NON - voumn ACIDS OF RED TART. am Thesis ”for the Dame '0? Ph D. MlCHlGAN STATE Vumvexsm Seshumani'xrishna Das 1 9 6 4 mas‘s WWWWWWM 3 1293 01008 5680 This is to certify that the thesis entitled Non-volatile Acids Of Red Tart Cherries presented by Seshumani Krishna Das has been accepted towards fulfillment of the requirements for Ph. D. Food Science degree in /¢fi%%“/ Manr/firofessor pm flit-mi 27, MW» (1 0-169 LIBRARY ' Michigan Stan: ' University. PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE W JAN 1 8 20m Ul ABSTRACT NON-VOLATILE ACIDS OF RED TART CHERRIES By Seshumani Krishna Das The non-volatile organic acids of red tart cherries, P,cerasus were identified and quantitatively determined in 1960, 1961 and 1962. The effect of degree of maturity and spray materials on the total titratable acidity and on the in- dividual acids was also studied. Acidified water extraction, lead precipitation, gradient elution column chromatography, paper chromatography and titration were used to identify and quantitatively determine the acids. The acids identified in these cherries are: aspartic, chlorogenic, citramalic, citric, fumaric, galacturonic, glyceric, glycolic, glucuronic, glutamic, glutaric, iso— chlorogenic, lactic, malic, malonic, neochlorogenic, phosphoric, quinic, shikimic, succinic, and tartaric. Malic acid represented 75 to 95 percent of the total titratable acidity. In 1961-1962, the total titratable acidity showed a marked decline as the fruit matured with a plateau at the time of the commercial harvest. In 1960, there was only a slight decline in acidity during maturation. The changes in concentration of all acids, except for phosphoric, succinic, and the uronic acids, during fruit By Seshumani Krishna Das maturation were similar in all three years for the fixed copper spray. The fungicidal sprays used in 1960 had no effect on the changes in individual acids during fruit maturation. In 1961 and 1962 the Cyprex spray treatment had the same effect on the individual acids as the fixed Copper spray. Ferbam and glyodin, however, affected citric, malic and the uronic acids differently than cepper and cyprex. There was a dip in the malic acid content in almost all treatments in 1960 and in all treatments in 1961 and 1962, either at the time of commercial harvest or a week prior to it. There is a possibility that this dip may be due to a malate- decarboxylation reaction. NON-VOLATILE ACIDS OF RED TART CHERRIES By Seshumani Krishna Das A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 196% ACKNOWLEDGEMENTS The author expresses her sincere thanks to Dr. Pericles Markakis for his guidance and helpful suggestions throughout this research. Special thanks are extended to Dr. Clifford L. Bedford for his advise and valuable help in this study. She expresses her appreciation to the members of the guidance committee, Dr. B. S. Schweigert, Dr. R. W. Luecke and Dr. Dorothy Arata for their help. Thanks are also extended to Mr. Jozef Bakowski for his assistance in the experimental part of this study and to other colleagues in the department for their help and encourage— ment. ******** ii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. . . . . 3 A. Acids of Red Tart Cherries . . 3 B. Non— Volatile acids in plant metabolism . . u C. Methods of qualitative and quantitative 9 determination of organic acids . 9 i. Paper chromatography . . . . . . 9 ii. Ion exchange column chromatography . 13 iii. Other methods. . . . . . . . 15 MATERIALS AND METHODS . . . . . . . . . . . 19 A. Preparation of the acid concentrate. 20 1. 1960 Method. . . 21 2. 1961-1962 Method 22 B. Column chromatography. . . . . . . 25 C. Paper chromatography . . . 28 D. Confirming tests . . . . . . . 29 RESULTS AND DISCUSSION. . . . 32 A. Qualitative Analysis 33 B Quantitative Analysis. . 38 C. Total titratable acidity . . 40 D. Changes in individual acids during fruit development. . . . . . . . . R3 E Effect of Spray treatments on individual acids. . . . . . . . . . . . . . . . . . 69 SUMMARY AND CONCLUSIONS . . . 78 BIBLIOGRAPHY. . . . . . . . . . . . . . . . 81 APPENDIX 0 o o o o o o o o o 92 iii LIST OF FIGURES FIGURE PAGE 1 Concentration gradient elution system .............. 2 Flowing chromatogram of pure acids ................. 3H 3 Titration of column chromatographic fractions of acids of Montmorency cherries - 1962 COpper treatment. 00000000000000000 .....OOOOOOOOOOOOCOOOOOO 36 4 Changes in total titratable acidity during fruit maturation - 1961-1962 Copper treatment. ........ ... M1 5 Effect of spray materials on the aspartic acid content during fruit maturation - 1960.. ........... he 6 Effect of spray materials on the shikimic acid content during fruit maturation — 1960....... ...... A6 7 Effect of spray materials on the quinic acid content during fruit maturation — 1960 ............. H8 8 Effect of Spray materials on the galacturonic acid content during fruit maturation - 1960.... ......... 50 9 Effect of Spray materials on the glucuronic acid content during fruit maturation - 1960........ ..... 50 10 Effect of Spray materials on the succinic acid content during fruit maturation - 1960............. 53 11 Effect of spray materials on the malic acid content during fruit maturation - 196 O. ............... ..... 55 12 Effect of spray materials on the citric acid content during fruit maturation - 1960...... ....... 55 13 Effect of Spray materials on the polyphenolic acids content during fruit maturation - 1960....... ...... 58 14 Effect of spray materials on the phosphoric acid content during fruit maturation - 1960..... ........ 6O 15 Effect of spray materials on the aspartic acid content during fruit maturation — 1961-1962. ....... 62 16 Effect of spray materials on the quinic acid content during fruit maturation — 1961-1962........ 62 iv LIST OF FIGURES (Cont'd) FIGURE PAGE 17 Effect of spray materials on the shikimic acid content during fruit maturation - 1961-1962.. ...... 63 18 Effect of spray materials on the glucuronic acid content during fruit maturation — 1961-1962........ 63 19 Effect of Spray materials on the galacturonic acid content during fruit maturation - 1961-1962... ..... 64 20 Effect of spray materials on the succinic acid content during fruit maturation — 1961-1962 ........ 66 21 Effect of spray materials on the citric acid content during fruit maturation - 1961-1962 ........ 66 22 Effect of spray materials on the malic acid content during fruit maturation - 1961-1962.... ............ 67 23 Effect of spray materials on the polyphenolic acids content during fruit maturation - 1961-1962 ........ 68 2% Effect of spray materials on the phosphoric acid content during fruit maturation — 1961-1962 ........ 68 TABLE 10 11 LIST OF TABLES PAGE Rf x 100 values in two different solvents, the % recoveries on Dowex- 1 and .50, and pK values of acids of red Cherries. ............... ..... .. 92 Rf x 100 values of other acids studied........... 9Lt Total titratable acidity for all treatments in 1960... OOOOOOOOOOOOOOOOOO 0 ...... 00 ...... 000.000.: 96 Total titratable acidity for all treatments in 1961and1962000 0000000 0. 0 ...... 0.00.00.00.00... 97 Non- volatile acids of Montmorency cherries during fruit development - 1960, Fixed COpper Spray, all season. 0.00000000000.000000000000000.0000.0000 Non-volatile acids of Montmorency cherries during fruit development - 1960, Copper spray, early (two applications); Ferbam and Glyodin spray, 99 late0000000000000000000000000000000000000.0000... Non-volatile acids of Montmorency cherries during fruit development - 1960, Ferbam and Glyodin spray, all season................................100 Non- volatile acids of Montmorency cherries during fruit development - 1960, Ferbam and Glyodin . spray, early (two applications); Fixed copper Spray, late......................................lOl Non—volatile acids of Montmorency cherries during fruit development - 1960, Ferbam and Glyodin spray, early (two applications); Nu-Iron and Glyodin sprays, late.............................lO2 Non-volatile acids of Montmorency cherries during fruit development - 1960, Cyprex spray, all Season.000000....0000000000.0.000.000.00000000000103 Non—volatile acids of Montmorency cherries during fruit development - 1960, Parathion spray, early (two applications); Actidione and Ferbam sprays, late.............................................10H vi LIST OF TABLES (Cont'd) TABLE 12 13 11+ 15 16 17 18 19 2O Non—volatile acids of Montmorency cherries during fruit development - 1960, Parathion spray, early (two applications); Actidione and Nu—iron sprays, late ......................................... ..... Non-volatile acids of Montmorency cherries during fruit deveIOpment - 1961, Ferbam and Glyodin spray. Acidity expressed as meq/100 meq of analyzed acidity .................................. Non-volatile acids of Montmorency cherries during fruit deveIOpment - 1961, Ferbam and Glyodin spray. Acidity expressed as mg/100 gm of fresh fruit ................................. . ...... ..... Non—volatile acids of Montmorency cherries during fruit development - 1961, COpper spray. Acidity expressed as meq/100 meq of analyzed acidity...... Non-volatile acids of Montmorency cherries during fruit development - 1961, COpper Spray. Acidity expressed as mg/100 gm of fresh fruit ....... ...... Non—volatile acids of Montmorency cherries during fruit deveIOpment - 1961, Cyprex spray. Acidity expressed as meq/100 meq of analyzed acidity...... Non—volatile acids of Montmorency cherries during fruit development - 1961, Cyprex spray. Acidity expressed as mg/100 gm of fresh fruit ............. Non—volatile acids of Montmorency cherries during fruit development - 1962, Copper Spray. Acidity expressed as meq/100 meq of analyzed acidity....... Non-volatile acids of'Montmorency cherries during fruit development - 1962, Copper spray. Acidity expressed as mg/100 gm of fresh fruit ............. vii PAGE .105 .106 .107 .108 .109 .110 112 .113 tad INTRODUCTION Organic acids are of great significance in plant and animal metabolism. In plants, these acids are early pro- ducts of photosynthesis and thus serve as precursors for the synthesis of other compounds. Some acids also arise as products of degradation of certain chemical compounds in plants. The close metabolic relationship of organic acids to fats, carbohydrates, and proteins emphasizes their key role in plants. In animals it has been shown that the tricarboxylic acid cycle releases energy and interrelates fat, carbohydrate, and protein metabolism. A number of workers have shown the functioning of Kreb's cycle in certain seedlings (1H), but the presence and importance of the cycle in mature plant tissues remains doubtful. Early investigations have been primarily concerned with the organic acids present in significant amounts and the acids in minute quantities were only determined in case of special interest. However, the acids present in very small amounts, either free or in combined state, may play an important part in the overall metabolism of the tissues and, therefore, it would seem desirable to investigate the nature and amounts of the minor acids. Academically this study would give us the knowledge of the acids present in the fruit tissue. From the practical ’1 point of view, since it is known that acids participate actively in fruit metabolism, we may be able to determine their possible role in non-enzymatic browning reactions, other type of discolorations and flavor, and on the basis of this knowledge, suggest modifications in the handling or processing procedures which might minimize these undesirable changes. The object of this study was to identify and quanti- tatively determine the non—volatile acids of red tart cherries (Prunus cerasus, Var. Montmorency) and to determine the effect of degree of maturity and fungicidal Sprays on the total acidity and individual acids present. This research was conducted on Montmorency cherries, not only because Michigan is the number one state in the production of this variety of cherries, but also because it is the only variety that is produced in substantial quantities in all regions that grow red tart cherries. The Montmorency variety is favored because of the excellent flavor, the color and size of the fruit, the long harvest season, and also because it is a dependable producer of good crops (82). .- iv REVIEW OF LITERATURE A. ACIDS OF RED TART CHERRIES The acidity of red tart cherries has generally been reported either as total titratable acidity or as malic acid. The first known study on cherry acids was made by Keim in 1891 (69), who identified oxalic and succinic acids. In 1901, Desmouliere (2h) detected salicylic acid in the fruit, and other workers (2%, 25, 39, 64, 117) also reported the presence of salicylic acid; but, since 1905, no one has reported its presence. Truchon and Martin—Claude (118) found tartaric acid in the juice of cherries, and other workers (18, 86, 88, 89, 90) reported its presence in fruit tissues. Jorgensen, in 1907, (66) found succinic acid and Arbenz, in 1917)(2) determined the presence of oxalic acid. Citric (31), malic (9, 32),pyrrolidone carboXylic (79), and phosphoric (12H) acids have been reported in the cherry fruit, glycolic acid in the peduncle (5) and lactic acid (85) in cherry juice. Kohman (70) lists the oxalic acid content of red tart cherries at 1.1 mg./100 gm of edible tissue. Bridges (11) reports the acid content of cherries (variety not Specified) as follows: malic - 0.56 to 1.99%, citric - 0 to 0.01%, succinic — 0.07%, lactic — 0.13%, oxalic, trace. n B. NON-VOLATILE ACIDS IN PLANT METABOLISM Terminal respiration in higher plants involves the oxidation of pyruvic acid to C02 and water with certain organic acids acting as intermediates in a series of reactions most commonly known as the Krebs or Citric Acid Cycle (72). In 1950, in a review of the metabolism of organic acids in plants, Thimann and Bonner (116) reported that the presence of citric and malic acids is quite common in leaves and fruits, oxalic fairly widespread in leaves and tartaric the main constituent of fruits of Vitaceae. Fumaric and succinic acids are also distributed widely in higher plants, while oxalosuccinic, isocitric, cis—aconitic, oxaloacetic and oc—ketoglutaric acids are generally found only in traces, if detected at all. Isocitric is present in substantial quantities in Crassulaceae (110). Organic acids have been shown to be involved both directly in a reducing sugar—organic acid reaction and synergistically in a reducing sugar-amino acid—organic acid type of reaction (#9, 68). Others have also indicated (7, 62) that these non-nitrogenous organic acids, in addition to the amino acids, may play a part in browning reactions and in after-cooking discoloration reactions. The blackening of potatoes after cooking is one of the most undesirable qualities of potatoes. This colored product is considered to be a complex of ferric ion and chlorogenic acid (50). Hughes and Swain (51, 52) showed that citric acid was the most important 5 factor in preventing blackening of potato tubers. Citric acid chelated the iron and prevented its complexing with chlorogenic acid. Citric acid has been shown to be a good inhibitor of browning, not only as an inactivator of trace metals but also as a synergist for true antioxidants (3, 103, 114). Qureshi's work (103) indicates that oxalic acid was more effective than citric acid in inhibiting the brown- ing of various tuber starches from Indian tubers. Malic acid in combination with ascorbic acid inhibited the browning of cherries (11h). Chlorogenic acid, composed of quinic and caffeic acids, has been shown to occur in many plants -— in leaves of red tart cherries, apricots, peaches, sweet cherry and grape-cherry (130); in fruits of sweet cherry (131) and sweet mountain-ash (75); in tea (113); in pear (121), in coffee (6) and many other plants. Its isomers, isochloro- genic acid and neochlorogenic have also been detected in many of the plants in which the presence of chlorogenic acid has been positively identified (6, 122). Recent interest in chlorogenic acid has centered on its function as a substrate for polyphenolase. Joslyn and Ponting (67) suggested that the darkening of fruit on injury may be due to the oxidation (enzyme catalyzed) of chlorogenic rather than the true tannins. In the early part of 1963, Maier (80) identified the main substrate for enzymic browning in dates as a caffeoylshikimic acid. This was the first time 6 a crystalline caffeoylshikimic acid was isolated from natural material, and the first demonstration that such acids will undergo enzymic browning. He suggested that these acids may be widely distributed in plants but may not accumulate as much as chlorogenic acids, possibly because of their greater metabolic activity. Caffeic acid has been reported to be present in the leaves of apricot, peach and grape (130), roots of Polygala senega (21), and in fruits of sweet mountain-ash (75) and sweet cherry (131). There has been no positive identification of this acid in either the leaves or fruit or any part of the tree of red tart cherry. Quinic acid was first found in apple fruits (53). It was originally suggested that quinic acid might provide a link between aliphatic and aromatic metabolism in plants and that shikimic acid might be the first stage in the "desaturation" of quinic acid (53). Weinstein et al. (125), using labeled quinic acid to determine its role in aromatic biosynthesis in higher plants, found that the major labeled products were always tyrosine, phenylalanine, C02 and often shikimic acid. In apples it was found that shikimic acid increases as the fruit ripens and becomes senescent (57). Weinstein and co—workers also studied the role of shikimic acid pathway in beans (126). They found that much of the C11+ from shikimic acid was incorporated into phenylalanine and tyrosine and r... r.; ;'¢ 7 only a negligible amount was found in free or bound trypto- phan. They also noticed the irreversible conversion of quinic acid to shikimic acid. Succinic acid was shown to inhibit oxidative processes (120), and it was indicated that it may be actually toxic to plant material (56). Ttdeslatterassumption was based on the fact that on paper chromatograms of acid fractions from apples suffering from carbon dioxide injury, a spot correspond- ing in position to that of succinic acid appeared. While in normal healthy apples, succinic was found only occasionally and in trace amounts. The widespread occurrence of malonate in plant tissues has been demonstrated and the pathways for its enzymatic utilization and synthesis have been described by Shannon et al. (108). Malonic acid was not found to be an inhibitor of the Kreb‘s cycle in soybean leaves (113). However, it has been Shown otherwise in the mitochondria from cauliflower buds, where the organic acid concentration is small (14). Quinic and citric acids,on oxidation, have been shown to yield small amounts of malonic acid (55). The appearance of uronic acids (especially galacturonic acid) is usually associated with pathological disturbances, physiological changes such as senescence, and various types of physical damage (cutting or crushing of tissue) (77). It is also believed that they are seldom found in a free state in intact plants. Loewus (77) states that the uronic acids 8 undergo the synthetic steps leading to ascorbic acid. This was shown in detached ripening strawberries where D-galacturonic acid-1-C112L was converted to L-ascorbic acid labeled in C-6. Pyrrolidone carboxylic acid (PCA) has been shown to occur in a number of fruits and vegetables (79, 107), includ- ing red tart cherries, variety Montmorency (79), but only after the product has undergone some heat treatment. PCA, a product of glutamine decomposition, seems to be responsible for the off—flavor in certain products. The presence of phosphoric (107, 131), pyruvic (12, 119, 131),dC—ketoglutaric (12, 119), citramalic (121), oxalic (107, 123), glutaric and aspartic (107, 121), glyoxylic (12), oxalacetic (12), glyceric (16) and fumaric (12, 16), tartaric (16, 105), lactic (105), etc. have been shown to be present in plant tissues by many investigators. Schwartz and co—workers (107) studied the relationship of organic acid concentrations to Specific gravity and storage time in potatoes. Concentration of glutamic, aspartic, PCA, malic, oxalic, and phosphoric acids varied inversely with the specific gravity. During storage the citric acid content of potatoes increased while that of malic acid de- creased, suggesting the conversion of one to the other. Such a conversion was also shown to occur in tobacco leaves (1%). Disks of Sweede root tissue treated with several acids separately (succinate, malate, citrate, pyruvate and dC-ketoglutarate) showed an enhanced protein synthesis, ..m; k. 9 possibly due to increased concentration of C skeletons in the tissue (119). C. METHODS OF QUALITATIVE AND QUANTITATIVE DETERMINATION OF ORGANIC ACIDS Most of the work,conducted prior to the development of chromatographic techniques, on acidity in fruits was confined to the determination of total titratable acidity or total acidity as malic, citric or tartaric (major acids in certain fruits). But since the ”acid fraction" is certainly involved in many reactions, measuring small changes in the concen- tration of a specific organic acid or acids is likely to be more valuable in elucidating metabolic pathways than would be measuring changes in total acidity. Hence, work on the fractionation or isolation of certain acids of the acid ex- tract of fruits and vegetables was initiated. 1. Paper Chromatography Following the original work on paper chromatography by Consden, Gorden and Martin (20), this method has become the most popular procedure for the separation of various substances in mixtures and biological fluids. The separation of acids on paper was first described by Lugg and Overell (78). They suppressed tail formation by adding formic or acetic acid to the solvent, thus keeping the acids un-ionized. To identify a substance one usually measures the Rf Value (the ratio of the distance traveled by the solute to 10 the distance traveled by the solvent front) with a number of suitable solvents. Also, Rf values of reference sub- stances run on the same paper at the same time, are measured. However, it is not possible to identify an unknown substance by Rf value alone. In view of the great biochemical interest of acids, a number of solvents and techniques have been developed for improved or specific separations. Whiting (127) examined aqueous extracts of acids by descending chromatography, using the following solvents: a) Benzyl alcohol : isopropyl alcohol : tert. butanol : water (3:1:1:1) and 0.2% formic acid b) n-propanol : conc. aqueous ammonia (7:3) c) Phenol, 3 gm : water, 1 ml : formic acid, 1% Some of the other solvents used in separating organic acids: d) Iso-propanol : pyridine : acetic acid water (80:80:10:40) (115) e) Formic acid : butanol : benzyl alcohol water (60:1H0:22:240) (91) (volzvol) f) Butanol : acetic acid : water (100:2H:100) (H) g) Butanol : formic acid : water (100:30:100) (H) h) n—Butanol : 3N formic acid (V./V.) (81) i) n—Butanol : water : diethylamine (100:15:1) (65) j) Ether : acetic acid : water (15:3:1) (65) e: rL. ... r—g ‘\. p.A .5 11 k) l—pentanol : 5 M aqueous formic acid (1:1) (65) l) 2-ethyl-l-butanol : 5 M aqueous formic acid (2:3) (65) m) 95% ethyl alcohol : water : conc. ammonium hydroxide (8:1:1) (65) n) 95% ethanol : ammonium hydroxide : water (20:1:H) (10) For the detection of the acids the volatile acid or alkali of the solvent is completely evaporated and the paper sprayed with an indicator, such as bromocresol green or bromophenol blue (0.0% g. in 100 ml. of 95% ethanol —- (7%)). When acetic acid is included in the solvent, evaporation takes longer than with formic acid; however, in either case drying should be carried out Slowly (for at least 2—3 hours) at room temperature. One-dimensional paper chromatography has been used to determine mono— and dibasic acids (83), di- and tricarboxylic acids (111), mixture of organic acids (132), citric and malonic acids on Ni(OH)2— impregnated paper (35), organic acids by use of papers containing ion—exchange resins and O—(carboxy-methyl)—cellulose (87), etc. There are a number of other techniques that have emerged from this simple one- dimensional paper chromatography. A two-dimensional paper chromatography has been developed for the separation of organic acids and used by several investigators (19, 29, H5). 12 A mixture of uronic acids was separated by circular chroma- tography (semi-circle technique) (115). Ascending chromatography has been used in combination with electro— phoresis for a study of non-volatile organic acids of biological media. Badrinas et al. (4) used a fluorescent indicator for the chromatographic development of non- volatile mono-, di-, and tricarboxylic acids. The reaction is based on the inhibition of the formation of the fluorescent complex, of aluminum-8—hydroxyquinoline, by organic acids. Also, organic acids can be separated and identified on papers impregnated with ion exchange resins, O-(carboxy-methyl)—cellulose (87), and Ni(OH)2 (35). A method has been developed for the separation of substances of very narrow Rf range. This is called ”fractionated one- dimensional paper chromatography" (106). Fractionated chromatography is conducted by developing and drying the chromatogram, then submitting it again to the same solvent system. This produces a second migration and a new series of Rf values. The first is called the primary Rf value, the others secondary. This can be repeated up to ten times, thus extending the length of the chromatogram. Paper chromatography has also been used for quantitative determi— nation of di- and tricarboxylic acids, with an accuracy up to 0.4—0.5% (111). Electrometric contact method has been applied to paper chromatography of organic acids (133) using Kamienski's 13 microelectrode. The method allows detection of acids studied in quantities of the order of 10712 mole on the active surface of the electrode (3.1% sq. m.m.). Both ascending paper chromatography (91) and descending paper chromatographic methods have been used (81, 127). ii. Ion Exchange Column Chromatography In ion exchange chromatography, an ion exchange resin serves as the chromatographic column. The solute mixture is first added to the resin in a vertical column and once the ions are exchanged on the resin, separation can be accom- plished by displacement, elution, and frontal analysis (26). In the past, anion exchangers were used for the separation of organic anions from cations and uncharged molecules present in biological extracts (7%). The use of anion exchangers for the separation of organic acids from each other has only been reported recently. Wilson (128) has used ion exchange resins to determine fruit acids; Busch et al. (15) have used the formate form of Dowex-l for the separation of the acids of the citric acid cycle. The organic acids were displaced with formic acid of constantly increasing concentration. Excellent separation of lactate, succinate, malate, fumerate,c<:-ketoglutarate, and cis- aconitate was achieved. Pyruvate, malonate, citrate, and isocitrate were not separated but could be resolved on a silica gel column. Reproducibility of the positions of the 1% peaks of known acids was fairly good. Following them, a number of investigators have used anion exchange resins for the separation of organic acids: Schenker and Rieman (105) have separated malic, tartaric, and citric acids from fruit extracts; Owens et al. (92) studied the organic acids in sugar beet liquors; Palmer studied the acids of tobacco leaves (93); Hulme and Wooltorton (59) investigated the acids in apples, strawberries, and sweet cherries (61). These investigations were followed by several others (16, A1, 129) who used either the Dowex—l or Duolite A—3 or A—M, or Amberlite IR-HB columns for the fractionation and determination of organic acids in various plant materials. Gradient elution chromatography was first described by Alm et al. (1). They showed that the successive zones into which the components of a mixture resolve could be narrowed considerably, and the elution peaks sharpened, by passing a gradually increasing concentration of eluting agent through the column. The separation of the various acids takes place primarily by elution chromatography on strongly basic anion exchange resins in the acetate or for- mate form (59, 81). The acetate form gives better separations of the weaker monobasic acids such as quinic and shikimic (59). The acids are displaced by "gradient elution." The principle of the method depends upon the fact that organic acid anions combine with positively charged groups on a synthetic organic resin of suitable composition 15 and remain fixed to it until displaced by an aqueous solution of an acid of increasing concentration which percolates through the column. Hulme and Wooltorton (59) believe that, in general, acids leave the column in the order of their pK values, but that there are exceptions. Also, unsaturated acids are more tightly bound to the resin than saturated acids of similar basicity and molecular weight. Most of the acids may be completely separated and a high percent recovery is obtained, but the yields of some acids are low, owing to decomposition or other unknown reasons. Acids with closely similar pK values cannot be separated quantitatively, especially if one of them is present in relatively large amounts. In such cases the mixed fractions may be resolved and the individual acids determined quantitiatively by other methods such as silica gel column chromatography or paper chromatography. Peterson and Sober (97) have described a device for the production of a wide variety of concentration gradients using up to nine chambers. iii. Other Methods In the 1930's, Fidler investigated several methods for the determination of individual acids in plant material (60), but he found that they were not sufficiently accurate for the determination of acids which were present in apples in very small amounts. 16 Later, Pollard et al. (101) and Prigot and Pollard (102) converted acids into their piperazinium salts as a method of identification of organic acids. Curl and Nelson (23) isolated citric, isocitric, and malic acids by distillation of their ethyl esters and characterized them as hydrazides. Oxalic acid was isolated and identified by crystallographic methods after precipi- tation as calcium oxalate. Mixtures of dicarboxylic acids were also separated by preparing their amides or imides which were then dis— tilled (8). Dunbar and Moore (27) studied carboxylic acids by preparing their P-toluidides and amides. The uronic acids, especially galacturonic and glucuronic, have been separated on anion exchange columns (73) and determined on thin silica gel layers, with naphthoresorcinol solution as a specific detecting reagent (95). These acids have also been identified by lactonizing and chromatographing (36). Characteristic migration rates, the hydroxamic acid—ferric ion test for lactones, and the Specific lead acetate test for galacturonic acid were used to identify these on paper chromatograms. D-galacturonic acid can also be determined colorimetrically with 2— thiobarbituric acid (134). The reaction in this case gives a yellow color which is measured at 400 m”. Uronic acids have also been separated by circular chromatography (115), paper electrOphoresis (4H), and using anthrone (H7). 17 Keto—acids can be separated in mixtures as the 2, h-dinitrophenylhydrazones (12, 30) and quantitatively deter— mined with the aid of a photometer (30). dC,—Ketoglutaric acid was determined fluorimetrically by Spinker and Towne (112) by treating it with o-phenylene diamine to give fluorescent quinoxaline. Lactic acid has been determined spectrOphotometrically by converting it to CHI3, dissolving this in CHCl3 and measuring at 347 m/( (34). Courtoisier (22) estimated lactic and tartaric acids by oxidation with permanganate and chromate in HQSOu and malic acid by oxidation with ceric sulfate. High-voltage paper electrOphoresiS has been applied to the study of organic acids (40). Also, electrochromato- graphy has been used as a method of direct identification of non-volatile organic acids of biological media (91). Following Isherwood's introduction (63) of column chromatography for the separation of organic acids on silica gel, the method has been adopted and somewhat modified by a number of investigators (13). Wagner and Isherwood (123) recently described the separation of some 25 different acids from peas, including lactic, succinic, oxalic, ciS-aconitic, malic, and citric. Carles et al. (18) employed Celite columns for the separation of organic acids of various parts of grape vines by partition chromatography. Columns contain— ing alumina (A1203) (38, #2) and cellulose (H1, 99, 122) 18 have also been used for the separation of organic acids. Thin layer electrophoresis has been applied to the separation of phenols and phenol carboxylic acids (96). layer chromatography has also been used in the separation of phenolic carboxylic acids on layers treated with chelate- forming anions (M3), and saturated aliphatic dicarboxylic acids (oxalic, malonic, succinic, glutaric, adipic, etc.) on silicic acid layers (98). Shibazaki (109) simplified a gas—chromatographic apparatus for the determination of inorganic and organic acids. Infra red spectroscopy as a method of organic acid determination is gaining popularity. Henshaw et al. very recently used it for the identification of organic acids of the rhizome of Iris pseudacorus (H8). MATERIALS AND METHODS MATERIALS Red tart cherries were collected during three seasons, 1960, 1961 and 1962. These cherry samples were obtained from the Horticultural Farm at Michigan State University. Cherries were picked at weekly intervals, starting with the H5th and 96th day after full-bloom. There were six such pickings in 1960, and eight each in 1961 and 1962. The trees were treated with eight different fungicidal sprays in 1960, three in 1961, and one in 1962, as shown below. The cherries were either washed with water, pitted, weighed, and extracted immediately or washed and stored at -1OOF until extracted. Fungicidal Sprays Used 1960 1. Fixed Copper, all season 2. Fixed Copper, early (2 applications); Ferbama and Glyodinb, late 3. Ferbam and Glyodin, all season H. Ferbam and Glyodin, early (2 applications); fixed copper, late 5. Ferbam and Glyodin, early (2 applications); Nu—iron and glyodin, late a Ferric dimethyldithiocarbamate b 2-heptadecylglyoxalidine acetate 19 20 6. Cyprexc, all season 7. Parathiond, early (2 applications); actidionee and ferbam, late 8. Parathion, early (2 applications); actidione and Nu-iron, late (Sevinf was also used for cherry fruit-fly control) Also, 1 through 7 were treated with lead arsenate all season. 1961 1. Ferbam and Glyodin 2. Copper (Fixed) 3. Cyprex (Organic) 1962 1. Fixed Copper METHODS A. Preparation of the Acid Concentrate Two different methods were used in extracting and concentrating the acids before their chromatographic separation. These methods are described as the 1960 Method and the 1961-1962 Method, indicating the years in which each one was applied. c n—dodecylguanidine acetate d 0,0 - diethyl O-p-nitrophenyl thiophosphate e Beta - (2-(3, 5—dimethyl-2—oxocyclohexyl)— 2-hydroxyethyl)—glutarimide f 1-naphthyl—N-methyl carbamate 21 1. 1960 Method: The procedure used was a modification of that described by Hulme and Wooltorton (59). Fifty grams of pitted fruit were blanched in approximately 75 ml. of boiling distilled water for one minute and then 400 ml. or sufficient 95% ethanol was added to give a 70% ethanol solution. This pulp—ethanol mixture was blended in a Waring blendor for three minutes at high speed. It was then cooled to room temperature, filtered through sharkskin paper, and the residue washed thoroughly with 50-60 ml. of 80% ethanol. Filtrate and washings were combined and concentrated to 40—50 ml. in vacuo in a flash evaporator, with the water bath at 400C. The con- centrate was then treated with 1.0 gm deactivated carbon for two hours on a shaker to remove the color- ing matter and filtered. The deactivated carbon was prepared by shaking 150 gm-of Darco, Grade G-60 activated carbon with 1 liter 5% acetic acid for an hour, filtering and washing with distilled water until acetic acid was removed, and drying at HOOC. The cherry filtrate was applied on a water- washed Dowex—50 WX8 cation exchange resin column (H+form 50-100 mesh, 15 cm long by 0.7 cm in diameter). 22 The organic acids were eluted by means of water. The column retained the amino acids and cations. The eluate from the column was concentrated in the flash evaporator and made to a volume of 25-H0 ml., depending on the amount of washing required to re- move the concentrate, both from the flash evaporator flask and the column. Representative aliquots were titrated with 0.1 N NaOH using phenolphthalein as an indicator to determine the acid content. Sample portions corresponding to a total acidity of 1.0 — 1.2 meq. were used for fractionation. The rest of the acid extract was stored at -1OOF. 2. 1961-1962 Method A modification of Hartman's lead precipitation procedure (#6) for the preparation of the acid con- centrate, was used. A weighed amount of pitted fruit (50-100 gm) was blanched and homogenized in a Waring blendor, with 75 ml. distilled water, at high speed for three minutes as in 1960. The homogenate was boiled for ten minutes, then 2 ml. of 1 N HNO3 were added and the acidified homogenate was cooled to room temperature. It was then made up to 250 ml. and filtered through a 6—1/2" single gauze milk filter disc. Two hundred ml. filtrate was concen- trated to approximately 50 ml. in vacuo, and diluted 23 to 250 ml. with 95% ethanol. The precipitated pectins and other alcohol insoluble material were removed by a milk filter disc and 200 ml. of the filtrate was transferred into a 250 ml. centrifuge flask contain- ing a Teflon-coated magnet. The pH was adjusted to 7.8 with 2N NHnOH and lead subacetate, dissolved in a few ml. of water, was added. The amount of lead subacetate used was equivalent to about twice the titratable acidity of the filtrate before the pH adjustment. Hyflo supercel (0.2 gm) was added to the mixture, it was stirred for five minutes, and centrifuged at 2500 RPM for ten minutes. The super- natant liquid was decanted and 50 ml. of 80% ethanol were added to the sediment, the mixture was shaken for complete dispersion, the sides of the flask washed and the contents were centrifuged as previously described. A second decantation, redispersion in 50% ethanol and centrifugation followed. If the third supernatant was not clear, it was not decanted, instead, the sediment was again dispersed and the pH readjusted to 7.5 with 2N NHnOH and recentrifuged. The sediment obtained after the third decantation was dispersed in 50 ml. of 50% ethanol and the suspension was saturated with H28, while constantly stirring with the aid of a magnetic stirrer (for five minutes). 2H The contents of the flask were centrifuged and the supernatant was checked for soluble lead with H28. If the precipitation of lead was complete, the super— natant containing the free acids was transferred to a flash evaporator flask. The precipitate (or sediment) of lead sulfide was dispersed in 50 ml. of 50% ethanol, centrifuged and the supernatant added to the previous one. This last step was again repeated to insure complete removal of the free acids from the centri- fuge flask. The combined solution of free acids was concentrated to 15-20 ml. in a flash evaporator and then passed quantitatively through a column of Dowex— 50 WX8 cation exchange resin, as described previously. During the passage of the solution of free acids through this column, pigments, cations, and amino acids (except part of aspartic and glutamic) were retained by the resin. The column was washed thorough- ly and the eluate concentrated and made up to 25 ml. in every case. Representative aliquots were titrated to determine the acid content and sample portions corresponding to a total acidity of 1.0 - 1.2 meq. were used for fractionation. The rest of the acid extract was stored at ~100F. —' ...L FIGURE 1. CONCENTRATION GRADIENT ELUTION SYSTEM Eluant Reservoir Capillary Glass Tube Mixing Flask Magnetic Stirrer Anion Exchange Resin Column Automatic Fraction Cutter Air Pressure Regulator 26 27 regulator (Moore Products Co., Philadelphia 29, Penna.) was connected to the separatory funnel by means of a rubber tubing to keep the pressure in the system at 150 inches of water. Mixing was accomplished by a Teflon—coated magnet in the flask and stirring by means of a magnetic stirrer beneath the flask. The level of the liquid in the mixing flask was kept just above the side arm. The first eluting solution consisted of 100 ml. 3N acetic acid, the second 50 ml. 6N acetic acid and the third 300 ml. 6N formic acid. Between 120 and 130 fractions of 9.0 ml. or slightly less volume were collected using an automatic fraction cutter (Rinco Instruments, Greenville, Illinois). The fractions were dried in a vacuum oven at 900C. For the quantitative determination the fractions were re- dissolved in hot water, kept in a hot water bath and titrated with 0.01 N NaOH, using phenolphthalein as indicator. The fractions for each peak were pooled, treated with Dowex-50 WX8 cation exchange resin to remove the sodium, filtered through cotton, and dried for qualitative determination. For the determination of the unknown peaks, 3% known acids were passed and eluted through the anion exchange column to determine the effluent volume of each acid and order of their emergence. To obtain sharper peaks only 3~H acids were passed through the column at a time. 28 C. Paper Chromatography The dried fractions were dissolved in 80% ethanol and paper chromatographed on Whatman No. 1 paper (#6 cm x 57 cm). The Spots were placed 2.5 cm apart and 7.0 cm away from the long edge of the paper. Sixty known acids were spotted in between the unknown fractions from cherry. Two different solvents, one acidic and one alkaline were used: (a) n— Butanol and 3N formic acid, 1:1 by volume; (b) Ethanol, ammonium hydroxide and water, 20:1:H by volume (10). The lower phase of the mixture (a) or, the mixture (b) was used for vapor equilibration. The spotted papers were irrigated descendingly by the upper phase of the mixture (a) or, mixture (b) for 15 and 12 hours respectively, at a constant temperature of 220 i 0.50. At the end of this time, the solvent front was marked and the papers were dried in an air draft for at least three hours and Sprayed with a 0.0H% solution of Bromphenol blue (BPB — Na salt) in 95% ethanol. The acid spots showed as yellow spots against a blue back- ground. The Rf values were determined for all acids. The cherry acids were tentatively identified with the known acids on the basis of having Similar Rf values. The presence and identity of chlorOgenic, isochlorogenic and neochlorogenic acids were determined by examination under UV light for fluorescence. Spot tests or color reactions were used for the various acids to confirm the identity of these acids. 29 Pure acids were obtained from commercial sources for the identification of the acids from red cherries and to determine the quantitative recovery using column chromatography. Neochlorogenic acid was obtained from Dr. J. Corse of the Western Utilization Research and Development Division, U.S.D.A., Albany, California. Isochlorogenic acid was isolated from green coffee beans by the author, using the procedure given by Barnes (6). D. Confirming Tests 1. Phosphoric acid test with molybdate (28): a mixture of 1 ml. of the test solution, 2 ml. of concentrated nitric acid, and 2 ml. of ammonium molybdate reagent was warmed and allowed to stand for at least five minutes. A fine yellow precipitate indicated the presence of phosphorus. 2. The fluorescent acids were further characterized by alkaline hydrolysis and subsequent paper chromatography of the hydrolysate. For the alkaline hydrolysis, 2 to 3 ml. of test solution were mixed with 2-3 drOps of KOH solution (13% w/v) and left for 15 minutes at 200C (5%). The sodium ion of the hydrolysate was removed with the Dowex-50 WX8 cation exchange resin and the free acids were paper chroma- tographed on Whatman No. 1 filter paper with n—Butanol: 3N formic acid (1:1) as the solvent, along with pure solutions of caffeic and quinic acids. 30 3. Glyceric acid test with naphthoresorcinol : 0.5 _ 1.0 ml. of test solution was heated with 0.75 ml. concentrated H2804 containing naphthoresorcinol (1 mg/10 ml), for 30—50 minutes in a water bath at 9000. A blue color appeared in the presence of 10 K or more of the acid (28). h. Glycolic acid test with 2,7-dihydroxynaphthalene (2,7-naphthalenediol) and sulfuric acid : a mixture of test solution (0.5 — 1.0 ml.) and 2 ml. of concentrated H2304 containing 2,7-naphthalenediol (1 mg/10 ml) was heated for 10—15 minutes in a water bath. A red to violet red appeared according to the amount of glycolic acid present. At least 0.22{ glycolic acid is necessary to give this color reaction (28)° 5. Lactic acid test with p-hydroxydiphenyl : 0.5 — 1.0 ml. of test solution and 1 ml. concentratengSOg were heated for two minutes in a water bath at 850C and cooled to 2800. A pinch of solid p~hydroxydiphenyl (phenylphenol) was added to it and swirled several times and allowed to stand for 10-30 minutes. A violet color was indicative of the presence of lactic and needs at least 1.5 K'for the reaction (28). 6. Galacturonic acid test : a drop of saturated basic lead acetate was placed on a test spot on a paper and heated for one minute on live steam. Brick red color appeared in the presence of galacturonic acid. 31 7. Quinic acid test with naphthoresorcinol : same test as for glyceric acid. A greenish color appeared in the presence of quinic acid (28). 8. Glucuronic acid test with naphthoresorcinol : same test as for glyceric acid. A yellow cxflxn‘ with a greenish fluorescence indicated the presence of glucuronic acid in the test solution (28). 9. Tartaric acid test with dinaphthol : 0.5 - 1.0 ml. of the test solution was treated with a little solid (5’15“ dinaphthol in concentrateiH280n and heated for half an hour in a water bath at 850C. When tartaric acid was present, a luminous green fluorescence gradually appeared during the heating and deepened on cooling. As little as 10 X’of this acid can be detected (28)° RESULTS AND DISCUSSION The 1960 method, using carbon for the removal of color and sugars, was not used after the first year because it was found that quantitative recovery of the aromatic acids could not be obtained. The deactivated carbon, although it removed the pigments more efficiently than the cation exchange resin, absorbed and did not release readily the aromatic acids, particularly chlorogenic acid. Using pure chlorogenic acid solutions, the maximum recovery of acid obtained was 66% while with the lead precipitation method 99 to 100% recovery was obtained. In the 1961-1962 procedure, the aqueous fruit homo- genate was acidified with nitric acid, extracted with cold 95% ethanol and the acids precipitated as lead salts with lead subacetate. This method resulted in better recovery of the organic acids because (a) the free acids were more soluble in ethanol than the salt forms, such as tartarates, (b) the precipitation of the acids as the lead salts per— mitted their separation from the sugars, which in concentrated solutions were difficult to handle and also gave acidic degradation products when passed through the Dowex»1 column,kflthe use of cold ethanol instead of hot ethanol eliminated partial esterification of the acids (17, 10%), and if no saponification was subsequently used, the quanti- tative determination would not be accurate. The pigment was 32 A ~— «‘9 A n. p _e 33 almost completely removed by the use of longer cation ex— change resin columns (20 cm). The modified ion exchange procedure used in this study was found to be more convenient and resulted in better fractionation and recovery of the organic acids of cherries than other methods. Palmer (93) has reported that the ion exchange chromatography has several advantages over silica gel methods for the determination of organic acids in plant materials. A. QUALITATIVE ANALYSIS The acids studied are listed in Tables 1 and 2 with their Rf values. The order of emergence of pure acids from the anion exchange column is shown in Figure 2. These posi- tions were determined by passing through the column a mixture of 8 — 10 known acids at a time. The identity of the acids of each peak was established by the use of paper chroma— tography and chemical identification wherever possible. It was found that in a mixture of acids, generally the indivi— dual acids left the column in a specific order (mono—, di-, and tri—basic acids) and that these separations were quite clear. These results were in agreement with those reported by Hulme and Wooltorton (59). The exceptions were certain unsaturated acids (chlorogenic acids) and keto acids (pyruvic acid). Of all the acids studied, only glyceric, glycolic, and glutaric acids did not separate into distinct peaks and FLOWING CHROMATOGRAM OF PURE ACIDS n C FIGURE 31+ A. OINHOOHOTHO EEO _ too 360 80 120 160 200 290 280 MO aisvwns OINEOOHOTHOOSI armssoaomuoosm ._ OIHIIO R ornoqvw - OIHVIHVI 4 V OITVW ‘ OITVNVHIIO OINIOORS OINOHDOHTO ‘ aravmnia OITOOATO OIHHOXTO OINOHHLOVTVO a ammo C OIIOVT ‘onIxIHs — OILHVdSV' omvmnrm R l I l m (\l ‘— HOEN N LO'O ‘ 'Iw _320 Volume - ml. 35 lactic acid came out with shikimic acid. The peaks obtained when the cherry acid extract was passed through the column are shown in Figure 3. Comparing the effluent volumes and the Rf values obtained with those of the pure acids, the following acids, in order of emergence from the column, have been identified in red tart cherry (variety Montmorency): glutamic, aspartic, lactic (occasionally), shikimic, quinic, galacturonic, glyceric, glycolic, glutaric, glucuronic, succinic, citramalic (occasionally), malic, tartaric (occasionally), malonic, citric, neochlorogenic, isochlorogenic, fumaric (occasionally), chlorogenic and phosphoric. Most of the groups of acids which could not be separated by ion exchange were subsequently resolved by paper chromatography. Such groups were the glyceric, glycolic and glutaric, succinic and glucuronic, citric and malonic, and neochlorogenic and isochlorogenic. Reducing the size of fraction in column chromatography in the hope of separating these groups was not very successful. Glycolic, glyceric, glutaric, and malonic acids were detected only when the paper was heavily Spotted. Since citric and isocitric acids cannot be separated by ion exchange and paper chromatographic methods, the fraction containing citric acid was chromato— graphed using silica gel chromatography (81). No isocitric acid was found to be present in the cherry extract. Glucuronic C) ’j— ,1” O C) [j OIHOHdSOHd .32 ornasoaoqno “’ -. S JIHVNRE -(g OINEOOHOTHOOSI (fl arussosoqnaoam rt? aruomvw ‘aiaiio C) rat OIHVIHVI --=:{{: o ‘10 “a nun-... a. OITVW OITVNVHIIO OINIOOOS OINOHLIOO’IO l 53 arsvrnis OITOOATO OIHHOATO C) OINOHOTOVTVO “EB OINIOO 80 OIIOVT ‘OIWIHIHS OIIHVdSV a mum) 3 v l l l I O O In Aitptae pezKIeue hem QQL/bew’ ml. 'Voliune — FIGURE 3. RRIES fl J TITRATION OF COLUMN CHROMATOGRAPHIC FRACTIONS OF ACIDS OF MONTMORENCY CHE COPPER TREATMENT 1962 — 3.7 acid usually gave double spots on the paper chromatograms. Partridge (99) has reported this due to the presence of glucuronolactone in the solutions. Three polyphenolic acids, neochlorogenic, iso- chlorogenic, and chlorogenic acids were identified not only by paper chromatography (comparing the Rf values of knowns and unknowns), but also by their fluorescence under ultra— violet light and the products identified after alkaline hydrolysis. 0n alkaline hydrolysis, all three chlorogenic acids showed the presence of caffeic and quinic acids in the hydrolysates thereby excluding the possibility of being caffeoylshikimic acids. Maier (80) recently indicated that it is impossible to differentiate caffeoylshikimic acids from caffeoquuinic acids on the basis of Rf values and ultraviolet Spectra. No caffeic acid was ever detected in red tart cherries. Either caffeic acid is absent in red cherries, or is present in such trace amounts that it could not be detected by the methods used. One reason why caffeic acid does not accumulate as the other phenolic acids, is possibly because of its greater metabolic activity. Phosphoric acid was the only inorganic acid and glutamic and aSpartic acids were the only amino acids that were deter— mined by this procedure. 38 B. QUANTITATIVE ANALYSIS The various fractions were titrated for total acidity and paper chromatograms of the titrated fractions were run, to confirm the identity of the acids in each fraction. Solutions of pure acids (of the acids identified in cherries) were prepared and divided into two equal aliquots. One aliquot was titrated directly and the other put through the column and then titrated. The recovery of each acid from the anion exchange column was then calculated from the titer values thus obtained. Recoveries were also determined with groups of two or three acids applied at a time on the column. There was no difference in the recovery figures obtained in the two ways. From these recovery figures correction factors were calculated to convert the acid concentration, obtained by titration, of some of the acids to actual concentrations. Table 1 gives the results of the recoveries for pure acids studied. From this table it is clear that this method was most precise for aspartic, shikimic, quinic, glyceric, glycolic, succinic, citramalic, malic, citric, malonic, fumaric, and phosphoric acids. It was fairly precise for glutamic, galacturonic and glutaric, but not for glucuronic, chlorogenic, and probably its isomers, in which case the necessary correction factor was applied. Isochlorogenic acid, isolated from green coffee beans, even after purifica— tion showed some contamination, both on the column and paper; 39 pure neochlorogenic acid was available only in very small amounts, so no recovery figures were obtained for them. Since isochlorogenic and neochlorogenic acids are isomers of chlorogenic acid, the same correction factor was applied to them as was applied to chlorogenic acid. A tentative structure for isochlorogenic acid has been given as 5—caffeoquuinic acid (6) (a position isomer of chlorogenic acid which is a 3-caffeoquuinic acid), while no structure has been yet pro— posed for neochlorogenic acid, except that it also is probably a position isomer of chlorogenic acid. No correction was made for glutamic and aspartic acids because of poor and variable recoveries from the Dowex-50 resin. The recoveries ranged from 12 to 1h% for glutamic and 2 to h% for aspartic acid. No explanation can be given for these poor recoveries. The use of Dowex-1 resin from different lots did not make any difference in the elution titration curve, as to the position of each acid peak and the recovery of the acid. For the quantitative determination of the acids, the titer values (ml. base used), under each peak, were added together and the necessary correction from the recoveries was applied. The pK values of all cherry acids are tabulated in Table 1. Since the pK values of all acids were well below 8.5, all carboxyls were titrated with NaOH using phenol— phthalein as an indicator. The phosphoric acid titration #0 values were multiplied by 3/2 because only two—thirds of this acid was titrated (81). After the corrections were made for the recoveries, the ml. base used for the titration of each acid peak was then converted to milliequivalents of acidity. This gave the milliequivalents (meq.) of acidity of each acid in the total acidity applied on the column. From this, the meq. of each acid per 100 meq. of total acidity was calculated. Also, the meq. of each acid per 100 gm of fresh cherries was calculated from this since the meq. of total acidity per 100 gm of fresh fruit at each stage of maturity was known. Using this last step and the equivalent weights of the acids, mg of each acid per 100 gm of fresh fruit was obtained. C. TOTAL TITRATABLE ACIDITY The cherries harvested 53 days after full-bloom, in 1960, were less deveIOped than those harvested in 1961 and 1962, #5 days after full—bloom. However, the total number of days required for fruit maturation for the 1960 fruit was less than those required in 1961 and 1962 because of slightly higher average daily temperatures. The changes in total titratable acidity (free and bound) as milliequivalents per 100 gm of cherries during the development of the fruit are shown in Figure H. The values are averages of 1961 and 1962 data for a similar treatment HzmzH I C? Aitptoe pezAIeue bem COL/Dew 65 in its quantity as the fruits matured. The maximum level of this acid occurred a week after the commercial harvest. (Figure 22) Citric Acid: Citric acid showed a certain pattern during fruit development in 1961 and another in 1962. There was a high concentration of citric acid in the green fruits in 1961, decreased until the fourth picking and then leveled off. But, in 1962, it started with a low concentration in the green fruits, showed peaks at the third and the fifth pickings, and then leveled off (Figure 21). In 1961, changes in citric acid showed an inverse change relation to those in malic acid (only in the first six pickings), while in 1962, there was a positive inverse change relation be— tween the two acids throughout the period of fruit development. Polyphenolic Acids: The combined values of the three polyphenolic acids, both in 1961 and 1962, Showed a high concentration in the green fruits, dropped gradually until the day of the commercial harvest, and then increased Slightly after that. (Figure 23). Phosphoric Acid: In both 1961 and 1962 seasons, phOSphoric acid showed a general decline until the fourth picking, a peak at the fifth, and then decreased, reaching a maximum in the overripe fruits (Figure 2%) In general, both in 1961 and 1962, phosphoric acid Showed an inverse change relation to malic acid and a similar trend to that of citric acid. 66 m©©eieome i onafimDH.U.\.. . ...\ of . 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