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The Coloring Matter of Lime Juice’ By F. Hardy a n d F. H. S. Warneford IMPERIAL DEPARTMENT OF AQRICULTURE, TRINIDAD, AND GOWRNXENT LABORATORY, LEEWARD ISLANDS, BRITISHWESTINDIES
URING the manu-
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with the calcium citrate. The color changes t h a t take place when lime juice is If calcium carbonate is subfacture of citric acid stored i n contact with air, or is concentrated or neutralcrystals, citric sirup, stituted for calcium hydroxized i n t h e presence of air, are due t o t h e oxidation of a ide, the color changes are and clear concentrated liqpolyhydroxyphenolic component. This coloring matter e x a c t l y reproduced, but uor from lime or lemon occurs mainly i n t h e outer yellow rind of the lime fruit there is no subsequent juice, considerable difficulty from which it is expressed during milling, becoming precipitation of coloring is generally experienced in mixed with t h e juice. It is a phlobatannin, closely rematter when the reagent is the removal of organic sembling, b u t not identical with, caffetannic acid in its added in excess. coloring mgtter. The deproperties and reactions. The lime fruit apparently conLime juice gives a conscription and approximate tains little carotinoid pigment or true anthroxanthin. spicuous color reaction with identification of the colorLime-rind phlobatannin yields a dark brown phlobatanferric chloride, the color ing matter of lime juice may nin when boiled with acids. produced being brownish Fusion with potash yields protocatechuic acid chiefly therefore lead to some modigreen. This reaction, toand hydrolysis with aqueous potash yields caffeic acid, fications in the processes a t g e t h e r with t h e color b u t t h e stability of t h e substance towards alkalies is present employed, or a t changes produced by oxileast might aid in a better characteristically high. It forms well-marked salts with dation, indicates that the appreciation of the behavior alkalies and alkaline earths. coloring matter of lime juice Precautions should be taken in t h e manufacture of of citrus juices during treatis a polyhydroxyphenolic citrus products to avoid oxidation of the coloring matter. ment in the factory. compound or compounds, Freshly expressed lime Especially should excessive liming be guarded against in resembling the tannins or t h e manufacture of calcium citrate, since t h e coloring juice is a turbid, pale greenanthoxanthins. ish yellow liquid, containing matter appears t o be particularly sensitive t o oxidation i n media of reaction’ more alkaline t h a n pH 7.3 to 7.8, 6 to 9 per cent of citric acid, Isolation giving rise to insoluble tarry products. The unoxidated and closely resembles lemon The extreme dilution a t coloring matter may, however, readily be removed from juice in composition and which the coloring matter freshly precipitated calcium citrate by thorough washing properties. If allowed to occurs in lime juice renders stand exposed to air, it very with hot water. this liquid unsuitable as a slowly darkens in color, exraw material from which hibiting various shades of yellow and brown and finally becoming almost black. The to isolate the color Drincide. An examination of the difrate a t which this color change proceeds is greatly accelerated ferent tissues of t h e lime *fruit demonstrated that the comduring the concentration of lime juice by boiling in open ponent which reacts with ferric chloride is located in the vessels. When the citric acid in lime juice is neutralized in outer yellow rind, and not in the inner white rind, nor contact with air by the addition of alkali, the liquid quickly in the juice-containing pulp of the fruit. To confirm assumes a dark sepia-brown color similar to that of concen- this, thin sections of the rind were placed in ferric chloride solution and then examined under the microscope. The trated juice. I n all probability these color changes are due to oxidation, vacuole contents of the cells bordering and surrounding the essential oil sacs became deeply stained. The almost entire as is indicated by the following facts: absence of chromoplasts in the rind cells was also noted. (1) Lime juice, the surface of which is covered by a layer of I n this respect the lime resembles the lemon and grapefruit, scum and essential oil that collects when the undefecated juice is allowed to stand, retains its pale color indefinitely. If access but differs from the orange and the mandarin, whose yellowto air is allowed through the removal of the scum, the customary red rind colors appear to be due mainly to abundant chromocolor changes immediately proceed. plasts carrying carotinoid pigments. (2) Lime juice concentrated i l z vucuo possesses a pale amber The paucity of carotinoid pigments in lime rind was color, but when air is subsequently allowed access, the color further demonstrated by the method of Willstiitter.a Five soon darkens. (3) Dark colored lime juice, concentrated juice, and neutral- grams of finely powdered air-dried lime rind were treated ized juice, when subjected to the reducing action of nascent on a Buchner filter with successive small quantities of acehydrogen, or of sulfurous acid, assumes a paler brown or yellow tone, without warming. The yellow solution was then run tint. into twice its volume of petrol ether contained in a separating The exact behavior of boiling lime juice on the addition funnel and an equal volume of water added. Any carotiof milk of lime, as a major operation in the manufacture of noid pigments present would be expected to pass into the citric acid, is noteworthy. The first visible result is the petrol ether. There was only slight coloration in the petrol precipitation of calcium citrate. This settles as a white layer, and this was largely removed by repeated washings deposit from a pale yellow mother liquor: When the point with water. The acetone-water runnings gave a marked of neutrality is passed, the color of the mother liquor rapidly positive reaction with ferric chloride, and therefore conchanges to deep brown. I n this respect, therefore, the tained most of the coloring matter of the rind. coloring matter serves as an indicator of alkalinity. Further When the rind of the lime is punctured in the Bcuelleprocaddition of calcium hydroxide causes the brown color even- ess for obtaining essential oil of limes, the phenol-containing tually to give place to a full yellow, with a concomitant cell sap escapes together with the oil, and may be removed deposition of yellow flecks of pigments, which become mixed Cambridge 2 Onslow, “Practical Plant Biochemistry,” 1923, p. 29. 1 Received
June 3, 1924.
University Press.
January, 1925
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therefrom by washing in a separating funnel. The oil itself contains no component giving a color reaction with ferric chloride. The reason why commercial lime juice contains phenolic coloring matter is therefore that, on pressing the fruits between the rollers of a mill, the cells of the rind are ruptured and their contents become mixed with the pulp juice. Such contamination might be lessened by previously peeling and washing the fruits before milling. I n devising a method for isolating the coloring matter of outer yellow lime rind, it is evident that water is not a suitable extracting agent, because it dissolves from the rind large quantities of pectin and pentosan which are difficult subsequently to remove. This difficulty is overcome lvhen alcohol is employed as extractor. The method finally adopted was as follows: A quantity of rind weighing about 2000 grams was separated from about two hundred and fifty fresh limes. I t was rapidly minced, squeezed in a tincture press, and extracted with 96 per cent alcohol in a large Soxhlet apparatus. During this process, and during the subsequent evaporation of the alcoholic extract under diminished pressure, a stream of sulfur dioxide was slowly passed through the apparatus to prevent oxidation. The concentrated extract was next dissolved ia water and lead nitrate added. This threw down a sticky white precipitate containing lead sulfate, phosphate, lead salts of organic acids, and possibly pectin, pentosan, and protein. The precipitate was removed by filtration, and basic lead acetate added to the filtrate. The phenolic coloring matter was thereby precipitated as a bright yellou7 lake. It was thoroughly washed with cold water by decantation, and decomposed by carefully adding sulfuric acid. After filtering from lead sulfate, the yellow solution was neutralized with barium hydroxide, filtered from barium sulfate, and concentrated by evaporation under diminished pressure in the presence of sulfur dioxide. The sirupy concentrate was dried in a desiccator over sulfuric acid. The anhydrous substance was a greenish yellow-brown vitreous solid with astringent acid taste, extremely hygroscopic, readily soluble in water, alcohol, acetone, pyridine, and acetic acid, slightly soluble in ethyl acetate, but insoluble in ether, benzene, petroleum, carbon disulfide, and chloroform. It showed signs of melting, accompanied by decomposition, when heated to 200” C. Reactions
The final product became sticky and rapidly darkened in color on exposure to air. If oxidation were allowed to proceed for some time, especially a t temperatures approaching 100’ C., the resulting substance was a deep brown resinous solid, insoluble in water, but readily soluble in dilute alkalies, alcohol, and pyridine. It thus resembles the phlobaphenes which are produced when certain tannins undergo oxidation or hydrolysis. A considerable amount of phlobaphene was also deposited as a brown, almost black, friable solid when some of the coloring matter \vas boiled for a long time with sulfuric acid. The remaining acid solution possessed a deep red color and a slight blue fluorescence. On cooling, it deposited crystals of calcium sulfate. Extraction with ether yielded a bright yellow liquid which, when evaporated, left a small amount of a dark brown tarry substance. This gave none of the positive reactions of flavone or flavonol compounds, but behaved towards reagents similarly to caffeic acid. The presence of a reducing sugar, possibly glucose, was demonstrated by decolorizing the acid solution with basic lead acetate, followed by animal charcoal, filtering, and then warming a little of the filtrate with Fehling’s solution. An osazone was also prepared from the solution. It is not concluded from this, however, that lime-rind coloring matter
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is a glucoside, since it is notably difficult to remove the last traces of reducing sugars from such preparations. Final positive evidence is afforded only when a suspected glucoside is found to contain sugar in definite proportions, and is demonstrably crystalline. A dilute neutral aqueous solution of the original lime-rind coloring matter gave an intense brownish green coloration with ferric chloride, which was changed to purplish brown and then to deep red on adding sodium bicarbonate, and to red-brown on adding sodium acetate. Iron alum gave a similar color reaction to ferric chloride. Ferrous sulfate gave a deep red. Bromine water yielded a yellowish precipitate which increased in bulk on standing. Formaldehyde in the presence of hydrochloric acid (Stiasny’s reagent), on warming, yielded a slight flocculent precipitate. These facts indicate that the coloring matter of the rind of the lime is a plilobatannin (catechol tannin).3 The following reactions of the substance aid in its further characterization. It did not precipitate gelatin or albumin from their solutions, but precipitated quinine sulfate. It was partly adsorbed by casein, and by a collodion membrane. The coloring matter, fixed in collodion, and stained with ferric chloride, was not decolorized by hydrochloric acid, resembling in this respect the behavior of catechol tannins in the goldbeater’s skin test described by Price.4 An aqueous solution of the phlobatannin gave no pink coloration with dilute ammoniacal potassium ferricyanide (Allen’s reagent), nor a precipitate with ammonium molybdate. It was not precipitated from solution by potassium bichromate, although the color of the mixture darkened considerably. It rapidly changed the color of dilute alkaline potassium permanganate to green without warming, thus proving its ready oxidizability. On treatment with sodium amalgam, a solution of the phlobatannin was rapidly decolorized. Limewater gave an orange-yellow coloration, which darkened somewhat on standing. The addition of an aqueous suspension of calcium hydroxide brought about a deposition of coloring matter on the solid particles, which then assumed a bright yellow color. Barium hydroxide and strontium hydroxide gave similar results. Caustic soda solution gave an orangeyellow, and ammonium hydroxide, a bright yellow coloration, turning brown in air. Evidence was obtained that lime-rind phlobatannin forms stable salts with the alkali metals and the alkaline earth metals. These salts could be precipitated by pouring their concentrated aqueous solutions into alcohol, in which the salts are insoluble. They are amorphous, pale brown substances. The phlobatannin also forms salts with aniline and with phenylhydrazine. A solution of the Eubstance quickly reduced ammoniacal silver nitrate without warming, yielding a black precipitate and a silver mirror. Fehling’s solution was slowly reduced in the cold, more rapidly on heating. Concentrated suifuric acid gave a reddish brown ring when slowly added to a dilute solution of the phlobatannin. When added to a concentrated solution of the substance in glacial acetic acid, this reagent gave a deep red coloration, but no crystals separated, even on standing many days, thus proving the non-flavone nature of the coloring matter.6 Note-This result is contrary to the positive reaction which Klein obtained with the yellow coloring matter, anthochlor, which he states occurs in citrus rinds.6 3
p.
Perkin and Everest, “The Natural Organic Colouring Matters,” 191P,
416. Longmans, Green & Co., London. 4 Biochem. J . , 16, 516 (1922);J . Soc. Chem. I n d . , 48, 1106 (1923). 6 Perkin, J . Chem. 506. (London), 67,644 (1895);69, 1439 (1896). 6 Bolan. Abslracfs, 12, 117 (1923);lS, 61 (1924).
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Since, however, Petrie7 has more recently described a coloring matter present in Acacia flowers, stated also by Klein to contain an anthochlor, which proved to be a rhamnoside of the flavonol, kaempferol, it is evident that lime-rind phlobatannin is not identical with Klein's pigment.
On pouring the mixture into water, a dense greenish black precipitate separated, having the properties of a ghlobaphene. Nessler's solution gave a yellow precipitate, which quickly turned brown and finally olive green on standing. Sodium nitrite, in the presence of dilute hydrochloric acid, gave an evanescent red-brown coloration. Uranium acetate gave a cherry red coloration; stannous chloride, a bright yellow precipitate; neutral lead acetate fi buff precipitate, soluble in acetic acid; and basic lead acetate, a bright yellow lake. Acetylation of the solid phlobatannin yielded a brownish black amorphous powder, which could not be obtained in crystalline form. Heating at 200" C. in glycerol for half an hour yielded no definite fission products. The behavior of the substance towards caustic potash under different conditions was therefore examined. Fusion with potash a t temperatures not exceeding 240" C., and subsequent extraction of the neutralized melt with ether, yielded a small amount of crystalline matter, which proved to be mainly protocatechuic acid. Hydrolysis with aqueous or alcoholic solutions of potash a t boiling temperature yielded ether extracts having a bright yellow color. On evaporation these left tarry residues, insoluble in cold water, moderately soluble in hot water, but easily soluble in alcohol. The largest proportional yield of ether-soluble matter was obtained when aqueous potash of 20 per cent concentration was employed. In no case, however, was the proportionate amount of residue very large, so that its exact identity could not be completely established. The stability of lime-rind phlobatannin towards caustic potash appears to be a highly characteristic property of the substance. After purification by treatment with charcoal and with suitable solvents, the small amount of crystalline matter that remained showed a melting point of approximately 216" C. Its aqueous solution gave a pure green coloration with ferric chloride, turning blue, violet, and red on addition of dilute sodium bicarbonate. It was therefore concluded that one of the products of alkaline hydrolysis of lime-rind phlobatannin is caffeic acid. It is noteworthy that lime leaves contain a yellow coloring matter identical with that isolated from the fruit rind. The reactions of lime-rind phlobatannin, and its behavior towards potash, suggest that it is closely related to caffetannic acid. Note-Caffetannic acid of coffee was first described by Rochleder.8 It was subsequently found to be widely distributed in nature. Kuntt-Krauseg proved that caff etannic acid yields caffeic acid on alkaline hydrolysis. Gorter'o later showed that the tannin is a mixture of two acids, chlorogenic and coffalic, of which the first yields caffeic acid and quinic acid on hydrolysis." Freudenberglz suggested that chlorogenic acid is a depside. The recent work of Oparin13 indicates that chlorogenic acid may be the chromogen of a respiratory pigment of the type described by Palladin.14 On the other hand, chlorogenic acid, by virtue of its o-dihydroxy constituent, caffeic acid, may function as one of the three factors which, according to Onslow16 comprise a plant oxidase system. Proc. Linnean Soc., N . S . Wales, 48, 356 (1923); C. A . , 18, 685 (1924). 8 Ann., 69, 300 (1847). 0 Arch. Pharm., 281,613 (1893); J . Chem. SOG.(London), 66,327A (1894). 10 Ann., 868, 327 (1908); J . Chem SOL.(London), 94, 186A (1908). 11 Ann., 869, 217 (1908); J. Chem. Soc. (London), 94, 345A (1908). I* Ber., 6SB, 232 (1920); J . Chem SOC.(London), 118,3228 (1920). 18 Biochem. Z . , 124, 90 (1921). P. Blakiston's Son & Co. 14 "Plant Physiology," 1918, p. 202. Bzochem. J . , 14, 535 (1920). 7
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Comparison with this substance, extracted for the purpose from fresh coffee berries, showed, however, that the two substances are not identical. For example, the color of solutions of caffetannic acid and of the phlobaphene derived therefrom is deep red instead of brown. Furthermore, caffetannic acid gives a deep green coloration with ferric chloride, whereas the color given with lime-rind phlobatannin is brownish green. It also yields a green compound with ammonium hydroxide (viridic acid), whereas lime-rind phlobatannin gives no such product. Practical Applications
Fresh lime juice intended for consumption as an article of food should be bottled and stored with complete exclusion of air, otherwise oxidation leads to the development of a dark color, and possibly also to some loss in antiscorbutic activity. Zilva,16has recently shown that oxidation, especially in the presence of alkali, destroys the antiscorbutic fraction of lemon juice. A preliminary heating to expel gases, and partly to pasteurize the juice, also aids in preventing darkening. If excessive, however, it may produce an undesirable flavor. Carbon dioxide or hydrogen may be used to replace air in storage vessels, or the vessels may be evacuated before ~ea1ing.l~ The coloring matter of lime juice intended for the manufacture of citric acid may be partially removed by longcontinued boiling in contact with air. This leads to the formation of insoluble phlobaphene, which may subsequently be removed by filtration. Partly for this reason, overseas manufacturers do not object to concentrated lime juice prepared by rapid evaporation in open vessels as a raw material for the manufacture of citric acid. There would thus appear to bc no advantage in the use of vacuum evaporators for concentrating lime juice, beyond the loss in citric acid sustained by the grower who exports that product. During the preparation of calcium citrate from lime juice, as an intermediate stage in the manufacture of citric acid,1* excessive liming should be strictly avoided. The autoxidation of polyhydroxy phenols probably does not become appreciable until the reaction of the solution is more alkaline than pH 7.3 to 7.8, but it is highly sensitive to further in- ' creases of alkalinity above this range. La Mer and Rideall9 have demonstrated these facts for the autoxidation of hydroquinone. At very high alkalinity the coloring matter of lime juice appears to be precipitated, but a relatively large amount of solid calcium hydroxide is required to produce this final result. Thorough washing with water, especially hot water, is very effective in removing the unoxidized coloring matter present in commercial calcium citrate prepared by careful liming. It is almost ine€fective if applied to calcium citrate that contains the tarry products of oxidation of the phlobatannin. To procure a high-grade product, the citrate should therefore be washed immediately after precipitation. Biochem. J . , 18, 182 (1924). McDermott, THIS JOURNAL, 8, 136 (1916); and University of Florida, Agr. Expt. Sta , Bull. 136 (1917). 1s Wilson, THIS JOURNAL, 13, 554 (1921), gives a full account of the manufacture of citric acid from lemons. 18 J . A m , Chem. Soc., 46, 228 (1924). 18 17
November Chemical Engineering Education in England-On 12, the Ramsay Memorial Laboratory of Chemical Engineering was opened at University College in London. Although chemical engineering has been taught a t other British institutions, the opening of this new laboratory marks the commencement of a definite department of chemical engineering in a British University. The syllabus of instruction drawn up by Professor Williams is very attractive, and it is noteworthy that the physical and physical chemical aspects of chemical engineering will be given the fullest possible treatment.
