mother liquor, from which it was precipitated by alcohol. This gave a much larger yield, 0.15-0.20 gm. per k. of dry leaves.

The preparation from carrots was carried out by extracting the dry material with petroleum ether in a percolator and purifying as above; 5000 k. of fresh carrots (473 k. of dry material) gave 125 gm. of pure carotin.

Carotin forms quadratic or four-sided reddish-yellow plates, which exhibit the phenomenon of dichroism, being orange-red by transmitted light and greenish-blue in refracted light. Dilute solutions are yellow, conc. solutions orange-red but solutions in carbon disulfid, or in other solvents upon the addition of carbon disulfid, are red. Analyses of carefully purified products indicate the formula (CH), which, from molecular weight determinations, becomes C40H56. Earlier workers gave C5H8, C18H240,5 C26H386 and other formulae." It should be mentioned that in the precipitation with absolute alcohol, a product is obtained which contains from 1/2 to 3/3 of a molecule of alcohol; this may be removed by recrystallization from petroleum ether. With conc. sulfuric acid it gives an indigo-blue color; upon diluting this solution, green flakes precipitate. Solutions of carotin readily absorb oxygen. Willstätter found that carotin took up 34.3 percent of its weight (II atoms), forming a colorless compound. Various other values, from 21 percent to 37.8 percent, have been given. Shaken with 1/3 of its weight of iodin in ether, a di-iodid is formed, C40H5612; rosettes of dark violet prisms. However, if benzene, carbon disulfid or carbon disulfid-ether is used, and a larger amount of iodin, a tri-iodid results; dark violet leaflets, melting at 136-7°. Carotin, shaken with bromin at oo, and then allowed to stand at room temperature, forms a bromid, C40H36Br22, decomposing about 171-174°. During the process, about 20 molecules of hydrobromic acid are evolved, so that probably 2 atoms of bromin are added and 20 atoms of hydrogen substituted by bromin.

1887.

Zeise: Ann. d. Chem., 62, 380, 1847.

5 Husemann: Ibid., 117, 200, 1861.

• Arnaud: Compt. rend. acad. sc., 100, 75, 1885; Bull. soc. chim., 48, 641,

Immendorf: Landwirtschaftliche Jahrb., 18, 507, 1889.

Carotin is of interest because of its probable physiological significance. The work of Tammes and Kohl10 shows that carotin absorbs certain rays of radiant energy, which can be made use of in photosynthesis. It may also be of importance in respiration, acting in a manner comparable to the hemoglobin of the blood. Palladin11 supposes that, by the action of an oxidase, carotin is changed into xanthophyll (C40H56O2), which in turn is acted upon by a reductase, yielding carotin. In cases where large amounts of carotin occur in organs of storage, such as the roots of the carrot, it may be of value as a reserve food material. Finally, where the colors of flowers are due to its presence, it is of importance in floral biology.

Experiments by Iwanowski, 11a in which chlorophyll solutions containing various amounts of yellow pigments (carotin and xanthophyll) were subjected to the action of sunlight, show that with the increase of the relative content of yellow pigments the stability of the chlorophyll towards light also increased. While this protective action is exercised by both carotin and xanthophyll individually, a more favorable effect is obtained by a mixture of the two. This action is probably due to the absorption of the blue and, especially, the violet rays, whose chlorophyll-destroying power is very high. It is not yet established whether the oxygen absorption of these pigments plays a rôle in this process.

Mention may be made here of the recent studies of Palmer and Eckles12 on carotin and xanthophyll. They have shown that the fat of cow milk owes its natural yellow color to the presence of carotin and xanthophyll (principally carotin), which are taken up from the food and subsequently secreted in the milk fat. The same pigments are found in the body fat, blood serum, corpus luteum, and human milk. Carotin is assimilated from the food of the cow in preference to xanthophyll, partly because of its greater stability toward the digestive juices. 13 It probably forms by far the greater part of the lipochrome of the cow body, chiefly on account of its ability to form a compound with one of the proteins of the blood. Xanthophyll apparently is not capable of forming such a complex. It is much more soluble in bile than carotin11 which accounts for its appearance in the fat of the blood. While Palmer did not isolate carotin, it has been separated by Escher, 15 who obtained 0.45 gm. of pure pigment from 10,000 cow ovaries (corpus luteum). Carotin has also been isolated from brown algae.16

Lycopin. Lycopin is the coloring matter of the tomato. Earlier investigators considered this pigment identical with carotin.17 Schunck, 18 by a careful spectro-analysis of the two compounds, showed that they were quite different and gave the tomato pigment the name lycopin. The next year Montanari19 confirmed the observations of Schunck; he recognized it as a hydrocarbon and ascribed to it the formula, C52H74

Willstätter and Escher 20 found that it was isomeric with carotin, having the formula, C40H56. They used tomato conserve instead of the fresh fruit for the preparation of the pigment. The conserve was treated with 96 percent alcohol (to coagulate it), pressed, dried and extracted with carbon disulfid. The concentrated extract was precipitated with absolute alcohol and then recrystallized several times from petroleum ether and carbon disulfid. The yield was about 0.2 percent of the dry substance, i. e., 74 k. of conserve (5.6 k. of dry powder), yielded 11 gm. of pigment.

Lycopin forms light, or dark carmine-red, long, microscopic prisms or hair-like needles, which cannot be mistaken for carotin. Dilute sol. in carbon disulfid have a bluish-red color, while those of carotin have a yellowish tinge. The two pigments show the same color reactions with sulfuric and nitric acids. Lycopin differs from carotin in the following points: Lycopin absorbs oxygen more rapidly and to a greater extent than does carotin. Under the same experimental conditions (in 10 days), lycopin absorbed 30 percent of the oxygen from the air; carotin 0.25 percent. Lycopin does not give a crystalline iodin addition product, but a dark green amorphous product with indefinite iodin content. It reacts with bromin with the evolution of hydrobromic acid, but differs from carotin in that it takes up far more bromin than corresponds to the hydrobromic acid evolved; the compound formed is probably C40H44 Br26. It is very evident from these differences that the two isomers must vary considerably in structure.

Xanthophyll. The existence of a second class of yellow pigments in leaves was first mentioned by Stokes, 21 who supposed the existence of two xanthophylls. Sorby22 believed that there were three such compounds. Borodin23 divided the yellow pigments into two classes: the carotins, soluble in benzine and slightly soluble in alcohol; and the xanthophylls, slightly soluble in benzine but soluble in alcohol. His observations were confirmed by Monteviede, 24 Tschirch, 25 Tswett, 26 and Schunck.27 Other writers thought that carotin was the only yellow pigment accompanying chlorophyll. 28 The question was partially settled by the isolation and analysis of a crystalline representative of the second class of Borodin, by Willstätter and Mieg. 29 The high yield of the two pigments, carotin and xanthophyll, makes it very improbable that there are any other carotinoids (this term includes both classes of pigments) accompanying chlorophyll in the land plants. Tswett, 30 on the basis of a chromatographic adsorption analysis (the pigments in organic solvents, filtered through a column of calcium carbonate, inulin or sugar, are adsorbed in different zones; each zone is considered a chemical substance, the test being a different adsorption spectrum) distinguishes four xanthophylls, a, α', α", and β. He believes the xanthophyll of Willstätter and Mieg is an isomorphous mixture of two or three xanthophylls, with the a-form predominating. Unfortunately the method does not seem to permit of the isolation of the individual pigments in quantity large enough for chemical investigation. It is entirely possible that Tswett is right and that there is present in the chloroplast a mixture of very similar isomorphic and isomeric xanthophylls, for the separation of which we have as yet no preparative method. This is all the more plausible when we consider the slight differences between carotin and lycopin; and the similarity of xanthophyll and lutein (described below). On the other hand, these compounds are rather easily oxidized and the slight differences in the absorption spectra may be due to changes in the xanthophyll by oxidation.

Xanthophyll is found in the alcoholic extract of the leaves. Attempts to obtain it pure, in which the chlorophyll was isolated as the magnesium-free derivative, pheophytin, by treatment of the extract with oxalic acid, always gave negative results. This is probably because the xanthophyll is changed by the acid into a more easily soluble and non-crystalline substance. Better results were obtained when the mother liquor of potassium chlorophyllin was used. For example, the extract from 100 k. of nettle leaves, after removal of the potassium salt by filtration and further precipitation with a large quantity of ether, was washed free from alcohol with water, the deep yellow ether sol. evaporated to about 6 1., washed repeatedly with alcoholic potash sol. and water, dried with sodium sulfate and mixed with 2 vol. of petroleum ether. The xanthophyll was purified by extraction with 1200 cc. of boiling acetone, and precipitated with 2 vol. of methyl alcohol. Recrystallized from methyl alcohol, about 12 gm. of xanthophyll were obtained.

Willstätter and Stoll have also described methods for the preparation of xanthophyll from the mother liquors of chlorophyll and of crystalline chlorophyll. These depend upon the removal of xanthophyll, from the petroleum ether sol., with dilute methyl alcohol (80-90 percent), the carotin and chlorophyll remaining in the petroleum ether. This is also the basis of the quantitative estimation of the various plant pigments. The two yellow pigments make up from 0.1 to 0.2 percent of the dry weight of the leaf, of which