The Chemistry of Chlorophyll (With Special Reference to Foods)1

The Chemistry of Chlorophyll (with Special Reference to Foods)l

.

BY S ARONOFF

Iowa State College. Ames. Iowa CONTENTS

. ............ I1. Nomenclature . . . . . . . . . . . . . I11. The Chemistry of Chlorophyll . . . . . . IV. Extraction a n d Isolation . . . . . . . . V. Analytical Methods and Criteria of Purity . . 1. Determination . . . . . . . . . . . 2. Spectrophotometry . . . . . . . . 3. Criteria of Purity . . . . . . . . . a . Extracted Material . . . . . . I Introduction

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155 155 162 163 163 163 164 164 164 164 164 165 165 172

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VI By-products of Chlorophyll 1 Industrial Uses a Pigments and Paints b Chlorophyll and Oil Oxidation c Chlorophyll as a Deodorizer 2 Medical Applications a Therapeutic Action b Antibiotic Action c Gonadotropic Effeet d Photodynamic Aspects e The F a t e of Chlorophyll on Mammalian Ingestion References

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(1) Chromatographic Homogeniety . . . . . . . . (2) Oxygen Uptake: Allomerization a n d the Phase Test (3) Methanolysis (4) Absence of Chlorophyllides . . . . . . . . . (5) The Cleavage Test b Isolated Material . . . . . . . . . . . . . . . (1) Ratio of Heights of Absorption Bands 4 Absorption Coefficients 5. Colorimetric Analysis . . . . . . . . . . . . . . . 6 Fluorimetric Analysis

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174

. . . . . . . 175 . . . . . . . . 175 . . . . . . . . . . . . . 175 . . . . . . . . . . . . 175 . . . . . . . . . . . . . 176 . .... . . . . . . . 177 . . . . . . . . . . . 177 . . . . . . . 178 ....................... 179 This is the last of three reviews on chlorophyll. The others are: Absorption spectra of chlorophyll and related compounds. Chem. Revs . 47. 175 (1950), and Chlorophyll, Botan. Rev . 16. 525 (1950). 133

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134

S. ARONOPP

I. INTRODUCTION

It is not the purpose of this review to summarize the changes in the chlorophyll moeity in various products as the result of technological operations. These are the practical problems associated with the particular worker in food research. Rather it is an attempt to survey those aspects of chlorophyll which should make an investigation into these changes more understandable or easier. Thus, it will be apparent that those processes which release free organic acids from the cell (e.g., blanching) will result in pheophytin formation ; that alkaline oxidations will cause pclrpurins to arise ; that cooking in copper kettles may cause the substitution in the chlorophyll molecule of magnesium by copper, etc. I n other words, here we assume a general approach, from which particular, practical applications may be deduced. The importance of chlorophyll to the research worker in food chemistry arises from three sources : (1) the degradation of chlorophyll during food processing; (2) the fate of chlorophyll in biological systems; and (3) the possible utilization of chlorophyll as a raw material for industrial and pharmacological purposes. Consideration of the first point invites a brief survey of the pertinent chemistry of chlorophyll, analytical methods used in its determination, and industrial and medical aspects of possible interest to the food industry. The second problem inquires essentially whether the ingestion of chlorophyll is harmful, to which we may answer that, to the best of our knowledge, under normal circumstances i t is not. Nevertheless, we are aware that, a t times, degradation products of chlorophyll may result in pathological conditions in stock, such as that caused by photosensitization i n light-colored animals. One inquires, further, whether on the other hand chlorophyll or a solubilized derivative may be helpful nutritionally. Because of conflicting evidence, there is no clearcut evidence to support such a claim. Considering the third point above, there are consistent reports of the possible value of chlorophyll in treatment of wounds, etc., and, on the industrial side, it has long been used, although probably not to its greatest extent, as a coloring matter. Of the various chlorophylls now known to exist in nature (Aronoff 1950b), by far the most important are the chlorophylls a and B , the common green matter of all higher and most lower photosynthetic plants. It is possible that, as our technology makes more use of oceanic flora, we will be concerned more than academically with the chlorophylls c and d, the bilins, and the bacterial chlorophylls. IIowever, in this discussion we will restrict ourselves to the a and b forms.

CHLOROPHYLL

135

There is uncertainty as to the actual mode of existence of chlorophyll (a and b ) in plants; the product characterized to date is that extracted from plant tissues. Any difference must be a subtle one, as very mild methods may be used. Even so, there is no single absolute method of determining the purity of the extracted material, and under certain conditions the crude chlorophyll may be only a minor part of the yield. By suitable methods of refinentent we may obtain products which appear to undergo no further change in properties with additional manipulations. It is these products we call chlorophylls a and b, and which we use as our standards. The chemistry of these compounds is essentially complete, and there is no reaeon why the chlorophylls cannot be used with confidence as raw materials of known purity f o r a variety of purposes.

11. NOMENCLATURE Every subject with a large history of research has developed its own language. A variety of terms and structures native to chlorophyll chemistry is appended below : Porphyrin. The general class of con,iugated, cyclic, tetrapyrrole compounds, including porphines, chlor ins, azoporphines, etc. Porphine. A porphyrin in which all four nitrogens are equivalent except for differences caused by @-substitutions.

2c(2d

3c

I

A numbering system based on the c1as:sical system used by Fischer is given in formula I. Since the carbons adjacent to the nitrogens have heretofore not been numbered, it has recently been suggested (Wittenberg and Xhemin, 1950) that a revision of the nomenclature of the porphyrin ring is desirable to permit identification of the indi-

136

S. ARONOFF

vidual carbons. With the above method there appears to be no necessity to dispense with the commonly used Fischer system. Pyrrole. The four cyclic components of the porphyrin nucleus; they result from reductive degradation of porphyrins.

I!

Maleic imides. The products of oxidative degradation of porphyrins, as in 111. H

I

III

Etioporphine III. 1,3,5,8-Tetramethyl-2,4,6,7-tetraethyl porphine. Rhodoporphine. Same as etioporphine 111, except 6-carboxy-7-propionic acid. Pyrroporphine. Same as etioporphine 111, except 6-desethyl-7-propionic acid. Phylloporphine. Same as etioporphine 111, except 6-desethyl-7-propionic acid, y-methyl. Chlorin. A dihydro-porphine. I n chlorophyll terminology, this usually implies a 2-vinyl substitution in addition. Phorbin. A chlorin containing an isocylic ring connecting Cy and C6 with two additional carbons (9 and 10). Purp&n. A phorbin with an ether linkage of Cg or Cl0, e.g.,

(@,r)

IV

Chlorins, phorbins, and purpurins are often provided with suffixes denoting the number of oxygen atoms in the molecule, e.g., chlorin e6, with six atoms of oxygen. An exception is purpurin 18, whose name is derived from its “acid number,” i.e., the percentage

CHLOROPHYLL

137

of aqueous HC1 required to extract two-thirds of the pigment from ether if equal volumes of ether and aqueous HC1 are used. Meso compounds. Chlorophyll derivatives in which the 2-vinyl group has been reduced to 2-ethyl. One thus speaks of mesopheophorbide, or mesochlorin e5, etc. Chlorophyll a. Mg chelate of 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9keto-10-carbomethoxyphorbin phytyl-7-propionate.

Chlorophyll b. Corresponds to chlorophyll a, except that the 3-position is substituted by a formyl group rather than a methyl group. It is therefore 1,5,8-tetramethyl-3-formyl-4-ethyl-2-vinyl-9-l~eto-l0carbomethoxyphorbin phytyl-7-propionate. Pheophytin. Chlorophyll minus Mg. Pheophorbide. Pheophytin minus phytol. Pheoporphyrin us. Isomeric with pheophorbide, but the two labile H’s (7 and 8 ) have migrated to the vinyl, converting it to an ethyl. Phytol. An alcohol of the following structure :

138

S. ARONOFF

111. THE CHEMISTRY OF CHLOROPHYLL The chemistry of chlorophylls a and b is complete in the sense that the molecules may be synthesized in stepwise fashion from simpler molecules of well-known structure. It is not complete in two other senses: (1)we are not yet certain that the extracted substances are not in some subtle manner different from those existing in the natural state; and ( 2 ) there is some doubt as to the “fine structure” of the chlorophylli.e., hydrogen tautomerism in the free bases, and the contribution of the particular substituents to the absorption spectra. The fundamental chemical similarity of chlorophyll and heme have been recognized for almost a century. Early studies were concerned with drastic oxidative and reductive degradation products of chlorophyll and hemin. Thus, hydriodic acid reacting on chlorophyll forms a variety of pyrroles : opso- (VIII), hemo- ( I X ) , crypto- ( X ) , and phyllo-pyrroles (XI) : H

VIIl

Chromate oxidation resulted in the formation of acidic and basic imides. The acid substances were hematinic acid ( X I I ) and carbon dioxide. The basic imides were methylethylmaleic imide ( X I I I ) , citraconimide (XIV), and hemotricarboxylic imide (XV). The origin of these imides and their relation to the chlorophyll structure is indicated in the diagram for pheophorbide (XVI). The correlation of products formed by chromate oxidation with their origin is given in Table I. Table I1 summarizes the oxidation products of a variety of compounds related to chlorophyll or resulting from its partial degradation.

139

CHLOROPHYLL

'? i

H3C

iH

COOH

i

R

O H3C

xv

XIV

Carbon dioxide

acid imide

HX H C,H, 0

XVI

140

S. ARONOFB TABLEI Correlation of the Products Fornied by Chromate Oxidation and Their Orlgin Product formed

Acid substances 1. Carbon dioxide 2. Hematinic acid

Origin Primarily from methylene carbons. Obtained only from porphines, not from chlorins and rhodins; therefore arises from ring IV, not 111.

Basic substances 3. Hemotricarboxylic imide

Obtained only from chlorins a n d phorbins, n o t from porphines, therefore arises only from ring

IV. 4. Methylethylmaleic imide

Not from chlorins and phorbins of the chlorophyll b series; therefore only from ring 11, not I.

5. Citraconimide

Only from those compounds possessing a b-methylp-H, such as deuteroporphine or pliyllochlorin, therefore not from ring I.

TABLEI1 Oxidation Products of Chlorophyll and Related Compounds Acid fraction Compound Chlorins a Phyllochlorin (6-free) Phorbins a Chlorins b Phorbins b Porphines, from chlorophyll Pyrroporphine (6-free) Porphines, from hemin Deuteroporphine (2,4-free) Protoporphine

Hematinic acid 0 0 0 0 0

+ + + + +

Hemotricarboxylic acid

+ + + + +0 0

0 0 0

Basic fraction CitraC'onimide

0

+ 0

0

0 0

+0 + +

Methylethylmn!cic imide

+ + +0 0

+ + +0 0

Although chlorophyll degradations of this type have not been studied by isotopic techniques, they have for hemin (Wittenberg and Shemin, 1950 ; Muir and Neuberger, 1949). Hemin is reduced to mesoporphyrin, so that hematinic acid is obtained from rings I11 and IV, and methylethylmaleic imide from rings I and 11. Wittenberg and Shemin (1950) carried out the further degradation of the imides as follows.

141

CHLOROPHYLL

(1) Hematinic acid

NH,OH, E t O H

p methylethylmaleic

175" C.

imide +CO,

NaCIO,

( 2 ) Methylethylmaleic imide -+ methylethyltartaric imide 080,

HIO,

(3) Metliylethyltartaric h i d e

pyruvic acid

+ a-ketobutyric

acid

acid H

H H XVII

(4a) a-Ketobutyric acid (4b) Pyruvic acid

Get'

+ CO, + acetic

propionic acid

+ CO,

acid

If desired, both the propionic and the acetic acids may be degraded further for the individual atoms. The knowledge of the degradation products plus the synthesis of various porphyrins from these degradation products identical with

142

S. ARONOFF

known degradation products of chlorophyll have been the primary basis for the elucidation of chlorophyll chemistry. The reactive positions in the chlorophyll molecule are depicted above (XVIII). I n this diagram an attempt has been made to segregate the main types of reactions, and each of these reactions has been given a number (Roman numeral) which serves as a basis of classification for the succeeding discussion. Obviously not all these reactions may be of biological significance, but, should chlorophyll become a prominent raw material industrially, the degradation products may assume significance. (For a more extensive summary of similar scope, see Fischer and Stern, 1940).

I. Metal Complexing Next to the alkali metal complexes (e.g., disodium pheophytin) those of the alkaline earths [e.g., Mg-pheophytin (chlorophyll) ] are dissociated most readily. The reaction occurs easily with carboxylic acids (e.g., oxalic or acetic) and is the most common cause of discoloration of green food products due to the natural presence of plant organic acids. Neutral or alkaline solutions are required to retain this chelation during processing. The rate of removal of the Mg from chlorophyll a (in aqueous acetone) exceeds that of chlorophyll b by ninefold (see Mackinney and Joslyn, 1940). A possible explanation of this difference in rate has been ascribed to the inductive effect of the formyl group, increasing the bond strength of the Mg (Aronoff, 1950a). A variety of metals may be complexed with the porphyrin ring; an example is given below. These result in compounds of varying stability. It should be noted that it is possible to prepare doubly complexed porphyrins, e.g., Mg-Cu porphyrins. Presumably in this case the additional complex involves the carboxyl groups as well as the central portion of the ring. The monovalent elements form complexes in which there are two alkali metal ions within each porphine ring.

143

CHLOROPHPLL

Example. Substitution of Mg by Cu in chlorophyll and related compounds. Dissolve 100 mg. chlorophyll in 13 ml. chloroform. Add 2 ml. of a boiling methanolic solution of cupric acetate (ca. 30 mg.). Boil the mixture 2 min., a n d then wash out the alcohol and excess cupric acetate with water. Remove the chloroform and recrystallize the metal porphyriii from ether-petroleum ether. Esample. Preparation of the pheophytins. FOR SMALL AMOUNTS. Add dilute acid to the ethereal solution of the chlorophylls. Wash out the excess acid with water. Dry the ether solution. Concentrate a n d make up to 10% ether in petroleum ether. Chromatograph on a sugar column according t o the method for chlorophyll separation (Zscheile and Comar, 1941). The bands a r e easily observed with a n ultraviolet lamp. FOR LARGE AMOUNTS. Dissolve 12 g. chlorophyll in 2 1. diethyl ether. Cool to 0" C. Extract the pheophytins with 2 1. ether-saturated 30% hydrochloric acid in four or five portions. Remove the b component b y shaking the acid with one-fourth i t s volume of ether. Transfer the pheophytin a (as well as the pheophorbide a formed) t o fresh ether by the addition of water. Extract the pheophorbide a from the ether with 25% hydrochloric acid. For complete removal of pheophytin b, the pheophytins should again be extracted with acid a n d the pheophytin b returned t o ether.

I I . Reactions of the Ester Regions The chlorophylls are diesters. The propionic acid residue in the 7-POsition is esterified with the long-chained phytol alcohol, and the carboxyl adjacent to CIOis esterified with a methyl group. (The sequence of carbons: C9,Cl0, and the carboxyl adjacent to Clo may well have constituted a propionic acid residue in the precursors of chlorophyll during its biological formation, analogous to the two propionic acid residues in hemin.) The hydrolysis of the phytol from pheophytin results in the compound pheophorbide (XIX) .

COOH

XIX

xx

That the phytol is attached to the propionic acid .residue has been shown in two ways: (1) Conant's proof and (2) Fischer's proof. 1. Conant's Proof. Pheophorbide has a methoxy group a s well as a carboxyl group. Pyrolysis of pheophorbide results in pyropheophorbide (XX). The methoxy is lost, but one carboxyl stiII remains. This re-

144

8. ARONOFP

sidual carboxyl must therefore be the propionic acid residue and have been originally esterified with phytol. 2. Fiischer’s Proof. Ethyl chlorophyllide ( X X I ) (the product resulting from the action of the enzyme chlorophyllase on chlorophyll in the presence of ethanol) is converted by 131 to the corresponding porphyrin, ethyl pheoporphyrin a5 ( X X I I ) , which in turn is readily decarbomethoxylated by heat to ethyl phylloerythrin ( X X I I I ) . The structure of phylloerythrin has been determined by synthesis and degradation ; i t contains one acid group, the propionic acid residue. Therefore the ethyl in ethyl chlorophyllide and the phytol in chlorophyll must be esterified with the propionic acid carboxyl.

XXI

XXIII

Probably in all green leaves, although to a greater extent in some than in others, the enzyme chlorophyllase occurs, which hydrolyzes the phytol alcohol from chlorophyll. For the most recent work on this enzyme (which has not been well characterized, though it has high optimum temperature and operates in unusual solvents) the reader is referred to the work of Mackinney and Weast (1940). If the reaction is performed in such solvents as ethanol o r methanol, instead of a hydrolysis there is merely an exchange of the phytol with the solvent. The ethyl chlorophyllide resulting from such a replacement reaction is that commonly referred to as crystalline chlorophyll. Chlorophyllase will exchange phytol with such compounds as bacteriochlorophyll, the chlorophyllides, and purpurins, but not with chlorins. The presence of such an enzyme is obviously of considerable interest to food processing, since the chlorophyll is no longer a fat-soluble pigment but rather a water-soluble one. It is thus more readily lost from the food matrix into the syrup or juice. The chlorophyll esters are readily hydrolyzable in strong alkali, forming the free carboxylate ions, from which salts of the alkali or alkali earth metals are prepared. These are often termed “soluble chloro-

CHLOROPHPLL

145

phyll." The methyl ester of pheophorbide is readily made through the use of diazomethane. Example. Preparation of pheophorbide a.

Dissolve 5 g. pheophytin a in 1 1. ether and shake with 1.6 1. of 34% hydrochloric acid until the phytol is hydrolyzed (take occasional samples and test the partition coefficients of the ethereal material with water). Let stand 45 min. a f t e r hydrolysis; then dilute with 100 ml. water and extract the phytol with ether. I f pheophorbide b is present, add water uiitil the HC1 is reduced to 18% (this will require ca. 750 ml.) and extract the pheophorbide b with ether. I f pheophorbide b is absent, dilute the acid-water mixture t o llyo HC1 a n d extract exhaustively with ether. Concentrate the ether until the pheophorbide begins t o precipitate out and then set in refrigerator f o r 24 hr. Yield, 1.3 g. Rhombic plates. Decomp. point, 190" to 200" C. Acid number, 15. Positive phase test. Example. Esterification of pheophorbide a t o methyl pheophorbide a. Dissolve 3 g. pheophorbide in 300 ml. acetone. Add a n ethereal solution of diazomethane obtained from 2.3 g. nitrosomethylurethane. After 2 min. pour into 2 1. ether and destroy the excess diazomethane with 10% HCl. Extract chlorin e, which is also formed with this 10% HCl until the only aqueous-acidic extracts a r e essentially colorless. Wash the ether with ammonia and water until the washes a r e neutral. Concentrate the ether until crystallization commences.

111. Migration of t h e Labile H y d r o g e n Pair t o t h e V i n y l Group It is not difficult to reduce the vinyl group to ethyl without disturbing the rest of the molecule. The resultant compounds are given the prefix meso-. It is also, however, readiIy possible to effect the migration of the hydrogens in the 7,8-positions (of the chlorophyll molecule) to the vinyl group, thereby converting the phorbin into the corresponding porphine. to v i n y l

Ezample. Isomerization of chlorins to rneso compounds.

Dissolve the chlorin in acetic acid and a d d a little colorless HI (sp. gr., 1.96). Heat 5 t o 10 min. at 60" C. (There is generally a pronounced a n d characteristic color change at this stage.) Transfer the pigment to ether and wash the ether with dilute ammonia, then with water until the washes a r e neutral. Let the ether stand 24 hr. to reoxidize any reduced pigment. Concentrate until precipitation occurs. The chlorins a r e thereby converted into porphyrins which a r e considerably more resistant t o oxidation and thus more color-stable.

146

S. ARONOFF

I V and V. The Carbomethoxy Group The hydrolysis of the methyl group from the Clo carboxyl does not proceed per se. That is, a free dicarboxylic pheophorbide is not known, and attempts to obtain it generally result in a simultaneous decarboxylation, as shown. Analogously, phylloerythrin is obtained from the corresponding porphyrins. Phylloerythrin, which has been demonstrated frequently as a biological degradation product of chlorophyll in the intestine, may be physiologically important, as when, under some conditions, it causes photosensitization by entrance into the blood stream and deposition in the skin. VI. Chlorin a d Purpurin Pornation Chlorin e6 (XXV) is readily obtained as the free tricarboxylic acid

XXV

from pheophytin and is easily re-esterified to the triester with diazomethane. Although it is not difficult to resynthesize the isocyclic ring, all attempts to form pheophorbide with the trimethyl ester of chlorin e have resulted in decarbomethoxylation and the formation of pyropheophorbide, identical with the pyrolyzed compound from pheophorbide itself. If, however, the Ce-carboxyl is esterified with glycol (X X V I), the resulting ester can be converted almost quantitatively to methyl pheophorbide.

147

CHLOROPHYLL

The treatment of chlorophyllide or pheophorbide with methanolic diazomethane results in the formation of chlorin e trimethyl ester. This process has been termed “methanolysis” by Fischer and Stern (1940). The reaction requires the presence of a carbomethoxy group on Clo and will not proceed with allomerized chlorophyll. Since the reaction is quantitative, it i s ilie most direct chemical test for t h e purity of chlorophyll and those derivatives which lend themselves to the determination.

VII. Clo as a Focal P o i n t of Oxidation. Chlorin e6 trimethyl ester is phase-test negative, whereas pheophorbide is phase-test positive. 10-Hydroxypheophorbide is phase-test negative, and the failure of this compound and of chlorin e6 (which is in the same oxidation state as pheophorbide) to undergo the phase test is thus ascribed to the necessity for : (1)a linkage between Cg and Clo (i.e., a n isocyclic ring) and (2) the Clo being C-H. Thus, the phase test, which results in the formation of chlorine, is pictured as involving a preliminary enolization of Clo-Cg. Nevertheless, since pyropheophorbide is

Phwphorbide

Pympheophorbide

phase-test negative, the carbomethoxy group is also involved, indicating a tautomerism of the type shown. That this consideration alone is involved in the phase test is almost obviously not true, since none of the possible intermediates involves a color change consistent with the pure yellow of chlorophyll a and the See p. 163 : Oxygen Uptake: Allomerization and the Phase Test.

148

S. ARONOFF

red of chlorophyll B . A more reasonable explanation involves a compound which results in a break in the conjugation of the porphyrin ring.

XXIX

Allomerization is the primary pathway of chlorophyll deterioration ow stmding. Essentially it consists in the oxidation of chlorophyll by oxygen, but it can be effected by other oxidizing agents such as quinone. The process involves initially an oxidation of Clo to hydroxy, followed by scission of the ring and the formation of a variety of purpurins, in the end and in the main, purpurin 18 (XXX). I n commercial chlorophyll preparations the primary contaminating components are frequently purpurins and chlorins.

Chlorophyll

Hypothetical epoxide

10-Hydroxyphwphorbide

Thus, where foods containing chlorophyll have been processed in alkaline media, the pigments may contain considerable quantity and variety of purpurins and chlorins. I n themselves they are presumably not harmful or less stable than chlorophyll, though they may well result

CHLOROPHYLL

149

in off-color products. The formation of purpurins, though not chlorins, can be obviously avoided by the exclusion of oxygen and minimized by the inclusion of antioxidants. VIII. Decarboxylation of the Isocyclic Ring

A type reaction is the hydrolysis of the trimethyl ester of purpurin 7 ( X X X I ) , which results in the formation of rhodochlorin (chlorin f, X X X I I ) . Rhodochlorin can, in turn, be converted to the Ce-H porphine, pyrroporphine, by heating a t the 240" to 250" C. in diphenyl.

Two other common porphines readily formed from chlorophyll are rhodoporphine ( X X X I I I ) and phylloporphine (X X X IV ) . IX. Reduction The porphyrins, including chlorophyll, are readily reduced by hydrogen. In the case of vinyl compounds, the initial uptake results in the saturation of the vinyl group (i.e., the formation of meso compounds). Two more moles of Hz are required to convert the porphyrins to leuco compounds. The leuco compounds are only partially reoxidizable to the original substances, with porphines giving a better yield of reoxidized material than chlorins. Even so, the chlorin regeneration occurs only in neutral (acetone or dioxane) or aqueous alkaline solution and not in acid solution. I n acid solution the leuco compounds are reoxidized only to por-

150

S. ARONOFF

phines, and this may indeed serve as a good method for the preparation of porphines from corresponding chlorins. Pyrroporphine and rhodoporphine are among the few porphines which may be catalytically reduced by hydrogenation to chlorins. As mentioned earlier, vigorous reduction with HI-HOAc results in the formation of pyrroles and homologous carboxylic acids.

IV. EXTRACTION AND ISOLATION A variety of solvents may be used to extract chlorophyll, the particular choice depending upon the needs of the investigator, e.g., whether one is interested in obtaining the pure chlorophylls, or whether one desires to measure the amount of chlorophyll with varying degrees of precision, or, indeed, if one wants merely to separate the chlorophylls from the carotenoids. Where carotenoids are sought, the chlorophylls are usually removed from the extraction solvent (ether, petroleum ether, Skellysolves, etc.) by alkaline hydrolysis and transferred to an aqueous solution. Both B a ( 0 H ) z and KOH have been used for hydrolysis, Ba(OH)z yielding an insoluble barium salt, and the KOH the soluble potassium chlorophyllide (Morgal et al., 1941; Petering e t al., 1940, 1941 ; Benne, 1942 ; Wall and Killey, 1946; Wood, 1941). The salts are removed from the aqueous solution by filtration through lime or diatomaceous earth. It is, of course, possible to determine the concentration of the chlorophyllide in the aqueous solution spectrophotometrically. P b S has been used (actually lead acetate HzS) to absorb chlorophyll (Bose and Bhattacharya, 1945), and no doubt a variety of other substances can be found for this purpose. A British concern obtained a patent f o r the extraction of chlorophyll by halogenated aliphatics, such as ethylene dichloride and trichlorethylene (British Chlorophyll Go., 1939), which is then evaporated. Care must be used with chlorinated solvents since their hydrolysis yields HC1, which, of course, converts chlorophyll to pheophytin. It is an interesting fact that, although the chlorophylls are very solu-

+

CHLOROPHYLL

151

b k in ether, one cannot extract them merely by titurating a leaf with ether. It is necessary to add a small amount of ionic material, such as sodium chloride. Even so, because of the general tendency of porphyrins to adsorb on proteins, use is generally made of a n initial extraction with a solvent mutually miscible with water and ether or a hydrocarbon. Acetone or alcohol are employed most commonly. Although acetone is to be preferred (because with alcohol allomerization and isomerization proceed more rapidly) the alcohols are commonly used with certain material such as algae. It is easier to obtain quantitative extraction of the chlorophylls from algae with methanol than with acetone. The isomerization, of unknown character, of chlorophyll a to ar and b to b' is a process the extent of which can later be determined chromatographically. To prevent excessive adsorption of the chlorophyll on the matrix, the alcohol and acetone are generally diluted with 15 to 20% HzO. Additional water results in the formation of colloidal chlorophyll ; in fact the moisture content of the system must not exceed 22% by volume. I n the case of the algae it is necessary to boil the mixture a few minutes. If fresh tissues of higher plants are used, they are frequently shredded in a Waring Blendor, or ground with washed sand in a mortar and pestle. Aqueous buffered solutions (slightly alkaline) are frequently used to avoid replacement of the Mg in acidic solution. Details of a satisfactory method for the preparation of quantities of the order of 1 to 3 g. are given by Mackinney (1940a). The stability of chlorophyll is enhanced considerably by drying in a high vacuum. A sample thus prepared was recently used over a period of ten years; chromatography of the last portion showed the only decomposition products to be the pheophytins, and these were present to no more than 2%. After the extraction process, the pigments are usually transferred to petroleum ether and the water-miscible solvents washed out. [See Mackinney (1940a) and Griffith and Jeffrey (1944, 1945) for a simplified procedure.] The complete removal of such substances as alcohol and acetone are generally essential to the subsequent chromatography. Various substances have been used for quantitative chromatography, and general procedural details are to be found in the books on that subject by Zechmeister and Cholnoky (1941) and especially by Strain (1942). Recent work in the field is still concerned with the choice of adsorbents, which include the carbohydrates (e.g., sucrose, starch, and inulin), mineral oxides (e.g., MgO), and even such substances as CaHPO4. Although the sucrose method has been used successfully (Zscheile and Comar, 1941), satisfactory results have been reported by workers using the starch procedure (e.g., Bukatsch, 1942). Further-

152

S. ARONOFF

more, the use of bone meal has been reported (Mann, 1944) and ureatalc (Masood et al., 1939) has been stated as superior to sucrose. I n all cases, special attention must be paid to the composition of the solvents used for adsorption and the subsequent elution, as well as the usual preliminary treatment of the adsorbent, e.g., drying or activation. For details the original papers should be consulted. The most commonly used procedure, however, is that of Zscheile and Comar (1941), which utilizes confectioners' sugar (which contains some starch). Here again one must stress that the efficiency of separation of the chlorophylls is dependent upon (1) the completeness of removal of the miscible solvent (alcohol or acetone) from the hydrocarbon and (2) the dryness of the sugar. The developing solvent is generally 10% diethyl ether in 30" to 40" C. petroleum ether. The chlorophylls may also be separated on paper chroma tog ram^.^ The tissue is extracted with 95% acetone or alcohol (the use of 80% solvents results in the extraction of a substance which causes excessive movement of the chlorophylls in step 1) and transferred to petroleum ether (30" to 40' C.). With small amounts, e.g., as in leaf punches, the acetone solution may be spotted directly on the chromatographic sheet (Whatman No. 1). The chromatographic sheet is formed into a cylinder, stapled, and ru n ascendingly. Step I. Develop the chromatogram with petroleum ether (e.g., Skelly B). This separates the carotenes completely, and effects partial separation of xanthophylls from the chlorophylls. There should be little movement of the chlorophylls. Xtep 2. Remove the chromatogram and add isopropyl alcohol to make 0.5%. Replace the chromatogram and develop in the same direction. This step separates chlorophylls a and b and, to some extent, the xanthophylls. S t e p 3. Remove the chromatogram and form the cylinder in the other direction. Develop in 25% chloroform in petroleum ether. (The chloroform should be thoroughly washed to remove alcohol and traces of acidity.) This step results in separation of xanthophylls and increased separation of the chlorophylls. The procedure as outlined results in considerable photooxidation, which may be overcome by performing the operations in an atmosphere of nitrogen. Its use as a quantitative procedure is not yet recommended. The method is, however, a very powerful one for the separa8This procedure was developed with the cooperation of Dr. E. F. Lind of the Botany Department.

CHLOROPHYLL

153

tion of small amounts of pigment. Furthermore, by the conversion of the chlorophylls to their respective pheophytins, we have shown the multicomponent nature of what is normally termed pheophytin a. I n addition, the pheophytins are more sharply resolved than the chlorophylls and less subject to photooxidation. Details of the findings on pheophytin will be published in the near future. Although the above procedure is acceptable and adaptable to tracer methods in amounts too small to be measured colorimetrically, it seems reasonable that a method based on partition rather than adsorption should yield even better results. An industrial method for the separation of the plastid pigments extracts the pigments from dry alfalfa with hexane in two stages, concentrates the extract, and adsorbs it on activated charcoal (Shearon and Gee, 1949). The material, adsorbed on the bottom of a column, is subjected to a head of hexane until the carotene appears on top and is eluted. Isopropanol is added to the hexane, and the xanthophylls are also eluted on top. The chlorophylls, however, are reversely eluted by a warm solution of benzene-isopropanol, pumped in from the top. Various aspects of the application of chromatography to industry and its application to the preparation of chlorophyll in particular have been reported. Of particular interest in this connection is the use of various "earths" for the clarification of soybean oils, in which it is desired to remove the chlorophyll (Hinners et al., 1946). It has become apparent in this case that color determination by means of the Lovibond colorimeter is not satisfactory. Bleaching earths vary in adsorptive capacity, one measure of which is their hydrogen ion exchange capacity (Bicliford et al., 1940). Some physical properties of chlorophyll a, analytically isolated, are listed (Fischer and Stern, 1940) : Melting point, 11'7" to 120" C., waxy, blue-black, microcrystalline plates. Readily soluble in EtzO, EtOH, Me2C0, CHC13, CS2, and benzene; poorly soluble in cold methanol ; difficultly soluble in petroleum ether. Some properties of chlorophyll b are : sinters between 86" and 92" C., begins to decompose considerably at 120" to 130" C. ; readily soluble in absolute EtOH, ether ; difficultly soluble in petroleum ether and cold methanol.

154

S. ARONOFF

METHODS AND CRITERIA OF PURITY V. ANALYTICAL The analysis of any material involves at least three steps-extraction, isolation, and determination. I n isotopic analyses a fourth step is now common-degradation. For the quantitative precision now available in the second and third steps we are indebted primarily to Zscheile and co-workers (1944, 1942, 1941) and to Mackinney and co-workers (1940, 1941). A review by the former summarizes the information then extant in analytical methods for chlorophyll (Zscheile, 1941). However, this information is for practical purposes still scattered, a condition which the following discussion is intended to ameliorate. 1. Determination

Although i t is obvious that any of the properties of chlorophyll may be used for analytical purposes (e.g., Mg o r N content, optical activity, etc. ), practical considerations direct one to the easiest or most sensitive. With chlorophyll both properties are found in the color of the pigments and the usual analytical methods are colorimetric, spectrophotometric, or fluorescent. Although the last is by far the most sensitive, the first two are the most commonly used and generally sufficiently sensitive for all purposes. 2. Spectrophotometry The absorption spectra of the chlorophylls are given in Fig. 1. ( F o r a review on the absorption spectra of chlorophyll and related compounds, see Aronoff, 1950a). It is apparent that the maximum sensitivity is obtained by measurement at the wavelength corresponding to the band maxima. Because of the absorption in the red, a t wavelengths where few other substances present in plantotissue will interfere, measurements are usually made at 6600 and 6425 A for chlorophylls a and 6, respectively. The spectra are drawn on the basis of obedience to the Lambert-Beer but the effect of the fluorescent light on the I term in the law is This law states that the logarithm of the ratio of the intensity of monochromatic light incident on and transmitted by an absorbing solution is proportional t o the concentration of the substance (per unit path length) or to the path length (per unit concentration). It says, in effect, that each unit of substance within a linear array in the solution absorbs the same fraction of the incident light upon it as the preceding fraction. Mathematically the law may be stated

CHLOROPHYLL

155

neglected. This omission is, however, of no serious consequence in those concentration ranges where most measurements are made. There is considerable deviation from Beer's law (see below) when colorimeters are used, and extreme care must be exercised when performing precise work with them. Spectrophotometers, using relatively narrow spectral regions, show that Beer's law is obeyed by the pigment in the region of optical density 0.2 to 0.8. The colorimetric error, which will be discussed more fully later, is then probably due to the use of non-monochromatic light by the eolorimeter. Since i t has been shown that the chlorophylls in solution possess spectra which are completely additive (Aronoff and Mackinney, 1943), it is possible to determine the concentration of both a and b directly by application of the Lambert-Beer law ; i.e., given two substances A and B, both of which absorb independently, the resultant transmission is thus the sum of their independent absorption : log

I I

(kAcA + kBCg)d

where the symbols correspond with those used above. mixture of chlorophylls a and 0, we have

I

log"= I

Thus, for the

(kaca f kbcb)d

for any wavelength. By choosing a,ny two wavelengths, we may insert the known values of k , and kb a t those wavelengths and solve for c, and cb. Thus, by using the respective red maxima for chlorophyll a (6600 A) and chlorophyll b (6425 A), the following equations may be formulated :

where the values for k , and kb are expressed in terms of concentration or

3

log = kca I where Z,,= intensity of initial light. I = intensity of transmitted light. c = concentration of substance. d = path length in centimeters. k = a constant (per wavelength) in units dependent upon the units used for eoncentration.

156

S. ARONOFP

of grams per liter. Since the path length, d, is common to both, it may be canceled and the equations normalized.

log

I I

= 102c,

+ 4 . 5 0 ~ ~ at 6600 A

= 102ca

+ 360cb

T

1

6.26 log0

I

at 6425 A

or 12.8 log

I I

+

a t 6600 €i

+ 57.5cb

at 6425 A

= 1 3 1 0 ~ ~5 7 . 5 ~

I0 log = 16.3~a

I

The two sets of equations are of course identical, and either may be used to solve f o r c, and cb. The solution of these equations gives the concentration of chlorophylls a and b in grams per liter. Typical examples of the use of this method are given by Zscheile and Comar (1941), Comar (1942), and Griffith and Jeffrey (1944, 1945). The method can also be used to determine the degree of contamination of chlorophyll in carotene preparations. The reverse is readily accomplished by measurement in the regions in which carotenoid absorption occurs, although, unless one is aware of the nature of carotenoid, this is an arbitrary correction (see, e.g., Singh and Rao, 1940). This technique is obviously also applicable to mixtures of chlorophyll and its derivatives, e.g., pheophytin. I n pheophytin, the loss of the magnesium results in the formation of a new absorption band in the green, small but usable (see Fig. 1). If all four components are present chlorophylls a and b and pheophytins a and b ) , four equations will have I0 : to be used ( D = optical density = log -) I

D50501 = D 6 2 5 0 i= D6425i= D 6 6 0 0 i=

+

1 . 9 5 ~ 2.8b 2 . 0 ~ + 3.08b 16.3+ ~ 57.5b 102a + 4.50b

+ 13.4 pheo-a + 6.3 pheo-b + 5.4 pheo-a + 12.6 pheo-b + 5.8 pheo-a + 3.2 pheo-b + 42 pheo-a + 20 pheo-b

where each of the wavelengths corresponds to an optimal maximum for one component. I n actual practice the reliability of the results obtainable with this method are severely limited because the rapid change (with wavelength) of absorption coefficients requires the spectrophotometer to use very isolated spectral regions (e.g., ca. 10 A). Mackinney and Weast (1940)

157

CHLOROPHYLL

have used a considerably modified approach in determining pheophytin in food products. Essentially they combine the chlorophyll and the pheophytins, treating the mixture as a two-component system. Advantage is taken of the common absorption of the two pheophytins a t a pheophytin maximum of 5350 d and the considerably lower absorption

160

II I I

-Component u ---- Component

b

0

Wovelength, A

RO.1. The absorption spectra of chlorophylls a and b (from Zscheile and Comar, 1941).

of the chlorophylls a t that wavelength.

Furthermore, for any sample the total concentration of the chlorophyll and pheophytin remained constant throughout the experimental period, that is, concentration of pheophytin (C,) concentration of chlorophyll ( C , ) = constant ( C , = 1). Then D =kcCc f kpCp

+

where D = optical density. k, = “absorption coefficient’’ for chlorophylls. k, = “absorption coefficient” for the pheophytins.

158

S. ARONOFF

Since the concentration of chlorophylls

D

= k,(l

- C,)

+ pheophytins

+ kp 1,

= constant = 1,

so that

C

D--k k, - k ,

kc-kk, --k , - D - k, D - kp

A=-.

C,

D

Thus by a single measurement a t one wavelength, the relative amounts of chlorophyll and pheophytin may be determined. This procedure is quite satisfactory when a kinetic study is being performed on a single sample whose pigment concentration remains constant, or on multiple samples of identical pigment concentration. Should it be desired to obtain the actual concentration of the chlorophyll and pheophytin, nieasurement at another wavelength is necessary. For this purpose Mackinney and Weast recommend use of an intersection of the chlorophyll and pheophytin curves at 5600 A. Obviously, a t this wavelength the absorption coefficients of the two groups of pigments are identical. Since this value may be calculated for any chlorophyll a j b mixture, it is possible to set up a pair of simultaneous equations: P C

=

C P

=

C, C,

kc -

- D a t 5350 A

D - k,

k-, - D at 5600 A - kp

D

from which C, and C, may be calculated. Mackinney and Weast actually use a graphical equivalent of this calculation, and reference should be made to the original publication for details. Stern (1938) showed moist heat to be the most prominent factor in pheophytin formation in plants during processing, less being formed than, for example, in air-dried material. Dutton e t al. (1943) have shown that in unblanched, dehydrated spinach the chlorophyll is completely converted to pheophytin in 16 weeks, although a lowered water content decreases the rate (also see Maclrinney and Weast, 1940). Blanching appears actually to increase the rate. Pepkowitz (1943) noted an increased loss in carotene in cooked vegetables on the addition of chlorophyll, a result which is somewhat at odds with that of Dutton et al. (Zoc. cit.), since they report no loss of carotene after blanching, though accompanied by the aforementioned increased rate of loss of chlorophyll. Wilson (1945) notes that, during silage fermentation, plants which

CHLOROPHYLL

159

have considerable nitrate present may suffer a loss of chlorophyll and other pigments, as well as vitamins (by the nitrous acid produced by the anaerobes). More recent studies on the action of nitrous acid on chlorophyll (Sapiro, 1950) has resulted in the preparation of a t least four well-defined, including three well-crystallized, differently colored materials. These are not yet identified, except as tetrapyrroles. Two additional points should be made with regard to the absorption spectra. Evstigneev e t al. (1949) have given evidence for an effect of oxygen on the extinction coefficient and the position of the maxima in such solvents as toluene. Small amounts of ethanol, acetone, or pyridine remove these effects, however. Thus chlorophyll a has a lower coefficient and a red shift of 2 to 3 my, while b has a higher coefficient in the red. The effects are essentially reversible. Typical data on absorption values a t the red maximum for a mixture of a and b are 0.570 before evacuation, 0.418 after evacuation, and 0.479 after readmission of air. I n this same sample the fluorescence changes in arbitrary units were, in the same sequence, 54, 29, and 49. An additional problem is involved in the change of the position of the maxima and the absorption coefficients with different solvents. This point has undergone some controversy, centering about the validity of the data of Kundt, which indicated that the absorption maxima of substances are shifted to the red, the higher the refractive index of the solvent. This has come to be known as Kundt’s rule. Such variations in the red (taken from Hubert, 1935 ; Egle, 1939 ; and Maclrinney, 1938, 1940b) are given in Fig. 2. The corresponding absorption coefficients, based on the value for diethyl ether as 102, are given in Table 111. (The values have only relative meaning, as they depend on equal weights of chlorophylls a and b.) It is an obvious fact that the maxima shift with various solvents and that, roughly speaking, the lower the index of refraction, the shorter the position maximum. There appears, in contrast to the literature (see Mackinney, 1938, 1940b; and Egle, 1939), to be no basis for disputation of the relative position of the maximum in acetone. Indeed the apparently wide divergence with 1,l-dichloroethane (nD = 1.41655) may in a large part have arisen from one of the authors’ using the nD for 1,2-dichloroethane (ethylene chloride, fiD = 1.44432). I n both references mention has been made of the possible error in estimation of the center of gravity of the maximum when determining positions of skewed bands visually or photographically. This is not solved merely by the use of photoelectric measurement, since variation of slit widths can also cause similar errors. A most important possibility, as will be indicated below, could have been that of polymerization of chlorophyll with increasing concen-

160

S. ARONOFF

0

Hubert

o Egle

A Mackinney

o pyridine "4enzena

1450-

:

0 chloroform

I $

dichloroethane (1,l) O A

3

$ : $

1400-

o butonol

o propanol

I +

1.350-

$ I

o toluene

ethanol

I

I

'0

0 hexane acetone %pentone

7f

diethylether

I

I

FIQ.2. Relations between the position of the red absorption maxima of the ehlorophyils and the index of refraction of the solvent. Compiled from Hubert (1935), Egle (1939), and Maekinney (1938).

tration. This has now been shown not to occur (Aronoff, 1952). The different maxima for strong, medium, and weak solutions in Egle's data are only apparent discrepancies, due to skewness, since the concentrations (100, 50, and 20 mg. per liter) are roughly all still within the spectrophotometric Lambert-Beer law region. Nevertheless, the fact that a thin

161

CHLOROPHYLL

TMLE I11 Variation of Corrected and non-Corrected Absorption Coefficients of Chlorophyll a + b (1-1) in Various Solvents Solvent Methanol Diethyl ether Acetone Ethanol Benzene Carbon disulfide

* It

nD

1.329 1.352 1.358 1.361 1.501 1.627

k 88.3 102 104 96.6 94.0 84.4

has been shown that in Beer’s law the constant term is not ke but ];[ck

n

(na

+ 2)’ k”

90.5 102 104 96.7 73.4 63.9 ln2

+

2)2

,

where n = index of refraction (Citerne, 1947). This calculation, with the figures again made relative to 102 for ether, show little improvement compared t o k itself. (n,, = index of refraction at the yellow wavelengths of sodium.)

film of chlorophyll possesses a maximum approximately that of the living tissue (- 6800 A), f a r beyond that found in any common solvent, suggested that the above effects may have been a t least in part due to differences in the extent of polymerization. The concentration of chlorophyll in the chloroplast is of the order of 0.1 M , or 90,000 mg. per liter. I n the grana i t may be even higher. Solutions of such concentration are essentially saturated. It is therefore not remarkable that dry films of solid chlorophyll and living tissue exhibit maxima a t almost identical wavelengths. By dissolving chlorophyll in Celvacence grease a t a series of concentrations (0.1 to 0.001 M ) and spreading the grease in a thin film on a glass plate, i t was shown that there was no optically apparent polymerization of chlorophyll a t any concentration, although with increasing concentration there was a slight shift toward the red. Nevertheless, even a film of solid chlorophyll did not attain a position as f a r in the red as that of the leaf, and it seems most reasonable to conclude that in the leaf chlorophyll is combined with some substance. The usual explanation, association with proteins, is not to be discounted ; however, a true chlorophyll-protein molecule-not merely an adsorption mixture -has still to be proved. 3. Criteria of Purity

What criteria may then be used to assay the purity of chlorophyll? The purity of any compound is, of course, a problem of fundamental importance in chemical philosophy and hinges on the definition of the word ((compound.’’ It is, in this respect, doubtful whether by any one test a compound can be stated to be pure. Thus here, too, Maclrinney

162

S. ARONOFF

(1940a), as well as Zscheile and Comar (1941), suggests a variety of tests. These may be divided into types which correspond roughly to semiquantitative and quantitative or, correspondingly, to those used on extracted material and those on isolated material. a. Extracted Matem’al (i.e., Crude Chlorophyll). (1) Chromotographic Homogeneity. The number of bands arising from chromatographic adsorption on a Tswett column gives the minimum number of substances present. For example, the presence of two bands means that there are at least two substances. There may be more, but the adsorption coefficient of only one is different from the rest. The use of t w o solvents minimizes the degree of coincidence. The same criteria do not always apply in partition chromatography, where multiple spots may be obtained for a single compound (Aronoff, 1949). (2) Oxygen Uptake: Allomerization and the Phase Test. The degradation of extracted chlorophyll usually involves a n oxidation, the rate of which depends upon the amount of available water and other solvents. As mentioned earlier, the susceptible oxygen is the C10, there being a variety of purpurins formed. Chlorophyll which has been SO oxidized is termed allomerized. The extent of allomerization can be estimated semiquantitatively by determination of the residual oxygen uptake. A very rough indication of the extent of allomerization can be attained by the so-called “phase test” in which alcoholic KOH is added to a non-aqueous solution of chlorophyll. Pure chlorophyll a goes through a p u r e yellow phase, then a greenish, and finally a blue-green similar to the original. Chlorophyll b goes through a pure red which changes through red-brown and brown back to a green similar to the original. Mixtures of a and b are initially brown in the phase test even if uncontaminated by other substances. The duration of the transitory phases, especially that of chlorophyll a, may be considerably shortened in the presence of water. Without water the yellow phase may last as long as 2 min. The addition of a relatively large proportion of water, e.g., one-fourth volume, results in almost instantaneous completion of the phase test. Uptake of oxygen in alkali is given by substances other than chlorophyll a or b. It is found, for example, with chlorophyllides and with pheophorbides. The phase test is, therefore, not a specific test for pure chlorophylls a and b. Obviously, unless the procedure is made semiquantitative by measurement of oxygen uptake, it tells only whether there is an appreciable amount of residual chlorophyll. It should be noted that the phase test colors of the purified chlorophylls are “pure,” whereas with contaminants the phase test colors are “dirty.” The phase test therefore indicates merely that the isocyclic ring is intact.

163

CHLOROPHYLL

It is believed, a t present, t o pass through the following sequence of known compounds :

1O.Hydroxypheophorbide

t+

0

Purpurin 7

Chlorophyll

$Il8 N

0

Although this sequence indicates some of the intermediates, it is apparent that it is not complete. For example, the compound responsible for the pronounced color change is certainly not among the above. Indeed, from the similarity of the color to the bilins, it may well be that a n initial intermediate responsible for the color may be a C,hydroxy, which then rearranges to the 10-hydroxy. The C,-hydroxy would, of course, be expected to show considerable diminution in color intensity, as in a bilin, because of the rupture in the conjugation of the porphine ring.

164

S. ARONOFF

( 3 ) Methanolysis (see pertinent section on chemistry). The most quantitative chemical procedure which can be used a t present to evaluate the purity of chlorophyll is the conversion of chlorophyllide o r pheophorbide to chlorin e6 trimethyl ester. (4) Absence of Chlorophyllides. The enzyme chlorophyllase (Weast and Mackinney, 1940; Willstatter, 1928) exists in a wide variety of plants, if not in all. The unique properties of this enzyme, including its high temperature optimum of 70" to 80" C., necessitates the checking of all extracts for the possible presence of the chlorophyllides [i.e., the phytol-hydrolyzed chlorophyll (chlorophyllin)1. I n the presence of ethyl alcohol, the beautifully crystalline ethyl chlorophyllide is formed (the so-called " crystalline chlorophyll7') . The presence of alkyl chlorophyllide can be confirmed by its extraction (with loss of Mg) from the non-aqueous phase with 22% HC1. (5) The Cleavage Test. This test is based on the fact that the hot alkaline oxidation of chlorophylls a and b under specified conditions results in chlorin e4 and rhodin g respectively (Willstatter and Stoll, 1913) whereas the allomerized chlorophylls do not yield these compounds. The porphyrins, driven into ether by careful neutralization with acid, can be extracted from the ether with 4 and 12% HC1, respectively. The material remaining in the ether is indicative of the extent of allomerization. b. Isolated Material. (1) Ratio of Heights of Absorption Bands. This method is extremely simple, though sensitive. It is, however, difficult to estimate the nature or character of the impurities. Examples of the use of this method are provided in : ( a ) A compasison of the reds/blues of Mackinney's (1940a) and Zscheile 's (1941) purified preparations. Absorption band red a/blue a * red blblue b

* red

a/blue a

=

Mackinney 's values 0.77 0.38

Zscheile 's values 0.76 0.33

ratio of band heights of the maxima i n the red and blue of chlorophyll a.

( b ) Similarly Zscheile and Comar suggest the checking of the presence of pheophytin band heights, measuring the ratios a t 6600/5050 for the a's and a t 6425/5200 for the b's. Their values for various preparations designed to determine optional conditions varied in the a's from 23 to 52.4; in the b's from 13.9 to 18.9. 4. Absorption Coefficients

Actually the absorption coefficients are themselves indicative of the purity of the material, especially, as is not uncommon, if colorless waxes

CHLOROPHYLL

165

are present as impurities. Although the instrumental errors are in the neighborhood of l%, the largest uncertainties, especially in the red end % of the spectrum, are due to slit widths, resulting in errors of ~ 5 in the blue and ~ 7 in % the red (Mackinney, 1940a). The deviation of the average preparation is less than 3%. No corrections for fluorescence are made on any absorption spectra. 5. Colorimetric Amlysis

Colorimetric procedure differs primarily from spectrophotometric in the width of the spectral field used in the procedure. With the advent of interference filters, possessing an average transmission band of 150 A, there is, in general, little reason for the employment of a spectrophotometer for measurement of crude “chlorophyll,” unless it is suspected that there are variations in the two chlorophylls or in their ratio. It is always a source of satisfaction if any set of data can by some formula be made to fit a straight line. It is apparent that in the Lambert-Beer relationship, log

I = k h cd I

a plot of log I o / I vs. c should, at some prescribed wavelength, result in a straight line. Under such circumstances the compound is said to follow Beer’s law. I n this connection there is, remarkably enough, still no certain understanding concerning chlorophyll in the published literature. [For example, Zscheile and Comar (1941) state that their data follow Beer’s law within the region of concentrations used in the spectrophotometric data ( 3 mg. per liter to 230 mg. per liter, yet the latter figure is f a r below linearity on a curve of log Io/I vs. concentration in a colorimeter as later given by Comar (1942) for purified and commercial chlorophyll (see Fig. 3) . 5 ] Hubert (1935) also gives spectrophotometric data for a mixture of commercial chlorophylls, showing a linearity in log &/I vs. c. Because of the unique absorption of the chlorophylls in the red, it should be possible to measure plant extracts of chlorophyll E- There appears t o be a n error in Comar’s data, since according t o the published curves the absorption coefficients of the chlorophylls in t h e plant extract were considerably higher than in the purified mixture. Although it is common f o r plant pigments t o have both position and coefficients of absorption bands altered by co-pigments, no compounds of this nature have yet been identified for the chlorophylls. The published curve for purified chlorophyll seems t o be in error, since a plotting of the numerical data of the same chlorophyll in Table 1 of Gomar’s publication results in complete coincidence of the curves corresponding to the plant extract a n d the Durified chlorophyll,

166

S. ARONOFF

Chlorophyll concentration,

pwml.

+

b ) concentration and optical density FIG.3. Relation between chlorophyll (a using a colorimeter (Cenco-Sheard). Data from Comar (1942), Petering et al. (1940), and calculated.

Petering et al., commercial sample of chlorophyll. Comar, plant extract. A Comar, laboratory preparation of chlorophyll ; curve drawn from numerical data (see text). 4 Calculated optical density of a chlorophyll a / b = 2/1. 0 Optical density, at 10 pg. milliliter graphically determined. I

in the presence of carotenoids, and such measurements have been made by a number of workers, e.g., Petering e t al. (1940, 1941). A word of explanation should be given concerning the method used to determine the calculated absorption coefficient. From a. calculated curve of the ratio of chlorophylls ul.3 = 2/1, the center of gravity of the integrated area was found. This area, 6400 to 6700 A, corresponds, of course, to the region of the spectra isolated for absorption. The

167

CHLOROPHYLL

optical center of density had a n absorption value corresponding to a = 23.3 ( a is the absorption coefficient if the concentration is expressed in grams per liter). At the concentration selected (10 pg./ml.), the resulting optical density was obtained by substitution in the Lambert-Beer law. An explanation of the discrepancy should include a large allowance for slit width differences. The causes of deviation from Beer’s law when using a colorimeter have not been noted precisely in the case of chlorophyll. The obvious fact from Fig. 3 is that in some manner the absorption coefficients are noticeably diminished in high concentration. It is well known that Lambert’s law is always rigorous, but the same cannot be said for Beer’s law. Deviations have been classified as follows (Citerne, 1947) :

A. Real deviations. 1: The term that should remain constant with concentration changes is not the absorption coefficient k , but the term kn/ (nz 2)2,where n = index of refraction a t that wavelength. (Example: If n changes from 1.43 to 1.44, n / ( n z 2 ) 2 changes from 0.874 to 0.866, resulting in a change of 1%in k.) 2. Intermolecular effects : solute-solute ; solute-solvent. B. Apparent deviations. 1. Chemical effects, e.g., ionization or association. 2. Errors due to nature of light source (non-monochromaticity) .

+

+

I n chlorophyll an additional factor is involved ; i.e., the chlorophylls are never corrected for fluorescence. This error may be considerable in terms of absolute values, since the fluorescent yield may be, according to solvent and concentration, of the order of 10%. Actually, it has been shown by Watson and Livingston (1948) that the fluorescent yield is not appreciably diminished in the concentration range generally employed for observation measurements (see Fig. 4 ) . If the yield is changed in any direction with higher concentration, it is diminished by self-quenching. This should result in a relatively higher absorption coefficient. Variation in fluorescent yield cannot, therefore, explain the deviations. The data of Weiss and Weil-Malherbe (1944) were interpreted in terms of increasing dimerization with increasing concentration. There seems, however, no basis now for a belief in polymerization with increasing concentration (Aronoff, 1952). It has already been shown that there is no compound or complex formation between chlorophylls a and b which results in spectral change. Neither can results be attributed to (reversible) photochemical effects (or bleaching), since these are so small (less than 1%)as to be of no material consequence.

168

S. ARONOFF

a

ic

20.I

I

I

I

I

I

I

I

FIG.4. Fluorescent yield of chlorophyll as B function of concentration (from Watson and Livingston, 1948).

The alternative to a solute-solute complex is that of solvent-solute effects. Since the positions of the maxima vary with different solvents, it is possible that a solvent-solute interaction will cause increasing solutesolute interaction with increasing concentration. Since it has been shown that Beer’s law holds, in the case of the chlorophylls, for spectrophotometry and not for colorimetry, it is almost certain that the deviations from Beer’s law must arise from systematic error associated with the use of the latter. The most probable source of this error is the lack of monochromaticity of the light associated with variations in the absorption coefficients within the wavelength region. The lack of monochromatic light has been approached by Kortum (1937) in the following fashion. Assume that (as in the case of chlorophyll), with the concentrations employed, a solution obeys Beer’s law when monochromatic light is employed. If the particular wavelengths contributing are Ao, hl, hz, * * h,, having corresponding intensities II Iz,* In,then

-

I = I0

+ + I1

I2

.**

In =

n

X

In

0

On passage through the solution, each Iiis diminished according t i Beer’s law : I!o = ~ , O . l O - a O c ~ where

= absorption coefficient a t A,,. I . = incident intensity of light wavelength lo. 1’0= transmitted intensity of light of wavelength lo. €0

169

CHLOROPHYLL

Then the over-all extinction of the absorbing solution is n

x I,

I = Zcd Optical density = l o g y = log I 2 I,O. 10 - n,cd 0

where Z is thus the over-all extinction coefficient. If the incident light is composed of only two wavelengths, this equation can be simplified to Optical density = log

1 f n 10-"4 n

where n = a2/al.

+ - lo-+

= Zcd

Consider, then, that a chlorophyll solution is illuminated with light of wavelength 640 mp ( a = 23) and 660 mp ( a = 68). Assume, for additional simplicity, that Ixl = Ixz, so that n = 1, and that d = 1. The above equation then reduces to Optical density = log 1 o - % c

2

+ 10-a,C 2 - 10g10-680 + 10-230

where c = concentration in grams per liter. Substitution in this above equation of a variety of concentrations results in the series of values tabulated in the first column below. I n the second column are the corrected values based on a lack of deviation a t a concentration of 0.010 g. per liter. The data are plotted in Fig. 3. TABLEIV Calculated Values for Chlorophyll Absorption Values &/I. 0.005 0.010 0.020 0.030 0.040 0.050 0.070

Optical density D 0.212 0.400 0.709 0.973 1.21 1.45 1.89

0.100 0.212 0.375 0.515 0.641 0.768 1.00

It is apparent that, despite the crudeness of the approach, it is possible to obtain a theoretical curve f o r the colorimetric determination of chlorophyll which (up to ca. 0.050 g. per liter) follows the empirical curve rather well.

170

S. ARONOFF

It therefore seems reasonable to explain the apparent deviation of chlorophyll from Beer’s law when using a colorimeter as due to the nonmonochromaticity of the light. A word should be mentioned concerning the nature of the filters. It seems highly desirable f o r workers in this field to possess a suitable standard of reference for colorimetry. The primary prerequisite in colorimetric work is agreement on the spectral region employed. The use of Corning filter No. 2408, which permits the passage of light from 6400 A

Algoe, cu. mm./ml.

FIG.5 . Relation between concentration of Scenedesmus D, and optical density.

red-ward appears quite satisfactory since (1) this region begins a t the onset and includes the region of maximum absorption of the bands of both chlorophylls ; (2) these spectral characteristics of the filters are presumably highly reproducible ; and ( 3 ) the filter is reasonable in cost. The necessity of the inclusion of a n extra infrared absorbing filter is of dubious value, since most colorimeters now have some provision to prevent heat deterioration of the system and the wavelength response characteristics of the photronic cells used in most colorimeters do not require such a filter. Interference filters, however, will undoubtedly replace dye filters in the near future.

CHLOROPHYLL

171

It should be possible to determine relative amounts of chlorophylls a and b colorimetrically by the choice of suitable filters. Thus by the use of an interference filter in the region 6600 to 6800 A, which isolates the red maximum of chlorophyll a, one can determine the amount of that substance. By measurement of a and b with Corning No. 2408, the b is obtained by substraction. For the use of those workers in photosynthesis using the alga Scenedesmus D3, a curve of approximate density vs. the concentration of the algae is given in Fig. 5. This is, of course, based upon the absorption of light by the chlorophyll in the algae and provides a rapid and simple means for determination of concentration of the algae. The curve has not been verified for other algae and should not be used without discrimination, since the scattering characteristics of spherical algae of Chlorella may be different from ellipsoidal algae as Scenedesmus.

\IChlorophyll (Mg/l)

FIG.6. Relation between width of absorption band in the red and concentration of chlorophyll (constructed from data of Schurtz, 1928).

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S. ARONOFF

A linear relation between the “width” of the absorption maximum of chlorophyll and the square root of the chlorophyll concentration has been suggested by Sapozhnikov (1941). Although no data are provided, Fig. 6 was constructed from the data of Schurtz (1928), and the relationship quoted appears to be valid. It would be of interest to determine the practical lower limit of this method with the aid of a visual spectrophotometer, since the graph indicates, as Sapozhnikov suggests, a wide range applicable to very dilute as well as to concentrated solutions. 6. BVuorimetric Analysis

The fluorescence curves of purified chlorophylls a and Z, as determined by Zscheile and Harris (1943) are given in Fig. 7. It was noted by Goodwin (1947) that the fluorescent yield of chlorophyll a exceeded that of chlorophyll b approximately tenfold a t 4047 A and threefold a t

FIG.7. Fluorescence spectra of chlorophylls a aiid b (Zscheile and Harris, 1943).

CHLOROPHYLL

173

4358 A. Measurement of the fluorescence of chlorophylls a and b is, therefore, essentially a measurement of the a, but by determination of the relative fluorescence of the two wavelengths a ratio of chlorophylls can be obtained, and, by reference to a standard, absolute amounts. The fluorescent yield of chlorophyll a is believed to be about 10%. As outlined above, the chlorophylls may be well separated by paper (adsorption) chromatography. Under these conditions the amount of chlorophyll may be too small for the usual adsorption spectrophotometry, but it may be readily determinable via fluorescence. Evistigneev et al(1949) noted that the relative intensities of a solution of chlorophylls a and b a t a concentration (0.25 to 0.30 x M ) exhibiting maximum fluorescence under their conditions of measurement showed relative intensities of 40.5, 50.5, and 32.0 in 95% ethanol, acetone, and pyridine, respectively. Furthermore, at 7' C., if the fluorescence of a n anaerobic solution were taken as 100, the fluorescence of a solution with oxygen was 80.5. Livingston et al. (1949) have reported the almost complete extinction of the fluorescence of chlorophyll in completely dry, pure hydrocarbons. To evoke the fluorescence it was necessary to have polar solvent (0.01% or more of the total solution). They explained the phenomena as the result of the formation of a fluorescence complex of the polar solvent and the chlorophyll by hydrogen bonding through carbon atoms 9 and 10. This may be a n explanation for chlorophyll, but it is obvious that a

similar explanation cannot be invoked for the pure hydrocarbon porphines. The degree of chlorophyll fluorescence has been used as a measure of the rancidity of fats and oils. Coe (1941) indicated that this value is an even better indication of the rancidity of an oil than the well-known peroxide test, since the peroxide test may become a significant figure

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S. ARONOFF

while the oil is still sweet organoleptically. The quenching of chlorophyll fluorescence is presumably due to peroxides, etc., developed during rancidity. French and Lundberg (1944) do not share this view, having found no evidence of a stoichiometric quenching reaction between chlorophyll and the acceptor substances. They suggest the decrease in fluorescence to be caused by the strong absorption of the near ultraviolet radiations by the various substances, especially peroxides, in oils and the intense blue-white fluorescence of various substances in the oils. It would seem as if there should be no cause for disagreement on the basis of the method of measurement, since all that would be required would be the illumination of the sample with a wavelength that is not appreciably absorbed by the oil, e.g., the green of mercury, and the measurement of the fluorescence a t a wavelength that is also not characteristic of a few substances except the porphines, i.e., in the red, a t ca. 7000 to 7500 A.

VI. BY-PRODUCTS OF CHLOROPHYLL

I. Industrial Uses Some aspects of the chlorophyll industry, such as its preparation in the crude or partially purified form for use as a coloring matter, e.g., in foods, soap, and candles, are well known (see Aries, 1946) ; others, such as its use in therapeutics and as a possible paint pigment, are becoming more prominent. Some recent aspects will be discussed below. a. Pigments and Paints. Bryson (1945 a,b) has recently discussed the difficulties involved in the preparation of a satisfactory paint with chlorophyll, especially from the point of view of military value, i.e., camouflage. A patent for the preparation of a chlorophyll paint has been issued to Thimann (1949). Although the papers by Bryson contain inaccuracies concerning some of the physicochemical aspects of chlorophyll and its derivatives, some points are of interest, such as the claim that Cu-chlorophyll-containing paints are excellent heat reflectors, thereby keeping the surface below them cooler than that of normal paints. Considering the known strong absorption of 3 to 3.5 p of Cupheophytin (Stair and Coblentz, 1933), this seems rather difficult to believe. Other factors aside from mere absorption of infrared are involved, however, and the point is well worth investigating industrially, since Cu-pheophytin is relatively stable as a pigment. Indeed, from this point of view, the phthalocyanines should be even more valuable, being available in a range of solubilities from water soluble to oil soluble (Urban, 1941).

CHLOROPHYLL

175

b. Chlorophyll a d Oil Oxidation. Ruchkin (1939) notes that the addition of chlorophyll and its derivatives to drying oil (containing linseed oil) accelerated its rate of drying. The autoxidation of unsaturated fats by chlorophyll in the presence of lipoxidase is, of course, well known. Diemair, Ludwig, and Weiss (1943)) using the peroxide test on highly purified ethyl oleate and methyl linoleate, found chlorophyll to have a definite oxidative action. Taufel and Muller (1940) note that crude chlorophyll contains some material, possibly xanthophyll, which makes it act as a n antioxidant, whereas the pure chlorophyll is in effect an autoxidizer. Similar aspects are discussed briefly by Henk (1941). Melzer (1942a) has patented a method whereby the solubility of fatty pigments (such as chlorophyll and carotene), is increased by the addition of vegetable oils containing unsaturated acids. c. Chlorophyll as a Deodorizer. It is difficult to see how chlorophyll functions as a n “air conditioner” merely by the addition of formaldehyde to its aqueous solution (Paschal and Adams, 1944), and it would be of interest to note whether a solution containing the formaldehyde but omitting the chlorophyll would not be as effective. The implications of the photosynthetic properties of chlorophyll as being utilized in the manufactured product are misleading. Water solubilization of chlorophyll and its derivatives is proposed in two different ways. Melzer (1942a) dissolves the Cu, Zn, or Bi chlorin halide in alkali. McBain et al. (1941) disperse chlorophyll in sodium desoxychlate.

2. Medical Application There are various aspects of the relation of chlorophyll to medicine. Most prominent are its reputed therapeutic action and its photodynamic action and subsequent disorders. There are also reports on its antibiotic nature, its effect on gonadotropic hormones, etc. a. Therapeutic Action. Gruskin (1940), using various water-soluble chlorophyllin chelates of the general formula below, advocated their use in the treatment of acute and chronic suppurative conditions. It should be noted that Gruskin’s explanation of the beneficial effect of chlorophyll is, so far as we know, entirely in error. H e believes that the chlorophyll in the wound acts as in photosynthesis, decomposing the water into active hydrogen and oxygen. The presence of oxygen results in the destruction of the harmful bacteria in the wound since they are obligate anaerobes. Although it is conceivable that the chlorophyll may act in such a manner as to affect the “redox” potential of

176

S. ARONOFF

the system and thereby diminish bacterial growth, there is no known reaction in which isolated chlorophyll will form oxygen from water or any other substance. Gruskin’s therapeutic results are supported by Burgi (1942d) who, having produced skin wounds artificially, found that, of the chlorophylls, pheophytin, chlorophyllin, hemoglobin, hemin, and bilirubin which were tested, only pheophytin was unfavorable in affecting healing rate. To this may be added hematoporphine, which, together with chlorophyll and derivatives (except pheophytin), was used favorably for such conditions as corneal ulcer, ulcera cruris, torpid wounds and burns, and decubital sores (Burgi, 1942a). Similarly Barnes (1946) noted that chlorophyll effected almost twice the rate of healing abrasions caused by sterile sandpaper than did the controls. The Lakeland Foundation has actually patented (British patent, 1944) the addition of chlorophyll to an oily carrier, designating it as a cell stimulant in the treatment of infections, a view that appears to have received support in (1) the elimination of the lag period, and (2) increased rate of growth of fibroblasts in tissue culture (Smith and Sano, 1944). However, the results of Sedyrin (1945), in which food containing chlorophyll was compared with that not containing it as a function of erythropoiesis and the absolute value of the hemoglobin or even the enhanced phagocytic powers, are probably more a n indication of the beneficial effect of various components of the diet than of the chlorophyll itself. Chlorophyll does not aid in hemoglobin regeneration. Aside from the review by Gruskin (Zoc. cit.), few summaries of current work are available. A brief review is given by Lesser (1944) and by Dabrowslri (1943), but that which appeared the most promising, by Borja (1941), was unavailable to the writer. An extensive bibliography is attached to a review by Voge (1948), but the factual, non-medicinal, material contains inaccuracies. b. Antibiotic Action. The antibiotic aspect of chlorophyll is not pronounced, but the reported inhibition of the growth of H37 and avian tubercle bacilli by chlorin e6 and chlorophyllin (0.025 and 0.05%) in glycerol broth is a matter of interest (Daly, IIeller, and Schneider, 1939). This is especially true inasmuch as the copper chelate of the chlorin and the deuteroporphine free base and its copper chelate, as well as the sulfonic acid of pyrroporphine, did not inhibit their growth. Unfortunately further work in this direction does not appear to have been published; however, Smith (1944), after noting that the injection (subcutaneous or intravenous) of chlorophyll is non-toxic for man or other animals, interprets antibiotic effects as a n interference with the

CHLOROPEYLL

177

redox mechanism of the bacterial respiration. Buttitta et al. (1946), however, report a decrease in total and reduced glutathione after the injection of chlorophyll. In this regard it is interesting to note the conclusion by Daly et aZ. (Zoc. cit.) from spectroscopic measurements that Cu chlorin e6 actually unites with bacterial protein. c. Gonadotropic Effect. The sole contribution in this respect with which the writer is familiar (aside from that of porphines with adrenals and pituitary) is that of Leathem and Westphal (1940). After the presumably adsorptive effects of inert material are accounted for, the addition of chlorophyll has a slight augmentation of the effect of anterior pituitary extract, does not affect a male-urine hormone extract, and inhibits completely mare serum hormone (a function of contact time and amount of chlorophyll added). Effects were measured as the increase in ovarian weights in immature rats. d. Photodynamic Aspects. The ability of fluorescent materials to cause erythema and otherwise exert a photodynamic effect is well known (Blum, 1941). Thus, for example, Rosickey and Hatschek (1943) show that the painting of skin with benzopyrene resulted in a n acceleration of tumor formation if chlorophyll was added, followed by illumination. No increase in rate occurred in the dark. Similarly, Lewis (1945) showed that various fluorescent materials, including chlorophyll, caused abnormalities in the dividing cells of chick embryos only in the light. On the other hand, by incorporating sufficient of the chlorophyll into a salve so that it absorbed all the effective incident light and was not in direct contact with physiologically active cells, it could actually be used as a protective agent against photodynamic effects (Wunderer, 1939 ; 1941; Roffo, 1944). An analogous photodynamic hemolysis of erythrocytes is caused by chlorophyll. Reggianini (1939) states that this hemolysis, which is caused by a variety of fluorescent materials, e.g., erosin and Bengal rose (Robuschi, 1941), is a variable one, changing in intensity from animal to animal and from species to species. Robuschi notes that the addition to uranyl acetate or nitrate in excess (20 to 1002) of the photodynamic material inhibits the hemolysis. Although one is thus tempted to base an explanation upon quenching actions, this worker also points out that uranyl salts also inhibit the hemolysis due to the non-fluorescent taurocholates and saponins. The explanation must thus involve a strengthening or “tanning” of the erythrocyte membrane. An interesting aspect of photolytic action of chlorophyll is in the detoxifying action of rattlesnake venom after 15 min. exposure to ultraviolet light (though not to sunlight) for 3 hr. or tungsten light

178

S. ARONOFF

for 1% hr. (Ribeiro Guimar'es, 1942). The same detoxifying ability occurs on standing for several months. The rabbits thus injected with the detoxified venom can still produce specific immune bodies. It may well be that some lethal material has been made ineffective by a photooxidation. e. The Fate of Chlorophyll o n Mammalian Ingestion. Quin e t al. (1935) and Clare (1944) have shown that in some animals digestive degradation products of chlorophyll, especially phylloerythrin, may produce skin lesions as a result of photosensitivity (e.g., with lightcolored sheep and cattle). I n most of these cases, as in this work, the porphyrin circulates in the blood and exerts its action on those lightcolored portions of the skin which are especially tender, i.e., near the eyes, etc. I n severe cases the effect may be fatal. Numerous studies have been concerned with the fate of chlorophyll in the animal organism. These have been summarized by Fischer and Stern (loc. c i t . ) . Thus, in ruminants, it may be shown that the bile contains compounds which on oxidation give rise to phylloerythrin, as well as a small amount of pyrroporphine. It may be that the preponderance of the porphine being present as a leuco compound, i.e., a reduced compound, is of physiological significance. Photosensitization resulting from deposition of the porphines in the epidermis may be the result of the in vitro oxidation of the leuco compound. Similarly, in the excreta of the ruminants pheophorbide and phylloerythrin as well as a mixture of other phorbides were found and demonstrated, by means of a bile fistula, to have arisen from chlorophyll in the intestines rather than from bile. I n addition, small amounts of rhodoporphine carboxylic acid, pyropheophorbide, and mesopyropheophorbide were noted. Data from Rothemund e t al. (1934) on 15 lrg. of contents from the fourth stomach of the cow include: Phylloporphine Pyrroporphine Rhodoporphine Phylloerythrin Purpurin 18 Pheophorbide Pheoplkytin

4 mg. 5 mg. 3 mg. 15 mg. 7 mg. 11mg. Traces

Extracted from ether with 0.35% HCI Extracted from ether with 1.3% HCl Extracted from ether with 470 HCI Extracted from ether with 870 HCI Extracted from ether with 18% HCI Extracted from ether with 30% HCl Extracted from ether with 3570 HCI

Brugsch and Sheard (1938) have studied the decomposition of chlorophyll in the human body. Their method consisted in acid extraction of feces, transfer of the extract to ether, and fractional extraction of the ether with graded percentages of HC1. Chlorophyll was administered orally, daily, in 100-mg. amounts from the second through the

UHLOROPHYLL

179

fifth day. The maximum rate of excretion of the chlorophyll derivatives thus occurred on the fifth day. The pheophytin fraction (37% HC1 extraction of ether) was by far the largest fraction of the isolated material. Of the 400 mg. administered, approximately 125 mg. (31%) was pheophytin. A smaller fraction of about 30 mg. (25% HC1) was the phorbide fraction. A 10% HCl fraction, which might conceivably have been phylloerythrin, appeared spectroscopically to be quite different : indeed, in contrast with other animal studies no phylloerythrin was detectable. It was shown that, despite the high acidity of the stomach, some of the chlorophyll itself emerged. I n general, it was concluded that almost half of the ingested chlorophyll may be excreted. The fate of the ingested chlorophyll was not determined, but it could be shown not to be additional excretion of coproporphyrin. The possibility of the formation of leuco compounds and any increased bilinoidal pigments was not investigated. Fischer and Hendschel (1933) appear to have found phylloerythrin in human bile as well as other substances in the excreta, indicating that there was no significant difference in the degradation of chlorophyll in the human organism from that of ruminants. Finally, Baumgartel (1947) has shown that the normal intestinal bacteria Ba. ptricus verrucosus and B. coli commune can convert chlorophyll to phylloerythrin anaerobically in the presence of cystine and phosphate.

REFERENCES Aries, R. S. 1946. Chlorophyll. Chemurgic Digest 5, 100-104. Aronoff, S. 1949. Separation of the ionic species of lysine by means of partition chromatography. Science 110, 590-591. Aroiioff, S. 1950a. Absorption spectra of chlorophyll and related compounds. Chern. Revs. 47, 175-195. Aronoff, S. 1950b. Chlorophyll. Botan. Rev. 16, 525-588. Aronoff, S. 1952. Light absorption by chlorophyll a t high concentrations. Plant Physiol. 27, 413. Aronoff, S., and Mackinney, G. 1943. The photoiixidation of chlorophyll. J . A m . Chern. Soc. 65, 956-958. Barnes, T. C. 1946. Electrical measurements of the healing rate of human skin. Hahnemannian. Monthly 81, 8-10. Baumgartel, T. 1947. Degradation of chlorophyll in the human intestine. Ned. Y o m t s c h r . 1, 401-403. Benne, E. J. 1942. The determination of chlorophyll and carotene in plant tissue. J . Assoc. O f i c . Agri. Chemists 25, 573-591. Bickford, W. G., Anderson, S., and Markey, K. S.. 1940. Stability of vegetable oils. I. Spectral transmittance of soybean oils. Oil & Xoap 17, 138-143.

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Blum, H. F. 1941. Photodynamic Action and Diseases Caused by Light. Reinhold Publishing Gorp., New York. Borja, V. 1941. De quelques aspects de la therapeutique chlorophylliene. E. Le Franpois, Paris. Bose, P. K., and Bliattacharya, A. 1945. A method f o r the removal of chlorophyll from extracts of plant materials. Ann. Biochem. and Exptl. M e d . ( I n d i a ) 5, 7-10. British Chlorophyll Co. 1939. British patent 514,061; C. A. 35, 2910. Brugsch, J. T., and Sheard, C. 1938. Determination and quantitative estimation of the decomposition products of chlorophyll in the human body. J. Lab. Clzn. Mea. 24, 230-240. Bryson, H. C. 1945s. Camouflage and chlorophyll. Oil Colour Trades J . 107, 520-522. Bryson, H. C. 1945b. Heat-reflecting pigments and paints. Oil Colour Trades J. 108, 734-736. Bukatsch, F. 1942. Chromatographic determination of the content of chlorophyll in leaves. 2. ges. Naturw. 8, 79-86. Burgi, E. 1942. Wound-healing properties of porphyrins. Schweiz. med. Wochschr. 72, 239-242. Burgi, S. 1942. The wound-healing action of the chlorophyll and blood pigments. z. ges. exptz. N e d . 110, 259-272. Buttita, P. L., and Silliti, S. 1946. The action of chlorophyll on blood glutathione. Boll. soc. ital. biol. sper. 21, 203-204. Citerne, M. 1947. Chemical analysis by the measurement of light absorption. Some particular considerations. Congr. groupe avanoe. mdthod. anal. spectograph. produits m t . Paris 8, 85-102. Clare, N. T. 1944. Photosensitivity diseases in New Zealand, 111. The photosensitizing agent in facial eczema. N e w Zealand J. Sci. Technol. 25A, No. 5, 202-220 ; C. A. 38, 5284. Coe, M. R. 1941. Photochemical studies of rancidity; chlorophyll value in relation t o sutoxidation. Oil & Soap 18, 227-231. Comar, C. L. 1942. Analysis of plant extracts for chlorophyll a and b using a commercial spectrophotometer. Znd. Eng. Chem., Anal. Ed. 14, 877-879. Dabrowski, E. 1943. Value of plant pigments as medicinal agents. Deut. Apoth. Ztg. 58, 313-316; C. A . 39, 2177. Daly, S., Heller, G., and Sclineider, E. 1939. Effect of chlorophyll derivatives and related compounds on the growth of Mycobaceterium tuberculosis. Proc. Soo. Exptl. Biol. M e d . 42, 74-78. Diemair, W., Ludwig, H., and Weiss, K. 1943. Study of the anti-oxidative effect of biological material on f a t spoilage. Fette u. Seifen 50, 349-354. Dutton, H. J., Bailey, G. F., and Kohake, E. 1943. Dehydrated spinach. Changes in color and pigments during processing and storage. Znd. Eng. Chem. 35, 1173-1177. Egle, I(. 1939. Chlorophyll (111). The validity of Kundt’s rule with regard to chlorophyll spectra. Sitzber. heidelberg. Akad. Wiss. Math. naturw. Elasse I Abh. 19-30. Evstigneev, U. B., Gavrilova, V. A,, and Krasnovskii, A. A. 1949. Effect of oxygeii on the absorption spectrum a n d fluorescence of chlorophyll solutions. Doklady Akad. Nauk S.S.S.R. 66, 1133-1136.

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Fischer, H., and Hendschel, H. 1933. The biological breakdown of chlorophyll. 111. Preparation of chlorophyll derivatives from elephant and human excrement. 2. physwl. Chem. 216, 57-67. Fischer, H., and Stern, A. 1940. Chemie des Pyrrols II:2. Akademische Verlagsgesellschaft, Leipzig. French, C. S., and Lundberg, W. 0. 1944. Fluorescence of chlorophyll i n f a t in relation to rancidity. Oil 4 Soap 21, 23-27. Goodwin, R. H. 1947. Fluorometric method for estimating small amounts of chlorophyll a. Anal. Chem. 19, 789-794. Griffith, R. B., and Jeffrey, R. N. 1944. Determining chlorophyll, carotene, and xanthophyll i n plants. Ind. Eng. Chem., Anal. Ed. 16, 438-440. Griffith, R. B., a n d Jeffrey, R. N. 1945. Apparatus for the rapid determination of chlorophyll and carotene. Ind. Eng. Chem., Anal. Ed. 17, 448-451. Gruskin, B. 1940. Chlorophyll; its therapeutic place in acute and suppurative disease. Am. J . Surg. 49, 49-55. Henk, H. J. 1941. Effect of the natural catalysts on oxidative rancidity of the oils a n d fats. Seifensieder-Ztg. 68, 312-313; C. A . 35, 5731. Hinners, H. F., Mecarthy, J. J., and Boss, R. E. 1946. The evaluation of bleaching earths. The adsorptive capacity of some bleaching earths of various pH f o r chlorophyll in soybean oil. Oil 4 Soap 23, 22-25. Hubert, B. 1935. The physical state of chlorophyll in the living plastid. Rec. trav. botan. ne'erland. 32, 323-390. Kortum, G. 1937. Lichtelektrisclie Spcktrophotornetrie. Angew. Chem. 50, 193-204. Lakeland Foundation. 1944. Composition for infections. British patent 564,282. Sept. 21; C. A . 40, 3232. Leathem, J. H., and Westphal, U. 1940. Influence of chlorophyll on the activity of gonadotropic extracts tested on normal and liypophysectomized immature female rats. Endocrinology 27, 567-572. Lesser, M. A. 1944. Chlorophyll. Drug 4 Cosmetic Ind. 55, 38-39, 111-114. Lewis, M. R. 1945. The injurious effect of light on dividing cells in tissue cultures containing fluorescent subatxnces. Anat. Becord 91, 199-208. Livingston, R., Watson, W. F., and NcArdle, J. 1949. Activation of the fluorescence of chlorophyll solutions. J . Am. Chcm. SOC.71, 1543-1550. McBain, J. W., Merrill, R. C., Jr., and Vinograd, J. R. 1941. The solubilization of water-insoluble dye in dilute solutions of aqueous detergents. J . Am. Chem SOC. 63, 670-676. Mackinney, G. 1938. Applicability of Kundt 's rule to chlorophyll. Plant Physiol. 13, 427-430. Mackinney, G. 1940a. Criteria for purity of chlorophyll preparations. J . Biol. Chem. 132, 91-109. Mackinney, G. 1940b. Kundt's rule. Plant P7~ysioZ.15, 359. Mackinney, G. 1941. Absorption of light by chlorophyll solutions. J . Biol. Chem. 140, 315-322. Mackinney, G., and Joslyn, M. 1940. The conversion of chlorophyll to pheophytin. J . Am. Chcm. SOC.62, 231-232. Mackinney, G., and Weast, C. A. 1940. Color changes in green vegetables, frozenpack peas and string beans. Ind. Eng. Chem. 32, 392-395. Mann, T. B. 1944. Separation of 0-carotene, neo-0-carotene, and xantliophyll of dried grass, pasture grass and silage. Analyst 69, 34-39.

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