Raffinose Preparation and Properties E. H. HUNGERFORD AND A. R. NEES, The Great Western Sugar Company, Denver, Colo.
W
HILE raffinose occurs
was removed from this precipiThe final molasses f r o m the barium desugarizingprocess is used as the of rafinose. The tate with carbon dioxide, and the wideIy distributed in nature in many grains solution containing little else method of preparation is described. Pure r a . than raffinose and was and plants, it is best known as a nose hydrate is obtained bY crystallization f r o m concentrated to about 55" Brix. minor constituent of sugar beets because of its troublesome effect water. The solubility of rafinose in water has Twenty-seven per cent barium on the crystallization of sucrose oxide on dry substance of the been determined. Other properties ape discussed. from beet juices and because of 55" Brix sirup was added in the the complication it adds to the form of a 20 per cent solution a t estimationof sucrose in sirups in which it occurs. Cottonseed 80" C. This amount of barium oxide was barely sufficient to meal has been the raw material from which most of the small precipitate the sucrose alone. The barium precipitate, after supply of pure raffinose has been obtained. The yield of removal of barium with carbon dioxide, contained 16 per raffinose from pressed cottonseed cake is 1 to 3 per cent. cent raffinose on dry substance, while the filtrate, after reThe raffinose content in sugar beets is variable but always moval of barium, contained 32 per cent raffinose on dry sublow, so low in fact that an exact determination is difficult. stance. This represented a considerable concentration of It is well known that beets grown in California always con- raffinose, but the loss in the barium precipitate was obviously tain relatively less raffinose than those grown in the Rocky very large. By concentrating the filtrate to 70 per cent dry Mountain region. The percentage raffinose in beets grown substance and adding three volumes of 70 per cent ethanol, in the latter locality varies from year to year in an inexplicable raffinose crystallized out and was further purified by t v o remanner, usually between the limits of 0.01 and 0.10 per cent crystallizations from alcohol. A pure product was obtained, on beets. but the yield was small. The observation of Saillard (6) and of Spengler ( 8 ) that Shortly after this work mas done, it was observed that raffinose is more abundant in beets grown in wet years does raffinose had crystallized spontaneously from a sample of not seem to apply to this region, where irrigation is practiced Johnstown molasses after standing several months in the to such an extent that the effect of the annual rainfall is un- laboratory. It was found that the crystals could be separated important, from the mother liquor by means of a centrifugal lined with monel metal cloth, and that the crude material could be puriPREPARATION O F RAFFINOSE fied by recrystallization from water. A few pounds of rafThe raffinose is extracted from the beets with the sucrose, finose having a purity of 97.5 per cent were prepared from passes through the refining process, and goes out in the final this molasses. Subsequently about 150 pounds (68 kg.) molasses, which may contain as little as one per cent a t non- of pure raffinose hydrate have been prepared in a similar Steffen factories and as much as 5 per cent or more a t Steffen manner. factories. Spontaneous crystallization of raffinose from the molasses Zitkoaski (9) was able to prepare raffinose from beet is slow but is accelerated greatly if the molasses is mixed with molasses of the latter type by a process based on the relative seed crystals and cooled. I n 40 days 78 per cent of the rafinsolubility of lead raffinosate and the relatively high solu- finose crystallized from one sample kept a t about 20" C. bility of lead saccharate a t high temperatures. However, Over 60 per cent crystallized from the same molasses in 20 the concentration of raffinose is too low to make this and other days when agitated slowly and kept a t a temperature of 15" processes feasible in recovering any appreciable part of the to 18" C. These samples were heavily seeded by the addition 3000 odd tons of raffinose contained in molasses produced of 5 per cent of their weight of an aged molasses containing annually in the Rocky Mountain region. a very large quantity of crystalline raffinose. One factory a t Johnstown, Colo., employs the barium It was soon learned that the crystals could be more satisprocess for desugarizing molasses and uses the discard molasses factorily separated by filtration than by means of the cenfrom the Steffen factories as its source of raw material. trifugal : About 40 per cent Of the raffinose is precipitated the The massecuite diluted to 65 per cent d r y substance by sucrose by this process and remains in a final molasses which cold water, 40 to 50 c., and pumped through a plate and frame has approximately the following composition: filter, dressed with 16-ounce (0.45-kg.) duck under a pressure of 35 t o 40 pounds er square inch (2.5 to 2.8 kg. per sq. em.). % One-inch (2.5-cm.7frames were completely filled in 15 to 20 minDry substance 75.0 Utes. Satisfactory washing cannot be carried out in the press 66 0 on dry substance Sucrose since the cake channels easily. The best results were obtained 22 0 on dry substance Raffinose by elutriating the unwashed cake with cold water and refiltering. When this type of molasses first became available, the This second cake was superficially washed and blown as dry as authors prepared raffinose from it by a method based On the possible with compressed air. The crystals obtained in this contained 80 to 82 per cent raffinose anhydride on dry factthat calcium raffinosateis less soluble than is calcium manner suhst ance. saccharate under the conditions of Steffen cooler operation, Purification of the crude material was carried out in the foland on the further fact that barium raffinosate is more soluble lowing manner: The crystals were dissolved in hot water to form than is barium saccharate. Powdered lime was added t o a solution containing 50 to 55 per cent dry substance, to which Was added about 3 Per cent vegetable carbon* The the diluted Johnstown molasses in a Steffen cooler a t 10" C. was heated and filtered, and the concentration of filtrate td'usted The resulting precipitate containing most of the raffinose and to 45 per cent dry substance. It was then cooled t o 37 and a large part of the sucrose was filtered and washed. Lime seeded with a small amount of pure raffinose hydrate. On furu
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April, 1934
rV D U S T R I A L A I\; D E N G I K E E R I N G C H E i VI STR Y
ther cooling t o 20” to 25” C., preferably with constant stirring, a heavy mass of crystals separated, forming a thick suspension, which, however, may be pumped through the filter press without dilution. The crystals are 94.5 to 96 purity. The filtrate is evaporated to 55 per cent dry substance and a second crop of crystals is obtained, which are about 90 purity. The filtrate from this second crystallization contains about 35 per cent raffinose on dry substance or about 10 per cent of the total in the original crude material, and mzy be concentrated and added to the molasses in the crystallizers. Raffinose of better than 99 purity can be obtained by the recrystallization of the 94 to 96 purity crystals. One more recrystallization gives a product of practically 100 purity.
From the measurement of the increase in the dimensions of crystals grown in pure sucrose solutions, the following ratios were obtained: TESTS
A
The yield of high-purity raffinose is kept quite high by the proper mixing back of “green sirups” and “raw sugar” in the same manner as in a refinery. One ton of molasses will yield about 175 pounds (79.4 kg.) of easily recorerable raffinose hydrate.
PROPERTIES OF RAFFIXOSE FORMO F SUof sucrose obtained on crystallization from sirup containing small amounts of raffinose are well known to beet sugar refiners. Slow crystallization of sucrose from such sirups is a problem also familiar to sugar boilers. Quantitative data measuring these phenomena, however, are few. Some results obtained in this laboratory are interesting in that they interrelate slow crystallization and deformed crystals in a roughly quantitative way. The sucrose crystal is hemimorphic monoclinic. The large faces (Figure 1) intercept the inclined axis, A - A’, and are parallel to the plane formed by the intersection of the vertical C - C’ axis and the horizontal B - 8’ axis. Being hemimorphic, the growth of the crystal is dissimilar at the ends of the B - B’ axis. Growth is much more rapid a t the right end. I n these experiments individual crystals were developed in supersaturated sugar solutions, with and without raffinose, and the growth was measured under a microscope in three directions-namely, along the B - IZ’ axis, along the C - C’ axis, and perpendicular to the plane formed by the intersection of the R - B’ axis and the C - C’ axis. Obviously the latter measurement is not parallel to the A - A’ axis since this axis forms an angle of 103’30’ with the plane of intersection of the other two axes. It was not possible to grow the crystals in pure sucrose solutions and in sucrose solutions containing raffinose under identical conditions of supersaturation because the influence of raffinose on the solubility of sucrose is not known exactly. It has been shown by Brown and Nees ( d ) , however, that the solubility of sucrose in water is reduced by the presence of raffinose. The supersaturation of the solutions containing raffinose used in these experiments was therefore higher by an unknown amount than in those containing no raffinose. EFFECTO F
R.4FFINOSE ON THE CRYSTAL
CROSE. The peculiar flat crystals
- A’
0 85
c -
B -B’ 1 000
1 2595
C’ 0 8782
Within the limits of error, the growth meabured was one axial length along B - B’ and C - C‘ axes and one-half axial length along A - A’. 9series of tests run a t 25” C. with solutionq, each containing 245 grams wcrose per 100 grams water but with quantities of raffinose varying from zero per cent to 5 per cent on sucrose, gave the results n-hich are tabulated in Table I. TABLEI. RELATIVE INCREASE IN DIMESSIONS OF SUCROSE CRYSTALS GROWNIX SOLUTIONS CONTAINING 245 GRAMSSnCROSE PER 100 OF WATERAT 25’ C.
9 SUCROSE CRYSTAL
1 00
According to Becker and Rose (1) the crystallographic axes bear the folloning length ratios:
2 2
“NORMAL”
R E L A T I ~IXCREASE E I N DI\IENYIONS 4’ B - B’ c - C’
a -
0 63
38
TESTS
FIGURE 1.
463
2 2 4 8 8 2 1
G R O ~ TOF H RAFFINOSE B - B‘ yo on w c r ( M m . p e r d o y 0.00 0.10 0.30 0.50 0.75 1.00 2.00
3.00
4.00 5.00
1.00 1.18 0.89 0.74 0.58 0.51 0.38 0.43 0.38 0.45
RATIOO F INCREASE TO INCREASE IN B A 4’ B - R C - C’
f3’
-
0.56 0.47 0.45 0.47 0.50 0.57 0.42 0.07 0.08 0.06
1.00 1.00 1.00 1.00 1.00
1.00
1.00 1.00 1.00 1.00
0.80 0.74 0.71 0.97 1.00 1.16 0.92
0.67 0.61 0.50
The relative rate of growth along the three axes is changed little with concentrations of raffinose up to 0.50 per cent, Between 0.5 and 1 per cent raffinose, the C - C’ ratio increases, tending to make the side of the crystal appear more nearly square. Above 2 per cent raffinose, however, the type of deformation changes. The ratio of growth along the A - A’ axis-i. e., in thickness-to growth along the B - B‘ axis (length) decreases rapidly with increase in raffinose concentration until a t 5 per cent no growth occurs in the direction of the A - A ‘ axis. Growth along the C - C’ a x i s also diminishes to about one-half its value in pure sucrose solutions. At t h e s e h i g h e r concentrations the c r y s t a1 s become very thin, narrow plates. Column 3 of Table I gives a rough measure of 1 70I i BOi j i IOi 1 20I i ,,‘A,h,i,,~BY:~liiHS the rate of crystal30 40 50 60 lization of sucrose FIGURE 2. SOLUBILITY OF RAFFINOSE u n d e r t h e condiARXYDRIDE I N WATER tions of the experiment. It will be observed that even 0.5 per cent raffinose diminishes the rate about 2.5 per cent, and, a t 2 per cent raffinose and above, the rate is reduced to a half or a third of its value in pure sucrose solutions. Summarizing these results, raffinose diminishes the rate of deposition of sucrose on sucrose crystals on all faces. High concentrations of raffinose eventually prevent deposition of sucrose on the pinacoid faces which intercept the inclined axis and greatly retard deposition of sucrose on the faces parallel to the inclined axis.
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464
SOLUBILITY OF RaFFINosE IN WATER. The solubility of raffinose in water has been determined from 0" to 78" C. At temperatures below 78" C. raffinose crystallizes from water with five molecules of water of crystallization. Above this temperature the hydrate is not stable.
The apparatus used for the determination of the solubility of raffinose consisted of a brass cylinder 2 X 7 inches (5.1 X 17.8 cm.) fitted at the open end with a removable cover plate carrying a bearing 4 inches (10.2 cm.) long, in which a stirrer shaft rotated. When in use the apparatus was submerged in a constant-temperature bath within one inch (2.5 cm.) of the top of the bearing. The constant-temperature bath was the one used by Brown, Sharp, and Dahlberg (3) in their work on the solubility of sucrose in beet house sirups. For the determinations at 0" C., the apparatus was placed in a double-walled Parr colorimeter apparatus and surrounded by finely chipped ice. Temperatures were measured with thermometers tested by the Bureau of Standards. Samples for analysis were drawn with the same sampling device used by Brown, Sharp, and Dahlberg (3). It consists of a cover plate similar t o the one used on the container, pierced by a thin co per tube on one end of which was soldered a disk-shaped filter o! monel metal screen. A tire valve and a stopcock were also threaded into this plate. It was found necessary to cover the monel metal screen filter with duck filter cloth. The operation was simple. Water and pure raffinose hydrate were placed in the container, the cover plate carrying the stirrer was secured tightly, and the entire apparatus was clamped in the bath. The stirrer was turned at a rate of 100 r. p. m. After 16 to 48 hours the apparatus was raised sufficiently to lift the cover plate from the water, and the top was dried carefully and removed. The sampling apparatus, previously dried and heated to the temperature of the bath, was quickly placed in position. Compressed air was admitted through the tire valve, forcing clear solution upwards from the filter into a weighed flask surrounded by cracked ice to minimize evaporation. This operation required but a few seconds. The flask was stoppered, cooled if necessary, and weighed. At the higher temperatures, the sample was diluted with a weighed quantity of water before analysis. Analysis was made by dry substance determinations according to Brown and Sharp's procedure, except that the temperature of the oven was held at 70" to 72" C., or by accurate polarizations, or both. Duplicate dry substance determinations varied no more than 0.02 per cent. Raffinose determination by dry substance and by polarization agreed very closely. At several temperatures solubility equilibrium was approached from both sides with very satisfactory concordance. The melting point of the pentahydrate was determined in the usual manner and found to be 78.0 with a probable error of +0.05" C. This was used as the temperature at which raffinose forms a saturated solution in its water of crystallization corresponding to concentration 84.84 per cent raffinose anhydride. The results given in Table I1 are expressed in terms of raffinose anhydride, though the solid phase in equilibrium with the solution is raffinose pentahydrate. OF RAFFINOSE IN WATER TABLE11. SOLWILITY
TEMP. RAFFINO~FJ A N H Y D R I D ~ TEMP. RAFFINOSE ANHYDEIDFJ * C. % on soln. G./lOO g. H?O C. % on s o h . U./lOO 8. H z 0 0.00 0.02a 10.00 16.38"
3.3
20.00 24.80a
12.0 16.10 16.28
16.90
25.05" b
6.2 9.48 9.77
3.4
30.00
6.6 10.47
40.00 50.00
3.42
10.83 13.6 19.19 19.45
20.26 16.86 Determined. Melting point of the pentahydrate.
26.5OO a
3.31
39.3Sa
53.20"
60.00
61.60"
70.00 78.0b
21.3 32.27 33.3
27.1 47.65 49.9
46.5
86.9 105.13
74.3
289.1
51.25 60.6 62.50 84.84
153.8 166.67
559.63
When plotted, these results lie on a smooth curve (Figure a), which from 24" to 78" C. is nearly a straight line. The solubility of sucrose according to Herzfeld (6) is shown on the same graph for comparison. These results agree roughly with the approximate ones reported by Browne (4) but are very different from the single result obtained by Schecker (7) a t 24" C. His value of
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28.4 per cent raffinose anhydride is nearly 13 per cent too high. The three results at about 25" C. reported in this paper lie exactly on the curve. Two of these points were obtained by approaching saturation equilibrium from an undersaturated condition and the other from a supersaturated condition. I n the latter case, a 32 per cent raffinose anhydride solution was brought to equilibrium over rafbose hydrate crystals in 48 hours. The raffinose used for these determinations was prepared from Johnstown molasses. Direct polarization and dry substance determination indicated that the material contained a t most 0.01 or 0.02 per cent sucrose. Very little is known of the solubility and other properties of anhydrous raffinose, although certain inferences may be drawn from the following series of tests: One gram of raffinose anhydride was weighed into each of six small dishes. These were placed in individual desiccators over sulfuric acid solutions of such strength that the vapor pressures of water in the desiccators were kept a t 7.3, 11.8, 14.0, 15.5, 17.6, and 18.8 mm. of mercury a t 25" C. These dishes were weighed a t frequent intervals for one month. After 3 days the sample kept in an atmosphere containing 7.3 mm. of water vapor had increased in weight 5.02 per cent and was caked. All the others gained from 11.5 to 15.1 per cent and were in solution. Those exposed to 11.8 and 14.0 nun. vapor pressure were like clear glass, and those exposed to 15.5 and more formed viscous solutions. At the end of 2 weeks all had gained weight, but only the ones exposed to 17.6 and 18.8 mm. had gained the theoretical amount to form the pentahydrate. These two had partially crystallized into a mass of needle-shaped crystals. Those exposed to 11.8, 14.0, and 15.5 were clear liquids. This is a striking example of the difference in solubility of a substance and of its hydrate. At 25' C. the solubility of the pentahydrate is 19.4 grams raffinose anhydride per 100 grams water, whereas the anhydride forms a solution containing about 1000 grams per 100 grams water a t this temperature. The solution formed in this manner is too viscous to permit a rapid transition to the pentahydrate, which is the stable form below 78" C. The difficulty encountered in drying raffinose hydrate at temperatures above 80" c. is related to the high solubility of the anhydride. At 80" or above, the hydrate is unstable. The solution of the anhydride evaporates to a glass, from which water is forced with difficulty. Raffinose hydrate is easily and completely dried under moderate vacuum a t 70" to 75" C. Its vapor pressure a t 25" is 2.3 mm. mercury. The anhydride does not show definite melting point. The uses of raffinose have been limited to small quantities required for the preparation of certain culture media, and to that employed by various investigators in studying its chemical and physical properties, Heretofore the price has been so high that no one has been interested in developing further useq. LITERATURE CITED (1) Becker, K., and Rose, H.. 2. Physik, 14,369-73 (1923). (2) Brown, R. J., and Nees, A. R., IND. ENG. CHEM.,25, 555-8, (1933). (3) Brown, R. J., Sharp, J. E., and Dahlberg, H. W., IND. ENG. CHEM.,20, 1230 (1928). (4) Browne, C.A.,Handbook of Sugar Analysis, p. 735,Wiley, 1912. (5) Herzfeld, 2. Ver. deut. Zuckerind, 42, 147 (1892). (6) Saillard, Compt. rend., 192, 1748-50 (1931). (7) Scheoker, 2. Ver. deut. Zuckerind., 74,82 (1924). (8)Spengler, O.,Chem. Zentr., 1931, 11, 506. (9) Zitkowski, Am. Sugar Ind.,13,8 (1911). RECEIVED October 23, 1933. Presented before the Division of Sugar Chernietry at the 86th Meeting of the American Chemical Society, Chicago, Ill., September 10 to 15, 1933.
