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MICROLITERS
Figure 5. volume
Peak height vs. sample
0 -
0 Trivinylarrine
0-
0
Dimethylbromoarsine
sorb mi. With a helium inlet pressure of 25 p.s.i. and a flowrate of 350 to 355 cc. per minute, dimethylbromoarsine and trivinylarsine were eluted in 4.1 and 5.2 minutes, respectively. Because of the high toxicity and water sensitivity of these compounds, a calibrated microliter capillary pipet was used in conjunction with the PerkinElmer liquid sampling introduction system. This choice of sampling was made recognizing that peak height is highly dependent on flowrate, temperature, sample size, and method of in-
jection-factors which affect efficiency and resolution. Peak area is much less dependent on these operating parameters. The linear relationship obtained between the peak height measurement and the volume of arsines injected is noted in Figure 5. The reproducibility of peak height measurements for seven injections of trivinylarsine from the same pipet mas &3%, A linear plot relating peak height to milligrams of dimethylbromoarsine present in the sample was also obtained. With this plot and a knowledge of the sample density, an analyst can make direct concentration determinations on a weight per cent basis. As cited b y other investigators, this peak height method yields more accurate results than peak area integrations when there is significant band overlapping. ACKNOWLEDGMENT
The authors are indebted to L. Maier, hlonsanto Research S. A,, Zurich, Switzerland, for the arsine samples, and to R. Steeves, hlonsanto Research Corp., Everett, hfass., for some of the chromatographic data reported in this paper. LITERATURE CITED
(1) Abel, E. K.,Sickless, G., Pollard, F. H., Proc. Chem. SOC.,288 (1960).
(2) Brinckman, F. E., Stone, F. G. A., J . Am. Chem. SOC.82,6218 (1960).
(3) Grant, D. W., Vaughan, G. A., J . A p p l . Chem. 6, 145 (1956). ( 4 ) Gudzinowicz, B. J., Driscoll, J. L., ANAL.CHEY.33, 1508 (1961). (5) Gudzinowicz, B. J., Smith, W. R., Ibzd., 32, 1767 (1960). (6) Ibzd., 33, 1135 (1961). (7) Herbrandson, H. F., Kachod, F. C., In “Determination of Organic Compounds by Physical Methods,” p. 16, Academic Press, Xew York, 1955. (8) James, A. T., Martin, A. hf. P., Biochem. J . 63, 144 (1956). (9) Kaesz, H. D., Phillips, J. R., Stone, F. G. A., J . Am. Chem. SOC.82, 6228 (1560). (10) Kaesz, H. D., Stafford, S. L., Stone, F. G. A., Ibid., p. 6232. (11) hlaier, L., Rochow, E. G., Fernelius, W. C., J. Inorg. & Nucbar Chem. 16, 213 (1961). (12) hlaier, L., Seyferth, D., Stone, F. G. A., Rochow, E. G., J . Am. Chem. Soc 79,5884 (1957). (13) Matsuda, H., Matsuda, S., J . Chein. Soc. Japan, Ind. Chem. Sect. 63, 1960 (1960). (14) Pecsok, R. L., in “Principles and Practice of Gas Chromatography,’’ p. 31, Wiley, New York, 1559. (15) Phiffips, C., in “Gm Chromatography, p. 70, Academic Press, Kew York, 1956 (16) Smith, R.R., Gudzinomicz, B. J., Preprints, pp. 111-115, Third International Gas Chromatography Symposium, Michigan State University, East Lansing, hlich., June 13-16, 1961. RECEIVED for review November 27, 1961. Accepted March 9, 1962. Division of Analytical Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962.
Gas-Liquid Chromatography of Some Methylated Mono-, Di-, and Trisaccharides MILDRED GEE and H. G. WALKER, Jr. Wesfern Regional Research laboratory, Albany, Calif.
b Gas-liquid chromatography can be used to study mixtures of methylated mono-, di-, and trisaccharides. The use of 2-mm. i.d. packed columns with 5 to 10% polar liquid phase concentrations permits rapid analysis and good separation of complex methylated sugar mixtures. Use of gasliquid chromatography provides a more rapid and complete estimation of the position of various linkages present in carbohydrates than has been possible by other means.
G
chromatography has been used to aid studies of carbohydrate structure (1,3, 9, 10, 1.6). The sensitivity and separation efficiencies achieved by this method make it possible to detect and estimate small amounts of closely related methylated compounds AS-LIQUID
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ANALYTICAL CHEMISTRY
that might be overlooked using either paper or column chromatography. The present work was performed to establish the composition of a mixture of sucrose monoesters ( 5 ) . Although attention was directed towards separation of different methyl-tetra-0- and tri-0methyl glucosides and fructosides, information was obtained about the gas chromatographic behavior of a variety of methylated carbohydrates. The gas chromatography of some methylated sugars has been reported by other workers (1, 3, 5, 9, 10, 12, f4). We have investigated the effect of the operating variables that affect the separations of methylated sugars including mono-, di-, and trisaccharides. To date, the results have been primarily qualitative, but we have found no abnormal results that would exclude the use of the technique for quantitative analysis.
The separation of acetylated (4, 6, 15), reduced ( 6 ) , isopropylidene ( Y ) , or trimethyl silyl (8) derivatives of sugars has not been studied in this work. EXPERIMENTAL
Apparatus. An Aerograph ilQO-CS thermoconductivity detector instrument (Wilkins Instrument and Res., Inc., Walnut Creek, Calif.) attached t o a Leeds & Northrup 1-mv. recorder was used. Columns. Columns were prepared using 3.2-mm. o.d., 2-mm. i.d. aluminum tubing, 5 and 10 feet long. Chromosorb W (regular), 60-80 mesh, was used as solid support with varying amounts of partitioning liquid phases, diethyleneglycol succinate, neopentylglycol succinate, and Carbowax 20M. Columns were packed by vibrating in the a?;ial direction. Chromosorb W (regular) was preferred
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Figure I . Separation of (2, 3, 4, 5) heptamethyl sucroses from sucrose monopalmitate with (1 ) octamethyl sucrose as a reference standard Five-foot, 5% neopentylglycol succinate column, 2 5 ml. o f H e p e r minute; 207' C. 0.25&I.sample
ovcr firebrick, because initial attempts with firebrick showed strong (probably irreversible) adsorption of the sample on the support, giving longer retention times and small detector responses. Operating Conditions. Samples were injected without ililution, if t h e viscosity permitted sampling with a 1-p1. Hamilton microsyringe; if not, they were diluted n-ith acetone or toluene. For furnace temperatures brlow 200' C., the injection port was maintained approximately 50 degrees above column temperature. When furnace temperatures exceeded 200' C., injection temperature was only 20 to 30 degrees above eolumri temperature. Helium n a s used as thc. carrier gas a t flow rates of 1.5 t(J 40 ml. por minute. Inlet pressures up to 35 p.s.i. were used. Ten-foot columns were iised only if the separation efficiency n as appreciably improved over that of a %foot column. Often the shorter retention times and
wider choice of gas flow range with a shorter column outweighed the increased efficiency of a longer column. Peak Identification. Chromatographic identification of unknown peaks mas made b y injecting a mivture of u n k n o m sample and known standard and noting whether peak enhancement occurred. Samples. All sugars investigated were methylated with silver oxide and methyl iodide in dimethylformamide (11, 16). As Bishop points out (d), simple glycoside formation can frequently lead to production of two anomers which appear as separate peaks on a chromatogram. When the anomeric identity of one of the peaks of the pair can be established, the anorneric identity of the other is evident. Methanolysis of fully methylated sucrose gives a mixture of fully methylated a- and 8-glucopyranosides and a- and p-fructofuranosides. Chromatographic identity of the methyl tetra-0-methyl a-n-glucopyranose peak n'as established b y peak enhancement with a standard derived from crystalline methyl a-Dglucopyranoside. The peak position of the fully methylated @-n-glucopyranoside anomer was established b y sample collection and rotational measurement. The peak positions of the methyl tetra0-methyl-(@ and @)-n-fructofuranosides from sucrose were establishcd by chromatographic comparison n-ith a fully methylated fructofuranoside preparation from which the beta isomer had been removed previously by invertase treatment and fermentation (13). The positions of the methyl tetra-0-methyl (a and B)-~-galactopyranosides were established by comparison of the t n o galactopyranoside peaks from fully methylated melibiose with a standard prepared from crystalline methyl-a-Dgalactopyranoside. The methyl tri-0-methyl-mono-OH glucopyranoside and fructofuranoside standards werp prepared by methanolysis of appropriate completely methylated di. tri, or oligosaccharides. These were readily synthesized from available crystalline sugars. Anomeric assignment of peaks for the glucopyranosidrs 2
I
was based on the data of Bishop; for the fructofuranosides, i t was based on our empirical observation that the alpha isomer has a smaller retention volume than the corresponding beta isomer. Anorneric assignments were not made for the fully methylated glucofuranosides which were derived from a methyl glucoside mixture prepared from glucose and methanolic HC1. The anomeric assignments of the methyl tetra-0-methyl-a and p-fructopyranosides from an equilibrated methyl fructoside mixture are tentative. Anomeric assignments were not made in the disaccharide series because suitable standards were not on hand. Sucrose esters were methylated, saponified, and deionized. Although the resulting heptamethyl sucroses can be separated (Figure l), i t was preferable to methanolize and chromatograph the mixture of methylated hexosides, since standards for identification were more readily available (Figure 2). RESULTS AND DISCUSSION
T o identify the many peaks observed when hepta-0-methyl, mono-OH sucroses are methanolyzed and analyzed b y gas chromatography, i t was necessary to methylate and methanolyze other sugars of known structure t o use as standards for identification. Thus, we have studied the gas chromatographic behavior of a variety of methylated sugars, including the heptamethyl SLIcrases; the fully methylated derivatives of sucrose, raffinose, melezitose, maltose, cellobiose, melibiose, glucose, and fructose; and the methanolysis products of the di- and trisaccharides. Initially our experiments were patterned after those of McInnes and coworkers (12) and Kircher (@-Le., using 4-mm i.d. columns packed with support containing 20 t o 25% b y weight of liquid phase. By reducing the column internal diameter to 2 mm. and the liquid phase concentration to ti to 10% of support weight, significant improvement in separation efficiency and column performance could be obtained ivithout any change in packing tech-
Figure 2. Sugars derived from a sucrose stearate sample after methylation, saponification, and rnethanolysis 5
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(1) methyl 2,3,4,6-tetra-O-methyI-~-D-glucopyranoside (2)methyl 1,3,4,6-tetra-O-methyl-~-D-fructofuranoside (3) methyl 1,3,4,6-tetra-O-methyf-~-D-fructofuranoside ( 4 ) methyl 2,3,4,6-tetra-0-methyl-a-D-glucopyranoside ( 5 ) (6) methyl 2,3,4-tri-O-methyl-~-D-glucopyranoside and methyl 1,3.4-tri-O-methyl-a-D-frudofuranoside (7) methyl 3,4,6-tri-O-methyl-a-D-fructofuranoside (8)methyl 2,3,6-tri-O-methyl-~-D-glucopyranoside ( 9 ) methyl 2,3,4-tri-O-methyl-a-D-glucopyranoside (1 0) methyl 1,3,4-tri-O-methyl-~-D-fructofuranoside (1 1 ) methyl . 3.4.6-tri-0-methyl-0-D-fructofuranoside .. (1 2) methyl 3,4,5-tri-O-methyI-D-fructopyranoside ( 1 3 ) methyl 2,3,6-t ri-0-methyl-a-D-glucopyranoside Separatior- wuus ---I"n a IO-foot, 5% neopentylglycol succinate column, 1 4 4 ' C., 2 4 ml. of H e p e r minute, 0 . 2 - 4 . sample
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VOL. 3 4 , NO. 6, MAY 1962
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centratioii made it possible to chromatograph successfully the methyl ethers of higher molecular weight di- and trisaccharides on polar columns at temperatures below 250' C., which is the maximum usable temperature for many polar substrates. VandenHeuvel and Horning (15) reported the separation of acetates of high molecular weight carbohydrates using thin films of stationary liquids on solid supports. We have found that the optimum range of liquid phase concentration for methylated sugars is 5 to 10% by weight. When the liquid phase concentration was below 570, peak shapes were poor, probably because of sample adsorption on the solid support. At extremely low liquid phase concentrations (1% or less), the capacity of the column was too low to use the sample sizes required for our detector. With Chromosorb W (regular) columns using low liquid phase concentrations (5 to lo%), the relative detector responses (measured by area) of methyl tetra-0methyl hexosides and methyl tri-0methyl hexosides were almost linear and the same for both classes of compounds. Hon-ever, a t higher liquid phase concentrations, the relative response varied greatly, depending on column conditions. Silicone and other nonpolar columns offer no advantage over polar polyester or p l y e t h e r liquid phases. The main attribute of the nonpolar materials is their thermal stability above 200" C.,
TIME (MINUTES) Figure 3.
Separation of various isomers
( 1 ) methyl 2,3,4,6-tetra-O-methyl-fl-D-glucopyranoside
(2) methyl 1,3,4,6-tetro-O-methyl-a-D-fructofuranoside ( 3 ) methyl 1,3,4,6-tetra-O-methyl-fl-D-fructof~ranoside ( 4 ) methyl 2,3,4,6-tetra-O-methyl-a-D-glvcopyranoside ( 5 ) methyl 1,3,4,5-tetra-O-methy~-a-D-fructopyranoside ( 6 ) methyl 2,3,5,6-tetra-O-methyI-D-glucofuranoside ( 7 ) methyl 2,3,5,6-tetra-O-methyI-D-glucofuranoside (8) methyl 1,3,4,5-tetra-O-methyI-fl-D-fructopyranoside Five-foot, 5% diethyleneglycol succinate column, 1 3 6 ' C., 16 ml. o f H e p e r minute, 0.05-pI. sample
nique. A number of factors appeared to interact to produce a desirable combination of high efficiency and fast retention times without reducing the separation properties. Reduced overall flow rates improved the sensitivity of the detector, permitting the use of smaller samples. Operating temperatures could be decressed 10 to 20
Centigrade degrees, leading to less column bleed and better resolution of peaks. The combination of smaller column diameter and lower liquid phase con-
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Figure 4. Sugars derived from methylated, methanolyzed melibiose ( 1 ) methyl 2,3,4,6-tetra-O-methyI-fl-D-galactopyranaside ( 2 ) methyl 2,3,4,6-tetra-O-methyI-a-D-galactapyranaside (3) methyl 2,3,4-tri-O-methyl-fl-D-glucopyranoside (4) methyl 2,3,4-tri-O-methyl-a-D-glucopyranoside Ten-foot, 5% neopentylglycol succinate column, 1 3 6 ' C., 3 3 ml. o f H e p e r minute, 0.4-pl. sample
652
ANALYTICAL CHEMISTRY
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Figure 5. Sugars derived from a methylated, methanolyzed inulin sample ( 1 ) (2)unidentified ( 3 ) methyl 2,3,4,6-tetra-O-methyl-fl-D-gIucopyranoside ( 4 ) methyl 1,3,4,6-tetra-O-methyl-a-D-fructofuranoside (5)methyl 1,3,4,6-tetra-O-methyl-fl-D-fructofuranoside ( 6 ) methyl 2,3,4,6-tetra-O-methyl-a-D-gIucopyranoside (7)methyl 3,4,6-tri-O-methyl-a-D-fructafuronoside ( 8 ) methyl 3,4,6-tri-0-methyl-fl-D-fructofuranoside Ten-foot, 5% neopentylglycol succinate column, 1 4 2 ' C., 24 mi. of H e per minute, 0.4-pl. sample
METHYLATED SUCROSE
METHYLATED MELEZITOSE 5 6
METHYLATED RAFFINOSE
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Figure 6. Separation of mixture of fully methylated disaccharides ( 1 ) sucrose ( 2 ) cellobiose ( 3 ) (4)cellobiose and melibiose (5) ( 6 ) melibiose and maltose (7) maltose Ten-foot, 5% neopentylglycol succinate column, 2 2 6 ' C., 2 3 ml. of H e per minute, 0.5-/.11. sample
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Figure 7. Separation of fully methylated trisaccharides with fully methylated sucrose as an internal standard Five-foot, 5% neopentylglycol succinate column, 2 3 6 ' C., 2 3 ml. of He pet minute, 0.4.pl. sample
LITERATURE CITED
(1) Bishop, C. T., Blank, F., Gardner, P.E. Can. J . Chem. 38,869 (1960). (2) Bishop, C. T., Cooper: F. P., Ibid., p. 388. (3) Ibid., p. 793. (4) Ferrier, R. J., Chem. & Ind. 1961, 831. (5) Gee, M., Walker, H. G., Jr., Ibid., p.
but the poor resolutions obtained a t obtained by niethanolyzing a sample of these temperatures prerents their use fully methylated melibiose. The correN ith complex methylated carbohydrate sponding methyl 2,3,Ftri-O-methyl-a829. (6) Gunner, S. W., Jones, J. K. N., Perry, mixtures. Greater separation effiand P-D-glucopyranosides are also shown M.B., Ibid., p. 255. ciencies n ere achieved when polar as a product of the methanolysis reac( 7 ) Hedgley, E. J., hl6r6sz1 O., Overend, liquids were used. Useful polar liquid tion. Figure 5 shows a methanolyzed W. G., Rennie, R., Ibid., 1960, 938. phases are diethyleneglycol succinate, methylated inulin sample. Figure 6 (8) Hedgley, E. J., Overend, W. G., Ibid., D. 378. Carbowax 20M, and neopentylglycol shows a mixture of fully methylated (Qj Kircher, Henry W., AXAL.CHEM.32, succinate on Chromosorb W (regular). disaccharides, and Figure 7 shows the 1103 ( 1960). Diethyleneglycol succinate gives the separation of methylated trisaccharides (10) Klein, E., Barter, C. J., Jr., Textile best separation of isomers formed by with fully methylated sucrose as a Research J . 31, 486 (1961). (11) Kuhn. R.. Trischmann. H..' Low. completely methylating glucose and reference compound. . I., Angew. Chem. 67,32 (1955). fructosr (Figure 3). Two of the sugars Glycosidatioii of the reducing methyl(12) McInnes, A. G., Ball, D. H., Cooper, tetra-0-methyl-0-n-ghco--methj 1 ated sugars produces a mixture of alpha F. P., Bishop, C. T., J . Chromatog.1 , 5 5 6 ~ ~ ~ ~ a n oand s i dmethyl e tetra-o-methyland beta anomers which can frequently (1958). a-D-fructopyranoside-had equal reten(13) Purves, C. B., Hudson, C. S., J . Am. be resolved by gas chromatography. Chem. SOC.56, 702 (1934). tion times. Seopentylglycol succinate The presence of the two peaks increases (14) Siddiqui, I. R., Adams, G. A., Can. J . gives the best resolution of the mono-OH the number of gas chromatographic unChem. 38, 2029 (1960). methylated glucosides, fructosides, and knowns to be identified. However, when (15) VandenHeuvel, W. J. A,, Homing, sucroses. Carboivav 20LI gives separaE. C., Biochem. Biophys. Res. Comnz. a mixture of two sugars gives a pair of 4,399 (1961). tions intermediate b e h e e n diethylenepeaks that coincide, the second anomeric (16) Walker, H. G., Gee, M., hIcCready, glycol succinate and neopentylglycol peaks often can be used to identify qualiR. Rf., J . Org. Chem. 27, in press. succinate, with about the same retention tatively the presence of the two sugars. times as neopentylglycol succinste. The coincidence of retention times w ~ s RECEIVEDfor review October 20, 1961. llethylated sugars in general are slower Accepted February 16, 1962. The Wesnot observed for both anomeric peaks. tern Regional Research Laboratory is a on diethyleneglycol succinate than on This is shown in Figures 2, 3, and 6. laboratory of the Western Utilization the other two polar liquid phases. Research and Development Division, Other typical chromatographic separaAgriculture Research Service, C . S. DeACKNOWLEDGMENT tions of methylated sugar- using these partment of Agriculture. Reference to a company or product name does n o t imply polar liquid phases are shonii in the The authors are indebted to Joseph approval or recommendation of the prodfolloning figures. Figure 1 shons the Corse, Western Regional Research Labuct by the U. S.Department of Agriculture separation of the alpha and beta anomcrs oratory, Albany, Calif., for a sample of to the exclusion of others that may be of fully methylated galuc~top~rnno~idcs neopentylglycol succinnte. suitable.
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