Determination of the Acids of Plant Tissue 11. Total Organic Acids of Tobacco Leaf GEORGEW. PUCHER, HUBERT BRADFORD VICKERY,AND ALFREDJ. WAKEMAN * Connecticut Agricultural Experiment Station, New Haven, Conn. of a s b e s t o s a r e a d d e d a n d The organic acids of plant tissues can be thoroughly incorporated, and the plant tissues have long quantitatively extracted by ether, provided the mixture is transferred, with the been known to contain aid of conical funnel constructed 1 with material is acidfied to approximately p H consider a b l e proportions of of pa er and impregnated with sulfuric acid. The extract so obtained contains p a r a h i n , t o a 26 X 60 mm organic acids. C i t r i c , m a l i c , Schleicher and Schull extraction’ tartaric, succinic, and oxalic no signficant amounts of organic bases or of thimble. Small particles of maacids are usually the most abunmineral acids, with the exception of nitric acid. terial remaining are brushed in, dant, and the wide distribution and the glassware and funnel are The quantity of organic acids can be determined wiped with a small piece of of these particular acids is well in the presence of the nitric acid by titration bemoistened surgical cotton which is recognized. Surprisingly little then used as a pIug for the thimble. tween the limits p H 7.8 and 2.6, according to the that is definite is known of the The thimble is placed in a siphon functions of these organic acids principle of the method of Van Slyke and Palmer, tube of the t y e designed for rubber analysis bimer and Amend in the plant; their chemical reby means of a quinhydrone electrode and potenticatalog No. 30754), a short thin lationships to each other and to glass rod is thrust between the ometer system. Under the conditions adopted the protein and carbohydrate paper and the glass tube t o prooxalic acid behaves as a monobasic acid, a m e t a b o l i s m a r e still largely vide a space for the accumulation of ether, and the tube is suspended method is therefore provided whereby the oxalic matters of speculation. by a Ioop of fine galvanized iron The d e t e r m i n a t i o n of the acid can be independently determined and the wire close under the spiral coil of o r g a n i c acids of plant tissues the metal condenser. The connecessary correction calculated. The other acids presents an analytical problem denser i s furnished with a soft usually encountered are titrated to the extent of cardboard gasket and is placed on of c o n s i d e r a b l e complexity the conical extraction flask, being approximately 90 per cent. A correction factor because of the wide variety of held firmly in position by three applicable to the acids that occur in largest acidic substances that may be spring paper clips. The flask conpresent. Furthermore the ortains about 150 cc. of specially amount in tobacco leaf tissue is given. Only a p r e p a r e d ether. Extraction is ganic a c i d s must, in general, minor modfication of this factor would be recarried out on an electric hot be estimated in the presence of plate, so adjusted that the ether quired to permit the application of this method more or less m i n e r a l a c i d , a siphons back at least 40 times an to other tissues. circumstance that places restrichour, for 17 to 20 hours. The ether for this extraction is tions on t h e u s e of o r d i n a r y repared by being washed three times with water to remove alcotitration methods. The present communication describes iol; each liter of ether is subsequently shaken vigorously with a method whereby the total acidity due to these organic 200 cc. of 10 per cent sodium hydroxide and 5 to 10 grams of acids may be estimated. This method, used in conjunction powdered potassium permanganate (excess), and is then distilled. with methods for the determination of the individual acids, This treatment is essential for satisfactory results, and it is imyields data of considerable value for the interpretation of portant that the ether should have been prepared not more than hours before use. results obtained in studies of organic acid metabolism. The 24The ether extract is treated with 25 cc. of carbon dioxidemethod is founded on the observation that the organic acids, free water (previously boiled and stored in a bottle protected by together with the nitric acid contained in tobacco leaf tissue, soda lime), and 2 cc. of carbon dioxide-free, approximately 5 N sodium hydroxide. The flask is gently agitated t o insure recan be extracted by ether in an efficient extraction apparatus, moval of the acids into the aqueous phase, and the ether is careprovided the tissue is previously acidified with sulfuric acid. fully distilled off. The aqueous solution is transferred to a 100The acidity due to the organic acids can then be determined cc. flask and diluted to the mark with carbon dioxide-free water. by titration between the pH limits 7.8 and 2.6, according to This solution is “the organic acid fraction.” Parallel the principle of the method of Van Slyke and Palmer (6) for the determination of the organic acids of urine, Owing with the determination an equal quantity of ether is refluxed to the dark color of the solutions usually obtained from plants, for the same period of time, and subsequently carried through this titration is best conducted with the aid of a quinhydrone the same series of operations; the solution obtained is referred to below as the “ether blank.” electrode.
A
QUEOUS e x t r a c t s of
ORGANIC ACID EXTRACT In order to ascertain the amount of 4 N sulfuric acid required to acidify the tissue to pH 0.G to 0.9, preliminary trials are carried out in which a small quantity (1 cc.) of the acid is mixed with 0.5 gram of the tissue and sufficient water is then added so that a thin suspension of the powdered material can be transferred to the quinhydrone electrode vessel for measurement; suitable changes in the quantity of acid are made as suggested by the result of the first test, and the amount required for 2.0 grams of the tissue is caIculated. A 2.00-gram sample of the dry and finely ground tissue is then intimately mixed with this amount of 4 N sulfuric acid, 3.5 grams PREPARATION O F
ELECTROMETRIC TITRATION OF ORGANIC ACIDS The potentiometer circuit used in this laboratory employs a Leeds and Northrup student’s hydrogen-ion potentiometer. A galvanometer shunt of approximately 8700 ohms resistance is provided; this is indispensable for rapid titration. The reference elctrode is a saturated calomel half-cell ; connection between this and the titration vessel is established by &nagar bridge prepared according to Michaelis and Fujita (3). The electrodes are constructed by sealing 1 cm. of No. 22 platinum wire into the end of a 10-cm. length of 4-mm. diameter soft glass tubing. These electrodes must be free from cracks. They are cleaned by being boiled a short
140
March 15. 1934
,INDUSTRIAL AND ENGINEERING CHEMISTRY
time in 50 per cent nitric acid, and then in 5 to 10 per cent sodium acid sulfite (I), and are stored under distilled water. Before use, each electrode is allowed to stand at least 5 minutes in water that contains a little quinhydrone. Each electrode should be checked against a known buffer solution, or by titration of a standard solution of an organic acid; any that show a sluggish end point should be discarded, the fault in such cases being usually a minute crack. To conduct the titration, a 10-cc. aliquot of the “organic acid fraction” is transferred to a 50-cc. beaker that has a calibration mark at 25 cc., and 2 drops of a 1 er cent alcoholic solution of phenolphthalein are added; 0.5 $acid is added from a pipet until the red color is discharged; an electrode is introduced and 10 drops of quinhydrone solution are added.’ The positive wire from the potentiometer is connected to the platinum electrode, the negative wire to the saturated calomel half-cell, and the electrical circuit is completed by means of an agar bridge. The potentiometer circuit is then checked for balance, and the potentiometer scale is set at 0.00 millivolt (pH 7.8). Dilute alkali (0.05 N) is added drop by drop until no deflection of the galvanometer is obtained after careful stirring of the contents of the beaker. The solution is now at pH 7.8. The potentiometer is adjusted to 301 millivolts (pH 2.6), and 0.1 N nitric acid is rapidly added from a buret with stirring until the galvanometer deflections become small, and then drop by drop to zero deflection. The solution is finally diluted to the 25-cc. mark with carbon dioxide-free water, and the titration is carefully completed. The 0.1 N nitric acid used represents the titration value of the organic acids, and of 25 cc. of water, between the limits pH 7.8 to 2.6. A 10-cc. ali uot of the “ether blank” solution is then titrated with the same iectrode, and the titration value obtained is subtracted from that of the organic acid fraction. OXALICACID CORRECTION Oxalic acid behaves as a monobasic acid under the conditions described, only the weaker acid group entering into reaction; consequently it is necessary to carry out an independent determination of oxalic acid in order to correct the final result for the whole of the acidity due to this acid. To this end a 25-cc. aliquot of the organic acid fraction is acidified t o Congo red with 0.5 N hydrochloric acid, the recipitate that forms is allowed to flocculate and is filtered offon asbestos in a Gooch crucible and washed with water. A drop of methyl red solution is added t o the clear filtrate. Ammonium hydroxide is added to a faint alkaline reaction, 2 to 3 cc. of glacial acetic acid are added, followed by 5 cc. of 10 per cent calcium chloride solution. After being allowed to stand at least 2 hours the calcium oxalate is filtered on asbestos in a Gooch crucible, and is washed with a little very dilute ammonium hydroxide. Crucible and contents are then transferred t o a 100-cc. beaker, 5 cc. of 50 per cent sulfuric acid and 20 cc. of water are added, and the solution is heated to boiling and titrated with 0.02 N potassium permanganate. The oxalic acid is calculated from the relation, 1 cc. of 0.02 N permanganate is equivalent to 0.9 mg. of anhydrous oxalic acid. Under these conditions oxalic acid can be rapidly and accurately estimated in the presence of a large excess of citric acid. Data in support of this statement have been given by Vickery and Pucher (6). CALCULATION OF TITRATION DATA The proportion of an organic acid that is titrated between two given hydrogen-ion activities can be calculated from the dissociation constants of the acid. Calculations with respect to the acids that require consideration in the titration of leaf extracts, and to the pH limits the authors have arbitrarily adopted, are shown in Table I. The titration value of a leaf extract obtained as described above, expressed in terms of 0.1 N acid, contains an error owing to the fact that none of the common leaf acids titrate to the extent of 100 per cent. A further correction for oxalic acid must also be applied. 1 Acetone is treated with aqueous sodium hydroxide and excess of potassium permanganate and dietilled: 0.167 gram of quinhydrone is dissolved in 3 cc. of this acetone and stored in a test tube fitted with a medicine dropper. The solution should be discarded if it has stood more than 3 days.
141
It is convenient to express the acidity in terms of milliequivalents per 100 grams of dry tissue; substitution of the analytical values into the following equation yields the desired result: [(A - B ) 5 0 - 0 ~ g ] 1 . ~ 9 + - = 2 c& f* . E . 0.09
A = Cc. of 0.1 N nitric aiid required in the titration of a 10-cc. aliquot of the “organic acid fraction’’ B = cc. of 0.1 N nitric acid required for a 10-cc. aliquot of the “ether blank” C = per cent of oxalic acid in the dry tissue M . E. = milliequivalents of organic acid in 100 grams of dry tissue A - B gives the titration value of the acids in one-tenth of a 2-gram sample of tissue in terms of 0.1 N acid; the factor 50 converts this to milliequivalents per 100 grams; division of C by 0.09 converts the percentage of oxalic acid ( M . W.= 90) to milliequivalents per 100 grams, if oxalic acid is considered as a monobasic acid. The factor 1.09 is calculated from the observations (Table I) on the titration of malic and citric acids and represents as close an approximation as is necessary to the ratio between the actual titration value over the selected pH range, and the true amount of organic acidity present in tobacco leaf tissue. I n the case of tissues that contain tartaric acid as an important constituent a somewhat larger factor would be required. The first term of the equation, then, represents the acidity due to the organic acids exclusive of oxalic acid; to this is added the acidity of oxalic acid calculated as a dibasic acid.
EXPERIMENTAL FOUNDATION OF METHOD The selection of the quinhydrone electrode rather than the hydrogen electrode scarcely needs justification in view of the convenience and wide applicability of this system. The choice of the upper limit of the titration (pH 7.8) was made because, a t reactions more alkaline than this, the quinhydrone electrode changes polarity and becomes increasingly unreliable. The lower limit was arbitrarily selected because sharp end points could be obtained and the blank correction was not unduly large. Titration to pH 2.3 was less satisfactory. TABLE I. PROPORTIONS OF CERTAIN ACIDS TITRATED BETWEEN LIMITSPH 7.8 AND 2.6
PROPOR-
DISSOCIA-
ACID-FREEAT EXPON~NTS PH 7.8 PH 2.6
TION
FOVNDBY
TION
ACID 2-Malic Citric Oxalic &Tartaric Fumaric Maleic Succinic Lactic Acetic Malonic Boric Phenol HsPO4[H(NaHPO4)]
TITRATION
PK
%
%
3.48 5.11 3.08 4.39 5.49 1.42 4.39 3.00 4.39 3.03 4.49 1.93 6.58 4.18 5.57 3.86 4.73 2.80 5.68 9.2 10.0 2 x 10-7
0.009
93.8
93.5
0.005
89.7
90.0
0.00
49.6
50.5
0.08
85.0
84.0
0.00 0.00
83.9 ,
58.8
83.6 ,
56.0
0.05
98.6
98.5
0.00 0,008
94.8 99.3 76.0
.... ..
100.0 100.0
5.0 0.2 90.0
0.00
96.4 99.3 7.3
100.0
The final adjustment of the end point must be made in the presence of a constant volume of water. Experiments in which different amounts of water were titrated showed that a variation of *l cc. of water involved a variation of t0.03 cc. of 0.1 N acid; consequently, if a precision of the order of 0.1 cc. of acid is sought, the final volume must be adjusted to within 3 cc. of the standard volume selected. This is easily accomplished by calibrating a 50-cc. beaker a t 25 cc., and making the final adjustment of the volume with a little
ANALYTICAL EDITION
142
care. The h a 1 increment of acid required, if the titration is conducted as described, is usually less than 0.5 cc. Nitric acid was selected as the titrating reagent because it is slightly stronger than hydrochloric acid. Table I gives the results of titrations of a number of acids, most of which may be encountered in work with plant extracts; of these the first four are commonly the most plentiful. The titrations were conducted on 0.1 N solutions of the acids. Oxalic and maleic acids titrate as monobasic acids; only the former need be considered here, however, as maleic acid has not hitherto been detected in plants. I n tobacco leaves malic, citric, and oxalic acids predominate, these three usually making up at least 70 per cent of the total organic acid acidity. The present discussion of the correction factor to be employed will be restricted to results secured with this tissue. The application of the method to extracts from another tissue of different organic acid make-up would require, however, only a slightly modified factor. The ether extract obtained as described from tobacco leaf contains no bases that can be precipitated by silicotungstic acid. The chief mineral acid present is nitric acid which is quantitatively extracted from the tissue under these conditions (4). Traces of other nitrogenous substances are also present, but these, on the average, amount to only 3.6 mg. of nitrogen in the extract from 2 grams of dry tissue, and their influence on the titration can be neglected. Carbonic, sulfuric, phosphoric, and hydrochloric acids, if present, would affect the titration. Carbonic acid is expelled during the extraction period, and its subsequent advent is carefully guarded against. A trace (0.4 to 0.6 mg.) of sulfuric acid is, in fact, extracted, but control experiments indicated that the titration of this between the limits adopted would consume only about 0.01 cc. of 0.1 N acid, Ordinarily hydrochloric acid is present in plant tissues in very small amounts and, since this acid is titrated only to the extent of 3 to 5 per cent, no significant error can enter. Many experiments designed to test the effect of phosphoric acid on the titration showed that the presence of this acid can also, in general, be neglected. Phosphoric acid is extracted to the extent of about 15 per cent under the conditions described. A 2-gram sample of tobacco leaf tissue usually yielded from3 to 6 mg. of phosphoric acid in the extract, and the titration of this quantity of phosphoric acid under standard conditions required from 0.5 to 1.0 cc. of 0.1 N acid. Thus the error in the titration of a 10-cc. aliquot of the organic acid fraction might be from 0.05 to 0.1 cc., or from 2.5 to 5 per cent too high, if the titration value is 2 cc. Ordinarily the titration value is somewhat more than this, so the phosphoric acid error is not great. Should occasion arise, however, it can be easily eliminated by the introduction of a correction. To this end an aliquot part (10 to 25 cc.) of the fraction is acidified with sulfuric acid, filtered, neutralized, and the phosphoric acid in it is determined by the method of Fiske and Subbarow (2). The correction is calculated from the relation mg'
* 3*16
5.9
= cc. 0.1
N acid required in aliquot used
and is subtracted from the titration value observed. In order to test the completeness with which citric, malic, and tartaric acids can be extracted by ether from aqueous solution and subsequently recovered by titration, a mixture of approximately equal parts of these three acids was prepared of such concentration that a 5-cc. aliquot part of the solution should contain the equivalent of 25.03 cc. of 0.1 N acid. Seven titrations gave an average titration value of 21.98 cc. with a maximum variation of *0.2 cc. tSimilar quantities of the solution were acidified to pH 0.8, and were mixed with asbestos in the usual way, dried in a vacuum desiccator until the material could be conveniently trans-
Vol. 6, No. 2
ferred to the extraction thimble, and were then extracted with ether for various times. The results are shown in Table 11. TABLE11. EXTRACTION AND TITRATION OF A MIXTUREOF MALIC, CITRIC,AND TARTARIC ACIDS (If these acids titrated to the extent of 100 per cent the titration value would be 25.03 cc. Data are expressed in cc. of 0.1 N acid and are corrected for water blanks.) TIMH~ OF TITRATION VALUP EXTRACTION BBTWEEN PH 7.8-2.6 RECOVERY Hrs. cc. % 0 21.98 8719 4 20.88 83.4 20.75 83.2 6 21.75 8 86.9 22.48 12 89.7 22.08 88.7 16 22.13 24 88.4 72 22.25 88.9 22.20 93 88.7 22.25 120 88.9
It is clear that most of the acid is extracted in a relatively short time and that constant titration values are obtained after 12 hours of extraction. A 16- to 20-hour period has therefore been adopted for practical work. The average recovery of the acids after 16 hours' extraction was 88.7 per cent. Calculation from the data for the dissociation constants of these acids shown in Table I indicated that a 90.8 per cent recovery might have been expected; evidently therefore these acids can be satisfactorily extracted and estimated by the technic described. As a further test of the method a standard solution of citric acid, acidified with sulfuric acid to pH 1, was treated as described, and extracted for 16 hours. The results are recorded in Table 111. The average recovery was 97.7 mg. from 96 mg. taken. Analysis of the extracts by the pentabromoacetone method indicated the presence of 94 to 97 mg. in the different samples. The method may therefore be expected to yield results of an accuracy of *3 per cent. TABLE111. EXTRACTION OF CITRIC ACID CITRICACID TAKE%
TITRATION VALU~
cc.
Mo.
CITRIC Acrn FOUND
CITRICACID FOUND"
CORRICTAD~
MQ.
MQ
.
14.2 90.9 100.0 96 96 13.8 88.3 97.1 96 13.7 87.7 96.5 97.5 96 13.8 88.3 Cc. 0.1 N acid X 6.4 = mg. citric acid. b Corrected on the assumption that 90 per cent of the citric acid is titrated between pH 7.8 and 2.6.
TITRATION OF ACIDS OF TOBACCO LEAP I n order to establish the conditions for quantitative extraction of organic acids from leaf tissue, 2-gram samples of cured tobacco leaf, and of fat-free tobacco-seed meal, were extracted before and after the addition of citric, or of oxalic acid. Although only B small quantity of acid was added to the leaf tissue, the recoveries, shown in Table IV, were satisfactory. TABLEIV. RECOVERY OF ORGANIC ACIDSADDEDTO PLANT TISSUE QAMPLB
(Data are expressed in cc. of 0.1 N acid) c ORQANICACIDS Originally present Added Found Recovered CC.
cc.
cc.
Cured tobacco Cured tobacco
11.1 11.1
1.25" 1.25
12.15 12.20
Curedtobacco Curedtobacco Cured tobacco Cured tobacco
9.9 9.9 9.9 9.9
2.50) 2.50 2.50 2.50
11.2 11.1 11.3 11.1
Tobacco-seed meal Tobacco-seedmeal Tobacco-seedmeal Tobacco-seed meal
1.2 1.2 1.2 1.2
6.25a 12.50 12.50 12.50
7.0 12.6 12.6 12.7
a
Citric acid added.
6 Oxalic acid added.
CC.
-
%
1.05 84 1.10 88 Av. 86 1.3 52 1.2 48 1.4 56 1.2 48 Av. 60.8 93 5.8 11.4 91 11.4 91 92 11.5
Av. 91.7
RECOVERY
TH~ORY
% 90 50
90
March 15,1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
143
TABLEV. ORQANICACID CONTENTOF CUREDTOBACCO data are presented to illustrate the very satisfactory reproLEAF ducibility of the results of the titration. TITRATION SAMPLE 627 628 623 624 625 626
TITRATION VALU~
cc.
4.50 4.55 5.45 5.50 4.60 4.45 4.80 4.80 4.95 4.95 3.90 3.90
VALUE$ ORQANIC ETHERB L ~ N B : ACIDS cc. M , E./100 Grams 0.70 190 0.75 190 0.75 235 0.75 237.5 0.76 192.5 185.0 202.5 0.75 202.5 0.70 212.5 0.76 210.0 0.75 157.5 157.5
Of a series Of duplicate determishows the nations on a number of samples of cured tobacco. These
LITERATURE CITED (1) Coons,C. C., IND.ENQ.CHIOM., Anal. Ed., 3, 402 (1931). (2) Fiske, C. H., and Subbarow, Y., J. Bid. Chem., 66, 375 (1925). (3) Michaelis, L., and Fujita, A., Biochem. Z.,142, 398 (1923).
(4) Pucher, G . W., Vickery, H. B., and Wakeman, A . J., J . Bid.
Chem., 97, 605 (1932). (5) Van Slyke, D. D., and Palmer, W. W., Ibid., 41, 567 (1920). (6) Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bull. 352, 669 (1933). RECEIVED October 30, 1933. The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Washington,
Pectin Studies 11. Sugar-Acid-Pectin Relationships and Their Bearings upon Routine Evaluation of Apple Pectin R. STUEWER, N. M. BEACH,AND A. G. OLSEN General Foods Corporation, Battle Creek, Mich., and Fairport, N. Y. The interrelationship of acid concentration, sugar in the same gelometer readings. The elasticity of the concentration, and pectin concentration in apple apple jellies prevents a sharp break and results in pectin jellies is outlined, and it is shown that the opti- high readings on the instrument and f o r this reason mum p H of apple pectin jellies prepared by the usual the standard curves previously given f o r citrus pectin method varies with the concentrations of acid and jellies are not suitable for evaluating apple pectin of pectin. The changes in the position of the opti- jellies. An average curve for 100-grade apple pectin mum p H with variation in pectin concentration indicates a value of 50 Tarr-Baker units for 2.6 change the slope of the logarithmic jelly-strength- grams of pectin in 555 grams of jelly. A method of preparing apple pectin jellies which pectin-concentration curves to such a n extent as to render exact evaluation dificult. apparently obviates most of the diflculties of mainApple pectin and citrus pectins of the same com- taining optimum acid conditions and which permercial grade, or of diferent grades but used in mits ready routine comparisons of samples is deequivalent amounts in standard jellies, do not result scribed.
I
NAN EARLIER publication (8) Olsen presented a simple routine procedure for the evaluation of citrus pectin, in which the acidity was maintained constant. With apple pectins it is, however, necessary to prepare test jellies a t the so-called optimum pH. Where several samples prepared by the same method are to be compared, this is usually accomplished by adding predetermined standard amounts of acid. The acid requirements, however, change with the age of the sample and with the method of preparation, hence when the history of the sample is unknown, it is necessary to determine the optimum acidity. This may involve an extensive series of jellies. A method which would obviate such prior determination should therefore prove of considerable help in the evaluation of samples for routine or research purposes. The well-known data of Tarr (6) and Baker ( I ) suggest such a high degree of sensitivity of apple pectin towards moderate changes in acidity that the routine method proposed for citrus pectins cannot be expected to be applied without modifications in the evaluation of apple pectins. The data of these workers, in so far as they relate to the optimum pH of apple pectin jellies prepared by the usual “hot” method (6), are in essential agreement with unpublished data accumulated in the authors’ laboratories over a period of years.
The general procedure described by Olsen (3) was used in obtaining the data here presented except for such modifications as are mentioned specifically. Two typical jellystrength curves for alcohol-precipitated apple pectin jellies containing 60 per cent of sugar are shown in Figure 1. Identical series were run with lactic and tartaric acids, using a newly prepared sample of pectin. The two curves coincide closely. Definite pan gelation or curdling occurred in each case a t a pH of about 2.55. The authors’ observations invariably indicate a progressive change in the optimum pH towards higher acid requirement during storage of samples at ordinary temperatures. Studies were subsequently undertaken to observe the interrelationships of acid, pectin, sugar, and temperature over a range sufficiently wide to permit drawing general conclusions and if possible provide a basis for the simplification of the study and evaluation of samples of apple pectin with varying histories, SUGAR-ACID RELATIONSHIP Spencer (6) has a t some length reviewed the more important data and current hypotheses concerning the roles of sugar and acid in pectin jellies. I n 1922 Singh (4) showed that with citrus pectin the amount of sugar necessary barely
