f possible, this substance, the indigo was extracted with boiling alcohol, and the resulting green coloured solution evaporated and poured into ether. The ethereal liquid was well washed with water, filtered, and the ether removed by distillation, The yellow residue was crystallised first from dilute alcohol and finally from acetic acid :

0.1018 gave 0.2350 CO, and 0.0360 H,O.

It formed glistening, yellow needles melting at 276---27'i0, and

The acetyl compound crystallised from alcohol in colourless needles

0*10S1 gave 0.2410 CO, and 0.0425 H,O.

This substance, after crystallisation from methyl alcohol, commenced

C = 62.95 ; H= 3.92. Cl,HloO, requires C = 62.93 ; H = 3-49 per cent.

dissolved in alkaline solutions with a pale yellow coloration.

melting at 181-182O :

C = 60.82 ; H = 4.36. C,,H,,O,, requires C= 60.79 ; H = 3-96 per cent.

* From I n d i y o f c ~ u arrccttc.

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online

PERKIN: CONSTITUENTS OF NATURAT, INDIGO. PART 11. 437

to melt a t about 116”, became fluid at 120°, but on further heating gradually solidified, and finally melted a t 181-1 82’. This behaviour, it has been previously pointed out (Trans,, 1902, 81, 587; ibid., 475), is characteristic of acetylkaempferol, and there could be little doubt that the colouring matter in question therefore consistad of kaempferol. An examination of its dyeing and other properties fully corroborated this view.

The amount of kaempferol present in this sample of indigo was unfortunately not determined accurately, but as much as 0.6 gram was certainly obtained froin it. I t was now interesting to examine other samples of Java indigo in this respect, and through the kindness of Prof. van Romburgh, of Utrecht, numeroils samples were obtained by purchase from Holland. The exact manner by which indigo has been manufactured in Java has been kept secret, except in so far that hot water and sulphuric acid are usually employed, but on t h e other hand it is known that three distinct methods have been or are in vogue, and may be described as the “new process with hot water,” “ the new process with cold water,” and ‘( the old process in which no chemicals are used.” An investigation of samples of the dye-stuff which had been prepared in these three different ways by extraction with alcohol or carbonate of soda solution revealed in each case the presence of kaempferol, but only in minute quantity, for the largest amount that could be isolated was approximately 0.2 per cent. It appears, therefore, tha t Rawson’s indigo was abnormal in containing such a large amount of yellow colouring matter, as in fact he indi- cates, and it is also probable that by recent improvements in manu- facture the Dutch chemists have been successful in preventing the contamination of their indigos with this impurity.

The GZucoside.-As there was some doubt as to the occurrence of kaempferol or its glucoside in the lndigofera arrecta, the plant from which this indigo is derived, it was investigated in this respect. The material employed consisted of the air-dried leaves of the Natal plant which had been grown in India, and it should be noted that there is no botanical difference between the Natal and Java varie- ties, for the Indigofera awectcc, originally native of Natal, was intro- duced into Java from there, For a liberal supply of this I am indebted to the kindness of Mr. W. P. Bloxam.

The leaves were extracted with ten times their weight of boiling water for six hours, the solution ovaporated to dryness, the residue incorporated with sand, and digested with boiling alcohol. Ths alcoholic liquid was concentrated, treated with water, and the mix- ture evaporated t o a small bulk and filtered. The filtrate, on stand- ing several days, slowly deposited crystals, which were collected, washed with chloroform, and recrystallised two or three times from

VOL. XCI. G G

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online

43s PERKIN: CONSTlTUENTS OF NATURAL INDIGO. PART 11.

water and finally from dilute alcohol. Analyses of the substance dried at 160' gave the following result :

0.1127 gave 0.2330 CO, and 0.0547 H20. C=56*14 ; H=5*39. 0.1117 ,, 0,2294 CO, ,, 0.0530 H,O. C = 56.00 ; H = 5.27.

C27H30O14 requires C = 56.06 ; H = 5.19 per cent. When air-dried, the glucoside has the formula C,7H,,0,,,3~H,0

(H,O= 9.83 per cent.), which is evolved by heating to 100' (found H,0==9.65, 9.68 per cent.), but is again absorbed by standing for some hours in a moist atmosphere.

This substance, for which the name kaempferitrin is proposed, con- sists of a glistening mass of almost colourless needles, which when heated congeal together at 190-192° and melt a t 201-203'. It is somewhat sparingly soluble in boiling water and in cold alcohol, and its solution is coloured pale yellow by alkalis. Aqueous lead acetate gives no precipitate, but with basic lead acetate a bright yellow deposit is formed. Ferric chloride produces a greenish-brown coloration.

The hydrolysis of this glucoside mas studied quantitatively, employ- i n g in each of the cases given below 200 C.C. of water and 2 C.C. of sulphuric acid. After boiling for a short time, the clear liquid com- menced to deposit pale yellow crystals, and when the digestion had continued for an hour, t he mixture was allowed t o stand over night. The product was dried at 160' :

0,6785 gave 0.3300 yellow colouring matter. Found 48.63 per cent.

The acid liquid from which this substance had been deposited was neutralised with barium carbonate, filtered, and the filtrate evaporated to a small bulk. The residue yielded an osazone, and this, after washing with chloroform and recrystallisation from dilute alcohol, mas obtained in yellow needles which melted sharply at 180-182O and had the properties of rhamnose osazone.

The insoluble product of the hydrolysis, which on examination mas found t o be quite homogeneous, melted a t 275-277", and gave, when dried at 160', C = 63.01 ; H = 3 * 6 8 per cent.

The acetyl compound, after cry stallisation froiii methyl alcohol, exhibited on heating the peculiar propertids of acetylbaempferol, and on analysis gave C = 60.77 ; H = 4.43 per cent.

On hydrolysis, therefore, kaempferitrin yields kaempferol and rhamnose, and this may be expressed by the equation :

C,,H3,0,, + 4H,O = C,,H,oO, + 2C,H,,O,. The reaction requires a yield of 49.45 per cent. of kaempferol, and

It has been previously shown that robinin (Trans., 1902, 81, 473) is

0.6125 ,, 0.3013 ,, 1 , 9 , ,? 49.19 ,,

this is in agreement with that ac tudly obtained and given above.

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online

PERKIN : CONSTITUENTS OF NATURAL INDIGO. P A W 11. 4%

a glucoside of kaempferol, and this in general appearance and melt- ing point somewhat closely resembles kaempferitrin. Again, both glucosides -lare almost devoid of tinctorial property, although the extremely feeble effects given in the usual manner with mordanted woollen cloth are practically identical in each case. On the other hand, robinin on hydrolysis gives kaempferol, two molecules of rhamnose and one of glucose (P) according to the equation :

C,,H,,O,, + 4H,O = C1,H,oO, + X&H,,O, + C6Hl2O,, and for this reaction but 37.73 per cent, of kaempferol is required. There can be no doubt, therefore, that robinin and kaempferitrin are distinct substances. The amount of kaempferitrin obtained from the leaf by the above described method was approximately 0.6 per cent. (1.5 grams pure glucoside from 250 grams of leaf). A n ex- amination, however, of the mother liquid from which i t had been deposited indicated tha t a considerable quantity still remained dissolved, for although this could not be isolated by the methods employed, its presence was evidenced by the fact that when boiled with dilute snlphuric acid kaempferol was obtained.

It was, however, possible to ascertain how much kaempferol could be produced from the leaf, and for this purpose an extract of 200 grams of the material in 2500 C.C. of water was treated with 10 C.C.

of sulphuric acid and digested a t the boiling point for one hour. On cooling overnight a claret-red coloured precipitate had separated, and this was collected, dissolved in a little alcohol and poured into a large volume of ether. The claret-red impurity being insoluble in the ether was thus removed, and the clear liquid containing the kaempferol was then washed with water and evaporated t o dryness.

The residue, after extraction with water, was dried, and then con- sisted of st yellowish-pink, crystalline mass which weighed 3.956 grams. Thus the air-dried leaf gave approximately 2 per cent. of kaempferol, and would accordingly contain about 4 per cent. of glucoside on the assumption, as is probable, tha t this consists entirely of kaempferi trin.

From the experiments of Rawson (Beport on the Cultivation and Manufactwe of Indigo , ilfuxufe?poi.e, 1904), it is probable tha t the quantity of kaempferol which the leaf is capable of yielding varies with the seatEIon of the year, for although no direct estimations 'of the yel- low colouring matter actually given by the plant appear to have been made, the amount present i n the indigo produced from i t is suggestive. Thus indigo prepared from ths young leaves contained much yellow dye, whereas in the old leaf product it was absent.

An examination was now made of the leaves OF the ordinary Indian plant, 1. Sumatrana, with the result that these were also found to

G G 2

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online

440 PERKIN: CONSTITUENTS OF NATURAI, INDIGO. PART TI.

contain a trace of a yellow colouring matter. The amount present was very minute, however, and from 2 kilos. of the leaves insufficient was isolated for its satisfactory characterisation. I t s dyeing properties were very similar to those given by kaempferol, so that it possibly consists of this substance.

This result seems t o indicate that Bolley and Crinsoz’s indigo-yellow (Zoc. cit .) cannot be identical with kaempferol. On the other hand, as Bengal indigos do not appear to give a yellow sublimate of the charac- ter they describe, it is possible that they were mistaken as to the source of the material they employed. It was ascertained that if indigo mixed with 10 per cent. of kaempferol is cautiously sublimed bet ween watch glasses, the sublimate produced contains appreciable quantities of the yellow colouring matter.

The hydrolysis of the kaempferitrin which exists in the leaf during the process of indigo manufacture may possibly be due to the presence of a specific enzyme, although at present preliminary experiments in this direction have been unsuccessful. The subject of the indigo enzyme has recently been discussed by Orchardson, Wood and Bloxam (J. Xoc. Chern. Ind., 1907, 26, 4), whose results with the air-dried leaf are in harmony with those of the Dutch chemists who describe an insoluble enzyme,rnther than with those of Bergtheil, who considers this to be soluble in water. A study of the behaviour of the insoluble preparation, and of one which had been very kindly forwarded to this laboratory by Prof. Beyerinck of Delft, both of which readily hydrolysed indican, was made a t varied temperatures on solutions of kaempferitrin with negative result.

Apart, however, from thequestion of enzyme hydrolysis, it is likely that the use of sulphuric acid when manufacturing indigo from the Java plant may result in the contamination of the dye-stuff with kaempferol. I n this case the wet indigo sludge or ‘‘ ma1 ” is boiled with a little sulphuric acid, and as a result any kaempferitrin pre- sent in the adhering water would naturally be transformed into the insoluble colouring matter and remain with the indigo. I f it is con- sidered that the fresh leaf will yield but 1 per cent. of indigotin, and that i t may also contain 1 per cent. of the kaemprerol glucoside, the presence of but 2 to 3 per cent. of the yellow colouring matter in the finished indigo cake seems quite easy of explanation in this way.

The author is indebted to the Research Fund Committee of the Chemical Society for a grant which has been in part employed to cover the expenses of this research.

CLOTHWORKERS’ RESEARCH LABORATORY, THE UNIVERSITY,

LEEDS.

Publ

ishe

d on

01

Janu

ary

1907

. Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Ir

vine

on

25/1

0/20

14 1

3:01

:26.

View Article Online