or upon prolonged evaporation (25). The latter was indicated in the case of methyl ethyl ketone in sample 2 ; earlier determinations showed a value some 50% higher than determinations made on the same solution after it had stood for some time. Although the quantitative results for all of the carbonyl compounds determined are lorn, those for formaldehyde are the most striking. It is apparent that much of the formaldehyde is lost before the sample is processed ( 3 ) . A third sample collected a t room temperature failed to show the presence of methyl ethyl ketone. I t is generally known that higher aldehydes and ketones react to only a limited extent to form bisulfite addition compounds ( I O ) , and Wilson (28) has shown that the rate of trapping methyl ethyl ketone as a bisulfite compound is considerably less at room temperature than in ice water. ACKNOWLEDGMENT
pure compounds and for helpful suggestions. We are also grateful to Lois Lage Leng for the collection of the samples. LITERATURE CITED
( 1 ) Allen, C. F. H., J. Am. Chem. SOC. 53, 2955 (1930). (2) Altshuller, A. P., Cohen, I. R., ANAL. CHEM.33, 726 (1961). (3) Altshuller, A. P., Cohen, I. R., Meyer, M. E., Wartburg, A. F., Jr., Bnd. Chim. Acta 25, 101-17 (1961). (4) Bradg, 0. L., Analyst 51, 77 (1926), J . Chem. SOC.1931, p. 756. (5) Braude, E. A., Jones, E. R. H., J . Chem. SOC.,1945, p. 498. (6) Buske, D. A., Owen, L. H., Wilder,
P., Jr., Hobbs, M. E., ANAL.CHEM. 28. 910-15 (1956). (7) DeJonge, ’A. P.; Rec. Trav. Chim. 74, 760 (195.5). \ - - - - I .
(8) DeJonge, A. P., Verhage, A,, Rec. Trav. Chim. 76, 221 (1957). (9) Ellis. R.. Gaddis. A. M.. Currie. G. T.. ANAL.’CHEM. 30. 475-9 (1958). ’
.,
(107 Fieser, L. F.,’ Fieser,’ M., ’“Organic
Chemistry,” Heath, Boston, 1957. (11) Gasparic, J., Vecera, M., Collection Czech. Chem. Commun. 22, 1426 (1957). (12) Homer. L.. Kirmse. W.. Ann. Chem. ’ 597, 48-68 (1955). (13) Huelin, F. E., Australian J. Sci. Res. B., 5, No. 3, 328-34 (1952). I
The authors are indebted to Eugene Sawicki for furnishing some of the
(14) Jones, L. A,, Holmes, J. C., Seligman, R. B., ANAL. CHEM.28, 191-4 (1956). (15) Jones, L. A., Kinney, C., Hancock, J., J . Am. Chem. SOC.82, 105 (1960). (16) Klein, F., DeJonge, K., Rec. Trav. Chim. 75, 1285-8 (1956). (17) Meigh, D. F., Nature 169,706 (1952). (18) Ibid., 170, 579 (1952). (19) Miller, J. M., Kirchner, J. G., ANAL. CHEM.25, 1107 (1953). (20) Mold, J. D., McRae, M. T., Tobacco Sci. 1, 40-6 (1957). (21) Neuberg, C., Grauer, A., Pisha, R. V., Anal. Chim. Acta 7, 238 (1952). (22) Neuberg, C., Strauss, E., Arch. Biochem. 7, 211 (1945). (23) Rice, R. G., Keller, G. J., Kirchner, J. S., ANAL. CHEM.23, 194 (1951). (24) Roberts, J. D., Green, C., J . Am. Chem. SOC.68, 214 (1946). (25) Smith, Ivor, “Chromatographie and
Electrophoretic Techniques, Vol. 11,” pp. 264-5, Interscience, New York,
1960. (261 Timmons, C. J., J . Chem. Soc., 1957, . p. 2613. ‘ (27) Wallgren, H., Nordlund, E., Acta Chem. Scand. 10, 1671-3 (1956). (28) Wilson, K. W., ANAL. CHEM. 30, 1127 (1958).
.
RECEIVED for review September 13, 1962. Accepted January 14, 1963.
Thin Layer Chromatography of Some Methylated Glycosides MILDRED GEE Wesfern Regional Research Laborafory,’ Albany, Calif.
b Thin layer chromatography on silica gel serves to separate anomers and isomers of methylated sugar glycosides. The area on the developed and dried plates occupied by the sugar derivative was revealed by spraying with dilute sulfuric acid and charring at 110” C. The entire procedure requires only about 1 hour.
T
EXPERIMENTAL
chromatography on silica gel is versatile in resolving many complex mixtures of compounds into single components. A number of review articles have appeared (1, 6-8), and recent studies in the field of carbohydrates include resolutions of free sugars (16-18) and sugar esters (9, 19). I n our studies of the structure and composition of carbohydrates, it became necessary to separate mixtures of methylated sugars. Methylated sugar mixtures have been separated by chromatography on columns (15), paper (5, I d ) , reversed-phase paper ( W I ) , and most successfully by gas chromatography (2-4, 10, 11, 13). Gas HIN
350
chromatography requires expensive apparatus, but its resolving power is sufficient to separate anomers and isomers of methylated methyl glycosides. Thin layer chromatography has now been applied to the separations of some methylated sugar glycosides on silica gel G into alpha and beta anomers and pyranose and furanose isomers.
LAYER
ANALYTICAL CHEMISTRY
Preparation of Chromatoplates. Double strength window glass (20 cm. x 20 cm. x 0.3 cm.) was coated with a layer of silica gel G (Merck, Darmstadt, Germany) at a thickness of 250 microns, using an R. S. Co. Model 200 Spreader (Research Specialities, Inc., Richmond, Calif.). The coating mixture was prepared by mixing one part by weight of silica gel G with two parts distilled water and stirring to a uniform consistency. If the gypsum binder should begin to set during the preparation, water must be added to maintain a consistency of thick cream. After the glass plates were coated, they were dried at room temperature for 1 hour and then in an oven at 100” C.
for 30 minutes. ThP coated chromatoplates mere cooled and used for chromatography as described. Compounds Examined. Mixtures of completely methylated a and p anomers of furanose and pyranose sugar glycosides were made by direct methylation of sugars (20) by Kuhn’s procedure (14). They will be referred to hereafter as methylated sugars. The configurations of the various sugars resolved by silica gel chromatography n‘ere assigned by comparing RIfs of methylated sugars of known identity, separated, and collected by gas chromatography. Comparisons were made by addition of a known standard to a mixture and noting enhanced concentration of a particular spot. Gas chromatographic standards were prepared by methylation of glycosides of known configuration. Acetone solutions of methylated sugars were applied a t a distance of 2 em. from the bottom edge of the chromatoplates in 1 to 2 1 A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U. S. Department of A4griculture.
pl. amounts delivered from a Hamilton micro syringe. The total amount of sugar mixture applied was 100 fig. for hexose and disaccharides and 200 #g. for pentoses. These quantities gave optimum detection of mixtures of sugars with the indicator described. Individual spots containing a few micrograms of methylated sugars were detected in these mixtures. Development of Chromatoplates. Rectangular battery jars (22 em. X 11 em. X 23 em. high), fitted with glass lids, were lined with filter paper for solvent equilibration of the tank The chromatoplates were developed, in a depth of one em. of solvent, in the ascending direction for a distance of 15 em. past the sample origin. The methylated monosaccharides were developed in a mixture of ethertoluene (2:l v./v.) and the methylated disaccharides in a solvent system of methyl ethyl ketone-toluene (1 :1 v./v.). After the solvent had migrated the required distance in 30 to 40 minutes, the chromatoplates were removed from the jars and dried in a ventilated hood. After evaporation of the solvent, the rhrnmatoolatps were snraved with a 5% solution of sulfuric' alid in water and then heated a t 110' C. for 20 minutes to reveal the sugar spots as dark areas on a white background. RESULTS AND
DISCUSSION
The sugars methylated as described (14, $0) produced mixtures of a- and 8-anomers of pyranose and furanose ring configurations. Some of these products bad been separated into anomers and isomers by gas chromatography (10). Chromatographic separations on thin layers of silica gel G resolved, some of the isomers more completely than did gas chromatography, but resolution of anomers and isomers was not achieved in some of the mixtures. Methylated arahinosides separated into four isomers with a predominance of faster moving arabinofuranosides (Fignre 1, #l). The methylated fructose (Figure 1, #2) gave one spot for the faster moving e- and @-furanosidesand complete separation of the a- and P-pyranosides. Methylated galactosides (Figure 1, #3) separated into three spots with the a-furanoside overlapping the @-pyranosideisomer. The methylated galactofuranosideswere faster moving than the corresponding galactopyranosides (Figure 1, #4). However, in the ether solvent system, the methylated glucopyranosides (Figure 1, #5) separated, hut the o and @ furanosides were not resolved. The four possible isomers from a methylated and methanolyzed maltose sample are shown in Figure 1 (#6) and Figure 2 (#2) as methyl tetra-O-methyl-(o and @)-glucopyranosides and methyl 2,3,6-tri-Omethyl-(a and, @)-glucopyranosides. The separation was complete and is shown as four distinct spots. The
Figure 1. Separation of some methylated monosaccharides on thin layers of silica gel G by ascending development in ethyl ether-toluene (2:1 v./v.) solvent (1 I methylated arabinosides; 121 methylated fructoiider; 131 methylated gcllclctorider; (41 melhylated goloctopyronorider; (51 methylated glucosides; (61 methylated, methonolyred moltore; 171 methylated monnopyronorider; 18) methylated iyloridei
methylated mannopyranosides (Figure 1. #7) and xvlouvranosides ( R a r e 1, #8)&ere each separated into two corn: ponents by thin layer chromatography. A slower moving third spot for methylated xylofuranoside is evident in sample #8, Figure 1. For most methylated sugars the order of mobility in silica gel chromatography showed close similarity to retention times observed by gas chromatography. The a-anomer was faster moving than the @-anomer for methylated mannose and arabinose for both pyranose and furanose , form. The @-anomer was faster moving than the a-anomer for methylated glucose, galactose, and xylose. The .furanosides were faster than the pyranosides for methylated fructose, arabinose and galactose; and for methvlated nlucose. mannose. and xylose, dhe pyranosides were ;aster moving than the furanosides. The methylated monosaccharides were also developed in methyl ethyl ketonetoluene (2: 1 v./v.) solvent mixtures. The best separation of methylated glucosides was obtained with toluene-ether and, therefore, the latter was used to illustrate the separation. The resolution of a particular mixture of isomers of methylated sugar might be improved by varying the ratio of solvent volumes. The optimum sample size for detection varied with the thickness of the coating of silica gel G. At a thickness of 250 p, about 100 pg. of a four-eomponent system was necessary for good detection with the 5% sulfuric acid-heat
indicator. The methylated pentosides were not so easily detected as the hexosides, and about 200 pg. samples were required for good detection. Figure 2 shows the separation of some methylated disaccharides containing glucose as one component. Due to lack of standards, the (Y and @ identities were not assigned to all of the spots. An example of a separation achieved on a multicomponent mixture is shown, in Figure 2, #1 by chromatography of the methyl tri-0-methyl hexoside fraction obtained from sucrose monostearate after methylation, saponification, and methanolysis. This sample had previously been examined, and the structure of these components was established, by gas chromatography (11). Some identity can be established bv comoarison with the methylated riethanoiyned maltose sample. " The chromatographic behavior of a methylated inulin after methanolysis WBS observed inFigure2, #8 t o demonstrate the usefulness of this technique with a polysaccharide. A small amount of faster moving methylated monosaccharides is present, but the major components appear as two spots corresponding to methyl tri-0-methyl fructofuranosides. Traces of faster moving impurities were present in the methylated raffinose (Figure 2, #7) sample used, but the majority of the material appeared as a single spot. Tables I and I1 show R, values for some of the methylated sugars examined. The R, values varied with amount of sample applied, presence of VOL 35, NO. 3, MARCH 1963
351
Figure 2. Chromatographic behavior ofsome methylated sugars on thin layers of silica gel G by oscending development in methyl ethyl ketone-toluene ( I : I v./v.) solvent 11 I methyl tri-0-methyl glucopyranotider and fructofuronarides from o methylated, saponified, melhonolyred I Y C ~ O I ~monostearote sample; 12) methylated, melhanolyred maltose; 131 methyloted a- and 6-melibiorider; 141 methylated a- and 6-moltorides; 15) methylated a- and 6-cellobiorider; 161 methyloted I U C I O I ~ ; 171 methylated raffinose; (8)methylated, methandyzed inulin
other components, as well as chromatographic environment such as vapor equilibrium in the head space. These values are therefore not reproducible
Table I. R, X 100 Values far Same Completely Methylated Sugars
Ethyl ether-toluene (2: 1 v./v.) solvent migration of 15 em. on 20 cm. x 20 em. plates) Methyl tri-0-methyl-a-o-arabino. furanoside 51 Methyl tri-0-methvl-B-o-arabinofuranoside 38 Methyl tetra-0-methyl-a and p-ofructofuranosides 39 Methyl tetra-0-methyl-a-o-fructopyranoside 23 Methyl tetra-0-methyl;B-D-fruetopyranoside 16 Methyl tetra-0-methyl-6-o-glucopyranoside 46 Methyl tetra-0-methyl-a-o29 39 31 44
gal&tofuranoside Methyl tetra4-methyl-0-o-
.” 352
.
ANALYTICAL CHEMISTRY
29 28 21
and cannot be used for chromatographic identification. The latter requires simultaneous separation of a standard and the unknown sample on the same plate. However, Tables I and I1 serve to illustrate the separations that can be achieved by the technique of thinlayer chromatography. With some mixtures of methylated sugar isomers, the resolution was incomplete and a better separation was obtained by gas chromatography, However, in other cases, such as the methyl arabinosides, the resolution into four isomers by thin layer chromatography was better than that obtained hy gas chromatography, I n Figure 1, #l the slower two spots are not ideally separated but by varying solvent ratios, a mixture of methylated arabinopyranoside anomers will form two distinct spots. Theresultspresented here deal mainly with completely methylated sugars. Methyl sugars with one or more free hydroxy groups can be satisfactorily chro matographed by this method, as shown by the methylated, methanolyzed sucrose monostearate, maltose, and inulin (Figure 2, #l, #2, and 48). Table I1 includes the R, value for methyl 2,3, di-0-methyl-a-n-glucopyranoside. As the size of the sugar molecule becomes larger or the hvdroxvl content of the methylated su& &-
creases, the chromatography of the components can be improved by increasing the ratio of the ether or ketone. The chromatographic behavior of partially and completely methylated sugars on thin layers of silicic acid provides a new, rapid tool for the study of polysaccharide structure and methylation reactions of sugars. LITERATURE CITED
(1) Applewhite, T. H., Diamond, M. J.. Goldblatt, L. A,, J . Am. Od Chemists’ Soe. 38, 609 (1961). (2) Bishop, C. T., Blank, F., Gardner,
P. E., Can. J . Chem. 38,869 (1960). (3) Bishop, C. T., and Cooper, F. P., Ibid., p. 388.
Table II. R, X 100 Values far Some Methylated Sugars Methyl ethyl ketonetoluene (1: 1 v./v.) solvent on 20 em. x 20 em. plates with 15 cm. solvent migration
MethGlated raffinose 19 Methyl Z,3,4,6-tetra-O-methyl-6o-glueopyranoside 49 Methyl 2,3-di-O-rnethyl-a-oclueoowanosi de 14 .“ I
( 4 ) Ibid., p. i93. (5) Brown, F., Hirst, E. L., Hough, L., Jones, J. K. N., Wadman. H., Nature 161, 720 (1948). (6) Demole, E., J . Chromatog. 1,24 (1958). ( 7 ) Demole, E., Chromatog. Revs. 1, 1 (1959). (8) Demole, E., J . Chromatog. 6, 2 (1961). (9) Gee, M., Ibid., 9, 278 (1962). (10) Gee, M.,Walker, H. G., Jr., ~ ~ N A L . CHEM.34, 650 (1962). ( I 1) Gee, M., Walker, H. G., Jr., Chem. & I n d . 829 (1961). (12) Hjrst, E. L., Hough, L., Jones, J. K. h ., J . Chem. SOC.928 (1949).
(13) Kircher, Henry W., ANAL. CHEM. 32, 1103 (1960). (14) Kuhn, R., Trischmann. H.. Low,, I.., Angew. .Chem. 67, 32 (1955). (15) Lemieux, R. U., Bishop, C. T., Pelletier, G. E., Can. J . Chem. 34, 1365 (1956). (16) Pastuska, G., 2. Anal. Chem. 179, 355 (1961). (17) Prey, V., Berbalk, H., Kausz, M., Mzkrochzm. Acta 968 (1961). (18) Ibid., 449 (1962). (19) Tate, M. E., Bishop, C. T., Can. J . Chem. 40, 1043 (1962). (20) Walker, H. G., Jr., Gee, M.,Mc-
Cready, R. M., J. Org. Chem. 27, 2100 (1962). (21) Wickbere. B.. “Methods in Carbohydrate Chumistry,” Whistler, R. L., Wolfrom, M. L., Eds., Vol. I, 31, Academic Press, 1962. RECEIVED for review October 1, 1962. Accepted January 16, 1963. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
Plate Height of Nonuniform Chromatographic Columns Gas Compression Effects, Coupled Columns, and Analogous Systems J. CALVIN GlDDlNGS Deparfment o f Chemistry, University of Ufah, Salt lake Cify, Ufah
b A theoretical treatment is given for columns which have nonuniform properties along their lengths. The theory is applied to coupled columns and to gas compression effects in gas chromatography. It is shown by a simple two-segment model (in addition to a more detailed approach) that even with a lengthwise constant plate height, the observed plate height may appear as something entirely different from its constant local value. This refutes the argument that the velocity gradients accompanying gas compression are of no significance in determining plate height and resolution contributions of constant plate height terms.
C
are Often operated in such a way that the physical properties change from one end of the column to the other. In gas chromatography, for instance, the finite pressure gradient required for gas flow causes a significant velocity gradient in the column. Different columns are occasionally coupled together to take advantage of several sorbents simultaneously or to seek advantages through a change in column diameter. Other nonuniformities may develop along a column’s length because of the failure to acquire a homogeneous packing. The object of this paper is to develop a simplified theory for those variations that occur strictly as a function of the distance along the column (this excludes such subjects as programmed temperature gas chromatography where the principal change occurs as a function of time), Of primary interest is the effect of gas compression in gas chromatography. HROMIATOGRAPHIC COLUMXS
The popular misconception that constant plate height terms (such as the gas phase terms, where changes in diffusivity and velocity exactly compensate) appear unchanged in a system with velocity gradients is refuted with the help of a simple two-segment model. G A S COMPRESSION
A number of authors have discussed the influence of gas compressibility on plate height and resolution in gas chromatography (GC) ( 1 , 2, 5-8). While the conclusions have not always been the same, widespread agreement exists on the point that plate height terms which are constant throughout the column ( A ,B/v, C,v) will contribute just this constant amount to the observed plate height whether a velocity gradient exists in the column or not. This view has been opposed by the author and his co-workers ( 6 , 8) who have derived the equation where
and
f,
=
9(P4 - l ) ( P- 1) 8 ( P 3 - 1)2
(3)
The term H , is the collective sum of all the previously mentioned plate height terms which remain constant in the column. This term is part of the local plate height-i.e., the plate height which exists locally in a_small section of the column. The term H , on the other hand, is the observed plate height, and
is defined in the usual way in terms of observable quantities, (4)
The terms f2 and fl, the latter of which is of main interest here, are the pressure correction terms, and are functions solely of the compression ratio, P = inlet pressurejoutlet pressure. If Equation 1 were written to agree with the majority of equations describing pressure and velocity gradient effects, it would be necessary to write f i = 1 in place of thefl value of Equation 3. Numerically, the two values differ a t most by 12.5%. However, this modest percentage is becoming more and more important as the agreement betrveen chromatographic theory and experiment becomes closer (4). In addition, because two conflicting conclusions have been presented, an incorrect set of principles must have been used to obtain the wrong conclusion, and such principles might lead to additional errors. Therefore, it is important to discuss further the differences that exist. Various arguments for fi = 1 are not difficult to find. In a completely uniform column with no velocity gradients (such as the liquid systems for n-hich the plate height theory was initially intended), the observed plate height and the local plate heights are all identical. This concept is perhaps more meaningful if we say that such a column, if cut in two (while maintaining an equal flow velocity), would yield two observed plate height values (as defined in Equation 4) identical to each other and to the full-column value. VOL 35,
NO. 3, MARCH 1963
353
