22 4 



NA TURE 



[July 3 1884 



THE COMPOSITION OF CHLOROPHYLL 



AMONG recent papers on chlorophyll those of Hansen, 

 ■^ assistant to Prof. Sachs, are worthy of notice. 1 



Dr. Hansen has applied the saponification method, found so 

 useful by Prof. Kuhne - in his researches on the ehromophanes, to 

 tin study of chlorophyll, and has been led to some very im- 

 portant conclusions. It may be remembered that Fremy tried 

 to show that the green chlorophyll colouring matter consists of 

 a blue and a yellow constituent. He mixed an ethereal chloro- 

 phyll solution with hydrochloric acid, when two layers formed — a 

 lower blue layer and an upper yellow ethereal layer. The blue 

 colouring matter was named by Fremy phylloryanin and the yellow 

 thvll xauthin. 



Hansen shows that this is not due to a splitting up of the 

 chlorophyll green into a blue and a yellow component, but only 

 an incomplete separation of the chlorophyll green from the 

 chlorophyll yellow, the former becoming changed to blue by the 

 hydrochloric acid, and he further shows that an ethereal solution of 

 pure chlorophyll green treated with hydrochloric acid does not 

 furnish any yellow constituent, the ethereal layer remaining colour- 

 less. Fremy himself, however, abandoned the view that 

 chlorophyll consists of two colouring matters. 



The views of Kraus are so well known that it is hardly 

 necessary to recapitulate them here, but I may be permitted to 

 recall to mind that he supposed he had decomposed chlorophyll 

 green into a blue green and a yellow component. He mixed an 

 ordinary alcohol chlorophyll solution with benzol, and obtained 

 two layers, an underlying yellow alcoholic layer and an upper 

 blue-green layer. The blue-green Kraus named eyanophyll, the 

 yellow xanthophyll? Hansen shows, however, that Kraus is 

 wrong in supposing that a decompo-ition of the green colouring 

 matter into a blue-green and a yellow has taken place, as it is 

 only an incomplete separation of the chlorophyll green from the 

 chlorophyll yellow. Kraus's eyanophyll therefore is nothing 

 more than an ordinary chlorophyll solution out of which a part 

 of the yellow colouring matter has been removed. Both Fremy and 

 Kraus were correct in assuming that a yellow and a green con- 

 stituent were present, but incorrect in supposing they existed in 

 combination ; the correct view now is that they exist side by side. 

 In other words, chlorophyll is merely a mixture of these colouring 

 matters 4 [1 think it necessary here to give Conrad's view, viz. 

 that Kraus had effected a decomposition of the chlorophyll by the 

 use of water, as Kraus used weak alcohol Conrad showed that 

 by using strong alcohol no yellow pigment could be got into 

 solution by means of benzol. Cf. Sachs' "Botany," 2nd 

 English ed. p. 760.] In the preparation of pure chlorophyll 

 Hansen used young plants of wheat at the time of 

 their growth when the fourth leaf is formed. Then the plant 

 contains only protoplasm, chlorophyll, and cellulose. The 

 spectra of the solutions were observed as in the experiments of 

 Kuhne, 5 on the pigments of vertebrate eyes, and of Kruken- 

 berg 6 on those of various animals, by means of sunlight thrown 

 into the slit by a heliostat, a large chemical spectroscope having 

 been used. 



The leaves of the plants are first boiled to remove extractive-, 

 the water poured off, and the material washed with water until 

 the wash water is quite clear. It is then quickly dried at a low 

 temperature, and afterwards extracted with alcohol. Hansen 

 states that the boiling does not alter the chlorophyll, since the 

 plant residue, after boiling, gives the same bands as the living 

 leaf. For the alcoholic extraction 96 per cent, alcohol was used, 

 and it was carried on in a dark room to avoid decomposition of 

 the chlorophyll by light. A second extraction was also carried 

 out, and the alcohol left in contact with the residue until the 

 firmer assumed a dark green colour. 



'I he united alcoholic solutionswere then concentrated and saponi- 

 fied. I lansen had previously found that he could separate out, in the 

 case of the colouring matter of blossoms, by means of saponifi- 

 cation, the yellow colouring matters from the fats in combination 

 with them, as Kuhne had previously done in the case of the 

 chromophmes and other pigments, and not only did he get the 

 pigments fat-free, but also in a crystalline state. 



' "Der Chlorophyllrarbstoff," von I>r. Adolph Hansen, Arbeiten des 

 /■/•tan. hfstituts ZH H'iinbir^ Bit. iii. Heft , : an. I Sitzniifsbericlite der 

 ph\sikal.-medicin. Gescllscka/t. \\'ur?burg, 18S3. Also, Die Farbstqffi 

 der Bliithen mid F> it I:te. I'eihaudlini^eu d, • flivsikalis ■■'i-medicinis, inn 

 CI, sellschaft .,< Wur burg N.F., Band xviii. No. 7, 1S84. 



-' Kuhne, I'ntersiteh. a. d. /iliysiclcriscbeu Institute der Uui-'. Ileidei 

 B ind i. Heft 4. 1878, and Band iv. Heft 3, 1882. 



1 " Zur Kenntniss der Oil. inililiylllarlistuite," ,s.. .... Sum-art, 1872. 



' Ibid 5 .Local. 



6 krukenberg, '' Vergleichend physiol. Studien," 1880-82. 



The saponifying was carried out as follows : — The leaf-extract 

 (alcoholic), after concentration, was treated with caustic soda solu- 

 tion in not too great amount, but the amount to be added has 

 to be determined by the quantity of chlorophyll present. As a 

 general rule Hansen used 40-50 c.c. (of a iNaHO to 5IFO 

 solution) to 2i c.c. chlorophyll solution obtained by concentra- 

 tion of 16-20 c.c. alcohol extract. When the alcoholic solution 

 boils, the caustic soda is added drop by drop, the liquid being 

 stirred. After the alcohol is driven off, water is poured in, and 

 the heating continued. After the evaporation of a great part of 

 the water, alcohol is added once more, and the saponification is 

 ended. When the alcohol has evaporated, the soap lees is 

 diluted with water and an excess of chloride of sodium added to 

 separate the soap, which precipitates in a granular form. It is 

 then shaken in a separating funnel with petroleum ether, which 

 assumes a dark yellow colour, since it removes only the yellow 

 constituent ; this extraction is repeated as long as the petroleum 

 ether is coloured. On evaporation of the latter, the yellow con- 

 stituent is left. 1 



The soap is now treated with ether, which removes various 

 impurities, and a little colouring matter, and then with ether 

 containing alcohol, which removes the green constituent from 

 the soap. 



Hansen asserts, and gives his reasons for the assertion, that 

 no change takes place in the pigments by the above treatment. 



The yellow constituent crystallises in dark yellow needles out 

 of the petroleum ether, and gives all the reactions of a lipo- 

 chrome, both as regards spectrum and chemical characters. 



The green constituent can be obtained out of the ether-alcohol 

 solution after occasional filtering and evaporation of the ether, and 

 any yellow colouring matter adhering to it can be removed with 

 petroleum ether. For the usual reactions this pigment answers 

 very well, but for further study it has to be purified from water, 

 &c, which is done by further treatment with ether-alcohol 

 solution. Finally the pigment crystallises out in spherical 

 crystals, which show a beautifully-marked cross with crossed 

 Nicols. Even a drop of the solution allowed to evaporate on a 

 microscopic slide allows the crystals to be seen, thousands of 

 small "sphaerocrystals" appearing on the evaporation of the ether. 

 Hansen shows that the idea that plants contain but a small 

 quantity of chlorophyll is erroneous, as he has obtained out of 

 450 grams dried wheat leaves as much as three to four grams 

 solid colouring matter. 



Chlorophyll green is opaque in the solid state, and appears of 

 a black-green colour, and in that state possesses no fluorescence, 

 but in solution possesses the usual red fluorescence. Its various 

 chemical characters are given at length in the original paper, 

 and it is shown that some of the changes with acids described by 

 authors are not due to their action on pure chlorophyll green, 

 but on other unknown bodies. It is free from sulphur and from 

 iron. The elementary analyses agree very closely, and calcu- 

 lated for the ash-free substance are the following: — 



1. 

 C. 67 '26 per cent. 

 H. 10-63 

 O. 16-97 ,, 

 N. 512 „ 



II. 

 67-94 per cent. 

 10-36 

 16-12 



5-55 

 99-97 



The amount of carbon is 1 per cent, too low in both cases. 



Chlorophyll yellow occurs in small quantity as compared with 

 chlorophyll green, in the proportion of about 1 to 100. Its solu- 

 tions show no fluorescence, and statements to the contrary have 

 been based upon deductions drawn from imperfect methods of 

 separation. It possesses the reactions of Krukenberg's lipo- 

 chromes? in the solid state, namely : a blue coloration with sul- 

 phuric acid, the same with nitric acid, and a green-blue with a 

 mixture of iodine in potassium iodide. It shows three bands in 

 the blue half of the spectrum, but no absorption of red, and 

 agrees in spectrum with the yellow colouring matter of etiolated 

 leaves [etiolin), which is incorrectly represented by some as pos- 

 sessing bands in the red part of the spectrum. Chlorophyll green 

 possesses four bands in the red half of the spectrum ; they agree 

 with the four bands of the ordinary chlorophyll solutions. 



With regard to Tschirsch's " pure chlorophyll," which, it may be 

 remembered, was described in (he Journal of the Chemical Society, 

 February 1S84, with the remark that the writer "reserved to" 



' Compare Kuhne, he. cit.. Band iv. Hefi 3, 188=. 



- Krukenberg, Inc. cit., "Zur Kenntniss der Verbreitung der Lipochrome 

 im Thierreiche " Zweite Reihe, 3 te Abth. 1882. 



