[Advances in Food Research] Advances in Food Research Volume 4 Volume 4 || The Chemistry of Chlorophyll (With Special Reference to Foods)

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

    BY S . ARONOFF Iowa State College. Ames. Iowa

    CONTENTS

    Page I . Introduction . . . . . . . . . . . . . . . . . . . . . 134

    I1 . Nomenclature . . . . . . . . . . . . . . . . . . . . . . 135 I11 . The Chemistry of Chlorophyll . . . . . . . . . . . . . . . . 138 IV . Extraction and Isolation . . . . . . . . . . . . . . . . . . 150

    155

    1 . Determination . . . . . . . . . . . . . . . . . . . . 155 2 . Spectrophotometry . . . . . . . . . . . . . . . . . . 155 3 . Criteria of Purity . . . . . . . . . . . . . . . . . . . 162

    a . Extracted Material . . . . . . . . . . . . . . . . 163 (1) Chromatographic Homogeniety . . . . . . . . . . 163 (2) Oxygen Uptake: Allomerization and the Phase Test . . 163 (3) Methanolysis . . . . . . . . . . . . . . . . 164 (4) Absence of Chlorophyllides . . . . . . . . . . . 164 (5) The Cleavage Test . . . . . . . . . . . . . . 164

    b . Isolated Material . . . . . . . . . . . . . . . . . 164 (1) Ratio of Heights of Absorption Bands . . . . . . . 164

    4 . Absorption Coefficients . . . . . . . . . . . . . . . . . 165 5 . Colorimetric Analysis . . . . . . . . . . . . . . . . . 165 6 . Fluorimetric Analysis . . . . . . . . . . . . . . . . . 172

    VI . By-products of Chlorophyll . . . . . . . . . . . . . . . . . 174 1 . Industrial Uses . . . . . . . . . . . . . . . . . . . 174

    a . Pigments and Paints . . . . . . . . . . . . . . . . 174 b . Chlorophyll and Oil Oxidation . . . . . . . . . . . . 175 c . Chlorophyll as a Deodorizer . . . . . . . . . . . . . 175

    2 . Medical Applications . . . . . . . . . . . . . . . . . 175 a . Therapeutic Action . . . . . . . . . . . . . . . . 175 b . Antibiotic Action . . . . . . . . . . . . . . . . . 176 c . Gonadotropic Effeet . . . . . . . . . . . . . . . . 177 d . Photodynamic Aspects . . . . . . . . . . . . . . . 177

    178

    References . . . . . . . . . . . . . . . . . . . . . . . 179 The others are: Absorption

    spectra of chlorophyll and related compounds. Chem . Revs . 47. 175 (1950), and Chlorophyll, Botan . Rev . 16. 525 (1950) .

    133

    V . Analytical Methods and Criteria of Purity . . . . . . . . . . . .

    e . The Fa te of Chlorophyll on Mammalian Ingestion . . . . . .

    This is the last of three reviews on chlorophyll .

  • 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 par- ticular 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 chem- istry arises from three sources : (1) the degradation of chlorophyll dur- ing 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 in- dustry. 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. Never- theless, we are aware that, a t times, degradation products of chloro- phyll may result in pathological conditions in stock, such as that caused by photosensitization in 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 clear- cut 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 ex- tracted 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 addi- tional 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 can- not 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 chem- istry is appended below :

    Porphyrin. The general class of con,iugated, cyclic, tetrapyrrole

    Porphine. A porphyrin in which all four nitrogens are equivalent compounds, including porphines, chlor ins, azoporphines, etc.

    except for differences caused by @-substitutions.

    3c 2c(2d 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 neces- sity 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. as in 111.

    The products of oxidative degradation of porphyrins,

    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-pro-

    pionic acid. Pyrroporphine. Same as etioporphine 111, except 6-desethyl-7-pro-

    pionic acid. Phylloporphine. Same as etioporphine 111, except 6-desethyl-7-pro-

    pionic acid, y-methyl. Chlorin. A dihydro- (@,r) -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.,

    I V

    Chlorins, phorbins, and purpurins are often provided with suf- fixes 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-9- keto-10-carbomethoxyphorbin phytyl-7-propionate.

    Chlorophyll b. Corresponds to chlorophyll a, except that the 3-posi- tion 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-l0- carbomethoxyphorbin phytyl-7-propionate.

    Pheophytin. Chlorophyll minus Mg. Pheophorbide. Pheophytin minus phytol. Pheoporphyrin us.

    Phytol.

    Isomeric with pheophorbide, but the two labile Hs (7 and 8 ) have migrated to the vinyl, converting it to an ethyl.

    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 mole- cules 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 chlorophyll- i.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 al