Unfermentable Reducing Substances in Molasses. Identification of d

Unfermentable Reducing Substances in Molasses. Identification of d-Allulose. F. W. Zerban, and Louis Sattler. Ind. Eng. Chem. , 1942, 34 (10), pp 1180...
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Unfermentable Reducing Substances in Molasses IDENTIFICATION OF &ALLULOSE

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INDUSTRIAL AND ENGINEERING CHEMISTRY

d-Allulose, .the epimer of d-fructose, has been isolated and identfied as a nonfermentable constituent of cane molasses. Neither a 3-ketohexose (‘Lglutose”)nor any other sugar could be obtained from distillery slop in the form of its phenylosazone. The term “glutose” should therefore not be used as a synonym for the unfermented reducing substances in cane molasses. d-Allulose is not the only reducing substance that may be present in sugar products after fermentation, as it accounts for only part of the reducing power of distillery slop. The presence of fructose is indicated in the unfermented residue from heated invert sugar sirup after two fermentations with baker’s yeast. This may be vaused by fermentation inhibitors, or even by secondary formation by bacterial action, of fructose from mannitol, which has been found in considerable quantity. Another substance, with a possible empirical formula of CJOHMOISNZ and with strong reducing properties, has been isolated. Tetramethyl-n,fl-methyl-d-alluloside has been prepared. I t is presumably the 1,3,4,6-isomer. 2-Tosyl-1.3-ditri tylgl ycerol has been synthesized. Until methods for the determination of each constituent in a mixture of unfermented reducing substances are available, it will be best to report the total reducing power either in terms of copper or, perhaps, of levulose.

Bruyn and Alberda van Ekenstein claimed to have obtained an unfermentable ketohexose by treating fructose with lead hydroxide a t an elevated temperature. They reasoned that corresponding conditions of temperature and alkalinity existed in the processing of cane sugar, and consequently glutose wm formed as the unfermentable sugar in molasses. I n view of the fact that glutose is considered to

GENERAL VIEW

OF A PLANT WHICH OXIDI,

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have about half the reducing power of glucose, the glucose equivalent of the copper found in an analysis is multiplied by 2 to convert it to glutose. It is clear that this interpretation of the reducing power of the unfermentable substances in molasses rests wholly on the unproved assumption that glutose is present. Moreover, glutose as an individual sugar is chemically completely uncharacterized, and the work of Spoehr and Strain (16)has shown that it is not a 3-ketohexose as had been assumed-in fact, that it is not even a homogeneous substance. For these reasons the glutose basis for interpreting analytical data involving unfermentable reducing substances becomes more and more nebulous and untenable. As part of the work that is being carried out by the writers on molasses (36,36), this paper is presented in an endeavor to throw light on the above question. A convenient source for the unfermentable substances in molasses is distillery slops, and a factory sample of this material was purified to remove the nonsugars. Although previously Nelson and Greenleaf (17) had failed to identify any carbohydrates in such a product, we have prepared the phenylosazone of a sugar known variously in the literature as d-ribo-2-ketohexose (18)and d-pseudofructose (+fructose), from which it was renamed d-psicose by Ohle and Just (18). Its osazone is identical with allosazone and altrosazone. Because of this relation of the ketohexose to allose and altrose we favor the name “allulose”, proposed by William Lloyd Evans at the Atlantic City meeting of the AMERICAN CHEMICAL SOCIETY in September, 1941. H-C=O H-C=O Hr-C-OH H-A-OH H-

(A -OH

H-

-OH

A

I

HO-A-H

H-

A1 -OH

C=O

H - L H

H-C-OH

H-(4-OH

H-LOH

H-L-OH

H-&-OH

H*-A-OH d-Allose

H2-C-OH &Altrose

I

(4

-OH d-Allulose

H2-

Theoretically, the unfermentable hexose present in cane molasses could be allose or altrose, but i t has been definitely identified as d-allulose. An examination of the mother liquors obtained in the purification of the crude allulosazone did not reveal any other osazone. The work of Steiger and Reichstein (18) had shown that allulose is soluble in alcohol, and SO i t was thought that dry commercial distilleryslop concentrates could be extracted with that solvent; thus we could simplify the labor involved in obtaining purified sirup containing t h e unfermentable reducing substances. A commercial product known as Curbay B-G was extracted for US. From 500 grams of the concentrated- extract, Courtesy, Pennsylvania Sugar Company 1.7 grams of d-allulosaPRODUCES REFINED SUGAR, ALCOHOL, SOLID CARBON DIAND OTHERBY-PRODUCTS zone were obtained. As

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before, there was no evidence of any other reducing sugar in the osazone mother liquor. For comparison of the physical constants, the values for d-altrosazone given by Levene and LaForge (14) were consulted. Their figures for the specific rotations of various osazones, through a typographical error, are 100 times too small. These values are widely quoted in standard reference works on sugars such as Tollens-Elmer, Vogel and Georg, and Micheel (31). I n Tollens-Elsner (31) the error is unconsciously underscored by the following statement about galactosazone: “Es dreht in Pyridin-Alkohol schmach rechts”, [a]?= +0.73”. With the 100 fold correction, our values checked excellently with those given by Levene and LaForge. The presence of allulose might have been brought about by an epimerization of the fructose present, although its occurrence in cane juice is not excluded. Kilp (11)reported that fresh cane juice contained glutose and that on clarification more glutose was formed from the fructose, while Waterman and van der Ent, a t the Java Sugar Experiment Station, and Coltof obtained small quantities (up to 0.11 per cent) of unfermentable reducing substances from raw cane juice; but it has not been proved that Courtesy, U.8.Industrial Chemicals, Ino. they contained reducing sugars. Molasses YEAST CULTURES ARE ADDEDT O S T E R I L I Z E D M.4sH IN THESE TANKS HOLDING MORETHAN11,000 GALLONS;SAMPLES ARE DRAWN FROM THE TA~YK FOR A obtained from Java and Cuba were shown FINAL CHECKBY THE LABORATORY BEFORE FERMENTATION BEGINS by Kilp to contain 6 to 9 per cent “glutose”, respectively. These figures are obviouslv fallacious. but thev have aualitative significance. According t o Fischer and Baer (7) d-fructose Pure allulose itself was finally prepared from the alcoholic and d-sorbose, synthesized from d-glycerose in the presence of extract of Curbay B-G by way of its known diacetone deriva0.01 M barium hydroxide, did not show the presence of either tive (28). This compound, on decomposition with dilute allulose or tagatose. At least, these writers conclude, if either acetic acid to form the free sugar, yielded an anhydrous sirup of these compounds was present, its amount was below 5 per which, after standing over phosphorus pentoxide in a vacuum cent of the total sugars. The observation by Steiger and desiccator, gave a specific rotation in sodium light of +2.9’ Reichstein (17) that I-allulose has 61 per cent of the reducing a t 20’ C. Steiger and Reichstein reported [a]’: = 3.1 ” for power of glucose toward Fehling solution is interesting when the synthetic sugar. it is coupled with the accepted reducing power of “glutose”; The pyranose or furanose structure of tetramethyl methyl but it loses its analytical significance when one takes into conalluloside cannot be decided from known rotation data in sideration the fact that the reducing power of the material connection with Hudson’s rules. As is well known, the solcalled “glutose” varies between wide limits (6). vent used has a large influence on the rotation of the methylRepeated attempts were made to isolate from the crude ated methyl glycosides. Consequently, the prediction of the unfermentable concentrate any other sugar that might be structure must be based solely on the method of preparation, present, in the form of the acetyl, benzoyl, and trityl derivaand by analogy our derivative may presumably be the tives, but all these experiments were fruitless. During this 1,3,4,6-tetramethylisomer. phase of the work a new compound (2-tosyl-1,3-ditritylUnfermented Residue of Heated Invert Sugar glycerol, melting a t 155-156” C.) was prepared from the Sirup glycerol present as a by-product of the fermentation. Methylation of the crude allulose concentrate by the methods of While it is true that in the manufacture of sugar from cane West and Holden (34) and of Levene and Tipson (16), with the juices are kept alkaline for some time, the products are usually on the acid side in the later stages of processing. To the apparatus used by Levene and Kuna (13) followed by a high-vacuum distillation of the product, yielded tetramethyldetermine the effect of such conditions on the production of a,@-methyl-d-alluloside which distilled as a thick oil a t a unfermentable sugars, pure sucrose was inverted with Conbath temperature of 105-140” C. and a pressure of 8 X vertit and then heated for 10 weeks a t 55” C.; during all this mm.: time the mixture was kept a t about pH 4.5 with acetic acid. After fermentation and refermentation of the resulting % C %H % OCHI residuum, followed by suitable chemical treatment and evapoCalod. for C~tHnzOs 52.77 8.86 62.00 ration in vacuo a t low temperature, a partially purified product Found (nab. 1.4572) 53.00 9.24 59.70 was isolated. On analysis by the method of Zerban and When the tetramethyl methyl alluloside was heated under Sattler (36), it contained 0.46 per cent “dextrose”, 13.17 per pressure for 2 hours in methanol containing a small amount of cent “levulose”, and 18.95 per cent “maltose”, based on actual hydrochloric acid, the product (probably an equilibrium mixdry substance. Complete analysis was: total sugars, 32.83 ture of tetramethyl methyl allulosides) gave [a]?= 36”. per cent; ash, 2.10; water, 26.66; and nonsugars, 34.41.

+

-

+

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When treated with phenylhydrazine, the alcohol-soluble portion of the sirup slowly yielded phenylglucosazone. Methylation was resorted to as a means of separating the alcohol-soluble sirup into compounds whose identity could be established. I n this way large quantities of methylated glycerol were obtained, and the identification was completed by a comparison with known compounds. When the high-boiling portions of the methylated product were studied, three promising fractions were obtained with the following characteristics: Fraction NO.

I I1 I11

Bath temp., a

c.

90-115 120-160

160-190

Pressure, Mm.

%C

%H

n”DO

4 X 10-4 4 X 10-4

53.22 53.62 53.20

8.67 9.20 8.89

1.4428 1.4479

6 X 10-8

1.4677

In another series of methylations of the alcohol-soluble portion, a fraction was obtained whose boiling point was 109-115’ C. at 1.03 mm., and which contained 52.69 per cent carbon and 8.69 per cent hydrogen; index of refraction at 21.5’ C. was 1.4449. Incidentally, boiling points of compounds are reported in the literature in which the pressure is given in the range 10-4 to mm. although the setup was not a true molecular distillation apparatus. Consequently, such published figures may be misleading. A molecular still was not used in the present experiments, and so the boiling points at 10-4 mm. are to be interpreted as observed experimental conditions and no more. Fortunately, an adequate amount of material was available so that a regular vacuum distillation could be run in a small Claisen flask on the sample whose constant boiling point fraction is recorded at 1.03 mm., as taken with a McLeod gage. Of this sample, 1.2797 grams in 25 ml. of methanol gave a specific rotation of +4.53”; and after being heated for 2 hours in a pressure bottle at 100’ C. in the presence of a small amount of concentrated hydrochloric acid, it gave an equilibrium specific rotation of +7.03’. At first it was thought that the slow formation of glucosazone in conjunction with this low specific rotation for the fully methylated derivative indicated the possibility of its being impure tetramethyl

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methyl fructofuranoside, inasmuch as Allpress (1) had observed a low specific rotation in methanol; but it is more likely that oc,P,y,6,e-pentamethylmannitol(11) or other methylated mannitols were present to obscure the observations. This conclusion is based on the fact that fully one third of the unferrnentable residuum was alcohol insoluble, and mannitol is actually only slightly soluble and not really wholly insoluble. The crude alcohol-insoluble portion of the starting product was a blackish brown tacky material whose study was put off until the nature of the alcohol-soluble portion had been cleared up. This unattractive substance was readily purified by one crystallization from glacial acetic acid, followed by recrystallization from water or dilute alcohol. Identification of the material as mannitol was made by conversion into mannitol tribenzoyl acetal. When mannitol is gently warmed with an equal amount of resorcinol and a few drops of concentrated sulfuric acid, it yields a reaction product which, when poured into a large volume of dilute sodium hydroxide solution, produces a cherry-red solution with a strong green fluorescence. The fluorescence disappears on standing overnight. Sorbitol was found to give the same color reaction. The finding of mannitol in relatively large quantities in a product made by the action of baker’s yeast on invert sugar was rather disturbing, but it had the virtue of illuminating the causes of many of the difficulties which had been experienced in the work with the methylated sugars. Although it can be postulated that a Cannizzaro reaction may account for the formation of mannitol and gluconic acid from glucose (@), or that during fermentation a reducing action is taking place and consequently mannitol is formed (3, %$),there are other completely unrelated circumstances which can account for the presence of mannitol. Stiles, Peterson, and Fred (90, 29) claimed that mannitol-forming bacteria are to be found in yeast; and Gayon and Dubourg (8) found a microorganism which converts only fructose into mannitol. At this time the full microbiological significance cannot be discussed, but lactic acid and methylacetylcarbinol are important indicators that fermentation side reactions are taking place along with the normal ethanol production by yeast. Particularly noteworthy is the fact that, despite our efforts to ensure complete fermentation of “fermentable” sugars, . a reducing sugar yielding glucosazone is still to b< found in the u&%rmentable portion of the treated invert sugar. It is generally known that levulose is fermented more slowly than dextrose, and that industrial fermentation products such as wines, vinegars, etc., often contain small quantities of levulose’. This may possibly account for the glucosazone obtained from invert sugar exposed for a long time to a high temperature, followed by repeated fermentation on a large scale. The formation of fermentation inhibitors during the heat treatment of the invert sugar may also have prevented complete fermentation. Although no formal explanation is offered, it is also possible that certain microorganisms are capable of converting mannitol into levulose, and that any levulose found 1 Rothenbach and Kleber [mochsckr Brau., (17, 63 (l940)l found the same amount of apparent levulose by reduction of alkaline

Courtesy, U. S . Industrial Chemicals, I n c .

MIXTURESOF MOLASSES AND YEASTARE FERMENTED IN THESEFERMENTERS, OF 134,000 GALLONS EACHWITH A CAPACITY

ferricyanide aolution in beer wort before and after fermentation with brewer’s yeast, and ascribed this to the presence of fructosans which behave analytically like levulose.

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in the unfermentable sugars owes its origin to such action ( 2 ) . These so-called irregularities in the action of yeast on sugars may play an important role in analytical chemistry because, for example, this is a specific way to determine lactose quantitatively in sugar mixtures. A portion of the unfermentable residue, when treated with glacial acetic acid and allowed to stand in a refrigerator for several weeks, yielded a white crystalline solid which mas acid to litmus, was soluble in water and glacial acetic acid, and vigorously reduced both Fehling and Tollens solutions. It gave no murexide test and melted with decomposition a t ~ ~ N ~ Pby 206.5" C. An empirical formula C ~ O H ~ ~ isOsuggested the analytical data: carbon = 45.11 per cent, hydrogen = 6.67, nitrogen = 8.57, and phosphorus = 3.79. Organic phosphorus was also found in distillery slops, and this would seem to confirm the belief that the compound is essentially a yeast product. From the data secured in this investigation, it appears that the function of yeast in quantitative analysis and the significance of mannitol formation in fermentations with yeast require further study.

Purification of Distillery Slops Twenty liters of distillery slops were treated with an excess of neutral lead acetate; after the precipitate had settled, the supernatant liquid was filtered with the aid of Filter-Cel on a large Bucliner funnel. The filtrate was concentrated in vacuo in a water bath, kept below 55" C., to a volume of about 8 liters. After cooling to room temperature, the excess lead was precipitated as lead sulfide and removed by filtration. The filtrate was aerated to remove most of the dissolved hydrogen sulfide, then warmed to 50" C. with activated carbon, and finally filtered to remove as much coloring and colloidal matter as possible. The filtrate obtained from the carbon treatment was further vacuum-concentrated as before t o a volume of about 2 liters. To obtain a water-free material, 95 per cent alcohol and a small quantity of benzene were added, and the mixture was distilled t o a thick sirup. This process was carried out three times, and finally the sirup was extracted repeatedly with hot ethanol. The combined alcoholic extracts were filtered and concentrated in vacuo to a small volume as before. I n place of the hydrogen sulfide treatment of the lead solution, it is possible to delead it satisfactorily for most purposes with a phosphate-oxalate solution containing 7 grams of hydrated disodium phosphate and 3 grams of potassium oxalate in 100 ml. of solution. A small excess of the negative ions from these salts seems to exert a favorable influence on the formation of osazones. The ketose nature of the unfermentable reducing sugar was confirmed by the sodium hypoiodite oxidation procedure of Kolthoff and Kruisheer (6),which determines quantitatively the amount of ketoses in the presence of aldoses. After such treatment a sample containing originally 0.1868 gram of dry substance from the alcoholic extract of a commercially available distillery-slop concentrate gave 32.5 mg. of cuprous oxide by the Munson and Walker method. This is equivalent to 7.33 per cent "dextrose". The unoxidized solution showed 15.58 per cent "dextrose". The Kolthoff-Kruisheer method errs on the side of giving slightly low values for levulose and indicates the possibility of too vigorous action on the part of the sodium hypoiodite solution. The experiment was repeated qualitatively, but threefold excess of iodine was used; in addition, the mixture was allowed to stand 2 days instead of 7 minutes. I n spite of all this, the resulting mixture reduced Fehling solution, again indicating the presence of ketoses. Further confirmation of the ketose nature of the unfermentable reducing sugar was obtained by oxidizing the alcoholic

Vol. 34, No. 10

extract with nitric acid. Oxalic acid was the main product and there was neither any saccharic acid, indicating the absence of aldoses, nor mucic acid, indicating the absence of galacturonic acid. Incidentally, no evidence appeared of the presence of pentoses.

d-Allulose Phenylosazone from Purified Distillery Slops To 20 grams of the alcohol-soluble sirup were added 40 grams of freshly distilled phenylhydrazine and 60 grams of sodium acetate dissolved in 400 ml. of water, and the mixture was heated for one hour in a boiling water bath. After standing for 2 days a t room temperature, a voluminous brown precipitate formed which was removed on a Buchner funnel and then air-dried. The purification of the solid was effected by a preliminary extraction with either nitromethane or nitropropane (solvents which seem well suited to the purification of certain osazones), followed by recrystallization from 30 per cent ethyl or isopropyl alcohol with a small amount of decolorizing carbon. I n this way bright yellow crystals were obtained which were finally recrystallized from water in which they were just moderately soluble a t the boiling point. The analytical data for the osazone are the following:

% ' N fo: C18H2204N4: calcd. 15.63, found 15.32

Mol. weight for C18Hz~OaN4: calcd. 358, found 368

[a12 (after 24 hr.)

x 100 = -22.10 2.08 X 1

= -0.46

On heating in a capillary tube, the substance contracted at 175" C., melted a t 178", and decomposed a t 183". These values agree with the figures given by Steiger and Reichstein (28) for the osazone of d-allulose. In making up solutions for polariscopic measurements, the osazone should be first dissolved in dry pyridine and then diluted to the proper volume with absolute ethanol. This obviates the need for heating when dissolving the osazone in the pyridine-alcohol mixture. The final solvent should contain 1 ml. pyridine for each 1.5 ml. of alcohol.

d-Allulose Phenylosazone from Curbay B-G The spray-dried commercial slops concentrate was treated with powdered barium carbonate in sufficient quantity to prevent acidity and then stirred with warm 95 per cent ethanol. The alcoholic solution was filtered and concentrated in vacuo to a thick sirup, keeping the bath below 50" C. The sirup was taken up in distilled water and made into a 10" Brix solution to which was added neutral lead acetate solution in slight excess so as t o precipitate various substances which had been dissolved by the alcohol. After the lead precipitate was removed and the solution deleaded with phosphate-oxalate solution, the filtrate was concentrated in vacuo. This product was heated to 90" C. for 8 hours with freshly distilled phenylhydrazine in the following proportions : 10 grams Phenylhydrazine, 10 ml. water, and an equivalent volume of the sirup. The mixture was allowed to cool to room temperature and then poured with vigorous stirring into 300 ml. of cold water. After standing 24 hours, this precipitate was filtered on a Buchner funnel and dried. The solid was transferred to a micro-Soxhlet apparatus and extracted with hot water. From the water solution bright yellow crystals were obtained on cooling which in one recrystallization from 30 per cent ethanol gave a product with the proper melting point and specific rotations for d-allulosazone. A mixture vith

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INDUSTRIAL AND ENGINEERING CHEMISTRY

a sample previously made (from another source) showed no change in melting point. Preparation of 1,3,4,6-Tetramethyl-ar,B-methyld-alluloside The material obtained from distillery slops was methylated according to the method of West and Holden (34) with variations in the concentration of the sodium hydroxide used or the rate of addition of the reagents. There was no appreciable change in yield. In one case the methylation ran 8 hours instead of the customary 4 hours, but again there was no significant change in the amounts of methylated material produced. The omission of carbon tetrachloride was tried in one run, and it had the effect of reducing the extent of the foaming without affecting the yield. There was little choice between the method of West and Holden and that of Haworth and Leitch (9) as far as convenience and yields were concerned. A solution of 0.3199 gram of the fully methylated fraction, mm. in a total volume boiling a t 105-140' C. and 8 X of 25 ml. of methanol containing 4 drops of concentrated hydrochloric acid, was read in a saccharimeter; its initial specific rotation was +25.4', and after 2 hours of heating at 100' C. in a pressure bottle, its equilibrium specific rotation was +36":

[a]Y =

4-0.67" S X 100 X 0.3462 1.2796 X 05.= .f360

Preparation of Diaoetone d-Allulose One hundred grams of the concentrated alcoholic extract of Curbay B-G were placed in a 1-liter heavy-wall Pyrex

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Erlenmeyer flask, two dozen a/d-inch porcelain balls were added to increase the surface of the material, and it was then heated a t 50' C. in a vacuum oven in the presence of phosphorus pentoxide for 100 hours. At the end of this time the phosphorus pentoxide, which was frequently stirred or replaced, had not become damp. About 125 grams of the drying agent were required. The flask was then stoppered and allowed to cool to room temperature. Six hundred milliliters of acetone, twice redistilled from anhydrous calcium chloride, were added and then 50 grams of anhydrous copper sulfate. To facilitate dissolution of the gummy solid, the mixture was refluxed on a water bath for 2 hours during which time the material was protected from contact with any moisture. The flask was placed in a shaking machine and after addition of iifty half-inch chrome steel balls and about two hundred 6-mm. glass balls, it was shaken until the solid matter adhering to the walls of the flask was completely in suspension. Fifty grams of anhydrous copper sulfate, 350 ml. of dried acetone, and 1 ml. of concentrated sulfuric acid were added, and then shaking was resumed for 40 hours. During this time the material in the flask became darker. The reaction product was filtered on a Biichner funnel and washed with anhydrous acetone. The filtrate was treated with a slight excess of powdered calcium oxide and 20 ml. of water, and shaken to remove the sulfuric acid. After filtering, a small amount of solid potassium carbonate was added to the filtrate to protect it from acidity. The filtrate was then concentrated in vacuo to a small volume. The solution was extracted with ether, and the extract, after drying over anhydrous sodium sulfate, was vacuum-distilled, The portion distilling in the range 139-161 O C. a t 2.5 mm. was then heated with dilute acid according to the method of

Courtesy, U.S.Induntrial Chemicals. Ino.

PRESEEDING TANKS ARE ANOTHER STEPIN THE DEVELOPMENT OF THB YBAST;AT EACH STAQEMOLASSES Is ADDEDTO THE CULTURP TO PERMIT THE ORGANISMS TO DEVELOP

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Steiger and Reichstein for the preparation of pure anhydrous allulose sirup. A solution of 0.3011 gram, dissolved in 25 ml. of water and measured in a saccharimeter, gave a rotation of f0.05" S in a 5-cm. tube. The specific rotation conse. quently was +2.9": 2o

-

+0.05" S X 100 X 0.3462 0.3011 X 4 X 0.5

=

+-2.90

Fermentation Residue from Heated Invert Sugar Sirup Two 10-gallon bottles were partially filled with a 60" Brix pure sucrose solution, and the pH was adjusted to 4.3 by adding 0.5 ml. of acetic acid per gallon of sirup. For each gallon, 1.5 ml. of Convertit were added, and the mixture was heated between 53" and 55.5" C. for 10 weeks, during which time the pH was kept around 4.5. Fermentations were set up in two 36-gallon mashes with the solution diluted to 13" Brix. After the pH was adjusted to 5,3 by adding 5 ml. of ammonium hydroxide to each mash, 72 grams of ammonium sulfate and 36 grams of disodium phosphate were added as yeast food to each fermenter, followed by 1 pound of Fleischmann's starch-free yeast. After the fermentation was complete, the "beers" were filtered and concentrated under a 24inch vacuum, mainly to remove the ethanol which had formed. The residual liquid was refermented in order to ensure the complete elimination of fermentable sugars. Finally the mixture was filtered on a stoneware suction filter with Hyflo filter aid to remove all suspended matter and then concentrated 89 before under a 24-inch vacuum. The product had the following analysis: 14.0" Brix; 4.33 per cent reducing substances as dextrose; 0.00 per cent sucrose (Clerget); 3.06 per cent ash (conductometric) and 0.67 per cent ash (carbonated); pH, 6.25. Sufficient neutral lead acetate solution was added to give a slight excess of lead ions in solution, and the precipitate which formed was removed with the addition of Hyflo filter aid on a large Buchner funnel. The clear filtrate was treated with hydrogen sulfide until all the lead was precipitated and then filtered to free it from the precipitate. The solution was concentrated a t 63.5-mm. pressure a t 60" C. to a 62.5" Brix solution. Its volume was 4650 ml. This concentrate, for extraction of the alcoholsoluble substances, was transferred to a 30-gallon tinned copper tank, and 95 per cent ethanol was added in 2-gallon lots with constant stirring until 22 gallons of alcohol had been added. There was a slight precipitation of a gummy material, and the supernatant liquid was dark and appeared to be holding considerable quantities of solids in solution. The alcoholic mixture was warmed t o 50" C., allowed to cool to room temperature, and SPRAY D R Y I N Q then filtered. A sample was de-

alcoholized under vacuum, made up to volume with distilled water, and tested for reducing sugars. The results of the analysis showed 1.04 grams of reducing sugars (as dextrose) per 100 ml. of solution, which meant 790-860 grams on the total alcoholic solution. The alcoholic solution was concentrated in varuo a t 60" C. t o a thick sirup.

Oxidation of Alcohol-Soluble Material with Nitric Acid One hundred grams of the concentrate were oxidized with nitric acid according to the method described in TollensElsner (SO). Large quantities of oxalic acid were obtained but no saccharic acid was found. This indicated the absence of glucose.

Action of Phenylhydrazine Treatment of the alcohol-soluble material with phenylhydrazine under the usual conditions produced a messy, black, gummy solid The product was filtered on a Buchner funnel,

Li. IN THE MANUFACTURE OF CURBAY FOR

B-G

S. Industrial Chemicals, Inc

ELIMIN.4TES THE

HIGHTEMPERATURES

XnED

I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY

October, 1942

washed thoroughly with water, and then allowed to air dry. The solid was extracted in a micro-Soxhlet apparatus with 99 per cent isopropanol. The orange-brown solution was boiled with activated carbon which had a pronounced decolorizing action. On cooling, a rather gelatinous mass of yellow crystals formed which, on further reerystallization from isopropanol, produced bright yellow crystals which melted a t 208.5" C. The specific rotations in pyridine-alcohol were the following: [ a]+3(initial) =

[ a ] y(after 24 hr.)

-0.61 X 100 = 2.0 X 0.6 =

-0.33 X 100 = -330 2.0 X 0.5

A sample of pure glucose phenylosazone under the same conditions had an initial specific rotation of -63" and a final rotation after 24 hours of -35". The observational errors permit a variation of 1 2 " ; hence we have a check that our substance is glucose phenylosazone.

Preparation of p-Bromophenylosazone A portion of the sirup was heated in a boiling water bath with p-bromophenylhydrazine hydrochloride and sodium acetate for 10 minutes according to the usual procedure. On standing overnight a t room temperature, a brown solid separated which was removed by filtration and dried. The solid was extracted in a Soxhlet apparatus with 99 per cent isopropanol; when cooled the solution deposited dark brown irregular crystals which, on recrystallization from a mixture of anhydrous pyridine and 99 per cent isopropanol, gave a product which was considerably lighter in color. The substance thus obtained was extracted with nitropropane which effectively removed foreign coloring matter. Recrystallization from a mixture of petroleum ether (boilingat 75-105" C.) and 99 per cent isopropanol produced bright yellow granular crystals which contracted a t 205' C., melted sharply at 206-206.5", and decomposed at 207": % N for ClsHaoOlNaBrz: calcd., 10.86; found 10.62 Tritylation of Alcohol-Soluble Sirup A quantity of the alcohol-soluble extract was prepared and concentrated to a thick sirup in vacuo. The concentrate was repeatedly distilled in vacuo with added quantities of isopropanol to remove any water present, and finally benzene was added in successive portions to the distilling material to remove the last traces of water. The sirup thus prepared was dried in a vacuum oven at 50" C. and stored in a desiccator over sulfuric acid. Three grams of this dried material were treated with 60 ml. of anhydrous pyridine and 10 grams of triphenyl methyl chloride, and the mixture was allowed to stand a t room temperature for 3 days with frequent shaking. It was then poured slowly with stirring into a liter of ice water. A white, oily material formed which later turned into a stiff gum. The water was decanted and replaced about eight times a t intervals of between 2 and 3 hours. At the end of this treatment the product became granular. It was filtered on a Buchner funnel and then air-dried. Recrystallization from 95 per cent ethanol (125 ml. per gram) and decolorizing carbon to constant melting point yielded 0.75 gram of a compound melting a t 173.5-175' C. The crystals were long prisms collecting into rosettes. On treatment with a drop of concentrated sulfuric acid, the characteristic orange color produced by triphenylcarbinol with sulfuric acid was obtained (32). The compound was the 1,a-ditrityl ether of glycerol, and this was confirmed by the unchanged melting point of a mixture of this substance and authentic 1,3-ditritylglycerol (10).

1187

Preparation of 2-Tosyl-1,3-Ditritylglycerol The tosylation was carried out according t o the general procedure of Oldham and Rutherford (29). One gram of the trityl derivative was added to 1.5 grams of tosyl chloride in 45 ml. of anhydrous pyridine in a 50-ml. glass-stoppered Erlenmeyer flask. The mixture was shaken on a machine for 4 hours and then allowed to stand a t room temperature for 3 days with occasional shaking. The reaction product was stirred into a mixture of benzene and water, and when all had been dissolved, the water layer was removed in a separatory funnel. The benzene layer was shaken four times with cold 2 N hydrochloric acid and then twice with 5 per cent sodium carbonate solution. The benzene solution was dried with anhydrous sodium sulfate, shaken with decolorizing carbon, and finally filtered. On evaporation of the solvent, crystals were obtained which were analytically pure after three recrystallizations from 99 per cent isopropanol. They came out in the form of long prisms, melting a t 155-156' C.; the yield was 0.72 gram. For analysis a sample was vacuum-dried in an Abderhalden dryer a t 65' C. Calod. for CtsH4nOsS Found for CaHaOsS

%C 78.87 78.50,78.65

%H 5.79 5.98,5.95

Alcohol-Insoluble Portion of Sirup

ISOLATION OF MANNITOL.The alcohol-insoluble material was black and gummy. It was treated with dry pyridine and stirred to form a thin paste. The mixture was filtered on a Buchner funnel; the residue was repeatedly washed with dry pyridine to remove the glycerol which was adhering to it, and then finally with absolute alcohol. The solid thus obtained was placed in a vacuum drying oven kept a t 50" C. where, on prolonged drying, it was converted to a tacky, blackish brown tar. The substance was very soluble in hot glacial acetic acid. On cooling, a thick mass of crystals deposited, which was filtered on a fritted glass funnel and washed with cold glacial acetic acid to remove the coloring matter. The resulting flesh-colored crystals were washed with 99 per cent isopropanol and then twice recrystallized from 30 per cent alcohol. Snow-white crystals were obtained which melted sharply at 166' C. Qualitative analysis indicated it to be mannitol, and this was confirmed by converting the compound with benzaldehyde and concentrated hydrochloric acid into mannitol tribenzoyl acetal by the method of Meunier (26). The melting point of 221-222' C. checked the value given by Pette (ai),who made the same compound with phosphorus pentoxide instead of hydrochloric acid and thus claimed his compound to be purer than Meunier's. A sample made by mixing our compound with pure mannitol tribenzoyl acetal showed no melting point depression. METHYLATION. The reactions were carried out as in the previously described manner. I n all cases the yields were poor, and repeated methylations by the method of Purdie and Paul (23) were necessary to raise the methoxyl content of the product. Acknowledgment The authors desire particularly to thank G. T. Reich of the Pennsylvania Sugar Company for his helpfulness in arranging for and supervising the large-scale production of nonfermentable reducing substances from sucrose and for making pictures available. A. A. Backus, of U. 5. Industrial Chemicals, Inc., graciously arranged to have his organization extract some of their Curbay B-G for these experiments. The writers also wish to express their appreciation to Martin Kuna of the Rockefeller Institute for Medical Research for his invaluable assistance.

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INDUSTRIAL AND ENGINEERING CHEMISTRY

Literature Cited (1) Allpress, J . Chem. Soo., 130, 1722 (1926). (2) Angeletti, Ann. Chim. applicata, 26, 234 (1936). (3) Browne, Am. Chem. J., 28, 462 (1906). (4) Browne, La. Agr. Expt. Sta., Bull. 91, 17, 96 (1907). (5) Browne and Zerban, “Phy31cal and Chemical Methods of Sugar Analysis”, 3rd ed., p. 902, New York, John Wiley & Sons, 1941. (6) Coltof, Biochem. Z., 243,192 (1931). (7) Fischer and Baer, Helv. Chim. A c t a , 19,519 (1936). ( 8 ) Gayon and Dubourg. Ann. Inst. Pasteur, 15,527 (1901). (9) Haworth and Leitch, J . Chem. Soc., 113, 194 (1918). (10) Helferich, Ber., 56,769 (1923). (11) Irvine and Paterson, J . Chem. Soc., 105, 920 (1914). (12) Kilp, Z . Spiritusind., 55, 188 (1932). (13) Levene and Kuna, J . Bid. Chem., 127, 51 (1939). (14) Levene and LaForge Ibid , 2 0 , 4 2 9 (1915). (15) Levene and Tipson, Ibid., 105,419 (1934). (15A) Lobry de Bruyn, C. A., and Alberda van Ekenstein, W. A., Rec. trau. chim., 16,258 (1897). (16) Meunier, Ann. chzm. phyn., [el 22,412 (1891). 21,858 (1929). (17) Nelson and Greenleaf, IND. EXG.CHEM., (18) Ohle and Just. Ber.. 68B,601 (1935). (19) Oldham and Rutherford, J . Am. Chem. SOL,54,366 (1932).

(20) (21) (22) (23) (24) (25) (2G) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36)

Vol. 34, No. 10

Peterson and Fred, J . Biol. Chem., 41,431; 42,273 (1920). Pette, Ber., 64, 1587 (1931). Prinsen Geerligs, Intern. Sugar J., 40, 345 (1938). Purdie and Paul, J . Chem. Soc., 91,293 (1907). Raistrick and co-workers, T r a n s . R o y . SOC.(London), B220, 171 (193 1). Spoehr and Strain, J . B i d . Chem., 85, 365 (1929). Spoehr and Wilbur, Ibid., 69,421 (1927). Steigor and Reichstein, Helv. Chim. Acta., 18,794 (1935). Ibid., 19, 187 (1936). Stiles, Peterson, and Fred, J . Biol. Chem., 64,843 (1925). Tollenfl-Elmer, “Kurzes Haudbuch der Kohlenhydrate”, p. 199 (1935). Ibid.. p. 340; Vogel and Georg, “Tabellen der Zucker und ihrer Derivate”, 1931: Micheel, “Chemie der Zucker und Polysaccharide”, p. 70 (1939). Valentin. Collection Czechoslov. Chem. C b m m u n . , 3,499 (1931). Waterinan and van der E n t , Arch. Suikerind., 34,11,942 (1926). West and Holden, J . Am. Chem. Soc., 56, 930 (1934). Zerban, J . Assoc. Oficial A g r . Chem., 23,563 (1940). Zerban and Sattler, IND. ENG.CHEM.,ANAL.ED.,10,669 (1938).

PREBEKTED before the Division of Sugar Chemistry and Technology at the Atlantic City, N . J . 102nd Meeting of the A M E R I C A N CHEnfICAL SOCIETY,

Vapor-Liquid Equilibrium of Methanol-Ethanol-Vater Mechanism of Ethanol Dehydration Ternary vapor-liquid equilibria at atmospheric pressure are presented for the system methanol-ethanol-water. No ternary azeotrope was found. Requirements of a dehydration agent for the manufacture of anhydrous ethanol by distillation are discussed. A systematic procedure is used to determine from the ternary data how methanol behaves in this respect, with the conclusion that it is not suitable as a dehydration agent.

URING the investigation of a new approach to the problem of dehydrating ethanol by distillation, a fairly complete set of equilibrium data for the ternary system methanol-ethanol-water was obtained. These data are useful in distillation calculations for the recovery of the alcohols from their water solutions such as certain antifreezes and pharmaceuticals.

D

Ternary Equilibrium Forty ternary vapor-liquid equilibrium determinations were made in the original type Othmer still (8) with the addi1

Present address, Humble Oil and Refining Company, Baytown, Texas.

JOHN GRISWOLD AND J. A. DINWIDDIE] T h e University of Texas, Austin, Texas

tion of an electrical resistance wire wound about the body of the apparatus to compensate heat loss to the surroundings and thereby avoid refluxing. The results are summarized in Table I. The analysis was by the refractive index, specific gravity, and boiling point method reported earlier (6). The data are plotted as Figures 1 and 2. Although a method of plotting the vapor-liquid equilibrium data on a single ternary diagram (1) could have been used, a scheme involving two plots mas developed which will be found somewhat easier to read. Figure 1 is a y-x or vapor-liquid equilibrium plot for methanol in the ternary solution. It was constructed by plotting the data of Table I on the coordinates indicated. The number opposite each point is the mole percentage of water in the equilibrium liquid. Lines of constant mole per cent water were then drawn in by interpolation. Fixing the concentrations of any two components of a ternary mixture fixes its composition. Figure 2 for ethanol in the ternary mixture is analogous t o Figure 1. The water concentration in the equilibrium ternary vapor is obtained by difference. (For example, a liquid containing 20 mole per cent methanol and 30 mole per cent ethanol contains 50 mole per cent water. From Figure 1, its equilibrium vapor contains 36.5 per cent methanol, and from Figure 2 the vapor contains 38.5 per cent ethanol. Water in the vapor = 100 36.5 - 38.5 = 25 per cent.) Inspection of Figures 1 and 2 shows that, with a few exceptions, the data fall within 2 mole per cent of their correct positions with respect to the lines of constant mole per cent water. Since the analytical accuracy was better than this,