A N A L Y T I C A L CHEMISTRY
1148 respects, so far as has been investigated. Experiment 2 indicates that, even under somen-hat unfavorable circumstances, the volumetric technique may be practicable n ith some economic advantage. Note. The methods of statistical inference applied to the data of Experiments 1 and 2 are part of a development that is not only more firmly founded but often (as a t present) simpler to apply than theory based on the assumption of the conventional normal distribution. These methods are sometimes called “nonparametric” but preferably “distribution-free” methods of statistical inference; the latter term v-as proposed by Wilks (2.4) in an extensive review of the subject, which is treated in many recent texts (4-6‘,21). LITERATURE CITED
English, S.,J . SOC.Glass Technol.. 2, 216-19 (1918). Findlay, Alexander, “Practical Physical Chemistry,” 3rd ed., London, Longmans, Green and Co., 1919. Hamilton, P. B., and Van Slyke, D. D., J . Biol. Cheni., 150, 231-50 (1943).
Johnson, P. O., “Statistical RIethods in Research,” pp. 117-19, 169-83, Sew York. Prentice-Hall. 1949. Kendall. M. G., “Advanced Theory of Statistics,” 2nd ed., Vol. 2, pp. 83-4, London, Charles Griffin and Co., 1948. Mood, A. ll.,“Introduction to the Theory of Statistics,” pp. 385-418. New York, McGraw-Hill Book Co., 1950. Osborne, N. S., and Veasey, B. H., B d l . Bur. Standards, 4, 553601 (1908).
Peffer, E. L.. and Mulligan, G. C . , \ h t . Bur. Standards, Circ. C434 (1941).
Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical
(10) (11) (12) (13)
Chemistry,” Vol. 2, “Methods,” Baltimore, Williams and Wilkins Co., 1932. Reilly, J., and Rae, W. iV.,“Physicochemical Methods,” 4th ed., Vol. 1, pp. 47-8, New York, D. Van Nostrand Co., 1943. Stott, Verney, J . SOC.Glass Technol., 8 , 38-43 (1924). Thompson, IT. R., A n n . Math. Statistics, 7, 122-8 (1936). Thompson, W.R., Annual Report of Division of Laboratories and Research, K. Y. State Department of Health, p. 18, 1940; pp. 26-7, 1942; pp. 20-2, 1943; p. 20, 1945; pp. 31-5,
1948. (14) Thompson,
W.R., “Assumption Economy in Scientific Statistical Analysis,” presented at a panel discussion on Application of Statistical Methods to the Evaluation of Biological Data a t meeting of SOC.Am. Bact., Cincinnati, May 17, 1949. (15) Thompson, 1%‘. R., ISD. EXG.CHEN., AXIL. ED., 14, 268-71 (1942). (16) Ihid., 15, 118 (1943). (17) Thompson, W.R., J . Bact., 47,582 (1944). 118) Thompson, W.R.. and hfurdick. P. P.. Annual
Report. Division- of Laboratories and Research, S . P.State Department of Health, p. 23, 1943. (19) Van Slyke, D. D., and Folch, J., J . Biol. Chem., 136, 509-41 (1940). (201 Van Slyke, D. D., and Neiil, J., Ibid., 61, 523-84 (1924). (21) Wadsworth, A. B., “Standard RIethods of the Division of
Laboratories and Research of the Sew York State Department of Health,” 3rd ed., Baltimore, Williams & Wilkins Co., 1947. (22) Ibid., chapter on “Volumetric Calibration and Microvolumetric
Measurement.” (23) Ibzd., 2nd and 3rd eds., hlethod F 78 B. (24) Wilks, S.S..Bull. A m . Math. SOC.,54, 6-50 (1948)
R E C E I V Efor D review November 21, 1951.
Accepted May 22, 1952.
Simple Fractionating Device for Chromatographic Analysis Application to the Study of Carbohydrates L. A. BOGGS, L. S. CUENDET, MICHEL DUBOIS, AND FRED SRIITH Division of Agricultural Biochemistry, University of Minnesota, S t . Paul, Minn. A simple automatic apparatus was needed for the quantitative chromatographic separation of mixtures of sugars and their methyl derivatives on a scale large enough to provide sufficient material for making crystalline derivatives. The construction and operation of the apparatus developed are described. By the use of phenol saturated with water and a column of cellulose powder, a number of simple sugars have been separated. Similarly with methyl ethyl ketone-water azeotrope as the solvent, mixtures of methylated sugars may be readily re-
I
SVESTIGATIOKS into complex polysaccharides have been largely concerned with the separation and identification of
partial and complete hydrolytic products of the polysaccharides themselves and their methyl derivatives, and as a result the main structural features of a number of these important naturally occurring substances have been established (31). There are still many unanswered questions concerning the structure of these substances, and it has become increasingly apparent that much more attention should be paid to the finer structural details of even such polysaccharides as cellulose, starch, and glycogen, which hitherto have been regarded as relatively simple. Already there is evidence that some structural revisions may be necessary (3,7 , 18). Such investigations into the fine structure of polysaccharides clearly require a procedure for the quantitative separation of small amounts of sugars of their derivatives in a pure form from mixtures in which the required compound map be a minor constituent. Some success has attended fractional distillation procedures in
solved into their components. In this manner, it has been possible to determine the composition of methylated and unmethylated polysaccharides. The apparatus provides a simple and very useful tool for the quantitative separation of sugars and their derivatives from mixtures in which some of the components constitute only a small fraction of the whole. It thus becomes possible to determine the finer structural details of polysaccharides. The apparatus is of general use for fractionating eluates from chromatographic columns.
the past (16, 25), but the present prohlems require the handling of much smaller amounts of material and also a greater precision. This can be achieved to a limited extent by partition chromatography on large sheets of filter paper, but until thicker papers become available this does not constitute a practical solution t o the problem. Consequently, it has been found more effective to replace the sheets of filter paper by tubes packed Lvith powdered cellulose (21, 63),starch (28),silica (4), alumina (24, 2 7 ) , and various silicates (14). Only in this wa.v has it been posRible to obtain enough pure material for complete identification of some of the minor but perhaps highly important constituents of complex polysaccharides; such a task was regarded until very recently as Kell nigh impossible. Flolving chromatography on columns of absorbents with or without the added partitioning effect ( I O , SO) can be a tedious operation, but it can be greatly facilitated if it is used in conjunction with an automatic fractionating device (11, 15, 21, 32, 34, 38). A device (21, 38) that has been extensively used for
V O L U M E 24, NO. 7, J U L Y 1 9 5 2
1149
F i g u r e 1. A p p a r a t u s quantitative work is hascd upon the principle of a rotating tshle
or tray carrying a series of receiver tubes arranged in a circle. By suitable eleetrioal means the table is arranged to turn through a small predetermined angle. so that each receiver is successively brought beneath the exit from the chromatographic column for a certain definite time, which may he varied st will in order to collect a certain volume of eluate in each tube.
F i g u r e 3.
Figure 2.
Lower Portion of A p p a r a t u s
Although this type of instrument undoubtedly works satisfactorily, it seemed t o he unnecessarily complicated and rather expensive for isolating quantitatively and characterizing small amounts of sugars and their methyl derivatives, and an attempt was therefore made to devise a simplified apparatus. This paper describes such an apparatus and records a summary of some of the rfw l t s obtained with it during the past two years. APPARATUS
T h e upper portion of the apparatus see Figure 1, consists of a ..-.. E -_-I__-_& " _ _ I " A n v'0LnL+..he Tha.h.nrh. , L.... y l y . y 4-liter...I.~.. SWVULI bulvfilly a.LIu LI ent (powdered Whatman ashless filter tablets) is tightly and L ..l.
YUUI.
~
D i s t r i b u t o r Arm and Test Tubes
different diameters, each of which can he connected to a wooden pulley, N . The upper hearing of the spindle connecting N to L rotates in a hall-race, .JL. A steel spring connects N to one of the pulleys, P. One revolution of C can he produced in 6, 12, 18, or 24 hours, depending an which pulley is engaged. T h e arm, D,of the distributor is 28 inches long and eluate from It ~9 delivered down a detachable glass pendulum, E (approximately l inch long) into receivers, F (st.andard 1.8 X 15 pm. test tubes having an'internally sealed rolection, C ) ,placed In holesdnlled in a fixed wooden table, 0, &at is hraoed to woid,warping. In order to avoid 1088 of eluate, the length of E is adjusted so that its disengagement from the rojection of one receiver occurs only when the end of the distriiutor arm is over the mouth of the next tube (see Figure 3). Before a chromatographic analysis is begun, arm D is rotated (it turns freely when the spring is disconnected) and the tubes are carefully arranged so that E makes . anit,nhle . . .. contact with the projection on each receiver tuht3. PROC
.."" .-
The chromatographic analysi. *yI _rr the same way described by Jones and hi8 asso(:iates (df).
_..I
The eluate from the column is run t o wad;e via tube M until 1 hour before the first component of the Eixture of sugars is about t o emerge from the column a t which time fractionation isnnmmoneaA hv ro+ai;na.d;s+d ",e+rrtinrr+ho "___ mtor and diverting the eluate via distributor C and tube D into the collection tubes,
."""......
~
____
ANALYTICAL CHEMISTRY
1150
F. The emergence of the first component from the column can be estimated from the R/ value of the component, as indicated
one turn in 24 hours; 5-ml. portions of eluate were collected in each tube in 7 minutes. Examination of the fractions showed by paper partition chromatography and the rate of movement that separation was complete. Isolation of the two components of the solvent down the column, as shown by a dyestuff which gave Grhamnose [96 mg. (anhydrous), recovery 104%], the moves with the liquid front. For most purposes the gearing is set to give one revolution in 24 hours, but for the more difficult monohydrate of which had melting point and mixed melting separations or when rapidly percolating solvents are used, the point 93-4", [ a ] g 9" equilibrium value in water (concengearing is modified to increase the speed of rotation. After D tration, 2.0) (after crystallization from methanol) and D-glucose has made one revolution and has provided 200 fractions (5 to 10 (112 mg., recovery 109%) melting point and mixed melting point ml. each) the operation is arrested unless there is still more 146', [a]g 52" equilibrium value in water (concentration, 1.0) material to come from the column, in which case fractionation is (after crystallization from methanol). continued by using a second circle of receivers. This continued fractionation is best done by arranging for the eluate to enter a When a mixture of D-fructose (125.5 mg.) and D-glUCOSe (125 second compartment of distributor C, which is connected to the mg.) was separated as above, the recoveries were: D-fructose (122 arm delivering into a second circle of receivers. mg., recovery 97%) melting point and mixed melting point 103" After the separation has been completed, the distribution of the C., [a]g- 91" equilibrium value in water (concentration, 1.0) component sugars in the tubes is determined by putting a few (crystallized from methanol); D-glucose (125 mg., recovery 103%) drops of the eluate from every fifth tube on a piece of paper and carrying out a suitable spot test for reducing sugars (7-9, 19, melting point and mixed melting point 146", [a]g 52" equilib22, 29, SO). hmmoniacal silver nitrate (30) proved to be exrium value in water (concentration, 1.0) (crystallized from methacellent for free sugars, while the N,N-dimethyl-p-aminoaniline nol). Some idea of the capacity of the cellulose column is seen reagent is useful for reducing methylated sugars and nonreducing from the fact that a mixture of D-glucose and D-fIUCtOSe obtained sugars such as sucrose and raffinose (7). When phenol is used as by hydrolysis of 1 gram of sucrose was separated into its crysthe solvent, it is advisable to extract the paper with ether, after talline components with little or no overlapping of the fractions. the spots have been applied to remove excessive amounts of phenol vhich cause interference ( 7 ) . Greater precision in the location of the component sugars is then obtained by testing additional Table I. Separation of Cleavage Fragments of Certain Polysaccharides and tubes. Methylated Polysaccharides The eluates from those tubes Weight of Composition containing the same component Hydrolyzate Mole are combined and in the case Put on Wt., ratio Recovery, of methyl ethyl ketone and NO. Polysaccharide 70 Column, Rig. Sugar mg. (approx.) butanol-ethanol-water, are evap1 Glucofructosan of rye grain D-Glucose 210 23 1 99 orated in vacuo to dryness D-Fructose 185 8 (Secale cereale) a to give the required com208 ponent. When phenol-water is 2 Hemicellulose of rye grain D-Glucose 141 used, the combined fractions D-Xylose (Secale cereale) are diluted with water and L-Arabinose extracted with carbon tetra136 chloride to remove phenol. 3 Hemicellulose of corncobs D-Glucose 131 The carbon tetrachloride layer D-Xylose (Zea mays) is then extracted with water L-Arabinose and the aqueous solutions are combined and concentrated in 4 D-Fructose Methylated glucofructosan 1445 vacuo to give the required 1,3,4.6-tetramethyl363 2 98.5 of ti root (Cordyline tercomponent. The components 1,3,4-trimethyl185 1 minalis) 3,4.6-trimethyl- * 534 3 are dried and weighed. After 3,4-dimethyl341 2 the purity of the components 1423 has been tested by paper partition chromatography, they are D-Mannose 5 Methylated galactomannan 540 540 2,3,4,6-tetramethyl22 19 1 1 .. .. of Kentucky coffee bean purified by recrystallization in 282 284 14 14 2,3,6-trimethyl(86) (Gymnocladus dioica) the usual way. 2.3-dimethvl118 116 6 6 99 98 D-Galactose Sugars separated with phenol2,3,4,6-tetrametbyl- 11_3 E 8 5 5 .. .. water may be freed from small 535 527 amounts of column impurities D-Mannose 6 Methylated galactomannan by extraction with ethanol, and ... 2,3,6-trimethyl 274 3 3 c ... of carob bean (Ceratonia the methylated sugars sepa2,3-dimethyl86 1 1 98' siliqua L.)d rated with methyl ethyl keD-Galactose 2,3,4,6-tetramethyl'07 1 1 ... tone can be purified by extrac467 tion with water.
+
+
+
7
RESULTS
Separation of ArtScial Mixture@ of Sugars Using PhenolWater as Solvent. 9 mixture of krhamnose hydrate (102 mg.)and D-glucose (103 mg.) was separated as described above, using phenol saturated with water a t 25' C. as the solvent. The solvent-front time as indicated by Sudan I11 was 1545 minutes. Thirty minutes after the front had emerged from the column fractionation was commenced with the distributor arm making
8
Methylated wheat straw hemicellulose (IS)
Methylated corncob heniicellulose ( I S )
420
477
D-Xylose 2,3,4-trimethyl2,a-dimethyl2-monomethylL-Arabinose 2,3,5-trimethylD-Glucose 2,6-dimethylD-Xylose 2,3,4-trimethyl2.3-dimethvl-
58
0.5 27 6
7 301
...
,..
98
32
3
...
13 -
1
...
14 335 61
22 4.5
411
27
1
2
... ...
93
...
...
1 7 r 4 a Column chromatographic analysis, using phenol saturated with water, has shown t h a t dahlia (Dahlia uoriabilis) inulin a n d ti root (Copdyline terminalis) glucofructosan contain 6 a n d 3% of glucose, respectively. b Separated after conversion to 1,2-monoacetone derivative. Glucose (approximately 1 mole of a trimethyl derivative) was removed by bromine oxidation a n d absorption of derived acid on anion exchange column. Results for mole ratio and recovery were determined by paper chromatography using submicro colorimetric procedure (13). d Derived from com onent of carob gum which is insoluble in water (5'6). T h e structural signifcanoe of t h e above findings n-ill be discussed elsewhere.
V O L U M E 2 4 , NO.
JULY 1952
In another experiment a mixture of o-fructose (30 mg.), sucrose (280 mg.), and raffinose (190 mg.) was quantitatively resolved into its components, using phenol saturated with water as the solvent. Separation of Artificial Mixtures of Methylated Sugars Using Methyl Ethyl Ketone a s Solvent. h mixture of 2,3,4,&tetramethyl-D-glucose (48 mg.), 2,3,6-trimethyl-~-glucose (48 mg.), and 2,3-dimethyl-~-glucose(50 mg. ) was separated, using methyl ethyl ketone saturat,ed with water a t 25" C. The solvent-front time (tested with Sudan 111) was 6 hours and fractionation was commenced after this time. Fractions of about 10 ml. were collected in each tube, with the distributor making one revolution in 18 hours. The recoveries were, respectively, 54 mg., 108%; 52 mg., 108%; and 49 mg., 102%. Each fraction crystallized and paper chromatographic analysis showed that separation was complete. Determination of the Composition of Polysaccharides. Some results of the application of the above method to certain methylated and unmethylated polysaccharides are given in Table I.
1151
glycosidically linked D-xylopyranose residues which give rise to the 2,3-dimethyl-~-xylose. Both hemicelluloses possess a branched chain structure; branching occurs a t all the glucose residues, as only the 2,6-dimethyl derivative of D-glucose was detected and at these xylose residues which yield 2-methyl-~xylose. ACKYOWLEDGMEhT
The authors wish to thank D. R. Briggs and R. L. Hossfeld for their suggestions in connection with the construction of the apparatus, and E. F. Greinke for his help with the glass blowing. They also thank the Graduate School of the Universitv of Minnefor financial assistance. L I T E R A T U R E CITED
DlSCUSSION O F RESULTS
Columnar partition chromatographic analysis with the apparatus described above appears to be sufficiently accurate for most structural studies and in general only a few hundred milligrams of material are required. Usually the procedure provides enough of each component for the preparation of derivatives; this is the advantage over the more rapid paper partition chromatographic analysis, n-hich furnishes equally accurate results when the components are known but seldom if ever provides enough material for identification purposes ( 2 , 6 , 12). Phenol saturated with water enables an excellent and fairly rapid separation of the free sugars to be carried out. Thus a complete separation of the following mixtures was achieved: u-glucose and D-fructose; o-glucose and L-rhamnose, D-glucose, D-sylose, and barabinose; D-fructose, sucrose, and raffinose. The polyfructoPan of rye grain and that from the roots of the t
