August. 1923
INDUSTRIAL A N D ENGINEERING CHEMISTRY
775
Effect of Dialysis on Direct Crystallization of Citric Acid from Lemon Juice' By H. D. Poore LABORATORY OF FRUITAND VEGETABLE CHEMISTRY, BUREAUOF CHEMISTRY, U. S. DEPARTMENT OF AGRICULTURE, Los ANGELES, CALIF.
N T H E commercial The water was changed Preliminary experiments using collodion sacs in dialyzing every 3 or 4 hours during the production of citric acid lemon juice, followed by dialysis on a large scale in an osmogene daytime, and the dialysis from lemons, the acid containing collodion-impregnated cloth membranes, gave the followwas continued until the is precipitated from the ing results: dialysate contained no acid. clarified juice with calcium ( I ) The small quantity of colloids present in lemon juice does The contents of the sacs, carbonate, and the calcium not affect crystallization, but the ash and other nonacid constituents which was colorless, was citrate formed is decomprevent satisfactory crystallization of concentrated fermented juice. then removed and evapoposed with sulfuric acid. ( 2 ) In the presence of ash and other impurities, needle and leaflet rated to dryness, and the The calcium sulfate is filmodifications of citric acid crystals are obtained: upon the remocal total colloids were obtained. tered off and the citric acid of half of the ash, characteristic citric acid crystals are formed. After ashing, the organic solution is concentrated ( 3 ) At the concentration necessary for crystallization, the mother colloids were calculated. and crystallized. Although liquor is so viscous that the crystals cannot be separated. Duplicate determinations only two major chemical checked closely. reactions are involved in the process, a large number of operations necessitating careful The effect of different methods of treatment and filtration supervision and laboratory control must be carried out in order upon the colloid content of lemon juice is shown by the reto secure acid complying with the Pharmacopeia require- sults given in Table I. mentse2 TABLE I-EFFECT OF METHODOF TREATMENT AND FILTRATION ON COLLOIDAL CONTENT OF LEMONJUICE The average quantity of total acid present in a ton of lemTotal Organic ons is 70 pounds. About 85 per cent, or 60 pounds, of this Lot KINDOF Colloids Colloids No. JUICE TREATMENT % % is obtained from the juice and maceration water. The final 1 Fresh Filtered through filter paper: still yield of crystallized acid varies from 40 to 45 pounds, showing cloudy 0.078 0.072 2 Fresh SameasNo. 1. dialyzedat2.5°C 0 , 0 7 9 0.071 a loss during the process of 15 pounds or more per ton of fruit. 3 Fresh Filtered repeatedly through as: bestos until clear 0.039 0.036 Lemon juice has approximately the following composition : 4 Fresh Filtered hot through paper with 6 per cent of total acid, nearly all of which is citric; 2 per 0.033 0.030 2 8 per cent kieselguhr 5 Fresh Filtered hot through paper with cent of sugars, mostly invert; 0.3 per cent of protein; 0.4 2.8 per cent kieselguhr and 1.6 per cent kelpchar 0 , 0 2 2 0.021 per cent of ash; and 0.8 per cent of undetermined matter. 6 Fermented, 0.036 0.034 The Undetermined matter consists largely of gummy and pecclear . . . . . . .. 7 Fermented, Filtered hot through paper with tic substances. clear 1.4 per cent kieselguhr 0.028 0 . 0 2 5 8 Fermented, Filtered hot through paper with Peratoner and Scarlata3 have devised a method of manuclear 2 8 per cent kieselguhr 0.027 0.024 facture in which many of the nonacid constituents of the juice are precipitated by a mixture of alcohol and ether. The colloid content of the juice varied somewhat with the At present, however, this process is not economically possible method of pressing the fruit, high pressures giving the greater in the Ynited States. It was thought that these impurities quantity. might be colloidal and that purification might be obtained I n order to determine whether it would be possible to through dialysis. separate part of the ash and sugars from the citric acid in lemon juice by fractional dialysis, the following experiment DETERMINATION OF COLLOIDS IK LEMON JUICE was conducted : A sample of 250 grams of fresh lemon juice, containing 4 review of the literature on diffusion and separation of colloids from solution showed that the most satisfactory 6.2 per cent of total acid, 2.1 per cent of total sugars, and membranes were made of ether-alcohol collodion. A com- 0.3 per cent of ash, was dialyzed. The dialysis was carried plete bibliography of their development is presented by on in a beaker containing 700 grams of distilled water mainBigelow and Gemberling. Many other investigators, tained a t 80" to 90" C. At given intervals the dialysate including Bechold,6 Schoep,' Walpole,g Fouard,g in the beaker was weighed and 10 samples were removed and Eggerth,Iohave deveIoped methods for producing graded for analysis. During the course of the experiment, which ran for 2.5 hours, water was added to the dialysate to reseries of membranes of definite permeabilities. For the determination of the colloids in lemon juice, place that which evaporated. Table I1 shows the results 500-cc. sacs that were impermeable to boiled starch, made of the analyses of the samples, the total quantities dialyzed, from 4 per cent collodion solutions, were used. One hundred and the total percentage dialyzed. By plotting the percubic centimeters of juice were added to each of two sacs, centages against the time, the curves in Fig. 1 are obtained. which mere placed in distilled water held a t 90' to 95" C. These show that the ash dialyzes more rapidly than the acid and sugar during the first hour, although the difference is 1 Received February 9, 1923. not great enough to offera. means of removing part of the ash. 2 Wilson, THISJOURNAL, 1 3 , 564 (1921). As might be expected, the curves of the invert sugars and 3 Molmari, "Organic Chemistry," 2nd ed , part 1, p 414. citric acid are practically the same. J . Am. Chem Soc., 29, 1576 (1907) 5 Bzochem. J., 9, 594 (1915); 11, 40 (1917). DIALYSIS OF PROTEINS Z p h y s i k . c h e m , 60, 257 (1907); 64, 328 (1908). ' K d O i d - Z . , 8, 80 (1911). As the quantity of colloids found in the lemon juice was 8 Biochem J . , 9, 284 (1916). less than a third of that of the proteins, it seemed that most 9 Bull ~ S S O C chzm SUCY dzst., 28, 756 (1911). of the protein was not present in the colloidal state. Ostl o J. Bzol. Chem , 48, 203 (1921).
I
INDUSTRIAL A N D ENGINEEBIXG CHEiWISTRY
776
Vola 15, No. 8
TABLE11-COMPARATIVE RATEOF DIALYSIS OF ACID,S U G A R S , AND ASH OF LEMON JUICE Acr--TOTAL SUGAR AS INVERT--, ASH-----Weight Removed Removed Removed of In E or In for In for Time Dialysate Dialysate Samples Dialyzed Dial sate Samples Dialyzed Dialysate Samples Minutes G. % G. % G. % % G. 10 675 0.50 0,085 21.8 0.16 0.027 20.0 0.033 0.006 20 6370 0.22 0.80 33.4 0.29 0.076 35.6 0.043 0.013 30 698 46.5 1.00 0.39 0.33 0.13 43.9 0,053 0.022 45 6530 1.35 0.62 59.5 0.47 0.21 60.5 0.067 0.033 60 645" 1.55 0.88 68.6 0.51 0.30 66.8 0.068 0.045 90 640 1.70 1.17 75.9 0.58 0.40 76.1 0.070 0.057 120 575 1.90 1.50 78.0 0.64 0.51 77.0 0.080 0.070 150 555 1.95 .... 79.5 0.67 ..... 80.1 0.087 a 100 cc, of water were added t o the dialysate at the end of 20 minutes, 50 cc. at the end of 45 minutes, and 50 cc. after 60 minutes. r
.
k
I
...
waldl' states that some proteins dialyze in acid solution, and the following experiment showed that this is true of the lemon protein: Two collodion sacs were filled with 100 cc. of fermented concentrated juice containing 18.4 per cent of acid and 0.62 per cent of protein. The sacs were placed in 100 cc. of water, maintained a t a temperature of 80" to 90" C. for 5 hours, and then left over night a t room temperature. The dialysates were diluted to 100 cc. and analyzed, with the results shown in Table 111.
Test No.
1 2
TABLE111 --DIALYSATEAcid Protein
Q
70
1217 11.8
0.47 0.52
Acid Dialyzed
Protein Dialyzed
69 64
76 84
%
70
I n order to determine the effect of temperature and agitation of the dialysate on the rate of dialysis of the acid, three tests were made. I n eabh, 100 cc. of fresh lemon juice, containing 6.5 grams of total acid, were pipetted into a sac, which was then placed in 250 cc. of water. Samples of the dialysate were removed a t intervals, and the total amount of acid dialyzed was determined. The results are shown in Table IV. TemperaTest ture No. OC. 20 1 2 82 3a 82 a Agitated.
TABLEIV ----------ACID DIALYZED FOR-----30Minutes 60 Minutes 120Minutes G. % G. % G. 70 ... .. 2.8 43' 3 8 58 3 3 51 4.5 .. 69 3.8 58 5.0 77 . .
..
These results show that agitation increased the rate of dialysis a t 82" C. about 12 per cent, and that 60 per cent more acid dialyzed in one hour a t 82" C. than a t 20" C. The combined dialysates obtained in these experiments were evaporated to about 55 per cent acid. The thick sirup obtained did not crystallize, even after it was seeded with fine citric acid crystals and allowed to stand for several weeks. I n the next test 3 liters of fermented juice which contained only a trace of sugar were dialyzed. Concentrating the dialysate to 68 per cent acid gave a thick sirup, which crystallized after standing for a week. The liquor from these crystals, however, was so thick that it was only partially separated on a Buchner funnel. Upon attempting to wash with ice water, some of the crystals dissolved and only part of the adhering sirup was removed. After filtering with charcoal three recrystallizations were required before pure white crystals were obtained. OSMOGENE AXD M E M B R l l K E
T o facilitate work upon a larger scale, a small osmogene of 13 frames, 25 cm. square, was used. In order to save the lemon juice dialysate, a countercurrent arrangement, whereby the lemon juice passed through the even-numbered frames and the water or dialysate flowed in the opposite direction, through the odd-numbered frames, was adopted. I n this manner, by regulating the flow and employing enough frames, the dialysate should leave the osmogene at the same 11 "Handbook
of Colloid Chemistry," p
270.
Dialyzed
% 32.4 40.7 55.7 66.7 68.6 71.7 75.1 80.4
acid strength as that of the juice entering. Both the juice and dialysate water were heated to about 90" C. Although parchment paper has been used in the sugarhouse osmogenes, it was found, after testing the best grades obtainable, that the life of the paper was too short and that the speed of dialysis was much too slow. Walpole,* Brown,s Hatschek,12 and others have experimented with sheet collodion and collodion-impregnated cloth and paper. Many methods of preparing membranes were tried in order to find one that would be satisfactory. TABLEV-CONCENTRATED DIALYSATES COMPOSITION RATIOOF FINALTO ---PER CENT----ORIGINAL CONCENTRATION-. Determination Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Average Acid 66.91 6 7 . 3 66.11 11.9 12.0 11.7 11.9 Ash 5.20 4.90 4.64 14.9 14.0 13.3 14.1 Sugars 1.05 1.13 1.15 1 3 . 1 14.1 14.4 13.9 Protein 2.31 2.06 2.25 1 2 . 1 10.8 11.8 11.6 Undetermined 10 91 10.07 9 92 12.8 11.8 11.7 12.1
The best membrane was obtained by moistening cambric or long cloth with alcohol, dipping it in 4 per cent collodion solution, and drying until it began to curl. The time of drying was 25 minutes a t a room temperature of 18" C., 20 minutes a t 23" C., and 15 minutes a t 27" C. This membrane was impermeable to boiled potato starch when tested with lemon juice containing about 0.1 per cent of the starch and held a t 85" C. The rate of acid dialysis through it was about 80 per cent of that of a collodion'sac and double that of parchment paper. It will last several times as long as the parchment paper, the danger of breaking being eliminated.
DIALYSIS I N OSMOGENE One hundred and fifty-five gallons of lemon juice were fermented until only 0.08 per cent of sugars remained, giving a product of the following composition: Total solids Total acid as citric Fixed acid as citric Total ash Protein ( N X 6.25) Invert sugar Undeter.mined
Per cent 7.49 6.02 5.63 0.35 0.19 0.08
0.85
"Laboratory Manual of Elementary Colloid Chemistry," pp. 25 and 70.
1143 USTRIAL AND ENGTNBERING CHEXIXTRY
August, 1923
---
TABLE VI-COMPOSITIOX OF CONCENTRATED LEMON JUICE Loti------. Lot 2------
____
DETERMINATION Total solids Acid Total sugars Protein Ash Undetermined Solids less acid Per cent acid Per cent solids Per cent ash Per cent acid
Before Per cent 86.4 66.9 1.05 2.31 5.20 10.9 19.5
After Per cent 81.7 64.0 0.86 1.81 1 80 13 2 17.7
Before Per cent 85.4 67.3 1.13 2.06 4.90 10 1 18.1
BEFORE AND AFTER
After Per cent 81 5 62 2 0.75 1.83 2.30 14 4 19.3
ALCOHOLIC PRECIPITATION
____Lot 3a--
Before Per cent 84.7 68 2
...
4 89
16
777
After Per cent 81.4 67.6 1 00 1.63 1.66 9.5 13 8
5 34
78 3
78 8
76 3
80 5
83 0
74.2
7.8
2 8
7.3
3.7
7.2
2 5
8.5
CRYSTALLIZATION OF COSCEKTRBTES
.
Very small needle crystals with blunt ends appeared in two driys in Lots 1, 2, and 3, and a t the end of 10 days the thick viscous sirups were completely filled with the needle crystals. These crystals were not pointed, but had a columnar shape wholly unlike the characteristic citric acid crystals, which are rhombic prisms. The same difficulty was experienced in trying to separate the crystals as in the experiinents on a laboratory scale. The concentrated liquors, were so viscous that it was impossible to separate and wash them. I n order to determine whether crystals could be obtained from less concentrated liquors, samples were removed from Lots 1 and 2, and water was added to give concentrations of acid ranging from 52 to 62 per cent. After seeding and standing for 6 weeks, the more highly concentrated samples were filled with crystals, about half needle-shaped and half rhombic prisms. The other samples contained a few crystals, some characteristic types, but largely needle-shaped. Evaporation had taken place until 65 per cent of acid was present. These results show that large quantities of crystals will not form below a 65 per cent concentration, and that thi,c. concentratioi? is too great to permit separation of the crystals from the heavy sirup produced. After 13 days, Lot 3a was filled with very small rhombic prisms, the mother liquor containing 65 per cent acid. The crystals were dissolved in a little water and allowed t o re-
7
After Per cent 83 8 66 9 1 14 2 19 35 2 16 9
21.9
77.4
Three lots of this fermented juice mere put through the osmogene. Lot 1, consisting of 97 liters of unfiltered but nearly clear juice, was dialyzed for 84 hours, although the juice and water were actually in motion only 21 hours. Kinetythree liters of dialyzed juice having an acid content of 3.6 per cent and 132 liters of dialysate having 1.9 per cent acid were collected. While the fluids were in motion,, a flow of approxiniately 6 liters per hour was necessary to maintain a temperature of 80" C. As the total area of the 12 membranes was but 0.75 square meter, four times this area would be required a t the same rate of flow to yield a dialysate of 6 per cent acid. An osmogene of 50 frames, 90 em. square, would deliver 75 liters per hour of dialysate of approximately the same acid strength. This dialysate was evaporated in a 40-gallon vacuum pan to 6 kg., filtered, and evaporated on a water bath, below 85" C., to approximately 67 per cent acid. It mas then allowed to crystallize. Lot 2 was put through in a similar way, except that the membyanes used in the osmogene were dried for 5 minutes a t room temperature. The dialysate was slightly turbid. Lot 3, consisting of 82 liters of fermented juice which had been roncentrated to 16.4 per cent acid content, was dialyzed. I n order to check the evaporations made in the vacuum pan where unavoidable overheating occurred, 5 liters of this lot were evaporated directly on the water bath a t 70" C. to 67 per cent acid content. This lot is designated in Tahle VI as 3a.
or--
---Lot
Before Per cent 84 9 . 63 0
I
79 8 3 Y
crystallize. The crystals formed were neither rhombic prisms nor needles, but were of an irregular shape resembling leaflets. Upon a second crystallization, needle crystals mere formed. When some of the mother liquor from the leaflets was touched on the microscopic slide, fine needle cr were formed. After a third crystallization, about thirds needles and one-third characteristic citric acid-cyystals were present. Leaflet crystals were formed on the fourth crystallization, and on the fifth crystallization rhombic prisms, with a few needles. Because of the minuteness of the crystals and their intimate mixture with the sirup, attempts to separate them on the microscopic slide and to identify them chemically were unsuccessful. Considering the large quantities formed each time, there is little doubt, however, that both the needle and leaflet crystals are citric acid. These modifications probably are produced by tee large proportions of ash, protein, and undetermined matter present. CRYSTALLIZAl'IOiY OF CLARIFIED
JUICE
In order to determine whether fermented juice treated with kieselguhr would crystallize as well as or better than the dialyzed juice, three lots (01, 02,and 03)containing 19.2 per cent of acid, were treated with kieselguhr, evaporated, and allowed to crystallize. R e s u h similar to those obtained with Lots 1, 2, and 3 were secured. Needle, leaflet, and rhombic crystals were again produced. I'pon seeding these samples with citric acid crystals, growths of needle crystals started on the seeding crystals and deyeloped in size until they were as large and of the same shape as the needle crystals previously formed.
RECRYSTALLIZATION AFTER REMCVAL OF ALCOHOL PRECIPITATE
Samples of about 30 grams removed from Lots 1, 2 , 3a, and O2 were made to 200 cc. with 95 per cent alcohol, and the heavy precipitate was filtered off. The filtrate was evaporated to a sirupy consistency, 100 cc. of water m e ~ eadded, and the solution was evaporated to about 65 per cent acid. After cooling, the material was seeded with citric acid crystals. At the end of 8 days, Lots 1 and 2 were Hilled and Lots 3a and O2 were partially filled with crystals. S o needle or leaflet crystals were present. All were highly viscous, so that it was impossible to separate the crystals. Table V I shows the analyses of these lots before and after alcoholic precipitation. The results show that more than half of the ash and small quantities of the sugars, protein, and acid were removed by the treatment. The proportion of ash to acid was lowered from an average of 7.7 to 3.2 per cent. The excessive amount of ash present before alcoholic treatment with the other nonacid constituents probably produced the needle and leaflet crystals. The process of Peratoner and Scarlata3 doubtless removes a large proportion of the ash. These results also agree with those obtained in the dialysis of mo-
INDUSTRIAL AND ENGINEERING CHEMXTRY
778
lasses where the excessive amount of salts prevents crystallization of cane sugar.
Sample 1 Sample 2 Total solids Ash Total acid as citric Undetermined
COMMERCIAL ACIDLIQUORS In order to compare the composition of the citric acid liquors obtained in this investigation with that of the concentrated mother liquors produced in citric acid factories, two sainpIes taken from lead crystallizing pans were analyzed. Sample l came from a pan containing liquor that had been reworked four times. This means that the mother liquor had been removed from a batch of crystals, mixed with some fresh acid liquor, reduced to 38" BaumB, and allowed to crystallize. This was repeated four times, so that Sa'mpIe 1 contains an accumulation of salts, sulfuric acid, and other impurities from five lots of acid liquor. Sample 2 had been reboiled three times. After liquors have been reworked more than three or four times, they yield such poor crops of crystals that it is customary to return them to the neutralizing tanks and recover the acid as citrate of lime. The composition of these saniples was as follows:
Vol. 15, No. 8 %
70
74.09
72.4R
67.81
65.29
3.oi 3.27
.4.OS
3.11
A determination of totaI sulfates indicated that about 3 per cent of the total acid was sulfuric. These results show that, for approximately the same acid content, the dialyzed liquors (Table V) contain over 10 per cent more of total solids than the factory liquors made by the decomposition of calcium citrate with sulfuric acid. The factory liquors had such a low viscosity that the crystals could be readily removed and washed. The quantity of ash present, together with the small quantities of other constituents, is not great enough to prevent crystallization. ACKNOWLEDGMENT The author wishes to express his appreciation of the valuable assistance of E. M. Chace, chemist in charge, Laboratory of Fruit and Vegetable Chemistry, under whose supervision the work was conducted.
T h e . Relation of Alcohol Precipitate to Jellying Power of Citrus Pectin Extracts' 9
By Ruth Johnstin and Minna C. Denton OFFICE OF HOMEECONOMICS, U. s. DEPARTMENT OF AGRICULTURE, WASHINGTON, D.
C.
-
N ORDER to ascertain
filtered through C 1 0 h The amount of alcohol precipitate is not a reliable measure of lvhether a relationship effectiue pectin, as shown by comparisons of pectin solutions prepared The combined filtrates amounted to 600 grams. in the open kettles and in an autoclaue. . could be established Extracting in the autobetween the pectin content, Determinations based upon jellying power giue the only satisclaw required considerable as determined by the usual factory means of estimating effectiue pectin content at present alcohol precipitation methknown to us. study before a procedure was adopted which gave od, and the jellying power, Citrus pectin extracts prepared in an autoclaue at 5 pounds solutions that were consida study has been made of a pressure for 30 minutes had 25 per cent less jellying power than simered comparable with those large number of Pectin exilar extracts prepared in the open kettle. Prepared in the open kettle. tracts prepared from the The addition of acid in open-kettle extractions of orange peel If Pectins are unstable a t white Peel of citrus fruits. produced an extract of about 100 per cent greater jellying power the temperature of boiling The work has involved the than when no acid was used. water, i t seems reasonable consideration of such details to suppose that they would as the preparation of the peel, the proportion of water to peel, the number of extrac- be less stable a t higher temperatures. However, if the tions, the methods of filtration, concentration, and storage heating period could be shortened, something might be of the extracts, and their use in combination with different gained by extracting in an autoclave. This idea seemed fruits and fruit juices in the making of jams and jellies. The to be substantiated by the fact that autoclave extracts results have shown that it is possible to prepare fairly con- always gave a higher weight of alcohol precipitate than sirncentrated pectin solutions from citrus peel, which can be ilar ones prepared in the open kettle. By reducing the proportion of water and opening the petused for making clear, transparent jellies without subjecting cock during cooling, it was possible to effect a concentration the extracts to a process of purification. The investigation has been confined to a study of pectin on the pomace equal to that obtained in the open-kettle procsolutions prepared in the open kettle and in the autoclave, ess. Three extractions were made in every case, using 330 cc, of water to 226.7 grams of peel. The combined filtrates vith and without the addition of acid. amounted to 600 grams. Extractions were made with presEXTRACTION sures varying from 3 to 20 pounds and heating periods ranging The method of procedure adopted in this series of experi- from 10 to 40 minutes. Although there are several referments for open-kettle cooking, was as follows: 226.7 grams ences in the literature to the use of the autoclave in making of ground, white peel of oranges were extracted three times, pectin extracts, which suggest its use as a means of increasing using a liter of distilled water for each extraction. The heat- pectin yield, yet in no case has it been possible to prepare ing was carried on in shallow pans of such a size that a con- by this method a pectin solution which had as great jellycentration to about one-half the original volume could be ing power as comparable extracts obtained by open-kettle accomplished in less than 20 minutes. The material was heating. Fellenberg2 called attention to the fact that protopectin 1 Presented before the Division of Agricultural and Food Chemistry a t the 65th Meeting of the American Chemical Society, New Haven, Conn., changes to pectin more rapidly in acid than in neutral solu-
I
April 2 t o 7, 1923, under the title, "Experimental Studies of Citrus Pectin Gels "
9
Biochem. Z.,85, 118 (1918).
