PURIFICATION OF SUGAR JUICES A symposium presented before t h e Division o f Sugar Chemistry a t t h e 117th Meeting o f t h e American Chemical Society, Houston, Tex., March 1950
Composition of Sugar Beet Liquors EFFECT OF PROCESSING F u t u r e advances in beet sugar technology depend to some extent on more complete knowledge of the nature of nonsucrose substances t h a t must be eliminated in processing for maximum production of sucrose and superior sugar quality. During the past campaign, a study of liquors was begun in the hope t h a t knowledge so gained would open new avenues of approach to the problem of purification and by-product recovery. Processing liquors from sugar beet factories were analyzed for various anionic constituents and colloidal material. Methods were developed for the identification of organic acids by paper chromatography and for their fractionation on ion exchange columns. Results show that, except for anions forming insoluble calcium salts and colloids t h a t are heat-denatured or precipitable in alkaline calcium-containing solution, few changes in composition occur during processing of sugar beet diffusion juice. Methods t o improve purification procedures are suggested for further examination.
J. B. STARK, A. E. GOODBAN,
AND
H. S. OWENS
Western Regional Research Laboratory, Albany, Calif.
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*
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RODUCTION of sucrose from sugar beets is a major chemical industry in the United States. Last year nearly 2,000,000 tons of sugar with a purity greater than 99.9% were obtained from about 12,000,000 tons of beets. This enviable record was achieved despite a paucity of knowledge of the detailed composition of processing liquors, and full credit is due the sugar technologists who have brought the sugar beet industry to its present stage of development when so much remains to be learned about the raw material. There are numerous analyses of beets and liquors, but these cover broad subjects such as ash, pectic substances, and “harmful” nitrogen, and have not been specific. One of the analytical difficulties has been the chemical complexity of the sugar beet which contains so many compounds that a detailed analysis was almost impossible without excessive expenditures of time and effort. Future advances in technology, however, will to some degree depend upon more complete knowledge of the nature of nonsucrose substances that must be eliminated in processing for maximum production of sucrose and superior sugar quality. With the advent of paper chromatography and ion exchange resins, a more detailed analysis of the nonsugars of sugar beet juices became possible. Also chemical changes which occur dur-
ing processing could be followed and possible new by-products indicated. During the 1949 campaign, a study of liquors was begun in the hope that knowledge so gained would open new avenues of attack on the problem of purification and by-product recovery. This paper presents preliminary work that has been accomplished with liquors from four factories. MATERIALS AND METHODS
The materials under examination were diffusion juice, thick juice, and molasses from Sidney, Mont., Rupert, Idaho, Centerfield, Utah, and Manteca, Calif., obtained a t the beginning and near the end of the campaign. The samples were composites representing hours of operation. They were concentrated in vacuo and preserved with toluene for shipment to the laboratory. On receipt they were stored a t a temperature near 3’ C. to inhibit microbial action and chemical change. Amino acids were analyzed by microbiological methods that have proved satisfactory in this laboratory for other materials. Sugar beet juice is a complicated medium, however, and the results are not considered as accurate as those obtained with simpler media.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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Total nitrogen, amino nitrogen, chloride, sulfate, citrate, oxalate, and phosphate were determined by published methods. Betaine was determined by a method developed a t this laboratory by Walker and Erlandsen (11). The alcohol-precipitable material was obtained by mixing 1 volume of juice (about 10% refractometric dry solids) with 3 volumes of 95% ethanol. The precipitate was washed with ethanol, dried in vacuo and total nitrogen, ash, and uronic anhydride analyses were run. The last was determined by the method of McCready, Swenson, and Maclay ( 5 ) . Organic acids other than amino acids were separated by ion exchange fractionation and determined by titration. The resins investigated were 8293-M and A-300 (American Cyanamid Co.), A-4, A-5, and -4-6 (Chemical Process Co.), D-735 and S (Permutit Corp.), IR4B and IRA400 (Resinous Products Corp.), and Dowex 50 (Dow Chemical Co.). The literature on use of ion exchange resins has recently been reviewed, and 'numerous publications on separation of metal cations and amino acids have appeared (3, 7'). Published work on separation of organic acids by means of exchange resins is relatively sparse, but it seemed that this method would be the best to fractionate quantities of acids of the order of 100 mg. A number of resins were examined with respect to capacity. Table I shows that of the resins tested, D-735 and S had high exchange capacity for oxalic, citric, and succinic acids. These acids are typical of the organic acids present in sugar beets. Resins D-735 and S were ground and the portions from 60 to 80 or 80 to 100 mesh were used for fractionations. They were regenerated with sodium hydroxide and washed with distilled water. -4considerable amount of carbonate was adsorbed on resin S during regeneration and washing and was released during elution with acid solutions. This caused the column to break and form channels. Expansion of the resins during conversion from base to salt form sometimes caused plugging of the columns. To overcome these difficulties and to improve fractionations the resins in later fractionations were used in the chloride or the formate form. The resin columns were 16 to 18 mm. in diameter and 30 to 55 cm. in length.
Table I . Capacity of Some A n i o n Exchange Resins for Oxalic, Citric, a n d Succinic Acids-25 M1. 1.0 N A c i d / C r a m Resin
Amount of ilcid Adsorbed, Me./Gram Oxalic Citric Succinic
FRACTIONATION OF ORGANIC ACIDS
Processing liquors were passed through a Dowex 50 column to remove amino acids and convert salts present to the corresponding acids. Acids remaining in the effluent were sorbed on a column of D-735 or S. The amount of acid was chosen to load not more than the first few centimeters of the column without considering the presence of chloride or formate. The remainder of the column Was left for fractionation. The rate chosen for loading, washing, and eluting was as high as 1 ml. per square em. per minute in the first experiments, but experience caused us to reduce the rate to approximately 0.1 ml. per square cm. per minute. It was found that the latter rate gave sharper separation bands and better fractionation. After loading, the column was washed with water and eluted with sulfuric or hydrochloric acid near p H 1.5. This pH was chosen to provide the maximum differences among the percentages of ionization of the acids as shown by calculated curves of p H versus the percentage of ionization. Boundaries could be followed either by changes in color or by absorption of the fluorescence of the resin under ultraviolet light. Fractions
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were collected in an automatic fraction collector. Selected fractions were chromatographed in solvents found satisfactory for the purpose, and spots v,-ere noted after development with bromocresol green (10). Th.: acidic fractions were titrated for total acid with standardized base and for chloride by the Volhard method. It is doubtful that the method is quantitative, but i t indicates the order of magnitude of the acid present. Continued research is increasing the accuracy. RESULTS
Typical fractionations obtained with the processing liquors from Manteca, Calif., are given in Table 11. The numbers correspond to those obtained from papergrams indicating the number of fractions containing the acid in question. The best fractionation was with the column in the formate form (initial p H of 2.7) with a flow rate of 0.1 ml. per square cm. per minute. The initial conditioning pH with formate yields a p H a t the surface of the resin which should favor separation of the w-eaker acids. The low pH of the eluting acid should improve the separation of stronger acids.
Table 11. Acid Lactic Glycolic Unknown Pyrrolidone carboxylic 3Ial1c Citric
Efficacy of Fractionation of Organic Acids i n Ion Exchange C o l u m n s Fraction Numbera Diffusion juiceb Thick juicec 1-1 1 1-37 Concentration t o o low 38-47 Concentration too low 48-52
Molassesd 1-131 271-386 406-527
585-627 >627 Fractionation discontinued Absent Fractionation Oxalic > 27 discontinued 0. T h e numbers refer to the fractions in which t h e acid appeared. b Exchange resin (D-735) in base form. C Exchange resin S in chloride form. d Exchange resin S in formate form. Absent 16-18 18-27
62-63 63-70 67-72
AMINO ACIDS
Quantitative data on the amino acids are shown in Table 111. The results with the juices collected near the end of the campaign are not shown because of the small differences observed. Amino acids go through defecation without significant change. Glutamine was not determined separately. Evidence indicates that it is the source of the pyrrolidone carboxylic acid, inasmuch as the glutamic acid hardly changes during processing. Although i t appears that a large amount of amino nitrogen is not accounted for, the unknown amount will be reduced appreciably when ammonia, glutamine, p-alanine, glycine, and ?-amino butyric acid (1) are determined.
Table 111. A m i n o Acid Content of Diffusion a n d Thick Juices Concentration, Mg./Liter (10% Refractometric D r y Solids) Centerfield, Rupert, Sidney, Manteca, Calif, Utah, Idaho, Mont., diffusion diffusion diffusion Diffusiona Thick juice juice juice juice juice 205 Present Present a-Alanine (56)O lo0 63 79 109 Aspartic acid 185 182 62 (224) 0 82 Glutamic acid 73 24 28 Present Q Isoleucine 71 28 20 Leucine 43 52 14 19 Threonine 57 59 13 32 11 Valine 1240 1420 1270 1180 Betaine 180 310 67 84 105 Van Slyke N 710 620 476 380 368 Total N
(ti)
...
a Small quantities of tyrosine, proline, histidine, a n d phenylalanine have been found in this juice. b From chromatograms. C Values in parentheses are single microbiological determinations.
INDUSTRIAL AND ENGINEERING CHEMISTRY
March 1951 Table IV.
Concentration of N o n a m i n o Acids i n Sugar Beet Processing Liquors
Centerfield, Utah Diffusion Thjck Juice juice Molasses Chloride Sulfate Phosphate Oxalate Citrate Lactate Pyrrolidone carboxylate Malate Glycolate Total acids, me./liter Known acids, me./liter Alcohol-insolubles Uronic acid anhydride a
329 171 278 92 700 1700a Present Present
...
60.5 54.3 942 157
280
2f; ... Pieiknt Present Present
...
... ...
1530 955 95
...
P&sLnt Present Present
... ... ...
19 2
Rupert, Idaho Sydney, Mont. Diffusion Thjck Diffusion Thick juice juice Molasses juice juice Molasses Concentration, mg./liter (10% refractometric dry solids) 40 150 1480 80 360 1180 110 180 1010 160 1390 1030 22 307 114 32 110 475
