Orr and Callen (8) described the separation of the methyl esters of fatty acids on a Reoplex 400 plasticizer substrate (Geigy Chemical Co.). Investigation n i t h this material indicated that resolution of the CS monoand Clo dibasic acids could be achieved, but that the column was unstable a t the temperature necessary for resolution. I t is possible that the Reoplex 400 varies in temperature stability from lot to lot. This use of a polyester substrate led to a polyester with a molecular weight of ca. SO00 (from 1,2-propylene sebacic acid), which mas availglycol able in this laboratory. A 4foot column containing 35y0 by weight of this polyester on Celite gave excellent resolution and stability. I n contrast t o the silicone grease the polyester substrate is polar, and the polyester column is not as versatile as the silicone grease. For example. the silicone column will yield acceptable resolution of octadienes, amines, hydrocarbons, and alcohols. The polyester column, on the other hand, gives the best results only n-hen the sample components have some
+
polarity. However, the decrease in substrate preparation time and improved resolution on shorter columns were sufficient to recommend it for this analysis. Two facts concerning the sensitivity coefficient curve (Figure 1) should be mentioned. First, the graph as shown is a log-log plot. If the data are plotted on Cartesian coordinates, two lines are produced, one each for mono- and dibasic acids. However, such a two-curve plot is not as useful as a single curve, as retention time alone cannot reveal the nature of the acid. Secondly, the graph presented is not linear even on the loglog plot. The sensitivity apparently reaches a maximum value at low retention times. Because the width of the base of the first CS peak is more than twice the recorder speed, one possible explanation for this curvature is slow detector response. The method described here is sufficiently accurate, precise, and rapid to serve as a routine analytical method. Moreover, it should be adaptable to a variety of mono- and dibasic acids with minor modifications.
LITERATURE CITED
(1) de Boer, T. J., Backer, H. J., Rec. trav. chim. 73. 229 11954). (2) Cropper, F.’R., Heywood, A., Suture
174, 1083 (19%). (3) Frank, C. E., Abstracts of Papers, Division of Industrial and Engineering Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958. (4)International Union of Pure and Applied Chemistry, Special Committee, Analytical Section, D. Ambrose, Chairman, JUIY1959. (5) James, A. T., Biochem. J . 6 6 , 515 (1957). 16) James. A. T.. Martin. A. J. P., l b i d . , 50, 679 (1952).‘ ( 7 ) Nowakoska, J., Melvin, E. H., Wiebe, R., J . Am. Oal Chemists’ SOC. 34,411 (1957). (8) Orr, C. H., Callen, J. E., J . Am. Chem. Soc. 80, 249 (1958). (9) Smith, L. I., Chem. Revs. 23, 193 (1938). (10) Trent, F. M., M. S. thesis, University of Cincinnati, Cincinnati, Ohio, 1958. (11) Trent, F. M., Miller, F. D., Brown, G. H., J . Chem. Eng. Datu 5 , 110 (1960). RECEIVED for review December 10, 1959. Accepted M a y 20, 1960. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 2 to 6, 1959. \
,
Gas-Liquid Partition Chromatography of Methylated Sugars HENRY W. KIRCHER Deparfment o f Agricultural Biochemistry, College of Agriculture, University o f Arizona, Tucson, Ariz.
,The methyl glycosides of tri-0methylpentoses and tetra-0-methylhexoses are separated on a number of commercially available gas-liquid partition columns. Better results are obtained with columns prepared from certain methylated polysaccharides. A mixture of the fully methylated methyl 01- and @-pyranosides of Darabinose, D-xylose, D-glucose, D-
mannose, and D-galactose separates into eight distinct peaks when put through a methylated hydroxyethylcellulose column. The methyl glycosides of di- and tri-0-methylhexoses also pass through the columns. Starch, cellulose, guar gum, and dextran are used as examples of the applicability of gas-liquid partition chromatography to polysaccharide structural studies.
of methylated sugars are volatile entities and should, thcrefore, be subject to analysis by gas-liquid partition chromatography. JlcInnes et al. (10) described the separation of a number of fully methylated pentose and hexose pyranosides on an hpiezon 11 column, and showed that these sugar derivatives pass through the column unchanged. Subsequent 11-ork by this group consisted of the separation of partially methylated glucoses and sugar alcohols whose free hydroxyl groups were acetylated (2).
This work was initiated to see if 2,3,4,6-tetra-O-methyl-~-$lucosecould be separated from the corresponding mannose derivative. Prior work on a glucomannan (6) had failed to discover a solvent system for the separation of these two methylated sugars by payer partition chromatography. The tetra-0methylglucose did not pass through the gas chromatograph, but the methyl glycoside of this sugar separated into two peaks, corresponding to the 01- and p-anomers. 4 number of methyl glycosides were
ETHYL GLYCOSIDES
obtained (Table I). They were exaniined by paper chromatography on several solvents and each yielded only one spot. Of all the anonieric pairs, only methyl a- and p-n-mannopyranoside separated from each other (Table I). The methyl glycosides were fully methylated by the Kuhn procedure (9). The columns that were examined are listed in Table 11. Their efficiency was gaged by their ability to separate the fully methylated derivative of p-Darabinopyranoside from p-D-glucopyranoside and that of e-D-mannopyranoside from a-D-glucopyranoside. Columns n-ere also prepared from methylated and acetylated cellulose, but would not pass the carrier gas. The films and hard clumps that formed by evaporation of solvent from the cellulose derivative-Chromosorb mixtures required extensive trituration t o pulverize them. The resulting particles were then too small and packed too closely in the column. VOL. 32. NO. 9, AUGUST 1960
1103
tion B-2-b, NRRL B-512-E (13). Hydroxyethylcellulose, Cellosize WP-09, Union Carbide Chemicals Co. Cellulose, Eastman secondary cellulose acetate. The polysaccharides were acetylated and methylated by the usual methods (1, 6). Dextran (50 grams), after two methylations with methyl sulfate and aqueous sodium hydroxide, was insoluble in water and common organic solvents. Further methylation of 45 grams of the partially methylated product with 300 ml. of methyl iodide and 150 grams of silver oxide in 500 ml. of dimethylformamide yielded a material that was readily soluble in chloro204' (c, 0.5; symform, [a]? tetrachloroethane); [CY]': 215' (I.@, theoretical OCH,: 45.5%',,found: 42.8% OCH3 (4). The carbohydrate ethers and acetates were mixed with Chromosorb in 3 t o 7 ratio in acetone or chloroform. The mixtures were stirred occasionally as the solvent evaporated. After they were dried a t 100' C. over phosphorus pentoxide in vacuo, the column packings were gently triturated, sieved (20mesh), and poured into l/Anch outer diameter copper tubing. Compression rings and nuts xere placed on the ends of the columns and they were coiled around a 2-liter stainless steel beaker for insertion into the gas-liquid chromatograph. Procedure. Samples, 0.5 t o 20 pl., were injected into the gas chroniatograph with a microsyringe. Solids mere dissolved in methanol for injection. The columns were heated to 190' to 220' C. and most of them were stable a t that temperature. The response from the katharometer was recorded on a strip chart moving at a rate of '/4 inch per minute. Samples were collected a t the exit of the gas chromatograph in bulbless medicine droppers packed with cotton.
Table I. Standard Sugars Me1ti:g Symbol Used in Sugar Point, C. RGa Figures 1 and 2 Methyl a-D-arabinopyranosideb 129-30 1.59 a-Arab Methyl p-D-arabinopyranosideb I70 1.59 ,&Arab Methyl a-D-xylopyranosideb 2.27 a-Xyl Methyl p-D-xylopyranoside" 2.26 P-XYl Methyl a-D-galactopyranosideb 100-5 0.73 a-Gal Methyl P-D-galactopyranosideb 176 0.69 &Gal Methyl a-D-glucopyranosided 1.00 a-Gluc Methyl @-D-ghcopyranoside* 1.04 p-Gluc Methyl a-D-mannopyranoside" 1.57 a-Man Methyl p-D-mannopyranosideb 0.71 &hian Methyl a-D-arabinofuranosideb 3.11 a-Arab-f Methyl P-barabinopyranosidec 1.59 b-L-Arab Mobilities relative to methyl a-D-ghcopyranoside; solvent, 8: 2: 1 ethyl acetatepyridine-water ; spray, 1yo KMnOa. S . H. Richtmyer, N. I. H., Bethesda, ?Ad. c John Green, Institute of Paper Chemistry, Appleton, Wis. d Corn Products Refining Co., Argo, Jll.
Portions of the methylated guar gum, starch, dextran, and cellulose were hydrolyzed t o their constituent sugars. The methyl glycosides of these were prepared and put through the gas chromatograph t o provide examples of the method and t o show the separation of the di- and tri-0-methylhexoses as their glycosides.
Table II. Columns Evaluated for the Separation of Fully Methylated Sugars (Length in feet) BEST POOR Methylated h>-Craig polyester," 5 droxyethylcelluApiezon," 10 lose, 6 Starch triacetate, FAIR 10 Methylated starch, Hydroxyethylcellulose triace10 llethylated guar tate, 10 gum, 10 Silicone,a 5 Methylated dextran, Ucon polar,a 5 10 Carbowax,; 5 Craig polyester," 10 Apiezon," o LAC-416," 5 a Commercial columns.
EXPERIMENTAL
Apparatus. Gas chromatograph, Aerograph A-100-C, Wilkins Instrument and Research, Inc., Walnut Creek, Calif. Carrier gas, helium, 5 t o 40 p.s.i. inlet pressure. Detector, katharometer, atmospheric pressure. Columns, commercial columns and Chromosorb (35,430 mesh) were purchased from the above manufacturer. Standards. One-half gram of each glycoside (Table I ) was fully methylated with methyl iodide and silver oxide in dimethylformamide (9) and the product was distilled. Each product gave a single symmetrical peak when passed through the LAC-446 column. Synthetic mixtures were pre-
pared from the methylated sugars in various combinations to test the abilities of the different chromatographic columns to separate them. Carbohydrate Derivative Columns. Starch, J. T. Baker Co., soluble powder. Guar gum, Jaguar A-20-A, Stein, Hall, and Co. Dextran, frac-
4
RESULTS
A11 of the separation diagrams shown were obtained with the &foot methylated hydroxyethylcellulose column.
0
z
E 4
r
4
A
c
J
> i r J
_1
r x
B
+
m
a
A
+
m
9
"
x
x
u 4
"
E
z
3
4
,
B
4
1
I 1
0
IO
I
I
1
1
,
I
20
3G
40
so
0
IO
Figure 1.
Gas-liquid partition chromatograms
A. 6.
Fully methylated methyl a-0-pyranosides Fully methylated methyl 8-D-pyrunosides 1 9 0 ' C., 10 p.s.i. He, 3 4 cc. per minute
1104
ANALYTICAL CHEMISTRY
20
30
40
50
T I M E I N MINUTES
TIME I N MINUTES
Figure 2.
Gas-liquid partition chromatograms
A. Fully methylated methyl arabinosides and galactosides 6. Fully methylated methyl a- and 6-D-pyranosides 190' C., 10 p.s.i. He, 3 4 cc. per minute
METHYL
:
2,3,4,6-lETRA-O
ME T H Y L - e - G A L A C
T OS10 E
n
Figure 5. Gas-liquid partition chromatograms
3
; i : p
M E T H Y L 2.3.6 - T R I - 0 -
METHYL-0-M A N N O S 1 D E
!
I
-
y _1
A.
Methyloted dextran glucosides
6. Second-methylated dextran glucosides 220' C., 2 9 p.s.i. He, 70 cc. per minute
d
e :
A METHYL
2.3.4-TRI-0-
METHYL-!-GLUCOSIDE
10
0
30
20
50
40
,
~
TIME IN MINUTES
M E T H Y L 2,4-01-05
METHYL-P-GLUCOSIDE
A
A
m
Figure 3. Gas-liquid partition chromatograms
I
I
I
I
A.
Methonolysis products of methylated guar gum 2 2 5 ' C., 20 p.r.i. He, 75 cc. per minute
TRI-0-ME TH Y L-
B
a- G L U C O S i D E M E T H Y L 2.3.4.6-
0
TETRA-O-METHYL-
I O
TIME
e-GLUCOSIDE
1-
I
L
20
30
UINUTES
1
,
L
20
IO
0
TIME
IN
30
MINUTES
Figure 4. Gas-liquid chromatograms
partition
A.
Methylated cellulose glucosides 6. Methylated starch glucosides C. Methylated cellulose glucosides showing methyl furanoside formation of 2,3,4tri-0-methyl-D-glucose 2 2 0 ' C., 2 0 p.s.i. He, 75 cc. per minute
The solvent peaks that occurred right behind the air peak in some cases were omitted from the figures. Standards. Figures 1 and 2 show the separation diagrams obtained from mixtures of the fully methylated methyl a- and 8-D-pyranosides of arabinose, xylose, galactose, glucose, and mannose. Xone of the columns was able to separate the a- and 8-anomers of arabinose or galactose (the melting points listed in Table I illustrate the differences between the anomers). The optically active carbohydrate derivative columns were also unable to separate fully methylated p-barabinopyranoside from its mirror image, the p-D-isomer, and fully methylated a-D-arbainofuranoside could not be separated from fully methylated 8-D-xylopyranoside. Guar Gum. Methylated guar gum
was examined by Rafique and Smith (If). Equimolar quantities of 2,3,4,6-tetra-O-methyl-D-galactoseJ 2,3,6-tri0-methyl-D-mannose, and 2,3-di-0methyl-D-mannose were obtained by hydrolysis. hlethanolysis of methylated guar gum followed by gas chromatography yielded the separation diagram shown in Figure 3. The sensitivity of the instrument was halved for the galactose peak; the areas underneath the peaks corresponding to the three sugars are in a 1.7: 1: 1 ratio to each other. The single peaks observed for methyl 2,3,6-tri-O-methylD-mannoside and methyl 2,3-di-Omethyl+-mannoside show that either one anomer is preferentially formed or that the a- and B-methyl glycosides of these sugars did not separate. I n the case of the dimethyl mannoside, the skewness of the peak suggests the latter possibility. Cellulose and Starch. Hydrolysis of methylated cellulose and starch followed by methyl glycoside formation and gas-liquid partition chromatography gave the separation diagram shown in Figure 4. The methyl tri-0-methylglucosides were collected. Material from the first large peak crystallized in the receiver, melting point 57' to 58' C. It was, therefore, methyl 2,3,6-tri-O-methyl-PD-glucopyranoside (3) and the sugar corresponding to the second large peak was the a-anomer. It remained a sirup and was hydrolyzed to 2,3,6-tri-Omethyh-glucose, melting point 119' c. ( 3 )'
When the 4-and 5-hydroxyl groups of
aldohexoses are both free, methyl furanosides and pyranosides can form. The furanosides have shorter retention times. This was the case initially observed with cellulose (Figure 4,C). The mixture of glycosides was hydrolyzed back to the free sugars and these were refluxed for 24 hours with methanolic HC1 solution. Gas-liquid partition chromatography then gave the separation shown in Figure 4 , A . Dextran. Methylated dextran NRRL-B-512 was examined by Van Cleve, Schaefer, and Rist (1.9). Hydrolysis of the dextran gave 2,4-di-0methyl+-glucose, 2,3,4-tri-O-niethyl-~glucose, and 2,3,4,6-tetraa-niethyl-~glucose in a molar ratio of approximately 1:21: 1. The original dextran had a molecular weight (weight average) of 30,000,000. The dextran used iri the present study had a molecular weight (weight average) of 75,000 (IS). A sample of the methylated dextran (experimental section) was hydrolyzed in 50 to 50 glacial acetic acid-1N hydrochloric acid and deacetylated with sodium methoxide (12). Methyl glycoside formation and gasliquid partition chromatography gave the separation diagram shown in Figure 5 4 . The first tn-o small peaks corresponded to methyl 2,3,4,6-tetra-Omethyl-a- and 8-D-glucopyranoside, the next peak was unidentified, the follovving two large peaks were presumed t o be methyl 2,3,4-tri-O-methyl-a and 8-Dglucopyranoside, and the last two, methyl 2,4-di-O-methyl-a- and P-Dglucopyranoside. When the hydrolysis of the methylated dextran was perVOL. 32,
NO. 9, AUGUST
1960
* 1105
"
A
C
Figure 6. 15
10
A. TIMES
ARE
IN MINUTES
a.
Methyl 2,3,4,6-tetra-O-methyl-~-D-glucoside
b. Methyl 2,3,4,6-tetra-O-methyl-cu-D-glucoside c.
B
Methyl 2,3,4,6-tetra-O-ethyI-a-~-glucoside
200' C., 20 p.s.i. He, 70 cc. p e r minute
a
4,6-O-Ethylidene-D-glucose 1,2,3-triacetate (mixture of isomers) b. Octamethyl sucrose 220' C., 40 p.s.i. He, 180 cc. p e r minute a. 3-O-Methy1, 3-O-ethy1, a n d 3 - 0 - v i n y l 1,2;5,6di-0-isopropylidene-D-glucose b. 1,2;5,6-di-O-isopropylidene-D-glucose 200' C., 20 p.s.i. He, 70 cc. per minute
8. a.
b Jc 0
Gas-liquid partition chromatograms
25
20
I
I
I
I
I
1
I
10
20
30
40
50
60
70
c a
C.
b
1. 0
1
I
I
IO
20
30
I
40
50
I
60
formed in 90% formic acid, the same of the methylated dextran. It would gas-liquid partition chromatogram was not have been detected during the earlier obtained. work (12) with paper chromatography Since the methylated dextran had less because it is nonreducing. than the theoretical per cent methoxyl When crystalline methyl 2,3,4-tri-0and was methylated by a new technique methyl-P-n-glucopyranoside !vas run (9), a second sample ( 5 grams) was through the formic acid hydrolysis and methylated. Three methylations with methanolysis cycles, about 10% of the methyl sulfate and sodium hydroxide resulting product gave the anomalous were followed by three methylations peak on the gas-liquid partition chrowith sodium in liquid ammonia (8) and matogram. Finally, the chromatogram one methylation with methyl iodide and of authentic 2,3,4-tri-O-rnethyllevoglusilver oxide to yield a product (2.64 cosan showed a single peak having the grams) freely soluble in chloroform. same retention time as the anomalous Found: 42.9% OCH3 (d), theoretical: peak. 45.5% OCHS. Hydrolysis of the tri-0The tri-0-methyllevoglucosan is, methyldextran in formic acid, methyl therefore, a n artifact, and the presence glycoside formation, and gas-liquid parof 2,4-di-0-methyl-~-glucose,2,3,4-tri-Otition chromatography gave the separamethyl-D-glucose, and 2,3,4,6-tetra-Otion diagram shown in Figure 5, B. methyl-n-glucose as the only structurally The compounds corresponding to all significant hydrolytic products (12) of of the peaks except the first two were tri-0-methyldextran is substantiated. collected from a number of runs. The methyl 2,3,4-tri-0-methyl-p-~-glucoCONCLUSIONS pyranoside, m.p. 92-4' C., methyl 2,4For the 2,4-di-O-methyl, 2,3,4-tri-Odi-0-methyl-8-D-glucopyranoside, m.p. methyl, 2,3,6-tri-O-methyl, and 2,3,4,6122' C., and the methyl 2,4-di-0tetra-0-methyl ethers of D-glucose, the methyl-a-D-glucopyranoside, m.p. 80methyl P-pyranosides had shorter reten1' C. (3) crystallized in their respective tion times than the methyl a-pyranoreceivers. Chromatographically pure sides. The same may hold true for the methyl 2,3,4-tri-0-methyl-a-~-glucoother methyl ethers of D-glucose, and a pyranoside remained a sirup ( 3 ) . similar relationship may be found for The material from the anomalous peak the D-mannose derivatives except that did not crystallize. Acid hydrolysis the a-glycosides d l have a shorter and paper chromatography (7) gave a retention time than the 0-anomers. faint spot having the same Rf as 2,3,4Other carbohydrate derivatives that tri-0-methyh-glucose. The hydrolytic have been passed through the gas-liquid product gave the same peak as did the chromatograph are shown in Figure 6. starting material on gas-liquid partition The column separated the anomeric chromatography. Methanolysis (1% pentamethylglucopyranosides better HC1) gave the original peak plus very than i t separated methyl 2,3,4,6-tetrasmall peaks corresponding to methyl 0-methyl-a-D-glucoside from the corre2,3,4-tri-0-methy1-au- and p-D-glUC0sponding tetra-0-ethyl isomer. It was pyranoside. The material was susunable t o separate the 3-0-methyl, 3-0pected to be 2,3,4-tri-O-methyllevoethyl, and 3-0-vinyl derivatives of 1,2;glucosan, arising from the 2,3,4-tri-05,6-di-0-isopropylidene-~-glucose. methyl-D-glucose during the hydrolysis 1106
ANALYTICAL CHEMISTRY
It appears, therefore, that the separation of carbohydrate derivatives by gasliquid partition chromatography on methylated polysaccharide columns is much more dependent upon the structure of the derivatives than upon their molecular weight. ACKNOWLEDGMENT
The author thanks Nelson K. Richtmyer and John Green for gifts of the methyl glycosides and Allene R. Jeanes for samples of dextran. He also appreciates the discussions of the work with Claude T. Bishop, John W.Van Clew, and James C. Masson, the latter having suggested the use of polysaccharide derivatives as column substrates. LITERATURE CITED
(1) Bates, F. J., Natl. Bur. Standards ( U S . ) Circ. C440, 506 (1942). (. 2.) Bishop, C. T., Cooper, F. P., Can. J . Chem. 38, 388 (1960): (3) Bourne, E. J., Peat, S., Advances in Carbohydrate Chem. 5 , 145 (1950). (4) Clark Microanalytical Laboratory, Urhana, Ill., Ref. Yo. 40195, 6, Jan. 12, 1960. (5) Falconer, E. L., iidams, G. ii., Can. J . Chem. 34,338 (1956). (6) Hamilton, J. K., Kircher, H. W., J . Am. Chem. SOC.80,4703 (1958). (T) Hirst, E. L., Hough, L., JoneP, J. K. N., J . Chem. SOC.1949,928. (8) Hodge, J. E., Karjala, S.A,, Hilbert, G. E., J . -4m. Chem. SOC. 73, 3312 (1951I . (9) Kuhn, R.,Trischmann, H., Low, I., Angew. Chem. 67, 32 (1955). (10) ;IlcInnes, 4 . G., Ball, D. H., Coopcr, F. P., Bishop, C. T., J . Chromalog. 1, 556 (1958). (11) Rafique, C. X I Smith, F., J . Am. Chem. SOC.72, 4634 (1950). (12) Van Cleve, J. W.,Schaefer, W. C., Rist, C. E., Zbid., 78, 4435 (1956). (13) Wolff, I. A,, Mehltretter, C. L., Mellies, R. L., Watson, P. R., Hofreiter, B. T., Patrick, P. L., Rist, C. E., Ind. Eng. Chem. 46,370 (1954). RECEIVED for review February 11, 1960. Accepted May 23, 1960. Division of Carbohydrate Chemistry, 136th Xeeting, ACS, iZtlantic Citv, N. J., September 1959.
