28
INDUSTRIAL A N D ENGINEERING CHEMISTRY
also by the modified adsorption method. Ethyl acetate ww used as the solvent for the oily matter, and methyl alcohol as the eluant for the sodium sulfonates. A comparison of the analyses is given in Table V1 Both methods gave essentially the same analyses, but the adsorption procedure was more rapid and convenient. A known blend containing 49.8 per cent oil, 15.5 per cent resin (65.3 per cent oily matter), and 34 7 per cent sulfonates was analyzed by the ethyl acetate-methyl alcohol adsorption procedure ; 66.2 per cent oily matter and 34.0 per cent soap was obtained. In the case of petroleum sulfonates which are not known to be free of resins, it is therefore advisable to introduce an ethyl acetate percolation a t the end of the petroleum naphtha wash, and before the methyl alcohol percolation, to test for the presence of resins. If the ethyl acetate displaces material which leaves practically no ash after ignition, the presence of resins is indicated, and one of the modified adsorption procedures described above should be adopted. DISCUSSION
The chief advantages of the adsorption procedure are freedom from emulsion difficulties, rapid convenient physical operations, and sharp separations of oil and sodium sulfonate components. Experience in this laboratory has demonstrated that a given adsorption procedure may fail t o give correct analyses on different types of samples. The procedure used will depend upon the adsorption characteristics of the components in the refined oilsoluble sodium soap. Unless the product t o be analyzed is known to consist entirely of oil and sodium sulfonates, as is normally the case, the mahogany soaps which are extracted by aque-
VoI. 16, No. 1
ous alcohol from caustic neutralized acid oil in the manufacture of medicinal white oil, it will be necessary to introduce an ethyl acetate percolation as a test for the presence of resins. The adsorption procedure can be applied t o crude oil-soluble sodium sulfonate products containing appreciable amounts of water and inorganic salts, after first removing these components This removal can be accomplished very conveniently in the determination of salt and water by the usual methods. For salt, a precipitation-type method such as A.S.T.M. Method D91-40 ( 1 ) may be used, and for water a modified Dean and Stark method is very convenient. Adsorption methods should find applications in grease and asphalt analyses, and in the separation of mixtures containing organic salts or acids and hydrocarbons. ACKNOWLEDGMENT
The author wishes to express his appreciation to S. W. Ferris, W. K. Griesinger, and C. E. Headington for advice arid assistance in the preparation of this paper. LITERATURE C‘TED (1) Am. SOC.Testing Materials, Committee D-2 Report 1941,
Method D91-40, p 236. (2) Archibald, F. M and Baldeschw eler, E L IND ENQ CHEM., ANAL.ED.,13, 608 (1941). (3) Strain, H. H., “Chromatographic Adsorption Analysis” New York, Interscience Publishers, 1942 (4) Strain, H. H I INDENOCHEM.,ANAL.ED.,14,245 (1942) ( 5 ) Zechmeister L.,and Cholnoky, L., “Principles and Practice of Chromatography” New York, John Wiley 6. Sons, 1941
Quantitative Determination of d-Galactose by Selective Fermentation W i t h Special Reference to Plant Mucilages LOUIS E. WISE
AND
JOHN W. APPLING Wis.
The Institute of Paper Chemistry, Appleton,
A simple method has been developed which permits the determination of small amounts of d-galactose in the presence of mannose, glucose, fructose, xylose, arabinose, and glucuronic acid with an accuracy of 92 to 98 per cent. It depends on differential fermentations with two yeasts, Saccharomyces carlsbergensis (N.R.R.L. No. 379) which ferments galactose, and S. bayanus (N.R.R.L. No. 966) which leaves galactose unfermented. The yeasts have little action on xylose, arabinose, or glucuronic acid, and these
R
ECENTLY, interest in the determination of galactose has beenjevived because of the newer technological applications of certain mannogalactan mucilages. The estimation of d-galactose which thus assumes a new importance, has always presented difficulties especially when other carbohydrates were present in quantity The van der Haar modification of the Kent-TollensCreydt method ( A ) , which depends on the oxidation of galactose to mucic acid, is not quantitative. Only when rigorous precautions are taken, and when relatively large amounts of galactose are present, does the procedure give fairly accurate results (14) Ever since the earlier investigations of Kluyver (7), the quantitative estimation of d-galactose by fermentation with certain yeasts has interested chemists and microbiologists. Kluyver
compounds do not interfere with the determination. Reducing values of galactore, mannose, and d-glucurone were determined using the Munson-Walker method of analysis. The fermentation techniques were successfully applied to the hydrolysis products of lactose and to certain plant mucilages. The possible applicstion of the method to galactose in the presence of galacturonic acid is being studied with a view toward its use in the analysis of other natural products.
found that galactose was fermented by two varieties of Sacharomyces cerevisiae and by a “milk sugar yeast“. He also showed that Schizosaccharomyces pombe, Torula monosa, and T. dattila did not ferment galactose. On the basis of these differences, he proposed a proximate method for the microbiological determination of galactose in the presence of other sugars Among those who used (and modified) Kluyver’s procedures were Schmidt, Trefz, and Schnegg ( I I ) , Hopkins, Peterson, and Fred (6), Sherrard and Blanco (IS), Kurth and Ritter ( 9 ) ,Kurth (8). Scott and West ( I d ) , Harding, Nicholson, and Grant (6),and, very recently, Menzinsky (IO). The last author showed that the strain of Saccharomyces cerevisiae which he used in galactose fermentation required preconditioning by culturing the yeast on a
ANALYTICAL EDITION
January 15, 1944
galactose-containing medium. Without such pretreatment the yeast was unable to utilize galactose. Besides the yeasts mentioned above, the following are known to ferment galactose: Saccharomyces pastorianus (S),S. m r x i a nus (3, 6),and S. fragilis (IO). Other yeasts known to ferment the common hexoses other than galactose are S. productivus ( 3 ) , S. apiculatus (a),and "Honey B" yeast (6). These lists are not exhaustive. Kluyver used the evolution of carbon dioxide as a meashe of fermentable sugars. Later investigators (6, 8) showed t h a t a quantitative measure of the reducing value simplified the analysis of fermented sugar solutions. Notwithstanding the extensive work on the selective fermentation of galactose, the results of relatively few experiments with pure sugar mixtures have been published and no attempt has been made to determine whether the methods could be applied in the presence of uronic acids. The limitations and general applicability of the fermentation methods are, therefore, indeterminate. The present study shows the usefulness of the microbiological method. EXPERIMENTAL
The objects ot the present investigation were to examine, more critically than heretofore, the application of differential fermentations to known sugar mixtures, noting their limitations, and to develop a proximate biochemical method for the determination of galactose in the hydrolyzates obtained from mucilages and hemicelluloses. Several strains of S cerevisiae, cultured ' r i the laboratories of The Institute of Paper Chemistry, had bcen shown to ferment galactose quantitatively in 1937 by Kurth (8). In 1942, however, qualitative experiments with these same strains showed 280
I
I
I
I
I
GLUCOSEe MANNOSE o GALACTOSE +
;-I O
REDUCING VALUES
OF SIMPLE SUGARS
(MUNSON 8 WALKER)
I
20
I
40
I
60
I
80
I
100
I
120
MILLIGRAMS OF SUGAR
Figure I
29
that none fermented 1 per cent galactose solutions completely within 190 hours. It is evident that strains of S. cerevisiae may lose their potency as galactose fermenters with time, unless special precautions are taken to recondition them. Torula dattila appeared a t first to give fairly promising results as a nonfermenter of galactose. About 92 to 95 per cent of the galactose (in a 1 per cent solution), when treated with Torula dattila, could be recovered after 2 days a t 30" C., but only about 80 per cent remained after a 5-day fermentation period. Whenever the concentrations of galactose dropped to 0.1 per cent, the sugar was rapidly destroyed. The use of these organisms was discontinued in favor of two interesting yeasts obtained from L. J. Wickerham, Northern Regional Research Laboratory, Peoria, Ill. These were Saccharomyces curtsbergensis var. mandshuricus, N.R.R.L. No. 379 (originally obtained from Charles N. Frey of the Fleischmann Laboratories in 1940), a highly fermentative strain acting on dextrose, galactose, and some of the common disaccharides, and S. bauanus, N.R.R.L. No. 966 (also originally obtained from Frey in 1940), which was known to ferment dextrose, sucrose, and maltose, but which, qualitatively a t least, had no effect on galactose. Neither yeast had (during the past three years) been kept on galactose media. These organisms proved entirely satisfactory. Neither had more than a slight effect on arabinose, xylose, and glucuronic acid. No. 379 fermented d-glucose, mannose, fructose, and galactose almost quantitatively within 48 hours. NO. 966 fermented the first three readily within the same time period and showed no action whatsoever on galactose. These differences led to the development of a satisfactory proximate method based on differential fermentations, in which the Munson-Walker reduction method ( 2 ) was used without modification. I n aliquot portions, taken from fermentation mixtures, 20 to 125 mg. of galactose could be determined with an accuracy of 92 to 98 per cent, even when other sugars were present originally in great excess. The yeasts were maintained in good condition by monthly transfers on glucose agar (Bacto-Dextrose agar, dehydrated). They have shown no decrease in potency over a period of 8 months. Agar slant cultures, 2 to 7 days old, were used for the preparation of the suspensions required in inoculating the sugar solutions. The following procedure was the same for either yeast. About 2 ml. of sterile water were pipetted into the tube containing the agar slant culture, and the surface growth was removed gently by means of the pipet, which also served to stir the suspension briskly. For each bottle slant of glucose agar required, 0.5 ml. of the suspension was removed and spread over the surface by tilting the bottle back and forth. Eight-ounce, narrow-mouth, square, flint glass bottles with molded screw caps were used as containers for sterile water, yeast suspensions, and bottle slants. Thereupon, the slants were incubated for about 48 hours a t 30' C. One bottle slant easily furnished enough inoculum for four subsequent sugar analyses, because a dense growth of yeast cells coated the agar surface a t the end of the 48-hour period. About 10 ml. of sterile water were added to the bottle slant, and the bottle was tilted back and forth to loosen the growth. About 5 ml. of the dense yeast suspension were pipetted into a sterile dilution bottle, and 20 to 30 ml. of sterile water were added. The exact amount of water depended upon the turbidity shown by a Cenco-Sheard-Sanford photelometer. Sus ensions whose readings fell within the range of 10 to 16 on t k s instrument when distilled water gave a reading of 90, subsequently fermented su ar mixtures satisfactorily. If the initial photelometer reading felf below 10, more water was added and thoroughly mixed with the suspension. Whenever the photelometer reading exceeded 16, more yeast suspension was added. In practice it was found better to work with too dense than with too light a suspension. The density range listed above was shown by plate counts t o approximate 35,000,000 cells per ml. This density also corresponded roughly to that of barium sulfate suspension prepared by mixing 5 ml. of a stock solution of 10 grams of barium chloride dihydrate per liter of water with 55 ml. of 1 per cent sulfuric acid and allowing the mixture to stand a t least a week in a sealed con-
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
30 T8ble
1.
Action of Yeasts on Pentoses after a 6-Day Incubation Period
Pentoses Present in Aliquot
Trpatment
CugO Obtained in Munson-Walker Determinations
?do. 12.5 (xylose) 12.5 (xylose) 12.5 (xylose) 12.5 (arabinose) 12.6 (arabinose) 12.5 (arabinose)
Control Organism Organism Control Organism Organism
379 966 379 966
26.3 20.8 22.3 28.6 26.7 26.7
tainer. It was found expedient to use 25 ml. of sugar solutions in the fermentations and to carry out all experiments in 125-ml. Erlenmeyer flasks. The total reducing sugar in such solutions never exceeded 2 per cent, and the galactose concentration was ordinarily kept within the range of 40 to 250 mg. er 25 ml. To the su ar solution were added 15 ml. of a filterefyeast extract. [Red Btar starch-free yeast cake was mixed with sufficient distilled water to yield a 10 per cent suspension. This was heated for 1 hour in an Arnold sterilizer a t about 100" C. and subsequently for 20 minutes a t 15 pounds' pressure (at 121' C.). The cooled suspension waa filtered several times through fluted filter paper using Cellite to clarify the solution. Ordinarily the yeast extract formed a slightly turbid solution. This was dispensed in 80-ml. portions into Erlenmeyer flasks, which were plugged with cotton and heated a t 15 pounds' pressure for 20 minutes. The cooled flasks of sterile yeast extract can be stored for months without change in a refrigerator.] The sugar and yeast extract mixtures, which showed a pH of about 5 to 6 (alkacid paper), were then sterilized a t 15 pounds' pressure for 15 minutes, cooled to about 30" C., inoculated under aseptic conditions with 10 ml. of the appropriate yeast suspension and incubated a t 30" C. for a minimum of 48 hours. The fermentations were always run concomitantly in pairs, under identical conditions, one flask being inoculated with organism 966 and the other with organism 379. Three or four times during the incubation period, the flask was rotated gently to bring the bottom yeasts into intimate contact with the sugar solution. A t the end of the fermentation period, each solution was diluted to 100 ml. with distilled water, thoroughly mixed, and filtered through two No. 50 Whatman filter papers. Twenty-five to 50ml. aliquot portions of the clear, pale yellow filtrates were taken for analysis by the usual Munson-Walker technique. (Care must be taken to digest the asbestos used in Gooch crucibles thoroughly with hot Fehling solution, and with concentrated nitric acid. To prevent later clogging of Gooch crucibles, such treatments should be continued until asbestos iilter pads ermit the rapid filtration of hot Fehling solution containing the gtered yeast extract referred to above.) The weight of cuprous oxide resulting from the fermentatioi with organism 379 was subtracted from that obtained by the use of organism 966. The galactose equivalent was calculated by the use of the galactose-cuprous oxide graph (Figure 1) drawn from data obtained experimentally with pure galactose. (The mannose values given in Figure 1 were obtained from pure &mannose; the glucose values were taken from Munson and Walker's tables.) The effects of S. carlsbergensis and S. bayanus on small amounts of xylose and arabinose were shown to be relatively unimportant, even after an incubation period of 144 hours instead of the usual @-hour period, This is indicated in Table I (and Table 11). Inasmuch as the net reducing values in all differential galactose determinations were obtained by subtracting the weight of cuprous oxide found after fermentation with organism 379 from that found after a fermentation with organism 966, the over-all error resulting from the presence of pentoses is negligible. Furthermore, the authors' quantitative fermentation periods seldom exceeded 2 days, which presumably would result in a lessened action on the pentoses. Because glucuronic acid may be a minor component of hemicellulose hydrolysis, the effect of d-glucurone in the galactose analysis was determined. Figure 2 shows the reducing values in milligrams of cuprous oxide plotted against the weights of glucurone (melting point 174-5' C.) taken for analysis. Over a fairly wide range, this curve is practically coincident with that of glucose. The action of organisms 379 and 966 on glucurone
Vol. 16, No. 1
was found to be very slight and their over-all effect in differential fermentations of galactose was almost negligible. This is indicated in the last row of Table 11. Orientating experiments were also carried out with purified galacturonic acid, which remains virtually unattacked by either organism, even when only small amounts of the acid are present in the usual fermentation mixture. The reducing value (Munson-Walker) of 12.5 mg. oi galacturonic acid was shown to be 23.0 mg. of cuprous oxide; when neutralized, sterilized, inoculated, and incubated in the usual manner, 20.7 mg. and 20.2 mg. of cuprous oxide were obtained with organisms 379 and 966. Here again the "error" is cancelled. The value and limitations of the selective differential fermentations in the determination of galactose are clearly shown in Table 11. The first and last columns of this table should be compared. Invariably, in the higher concentrations, galactose shows a slight but persistent residual reduction after fermentation with organism 379. Whether this is due t o very small amounts of unfermented galactose or (what is more probable) to the slight reducing power of the products of the fermentation is not known. The error is never very appreciable, but it accounts for the fact that galactose recoveries are usually somewhat low. Organism 966 is without effect on galactose.
"/ / O
'+
/i
./
REDUCING
VALUES OF
4
GLUCURONE
1
(COMPARED WITH THOSE OF GLUCOSE)
1 0 '
I
20
I
40
I
I
80
60
I
I00
i
I
120
1
140
MILLIGRAMS OF GLUCURONE
Figure
P
In order to determine whether galactose could be satisfactorily determined in the hydrolyzate of a disaccharide, pure lactose WBB heated with 2 r cent sulfuric acid a t the boiling point for 2.75 hours. The soctions were nearly neutralized with solid sodium carbonate (pH about 5, alkacid test paper). Aliquot portions representing, in each case, 125 mg. of lactose (hydrate) were sub-
January 15, 1944
Table
II.
Galactose Determination A l o n e and in Mixtures of Pure Sugars 2 Other Componenta
1 Gdactose Taken
Me.
MB.
125 125 126 150 (Fermented 72 hrs.) None None
Kone None None None
Mannose 125 Mannoee 125 Mannose 62.5 Mannose 80 Mannose 100 Mannose 230 Mannose 40 Glucose 40 Xylose 5 Mannose 20 Glucose 20 Xylose 45
ea.5
46 26 !&I
40
ANALYTICAL EDITION
J
40
3 CunO Obtained after Fernientation with No. 966
MS. 247.0 250.1 247.3 292.6
None Negligible
