264
ANALYTICAL CHEMISTRY
only when the distribution is uniform and when the quantity of solution per unit quantity of paper is the same in the two papers. .4lso comparison of R values of the same solute between different experiments can be made only when the quantity of solution per unit quantity of paper is the same in the different experiments. This is particularly important for radial flow. If R is not a constant, then its rate of change with t becomes important. From Equation 8, it is seen that the absolute value of dR/dz must decrease with increase of z unless the absolute value of Z - R increases rapidly with t . For further insight into this question, Equation 8 may be written as
For flow in a strip of paper of uniform cross section, L,P, and are proportional to the quantity of solution crossing the boundary a t y and z , respectively, per second. Consequently u,p, will be equal to or greater than v,p,. For radial flow v,p,y and z ~ ~ p ,are z proportional to the quantity of solution crossing the respective boundaries and again v Y p Y is equal to or greater than z',p,. On the other hand pz f(p,) will be less than p u f(c)(pole) and consequently the whole term could have values less than unity but not necessarily so. If it does have values less than unity as R must have, then the difference between this term and R may be small so that R does not change rapidly vith 2. For the systems examined in this paper, R does not appear to be a sensitive function o f t . With downward flow, for which the distribution is rather uniform, R for methyl orange in lactic acid solution is a constant within experimental error. With upward flow, for which the distribution decreases with z , R decreases slowly. With radial flow for which the distribution markedly drcreasc~swith z , R for methyl orange appeared to be constant.
Since this could possibly be a fortuitous case, the experiment using fluorescein was made in which R was found to decrease. These considerations throw new light on the behavior of chromatographic zones in circular paper chromatography. For radial flow we have pointed out that v,p, > u,p, and therefore vy/v. 2 zp,/ypY. It appears from this equation that o y is probably greater than vI. Accordingly the migration of the solution at any given boundary with a certain radius would be greater than that at any other boundary with a greater radius. If then a rather wide zone of a solute is present on the paper, the velocity of the solution a t the forward edge would be less than the velocity at the trailing edge. I n such cases the zone would become narrower and would appear better defined as it moves outward but the concentration of the solute in solution in the zone would tend to remain constant.
ilpI
+
LITERATURE CITED (1) Austin. C. R., and Shipton, J.. J . Council Sci. Ind. Research, 17,
+
I
115 (1944). (2) Cassidy, H . G., A x ~ L CHEM., . 24,1415 (1952). (3) Fujita, H., J . P h y s . Chern,, 56, 625 (1952). (4) Holmgren, A. I., Biochem. Z., 14, 181 (1908); Kolloid-Z., 4 , 219 (1908). ( 5 ) Kowkabany, G. N.. and Cassidy, H. G., ANAL.CHEM.,24, 643 (1952). (6) Krulla, R., 2. p h y s i k . Chem., 66,307 (1909). (7) Le Rosen. A. L., J . A m . Chem. Soc.. 69,87 (1947). (8) Le Rosen, .4.L.. and Rivet, C. A . , Jr., AN.AL.CHEM.,20, 1093 (1 948). (9) lluller, R. H., and Clegg, D. L., Ibid., 23, 403, 408 (1951). (IO) Peek, R. L., Jr., and McLean, D. A., IND.EXG.CHEM.,ANAL. ED.,6 , 8 5 (1934). (11) Schmidt, H., Kolloid-Z., 24, 49 (1919). . 22,41 (1950). (12) Strain, H. H., A N . ~ LCHEM., (13) Takahashi, A,, Kagaku, 20,41 (1950). RECEIVED for review August 20,
1953.
4ccepted Sovember 21. 1953.
Chromatographic Separation of Sugars with Hydrocellulose 1. D. GEERDES, BERTHA A. LEWIS, REX MONTGOMERY, and FRED SMITH Division
o f Agricultural
Biochemistry, University of Minnesota, St. Paul, M i n n .
Although cellulose and certain types of modified cellulose have been used successfully for separating various mixtures of sugars and their methyl ethers, there is need of an adsorbent with a better resolving power and a greater capacity. The process of dissolving cellulose powder in phosphoric acid, followed by precipitation with water, produced a desirable hydrocellulose for use in the chromatographic separation of methylated sugars. Both the resolving power and capacity of this hydrocellulose were superior to other cellulose adsorbents. However this
P
-1RTITION chromatographic analysis (6) has become a standard technique for the separation, identification, and quantitative determination of sugars (5, 8, 11 ) and cellulose has been extensively used as the adsorbent (f2). For preliminary identification purposes and the separation of small quantities of compounds, cellulose in the form of filter paper is generally satisfactory. I n order t o obtain larger quantities for complete characterization, the paper has been replaced by columns of cellulose powder (3, 8). Usually, for column Peparation a single solvent mixture, is preferred ( 2 ) , since the romplete separation of a mixture of sugars can be effected by means of an automatic fraction collector without attention once the process has been started, but in some instances it appears to be an advantage to use R series of solvents (4).
improvement did not extend to the separation of free sugars. It is believed that part of the success of the modified column is due to the top of the adsorbent being kept in place by a glass-enclosed metal weight. The anomeric forms of ethyl-crhamnofuranoside have been separated. The increased capacity and improved resolution obtained with the modified columns make it possible to effect separationof practical amounts of methylated sugars without being involved in the slow development rates of larger columns. Although cellulose and certain hydrocelluloses have been used successfully for separating various mixtures of sugars and their methyl derivatives ( 2 , 8 ) , there is need of an adsorbent with a better resolving power and a greater capacity. On the assumption that there is considerable association of cellulose molecules through hydrogen bonding (IO), a considerable portion of the surface of the cellulose plays little or no part in chromatographic separations and hence the capacity of unmodified cellulose is low. Some support for this view is forthcoming from the observation that modification of the cellulose by acid (9) and by oxidizing agents ( 1 ) acting in heterogeneous systems has been shown t o improve its chromatographic properties for certain purposes. I n view of this it seemed that if the orderly arrangement of the cellulose molecules was disrupted completely by dissolving the
V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4
265
line to litmus. After stariding for about 2 hours under the alkaline conditions the hydrocellulose w a s w a s h e d Composition Front Rc Value of Sugar Band until the washings were neuColumn Weight, Time, Tube Leading Trailing Separation tral. Filtration, followed by Adsorbent Sugars mg. Min. No.' edge edge (Rt units)* washing with absolute ethanol and light petroleum ether Cellulose, 2 , 3 4.6-Tetra-O10.0 X 1 . 4 c m methyl-D-glucose 3 50 2-5 0.91 0.67 ,. (boiling point, 30" to 60" e.) 2,3,6-Tri-O-methyIand drying in vacuo yielded a 3 50 5-10 D-glucose 0.71 0.50 0 . 0 4 oveilap fine w h i t e p o w d e r w h i c h 2,3-Di-O-niethyl-~50 14-24 0.44 elucose 0.30 0 06 swelled but did not diseolve in -3 Total 9 water. Comparison of HydrocelluHydrocellulose2,3,4.6-Tetra-Olose and Cellulose in Column cellulose (1 : 1) methyl-D-glucose 3 65 4-8 0.81 0.62 10.0 X 1.4 cm.' 2,3,6-Tri-O-methylP a r t i ti on Chromatography. D-glUCOSe 3 6.5 13-20 0.52 0.39 0.10 The cellulose adsorbent conZ,a-Di-O-methyl-~sisted of Whatman No. 1 celluglucose -3 65 38-48 0.26 0 21 0.13 lose powder. The adsorbent Total 9 and the hydrocellulose were Hydrocellulose2.3,4,6-Tetra-Oseparately ground and passed cellulose ( 1 : l ) C methyl-D-glucose 720 260 8-21 0.79 0 . S .. through an 80-mesh screen. 40.0 X 3.0 em. 2.3.6-Tri-0-methyl33-60 0.45 0.33 D-glucose 652 260 0.10 When a column was packed 2,3-Di-O-methyl-~only with the hydrocellulose 110-136 0.20 glucose 60 260 0.17 0.13 the rate of flow of liquids Total z 2 through it was much too slow a Tubes were numbered from front time: collection time for each fraction of approximately 0.7 nll. w a s 5 minutes for most practical purposes. for two small columns. For larger column, 4- to 5-ml. fractions were collected a t 10-minute intervals. Hence the hydrocellulose was b Separation in Rt units corresponds t o numerical difference in Rt value between trailing edge of one component and leading edge of the one following. mixed with an equal amount C Recovery of three components was quantitatire. of Whatman KO. 1 cellulose powder. Two columns oi adsorbent. 10 cm. long and 1.4 cm. in diameter, were each packed with 7 grams of dry powder, the one with cellulose and cellulose and then reprecipitating it, the product would have a the other with the mixture of cellulose and hydrocellulose. The greater surface area and consequently it might be more effective columns were maintained a t 30" C. by water circulated from a than forms of cellulose modified in heterogeneous systems. thermostat. Accordingly, cellulose powder was disPolved in phosphoric acid A constant boiling methyl ethyl ketone-water azeotrope was used as the developing solvent for separating methylated sugars. and precipitated by water. The material %-as then compared Before commencing separations, prewashing of the column adsorbwith untreated cellulose with respect to its capacity and resolving ent was carried out for a sufficient time (1 to 2 days) to establish power for the separation of certain sugars and their methyl equilibrium. A constant head of solvent was maintained above derivatives. the adsorbent during the course of the experiments except \Then the mixtures were being put on the column. A mixture of 3 mg. each of 2,3,4,6-tetrs-,2,3,6-triand 2,3-di-0EXPERIM EiYTA L methyl+-glucose was dissolved in the developing solvent (0.5 Hydrocellulose. Five ml.). The solvent above the column was withdrawn with a pipet grams of powdered cellulose and the solution of the mixture of methylated sugars was added (Solka floc obtained from the dropwise to each column, care being taken to see that a layer of Brown Co., Berlin, N. H.) liquid did not form above the adsorbent; this was achieved by was quickly dissolved in 8 5 % adding the solution of the sugars a t about the same rate that the phosphoric acid (250 ml.) solvent left the column, When all the solution had soaked into with the aid of a Waring the top of the column, a constant head of developing solvent was L Blendor ( I S ) . T h e t h i c k arranged and the effluent was collected a t suitable intervals by means of a fraction collector. transparent solution was poured immediately with stirA small amount of effluent from each tube was spotted on paper ring into 3 liters of water. by means of a platinum loop and the distribution of the componBLarger quantities of cellulose ent sugars in the effluent was determined by spraying with either p-anisidine (9) or .Y,.V-dimethyl-p-aminoaniline reagents (2, 3). required a longer time to dissolve in the phosphoric acid The Rt value of the sugar was taken as the time required for the and this resulted in too much solvent t o travel the length of the column divided by the time redegradation. The p r e c i p i quired for the sugar to appear in the effluent. The time re-c tated h y d r o c e l l u l o s e was quired for the solvent to travel down the column was ascertained washed repeatedly by decanby adding a small amount of Sudan IV dye with the sugar mixture tation with water until the and noting when the dye emerged from the column. Movement washings were neutral to of the dye as a narrow band indicated that the column had been litmus. It was suspended in correctly packed. u-ater (3 liters) and ammonium hydroxide was added Comparative results for the separation of the representative until the solution was alkamixtures of methylated sugars are given in Table I. A series of experiments carried out with the free sugais ( D xylose, D-fructose, L-rhamnose, and D-glucose) using l-butanolethyl alcohol-water ( 4 t o 1 to 5 ) as the developer showed that 7 D the mixture of hydrocellulose and cellulose was no better than cellulose for separating these sugars. Figure 1. Diagram of Upper Portion of Column E Preparation of the Larger HydrocelluloseCellulose Column. After having established with the small columns that addition of A . Solvent level -F B. Metal weight enclosed i n hydrocellulose to cellulose facilitated the separation of methylated glass sugars, a larger column was prepared t o separate greater quantiC. Water jacket D . Perforated ceramic disk ties of methylated sugars. This enabled a comparison t o be E. Filter paper disk F . Column packing made of the small and large columns and i t also became possible t o ascertain the approximate capacity of the new type of column.
Table 1. Comparison of Separation of Methylated Sugars on Columns of Cellulose and of Hydrocellulose
, ,
-
...... ...... . . e . . .
.. ..+ ....
...... ...... . a . . . . . a
>
ANALYTICAL CHEMISTRY
266 A glass tube 3 cm. in diameter and 64 cm. long was packed dry with 160 grams of a mixture of equal parts of cellulose and hydrocellulose adsorbent to give an adsorbent column 40 cm. in length. A filter paper and a perforated ceramic plate were put on the tightly packed column. A JVood's metal cylinder, sealed in a glass tube (weight 320 grams, diameter 2 cm., length 20 cm.) was placed on top of the ceramic plate to keep the adsorbent firmly in position (see Figure 1). The column was surrounded with a water jacket and maintained a t 30" C. Before use the adsorbent was prewashed in the usual way until equilibration had been established. I n order to minimize spreading of the bands the concentrated solution of the mixture of sugars was added dropwise avoiding, as with the small column, the formation of a layer of liquid above the adsorbent. Results obtained with this column are also recorded in Table I. The approximate capacity of the column was determined by adding increasing amounts of 2,3,4,6-tetra-0-methyl-n-glucose and determining the increase in the number of tubes of the effluent in which sugar mas detected. Synthesis of a- and p-Ethyl-L-rhamnofuranoside and Their Separation on a Hydrocellulose-Cellulose Column. A jield of 4.5 grams of rhamnose ethyl mercaptal] prepared from L-rhamnose hydrate (9.5 grams) by the method of Fischer (6) had a melting point of 1'13' to 134.5" C. after recrystallization from ethanol. The rhamnose ethyl mercaptal (3.6 grams) was transformeJ into a mixture of the a- and p-ethyl-L-rhamnofuranosides by reaction with ethyl alcohol, mercuric chloride, and yellow mercuric oxide according to the method of Green and Pacsu ( 7 ) . After removal of salts, neutralization of the reaction mixture, and concentration to dryness in vacuo, the residue was extracted with ether. Concentration of the ethereal extract in vacuo gave a sirup (1.95 grams) which had [a12 - 18.9" in water (c, 1.2). Paper chromatography of the sirupy mixture of glycofuranosides using methyl ethyl ketone-water azeotrope as the developer and p-anisidine-trichloroacetic acid as the spray reagent indicated three components-the 01- and p-glycofuranosides and a trace of a slower moving component which was probably the free sugar. The a- and p-glycosides did not separate completely on the paper. A solution of the sirupy glycosides (1.6 grams) in methyl ethyl ketone-water azeotrope (2 ml:) was added dropwise to the hydrocellulose-cellulose column and developed with the same solvent. The fractions were collected a t 15-minute intervals and the glycosides were detected by spotting the effluents on paper and spraying the p-anisidine; a yellow-brown color indicated the presence of the glycoside. The glycosides appeared in tubes 10 to 30 (R: = 0.69 to 0.40). The material collected in tubes 10 to 14 (Rt = 0.69 to 0.59) crystallized spontaneously and gave a-ethyl-brhamnofuranoside (450 mg.), with a melting point of 56.5" to 57.5' C. and [ a ] g98" in water ( e , 0.74) after recrystallization from ethyl alcoholpetroleum ether. The product obtained from tubes 15 to 16 (Rt = 0.59 to 0.56) was nucleated with the a-form but crystallization proceeded slowly since it was contaminated with the p-form. The material was therefore combined tvith that from tubes 17 to 30 ( R , = 0.56 to 0.40)and separated on the column as before. The glycoside appeared in tubes 10 to 28 (Rt = 0.69 to 0.42). Removal of the solvent from the effluent in tubes 10 to 14 ( R t = 0.69 to 0.59) gave the a-anomer (300 mg.). The remaining material in tubes 15 to 28 was rechromatographed. I n this final fractionation the separation was almost quantitative. Thus tubes 11 to 16 ( R : = 0.67 to 0.56) gave the pure a-isomer (130 mg.), tube 17 ( R t = 0.56 to 0.54)contained mainly the p-form 82" in water ( e , 1.0) a8 shown by its specific rotation [a]: while tubes 18 to 28 ( R , = 0.54 to 0.42) afforded the 0-form (570 mg.), specific rotation [ a ] : 105' in water (c, 1.0). The latter crystallized spontaneously when cooled to 5' C. and had a melting point of 21" to 24' C. Application of Hydrocellulose-Cellulose Column to Separation of Cleavage Products of Methylated Polysaccharides. The column has been used successfully for the separation of 2,3,4-
+
+
tri-O-methyl-n-xyloseJ 2,3-di-O-methyI-n-xylose, 2-0-methyl-Dxylose, 3-O-rnethyl-n-xylose, and n-xylose, obtained by hydrolysis of the methylated hemicellulose from the straw of flax (Linum ?Lsitatissim?bmsp.). The cleavage fragments of methylated aspen wood hemicellulose have also been separated in a similar manner. Further details of these investigations are as pet unpublished. RESULTS AND CONCLUSION
Examination of the results in Table I shows that with methyl ethyl ketone-water as the developing solvent a column containing hydrocellulose and cellulose is better than one containing only cellulose for the separation of methyl sugars. Increasing the amount of 213,4J6-tetra-0-methy~-~-g~ucose from a few milligrams to 2 grams did not result in an increase in the number of tubes in which the sugar could be detected so that 2 grams did not represent the capacity of this relatively small size column. When 4.5 grams of 2,8,4,6-tetra-O-methyl-~-glucose were added to the column, overloading became evident. The Rt values for the leading and trailing boundary were 0.80 and 0.48, respectively, as compared with values of 0.79 and 0.60 for the leading and trailing boundaries before saturation point had been reached. Colorimetric determinations showed, as expected, that the sugar \vas concentrated in the leading portion of the solvent boundary carrying the sugar. I t is believed that part of the success of this modified column is due to the top of the column being kept in place by the metal weight. The new column has been succe~sfullyused for the separation of the anomeric forms of ethyl-L-rhamnofuranoside, a resolution which is normally difficult by the usual classical procedures. I t is valuable for separating the cleavage products of methylated polysaccharides. I t is surprising, however, to find that there was no significant difference between the two types of adsorbent as far as the separation of free sugars was concerned uving 1-hutanol-ethyl alcoholwater as the solvent. ACKNOWLEDG,MENT
The authors wish to thank the Graduate School of the University of Minnesota for a research grant. LITERATURE CITED
dstwood, E. B., Raben, M. S..Payne, R. W., and Grady, A. B., J . Am. Chem. Soc., 73, 2969 (1951).
Boggs, L. A., Cuendet, L. S.,Dubois, l f . , and Smith, F., -4NAL. CHEM.,24, 1148 (1952).
Roggs, L.. Cuendet, L. S.,Ehrenthal. I., Koch, R., and Smith, F., Suture, 166, 520 (1950). Chanda, S.K., Hirst, E. L., Jones, .J. K. N , , and Percival, E. G. V., J . Chem. SOC.,1950, 1289. Consden, R., Gordon, A. H.. and Sfartin, A. J. P., Biochem. J.. 38,224 (1944).
Fischer, E., Ber., 27, 673 (1894). Green, J. W., and Pacsu, E., J . Am. Chena. Soc.. 60, 2288 (1938). H o u g h , L., Jones, J. K. N., and Wadman, W. H., J . Chem. SOC., 1949, 2511. Ihid., 1950,1702.
Pacsu, E., Fortschr. Chem. org. .Vutursto.fe, 5, 128 (1948). Partridge, S. hI., and Westhall, R. C., Biochem. J . , 42, 238 (1948).
Williams, R. T., and Synge, R. L. M,, Ed., "Partition Chromatography," Biochemical Society Symposia No. 3, London, Cambridge University Press, 1950. Stamm, .4. J., and Cohen, W.E., J . Phus. Chem., 42, 921 (1938). RECEIVED for review June 26, 1953. Accepted Kovernber 16, 1953. Paper No. 3024, Scientific Journal Series, hlinnesota .4gricultural Experiment Station.
