nciae, :ir:iljiiiose, and xylose were dificultly visible under daylight. These coiiipouncls may have been present' in conc>eiitrations higher than those used by Godin. giving rise t o a n apparent dis:igreement wit,h Godin's report' (6). To determine the quality of the reaction :it lower concentrations, some of the foregoing compounds, along with others t h t seemed bo give rather pale reactions, were examined a t lower concentrations (Table 11). These were unirrigated spots placed on the paper with the ultr:iiiiicrol,uret. The small spots 11-ere enlargetl to an area of approximately 1 sq. cni. 13-: superimposing a droplet of tlistilled water and alloir-ing the chromatogram to dry thoroughly before q)r:i>-ing. All the spots could be seen untler ultrxriolet light a t a concentration (Jf 25 y and two compounds could be picked out a t a concentration as Ion- :is 10 y. Color reactions observed untler daylight for dihydroxyacetone and fi1co.e may be due to the presence of impurities.
Table
II.
Effect of Decreasing Concentration"
Concentration 100 y Day. UV
Compound Diacetone fructose G Br Dihydroxyacetone Pk-T Y-F Fucose T IT-F Rhamnose T IT-F W-F Ribose T Sorbose XJ lose IT-F a-Methyl-omannoside T IT-F a Symbol. same as for Table I.
pz
50 Day. LV 1G Y-F Pk T-F pT W-F pT W-F
pz
The Godin reagent - could be used to develop spots of carbohydrates suecessfully after chromatograms had been exposed to ninhydrin or iodine crystals, or both. However, the color of the suot was not alviavs identical with that developed on a chroniatogram not previously sprayed with ninhydrin or exposed to iodine. I
TV-F T IT-F
Day.
UV
pT
T-F
25 y
Day. T-G Pk
10
UV T-F Y-F W-F IT-F IT-F T K-F
G
IT-F
"/
IT-F
K-F
LITERATURE CITED
( 1 ) Albon, N., Gross, D., L 4 7 W s t 77, 410 (1952). ( 2 ) Godin, P., S u t u r e 174, 134 (1954). (3) Lambou, 11. G,, unpublished manuscriut. RECEIVED for review Februarv 23. 1957. Accepted RIay 6, 1957. The "mention of
trade names does not imply their endorse-
ment by the
L
)
of Agriculture ~
over similar products not mentioned.
Separation and Quantitative Determination of Methyl Mannosides in an AutomaticaIIy Controlled Cel Iulose Partition Column DWIGHT
F. MOWERY,
Jr.]
Ripon College, Ripon, Wis.
bA
reliable quantitative analytical method for nonreducing carbohydrates utilizes an automatically controlled cellulose powder column and the nonspecific anthrone color reaction. The method has been tested b y analysis of mixtures of known composition containing methyl oc-D-mannopyranoside, methyl @-D-mannopyranoside, methyl a-E-mannofuranoside, methyl /3-Dmannofuranoside, and D-mannose. In most cases the percentage of a component found i s within 2 units of that actually present over the range from 1 t o 5770, even though the components present in small quantity are immediately preceded and followed in the column b y relatively heavy bands of material.
A
many successful methods h a w been developed for the paper chroniatographic separation and identiPresmt address, X e x Bedford Institute of Technology, K e a Bedford, Mass. L~HOCGH
fication of nonreducing carbohydrates (3, 4,6, 9, 1 1 ) , the quantitative determination of these substances has proved more difficult. Direct densitometric measurements on paper chromatograms have been advocated b y some chromatographers (11, I S ) , but nonreducing, unlike reducing sugars, suffer from lack of good nonspecific spray reagents. This means that for reasonable accuracy known standards bracketing the quantity of each unknown present must be chromatographed simultaneously with the unknon-n mixture. K i t h mixtures of five unknowns, IT hen percentages may vary over nide limitq. the labor of standard preparation, spotting, and adjustment to the correct range for each unknown can become considerable. Methods requiring spot elution and microanalysis ( 1 , 7 , 11, 12, l 7 ) , although allowing greater freedom in choice of analytical reagents and eliminating the need for simultaneous processing of known standards. are generally laborious and require considerable care for
good results. A starch column was utilized by Gardell (8) for the separation of pentoses. niethylpentoses, and aldohexoses. The column effluent n a s collected in small fractions, the sugar in each fraction was determined colmimetrically, and the quantity of rach sugar in the original mivture n a s calculated by a process of summation. The main disadvantnge of this nietliod n a s the necesqity of packing a fresh starch column for each dctrrminf'1 t'ion. The present article describes a cellulose column n-hicli has been used for an indefinite number of separation; of mannose and methyl mannosidcs by the flowing technique and anthrone analysis of each automatically collected fraction. The chromatographic column is shown in Figure 1. Percentages of individual methyl niannosides can msily be determined from the relati1-c areas under the peaks in a n absorbance plot. Anthrone in 9570 sulfuric acid was selected as the best nonspecific analytical reagent for hexoses and methyl hexoVOL. 2 9 , NO. 10, OCTOBER 1957
1451
~
sides. Phenol in sulfuric acid was also tried but, although somewhat more sensitive to carbohydrates than anthrone, tended to deviate from Beer's law and I$-astherefore discarded. Of several chromatographic solvents capable of separating methyl manno3, sides, butanol-pyridine-water-10, 3 (volume)-gave the most satisfactory color reaction n-ith anthrone and was adopted in subsequent work. Good adherance to Beer's law is observed with glycoside solutions of concentrations up to 0.15 mg. per ml. The sensitivity of the test using the butanol-pyridinewater solvent is about one half that using water, but the test is entirely satisfactory. The absorbance figures obtained with mannose and the four methyl mannosides in solutions of equal weight per cent are so nearly identical t h a t it is considered unnecessary to use more than one factor (0.300) for conversion of absorbance to milligrams of mannose or methyl mannoside per milliliter of solution. Figure 2 shows the elution curves obtained upon chromatographing two typical standard mixtures (A and E of Table I) prepared from crystalline methyl a-D-mannofuranoside, methyl a-D-mannopyranoside, and n-mannose and chromatographically pure methyl 6-D-mannofuranoside and methyl p-Dmannopyranoside gums standardized by optical rotation. The various isomers were identified by paper chromatography of the chromatographically pure methyl mannosides obtained by quantity separation on a large cellulose powder column. The above-mentioned chromatographic solvent was used and the spots were detected with a periodate, followed by a benzidine spray (14). I n addition, the furanosides could be easily distinguished from the pyranosides b y hydrolyzing the water solution of the mixture with a strongly acidic ion exchange resin and then rechromatographing the hydrolyzate. Upon such treatment the areas under the furanoside peaks showed marked reduction while the area under the mannose peak increased. Table I presents the results obtained upon analysis of six mixtures of known composition, providing an over-all test of the equipment and procedure. The percentage of a component found is usually within two units of that actually known to be present. The relatively large errors, percentagewise, of components present in small quantities when preceded and followed b y relatively heavy bands of material are to be expected in view of the incomplete separation attained. This is not believed t o be a serious objection to this method and the same source of error would be present to some extent in other quantitative chromatographic methods. 1452
ANALYTICAL CHEMISTRY
EXPERIMENTAL
Analytical
Chromatographic
Col-
umn. T h e small column used for analytical work is s h o m in Figure 1. It is approximately 1 X 30 inches and is packed with 130 grams of Whatman standard cellulose powder. Improved
Figure 1.
separation was obtained by giving the column a final packing a t a pressure of 20 pounds per sq. inch after the cellulose slurry had been poured in and allowed to settle. The fraction collector is a Reco instrument set for timed fractions. A constant f l o ~rate through the col-
Analytical chromatographic column equipped for automatic operation
Insert. Detail of Fisher Electro Hosecock modification
1
I
I
I
I
1
1
I
stondard mixture A
standard mixture E
fraction number
Figure 2. Typical elution curves of standard mixtures of D-mannose and methyl D-mannosides
-_
umn is maintained by utilizing a constant solvent head and a 25" C. jacket and allowing the column to run unthrottled. The column is equipped for completely automatic operation, so that it may be started a t night and, after a designated forerun, will start collecting fractions and finally shut itself off after the last fraction. The forerun and fraction time are determined b y timing the flow of 25 ml. from the column. The former is set on a GraLab timer, a, and the latter on the fraction collector timer, b.
b y using a Coleman Junior Spectrophotometer, arid the areas under the various peaks of the absorbance plot were determined with a planimeter. The percentage of a given isomer could easily be calculated as 100 times the area under the peak produced b y the isomer divided b y the sum of the areas under all the peaks. This simple relationship is approximately true, because all the isomers were found to be completely eluted and in each case to produce nearly the same specific ab-
Table I.
A
B C
D E F
Standard Mixture Present Found Present Found Present Found Present Found Present Found Present Found
on
the
analytical
ACKNOWLEDGMENT
The author wishes to express his thanks for the generous grants which made this work possible, from the Research Corp., Kew York, for equipment and from the Marathon Corp. for summer remuner nt'ion. LITERATURE CITED
( 1 ) Acker, L., Diemair, W., Pfeil, D., Die Stiirke 6, 241-6 (1954).
Percentage Compositions of Known Mixtures
a-Furanoside
a-Pyranoside
21 20 1 1 25 25 30 30 26 27 50 49
20 20 57 56 2 3 5 5 13 12 9 9
The heart of the automatic operation is a Fisher Electro Hosecock, c , modified as shown in the inset of Figure 1. A microswitch, d, was installed in the hosecock in such a way that when the hosecock closes the microswitch snaps open and locks it closed, a t the same time switching off both the hosecock and the fraction cutter. The plug, e , is plugged into the timer and the fraction collector is plugged into a receptacle, f, installed in the hosecock. The microswitch, g, makes contact and activates the hosecock when a knob, h, attached to the collector turntable, just after the last tube to be collected, comes in contact with it. The hosecock can be opened by depressing a specially installed plunger, i, which pushes down the spring arm of the microswitch. d, inside, allowing the hosecock to open. Kinety 13-mm. tubes were used and a large rubber disk, j , mas fastened to the top of the turntable so as to cover the tubes except when they are being filled, thus preventing dust contamination and evaporation. For carbohydrate tests 1 ml. of each of the 5-ml. fractions collected was transferred by an automatic pipet to one of a set of standardized 13-mm. tubes whose diameters did not vary b y more than 2y0. Two milliliters of 0.2% anthrone in 95% sulfuric acid, delivered by another automatic pipet, was then allowed to run down the wall of the tube, held a t a n angle. The solutions were mixed by a glass stirring rod fitted a t the lower end with a small snugly fitting Teflon washer. The absorbance a t 620 mp was determined after 1 hour,
chromatographed column.
,%Furanoside
pP3;ranoside
Mannose
17 18 39 39 2 4 4 5 10 9 22 23
22 22 2 2 46 44 30 31 26 27 4 4
20 20 1 2 25 24 31 29 25 25 15 15
( 2 ) Augestad, I., Berner, E., Acta Chem. Scand. 8 , 251-6 (1954). ( 3 ) Balston, J. N., Talbot, B. E., "Guide to Filter PaDer and Cellulose Powder Chromatography," pp. 59, 63-4, H. Reeve Angel & Go., London, 1952.
sorbance lvith the anthrone reagent. The working time required for a complete analysis is approximately 2 hours. The chromatographic separation usually required about 12 hours with a flow rate of about 1 ml. per minute.
Preparative Chromatographic Column. A 4.5 X 40 inch cellulose powder column developed with ethyl acetate-n-propyl alcohol-water-5, 3, 2 (volume) (2)-was used for t h e separation and purification of t h e four methyl mannosides used in preparing the standard mixtures. The methyl mannoside mixture was made by refluxing mannose in methanol with a strongly acidic ion exchange resin, Dowex 50 (15). The progress of the separation was followed by passage of the efluent from the chromatographic column through a 4-dm. polarimeter tube. A charge containing 50 grams of methyl mannosides could be roughly fractionated on this column and each fraction rendered chromatographically pure b y one pass through a 2 X 40 inch column. Standard Mixtures. Six standard mixtures were prepared from crystalline n-mannose (Pfanstiehl + 14.25'), methyl a-D-mannofuranoside (+I10 "), and methyl a-D-mannopyranoside ( +SO0), and chromatographically pure gums of methyl p-D-mannofuranoside ( - 6 8 " ) (6,10) and methyl p-n-mannopyranoside ( - 113") (16), standardized by optical rotation. Samples of 200pl. size containing approximately 20 mg. of the carbohydrate mixtures were
( 4 ) Block, R. J., Durrum, E. L., Zxeig,
G., "Manual of Paper Chromatography and Paper Electrophoresis,', pp. 150-1, Academic Press, New York, 1955. ( 5 ) Bott, H. G., Haworth, W.S . ,Hirst, E. L., J . Chem. SOC.1930, 2653-9. (6) Cerbulis, J., ANAL.CHEJI.27, 1400-1
(1955). (7) Frommhagen, L. H., Ibid., 28, 1202-4 (1956). ( 8 ) Gardell, S., Bcta Chem. Scand. 7, 201-6 (1953). (9) Gordon, H. T., Thornburg, W., Rerum, L. N., . ~ N A L . CHEW 28, 849-55 (1956). (10) Jackson, E. L., Hudson, C. S.!J . Am. Chem. SOC.61, 959-60 (1939). 111) Kowkabanv. G. N.. Advances in Carbohydrate 'Chem. ' 9 , 321-2, 324-6, %
,
341-4 _ _ _ - 119541. \----,.
(12) Laidlaw, R. -4.,Reid, S. G., J . Sci. Food Agr. 3 , 19-25 (1952). (13) bicCreadv, R. M.>McComb, E. A., A h . 4 ~ .CHEJI. 26, 1645-7 (1954). (14) Mowery, D. F., Jr., Zbid., 29, 1560 ( 19 57 - 1. (15) Mowery, D . F., Jr., J . Am. Chem. SOC.77, 1667-9 (1955). (16) Scattergood, A, Pacsu, E., Zbid., 62, 903-10 11940). (17) Smith, P. ~ B ,Pollard, , A . L., J . Bacterid. 63, 129-32 (1952). \ -
I
RECEIVEDfor review January 24, 1957. Accepted May 24, 1957. Division of Carbohydrate Chemistry, 130th Meeting, .4CS, Atlantic City, X. J., September, 1956. VOL. 29, NO. 10, OCTOBER 1 9 5 7
1453
