Chapter XIII

) united with about 4 per cent of _hæmatin_, a brilliant red pigment which has the formula FeClC_{32}H_{32}O_{4}N_{4}. When treated with acids, the iron (and its accompanying Cl) is removed, and hæmatoporphyrin, C_{32}H_{36}O_{4}N_{4}, is obtained. When either hæmatin, or hæmatoporphyrin is oxidized, hæmatinic acid imide identical with that obtained from ætioporphyrin is obtained. Also, when hæmatoporphyrin is reduced, hæmopyrrole identical with that from ætioporphyrin is obtained. Thus, it would appear that the unit structural groups in hæmatin and in chlorophyll are identical; although chlorophyll may exhibit more variations in isomeric arrangement of these structural units than have been found in hæmatin. Hence, it is apparent that the only essential difference in composition between chlorophyll and hæmatin is that in the former the structural units are linked together by iron, while in the latter, the same units are united through magnesium as the linking element. Further, it is known that while iron is not a constituent element in the chlorophyll molecule, it is, in some unknown way, absolutely essential to the production of chlorophyll in plants; plants furnished with an iron-free nutrient solution rapidly become etiolated and photosynthesis stops.

The following skeleton formulas have been suggested to indicate the way in which these elements are linked between the structural units in their respective compounds.

-C C- -C C- \ / \ / N N N N / \ / \ / \ / \ -C \ / C- -C \ / C- M g F e -C / \ C- -C / | \ C- \ / \ / \ / | \ / N N N C l N / \ / \ -C C- -C C-

Chlorophyll Hæmatin

It is understood, of course, that the mineral element does not furnish the definite means of holding the structural units together as otherwise it would not be possible to remove the iron, or magnesium, without breaking down the molecule, as is done in the case of the porphyrins. The actual binding linkage is undoubtedly between carbon atoms, as indicated in Willstätter's formulas for ætiophyllin and ætioporphyrin (see page 109). The attachment of the magnesium to each one of the four nitrogen atoms in the skeleton formula assumes the existence of subsidiary valences of 2-4 for magnesium (and of 3-5 for iron), or of possible _oscillating_ valences similar to those supposed to be exhibited by carbon in its closed-ring arrangements.

PROPERTIES OF THE CHLOROPHYLLS

The phytyl esters, or natural chlorophylls, are amorphous solids; while the methylethyl esters (chlorophyllins) and the free acids (phyllins) are crystalline compounds. All of these compounds are easily soluble in ether and alcohol, but insoluble in water. The chlorophylls and chlorophyllins are practically insoluble in petroleum ether and chloroform; but the monobasic acids (pyrrophyllin and phyllophyllin) and the neutral ætiophyllin dissolve easily in chloroform.

Solutions of the chlorophylls are fluorescent, being green by transmitted, and red by reflected light.

Chlorophyll _a_ is a blue-black solid, which gives dark green solutions in all of its solvents. Chlorophyll _b_ is a dark-green solid, which yields brilliant green solutions. Solutions in ether of glaucophyllin and of cyanophyllin are blue; of rhodophyllin, deep violet; of rubiphyllin, light violet; of erythrophyllin, red; and of pyrrophyllin and phyllophyllin, bluish-red. Solutions of the porphyrins are all red, the di-basic ones being usually a bluish-red, and the simpler ones a brilliant red to deep brownish-red in color.

The several chlorophyll derivatives are further distinguished by characteristic differences in their absorption spectra. These differences have been pictured by Willstätter in his book dealing with the results of his investigations concerning the chlorophylls, and reproduced in one or two other texts which treat in detail with the physical-chemical properties of these pigments, but need not be presented in such detail here.

THE CAROTINOIDS

The characteristic brilliant green of healthy plant tissues is due to the fact that there are always associated with the dark bluish-green chlorophylls two (or more) yellow pigments. These are known as the "carotinoids." This group includes the two brilliant yellow pigments, carotin and xanthophyll, and the reddish brown fucoxanthin and the brilliant red lycopersicin, which are similar in their chemical composition. The first two are found universally distributed in plants, associated with the chlorophylls, and may be regarded as vegetative pigments, although the characteristic ornamental yellow and orange colors of many flowers and fruits, as well as that of the roots of carrots, etc., due to these pigments.

=Carotin.=--This pigment occurs in various forms in plants, both amorphous and crystalline. It crystallizes out of solution in flat plates, which are orange-red by transmitted light, and greenish-blue by reflected light, and have a melting point of 168°. Carotin is insoluble in water, only very slightly soluble in acetone or cold alcohol, readily soluble in petroleum ether, ether, chloroform, and carbon disulfide. Its solutions are strongly fluorescent.

Its molecular formula is C_{40}H_{56}. It is, therefore, a hydrocarbon of a very high degree of unsaturation. On exposure to dry air, it absorbs 34.3 per cent of its own weight of oxygen, which corresponds to 11½ atoms of oxygen, computed on the basis of the molecular formula C_{40}H_{56}, and would indicate a formula of (C_{40}H_{56})_{2}O_{23} for the oxygenated compound; this being three oxygen atoms less than would be required to bring the compound to the theoretical stage of saturation represented by the unimolecular formula C_{_n_}H_{_2n+2_}. In moist air, two more oxygen atoms are absorbed, probably forming two OH groups in the molecule. Moreover, carotin absorbs iodine. When the calculated amount of iodine is used, a definite compound having the formula C_{40}H_{56}I_{2} is produced; but in the presence of an excess of iodine another compound having the apparent formula C_{40}H_{56}I_{3} (or 2C_{40}H_{56}I_{2}+I_{2}) is obtained. (Note that 2 atoms of iodine plus 12 atoms of oxygen, or 3 of iodine plus 11 of oxygen, produce the degree of saturation required by the formula C_{_n_}H_{_2n+2_}.) It is evident from these experimental data, that a part of the unsaturated linkage in the carotin molecule is of a type which can easily be saturated by direct addition of oxygen, while the remainder may be saturated by iodine.

The reaction of carotin toward bromine is peculiar. With this element, it forms a compound having the formula C_{40}H_{36}Br_{22}, indicating the direct addition of two atoms of bromine and the substitution of twenty atoms of this element for the same number of hydrogen atoms.

The oxygenated carotins are colorless substances, while the iodide crystallizes in beautiful dark-violet prisms, having a coppery red fluorescence.

=Xanthophyll= is closely related to carotin. It has the molecular formula C_{40}H_{56}O_{2}. It absorbs 36.55 per cent of oxygen (corresponding to 13 atoms, which would indicate the formation of two OH groups in addition to the saturation required by the C_{_n_}H_{_2n+2_} formula); and an iodine addition product having the formula C_{40}H_{56}O_{2}I_{2}, which crystallizes in dark-violet needles.

Xanthophyll differs markedly from carotin in its solubilities, being insoluble in petroleum ether and only sparingly soluble in carbon disulfide. It may be fairly easily reduced to carotin. This transformation is reversible, and suggests a similarity to the change from hæmoglobin to oxyhæmoglobin, and the reverse, in the blood of animals, as a part of their respiration process.

=Separation of the Chlorophylls, Carotin, and Xanthophyll.=--These pigments, which exist together in most plant tissues, may easily be separated from each other by taking advantage of the differences in their solubilities, according to the following procedure. Grind up a small quantity of the fresh tissue (leaves of the stinging nettle furnish a conveniently large supply of each of these pigments) with fine sand in a mortar. Cover with acetone, let stand a few moments and then filter on a Büchner funnel. Pour the filtrate into a separatory funnel, add an equal volume of ether and two volumes of water. Shake up once and then allow the ether layer to separate; the pigments will be in this layer. Drain off the water-acetone layer. Now to the etherial solution, add about half its volume of a concentrated solution of potassium hydroxide in methyl alcohol. Shake well and allow to stand until the mixture becomes permanently green. Now add an equal volume of water and a little more ether, until the mixture separates sharply into two layers. The chlorophylls will now be in the lower dilute alcohol layer, and the carotinoids in the upper ether, and may be separated by draining of each layer separately. To separate the carotin from xanthophyll place the ether solution in a small open dish and evaporate to a small volume. Now add about ten volumes of petroleum spirit and an equal volume of methyl alcohol, stir up well, transfer to a separatory funnel and allow the two layers to separate. The carotin will now be in the upper layer of petroleum ether, and the xanthophyll in the lower alcohol layer; these layers may be drained off separately and the solvents evaporated in order to recover the pigments in dry form.

=Lycopersicin= (or lycopin) is a hydrocarbon pigment having the same formula as carotin. It is, however, brilliantly red in color, and crystallizes in a different form and has a different adsorption spectrum from carotin. It is the characteristic pigment of red tomatoes, and is found also in red peppers. Yellow tomatoes have only carotin as their skin-pigment, while lycopersicin is usually present in the flesh of the ripe fruits of all varieties and in the skin of red ones. It has been shown, however, that if varieties of tomatoes which are normally red when ripe, are ripened at high temperatures, 90° F. or above, their skins will be yellow instead of red when fully ripe. Hence, the occurrence of carotin, or of lycopersicin, as the skin pigment is determined in part by the varietal character (being different in different varieties when ripened at normal temperatures) and in part by the temperature at which the fruit ripens. The two pigments are, of course, isomers; but the difference in their structural arrangement is not known.

=Fucoxanthin=, C_{40}H_{54}O_{6}, is a brownish-red pigment, found in fresh brown algæ, and in some brown sea-weeds. Its formula indicates that it is an oxidized carotin. With iodine, it forms a compound having the formula C_{40}H_{54}O_{6}I_{4}. It is unlike carotin and xanthophyll in that it has basic properties, forming salts with acids, which are blue in color.

PHYCOERYTHRIN AND PHYCOPHÆIN

These are the principal pigments of red and brown seaweeds, respectively. Their most characteristic difference from the pigments of non-aquatic plants is that they are easily soluble in water, and insoluble in most organic solvents, such as alcohol, ether, etc. At first thought, this would appear to be impossible, since the plants grow in water and it would seem that their water-soluble pigments would be continuously dissolved out of the tissues. The reason why this does not occur lies in the fact that these pigments exist in the cells of the seaweeds in colloidal form (see