Gas Chromatographic Determination of the Carbonyl Forms of Sugars ERNST BAYER and RUDl WIDDER Chemisches lnsfitut der Universitat Thbingen, Germany The different isomeric forms of sugars in solution were investigated b y formation of glycosides followed b y methylation and chromatographic separation of methyl ethers. It was demonstrated that the seven-memberedring septanose occurs in solutions of galactose. The question concerning the presence of carbonyl forms in equilibrium with ring isomers in solution was resolved b y detecting 3.470 in the case of fructose, 0.770,in galactose, and lesser quantities, (o.3~0), in arabinose and glucose solutions.
T
HE TRANSITIONof
the different ring forms of sugars during mutarotation is esplained by the presence of the carbonyl form in the equilibrium misture. Heretofore it has not been possible to determine directly these forms in solutions of aldoses by spectroscopic methods (5, 7 , 1 1 ) or polarography ( I O ) or in the solid state by s-ray investigations (4). I t was therefore concluded that less than 0.37, of the aldehydic forms exist in solution. Gas chromatography was selected as a method by which this problem could be investigated. The application of gas chromatography to sugar chemistry has been described by various authorh (1, 22, I S ) . In this study it wa,s proposed that the carbonyl forms partici1)ating in inutarotat~ion could be detccted after formation of the rnr~th!.lglycohides followed by permethylation or after direct methylation of th(, sugars. The methylglycosides rcsultiny after treatment with methano1,'hytlrochloric acid are no longer cal):thlc of mutarotation and may thcn IF methylated. For purposes of conipari~*onthe sugars may be directly rnr~thylatcd. 'The amounts of 2,3,4,5,6~~t~rit:tmc~th~~1-aldo-hesoses in the isoiiirrica mixtures should then he readily c~~tiinatcd bj- ga3 chromatography. Rtstcrition valucs of 2,3,4,5,6-penta~ i ~ c t l i ~ ~ l - a l t l o - ~ ~ - ~2,3,4,5,6-pentaliico~e: arnc~tli~~l-altlo-i~-galactose ; and 1.3,4,5,6p(w t niiwt h y 1-kct o- 1)- fruc t oic \r.erc obt a i n t d front 1)iire substance? recently ~ ) r ( ~ ~ x ~l)yr ( vm"ans l of preparative gas c~hloiiiatoprwl,hy ( 3 ) . 'The synthesis rr~imrtcd 1))- 1,evcne and 1Ieyer (8) Ir,atla t o niisturr,~containing no more than 95; of the carbonyl forms. 1452
ANALYTICAL CHEMISTRY
To achieve an esact identification of carbonyl forms, it was necessary to determine the retention values of all isomeric furanoses and pyranoses which might be present. In the case of galactose the retentions of the septanoses had to be determined as well, because these seven-membered-ring forms participate in the equilibrium system. EXPERIMENTAL
The pure samples of the different methyl ethers of sugars were synthesized according to the methods reported by Bayer and Widder ( 3 ) . D-glucose, Dgalactose, D(-) -arabinose, and D-fructose were investigated. Formation of Methylglycosides and M e t h y l Ethers. One-tenth gram of
the sugar was dissolved in 6 ml. of absolute methyl alcohol which con-
Table 1.
tained O.O13q", (or the amount listed in the tables) hydrochloric acid. T h e solution was reflused for 6 hours and then neutralized with silver carbonate. The filtered solution was then evaporated in vacuo a t 20" C., and the resulting syrup was dissolved in 3 ml. of dimethyl forniamide (D1IF). The methyl ethers were prepared analogous to Kuhn'r method ( 6 ) by adding 0.6 gram of barium oxide, 0.03 gram of barium hydroxide and 0.8 ml. of methyl iodide to the solution of 0.1 gram of methylglycoside in 3 ml. of DJIF. The solution was stirred continuously and maintained a t 45" C. for 1 hour with occasional cooling and was then stirred for an additional 10 hours a t 20" C. I t was then poured into 5 ml. of chloroform, filtered, and washed three times with 1.5 ml. of a a t f r . The chloroform solution was dried with anhydrous sodium sulfate, and the chloroform was removed under vacuum.
Relative Retentions of Isomeric Sugar Methyl Ethers
(Retentions measured relative to ethyl ether of succinic acid; conditions: see test) Relative retention Substance Ara hinose
2,3,5-Trimethyl-a-rnethyl-~-arabinoside ?,3,5-Trimethyl-p-methyl-~-arabinoside 2,3,4,5-Tetrarnethyl-al-~-arabinose
2,3,4-TriniethyI-&nie thy]-D-arabinoside 2,3,4-Trimethyl-a-methyl-u-arabinoside
1 .GO 2.04 2.50 3.05
3.20
Glucose 2,3,4,5,G-Pentaniethyl-al-~-glucose 2,3,4,G-Tetraniet~hyl-@-niethyl-~-glucoside 2,3,5-TrirnethyI-~-glucosane-a(1.4)+3(1.6) 2.3,4,G-Tetramet hyl-~-rnethy1-~-glucoside 2.3,5,G-Tetran~ethyIl-~-niethyl-~-glucoside 2,8,5,G-Tetraniethyl-a-methyl-~-glucoside 2,3,4-Trirneth,~l-~-gluc~osane-cu(l.5)-3(1.6) 2,~~,4-Triiiiethyl-~-riieth~l-~-gluropyranoside
2.3,G-Trimethy1-~-11iet hyl-~-gluc~c~furanoside
2,3,G-Tririiethyl-~-niethyl-~-glucofuran~~side
2,3,4-Trimethyl-a-met hyI-~-glucop~ranciside 2,3,G-TrimethyI-~-niethyl-~-glucopyranoside 2,3,G-Trirnethyl-cu-methyl-~-glucopyranoside Galactose 2,3,4,5,G-Pentaniethyl-al-~-galactose 2,3,4,5-Tetramethyl-B-methyl-D-galactoside 2,3,5,G-'I'etraniethyL~-rnethyl-~-galactoside
?,3,4,G-Tetraniet hyl-met hyl-u-galactoside 2,3,5,G-Tetrarnethyl-@-methyl-~-galactoside
2,:3,~,~-'~etramethyl-~-riiethyl-~-galactoside Fractose 1,3,.f,5,6-Pentaniethy~-keto-~-frurtose 1,3,4,5-Tetraniethyl-a-methyl-D-fructoside 1,3,4,G-TetramethyI-a-inethyl-D-fructoside 1,3,4,6-Tetramethyl-/3-1iiethyl-~-frurtoside 1,3,4.5-Tetrarnethyl-3-methyl-~-fructoside
2.32 2.80 3.41 3.88 4.25 4.87 5.25 6.61
7.01 8.03 9.87 10.84
14.42 2.71 3.02 3.81
4.58 4.76 5.30
2 16 2 75 3 05 3 83
6 75
Table II.
Content of Different Ethers in Methylated D-Fructose
(Conditions: aee Figure 1) Substance, 7, Dir. meth. 0.17, HCI BaO/ Ag,O/CH3I CHsI/DMF 2.4 ... 3.4 1.6 Glyc.
Peak No. in Figure 1 I
2 3
4 5 6 7
1
siart
J
TIME
Figure 1. Chromatogram of methylethers by glycosidification of D-fructose with 0.1 methanolic hydrochloric acid and methylation with AgzO/dimethylform a mide
70
2-m. column with 20% polyglycol on kieselguhr; T = 180' C.; standord: 6 = succinic acid ester; the numbering of peaks corresponds to numbers in Table II
Silver oxide may be used in place of barium oxide. Direct Methylations. These were achieved by dissolving 0.1 gram of the sugar in 3 ml. of D l I F and methylating as above with 0.8 ml. of methyl iodide, 0.6 gram of barium oxide, and 0.024 gram of barium hydroxide or kvith 0.3 gram of silver oside. G a s Chromatography. A PerkinElmer 11odel 116E was used for t h e analytical separation, and the apTable Ill.
Substance Unknown 1,3,4,5,6-PentamethyIketo-D-fructose 1,3,4,5-Tetramethyl-amethyl-D-fructoside 1,3,4,6-Tetramethyl-amethyl-~-fructoside 1,3,4,6-Tetramethyl-pmethyl-D-fructoside Unknown 1,3,4,5-Tetramethyl-pmethyl-&fructoside
paratus described by Bayer, Hupe, and Witsch ( 2 ) was used in the preparative work. Columns. Tubing, 0.4-cm. i.d. and 2 meters in length was filled with 20Y0 polyethylene glycol 1500 on kieselguhr (0.2-0.3 mm.). T h e preparative columns, 2 meters inlength and 2 cm. in diameter, were packed with 20Y0 polyethylene glycol 4000 on kieselguhr. Other liquid phases-e.g., Apiezon or polyesters-did not give results comparable to the polyglycols. For some separations 50-meter steel capillaries impregnated with polypropylene glycol were used. I n the latter cases the thermal conductivity detector was replaced by a flame ionization detector. Separations. Retention values were measured relative to t h a t of the ethyl ester of succinic acid. T h e methyl ethers of arabinose were separated on polyethylene glycol a t 150" C. with a carrier of gas flow of 220 ml. of H2 per minute. Others of glucose, galactose, and fructose were chromatographed under the same conditions
Relative retention 1.70 2.16 2.75
6.3
3.05
35.2
6.3
3.83
31.2
39.7
4.45 6.75
2.1 25.5
6.9 38.1
a t 180" C. T h e relative retention values of the pure compounds obtained under these conditions are listed in Table I. RESULTS
Fructose. T h e sugar was converted
to t h e methyl fructoside by t h e described method using O.lYO HCl. T h e methyl ethers were then formed and separated on the polyethylene glycol column. T h e chromatogram (Figure 1) exhibits three main peaks and t'hree components present in minor quantities. I n Table I1 the ethers correspmding to t,hese peaks are summarized together with the contents of the individual substances. The two furanosides were present in concentration greater than 30%. A comparable amount of the P-l)yranoside was present, and the a-pyranoside was absent. Although the latter substance is eluted quite closely to the
Content of Different Ethers in Methylated D-Galactose
(Conditions: see Figures 2 and 3) Band No
in Figures 2, 3 1
I
Substance 2,3,4,5>6-Pentamethy1al-D-galactose 2,3,4,5-Tetramethyl-p-
Relative retention 2 71
0 13% HCI 0 137, HCI
0 47, HCI
AgzO/CH31 BaO/CHd 0.3% 0 2%
AgzO/CHJ 0 6%
0 1370 HC1 0 13% HCI
20" C 40" C. AgzO/CH3I AgzO/CHJ 0 7% 0 5%
Dir meth BaO/CHJ 2 1%
3 02
0 7c/;.
methyl-o-galac toside 2
3a 3b 4 I1 5
-
6
8
2,3,5~G-Tetrameth~.l-pmettivl-~-ealactoside 2,3,4,6-Tetramethl 1-4met hvl-u-galactoside 2,3,4,6-Tetrarneth>I-ameth? I-n-galartoslde 2,3,5,6-TetraniethyI-amethx I-n-galactoside 2 3.4.5-Tetranieth\ I-@meth?l-n-~alactoside Trimet hyl-pniethyl-~galactofriranoside ,
/
I
Trimeth?.l-cu-nieth3.1-D-
galartofuranoside Trimethyl-p-me t hyl-Dgalactopyranoside Trimethyl-a-met hy1-Dgalartopyranoside
3 81
45 0 7 ,
45 3%
17 1%
4 50
39 9%
34 4y0
29 4%
4 58 4 76
14 1%
32 87, 36 6%
66 1% 14 8%
20 0%
3 3%
42 8%
25 1% 15 17,
5 30
64 2q-
9.27
0 9%
0 6%
3 47,
10.36
0 9%
14%
3 27,
11 42
2 77,
5 7%
3 9yc
0 85
12.36
5 5%
11 3%
4 4%;
3 orc
VOL. 36,
NO. 8, JULY 1964
1453
Table IV. Content of Different Methylethers in Methylated D-Glucose 2-rn. Column (0.4 cm. diameter) with 20% polyethylene glycol on kieselguhr (0.2-0.3 rnm.). T
Retention relative to succinic acid ester Relative 0 013:& HCI retention BaO/CHJ 2,3,4,516-Pentarnethyl-al-~-glucose 2,3,4,6-Tetraniethyl-p-methyl-glucoside 2,3,5-Trimethyl-glucosan-a 1,4 -p 1,6 2,3,4,6-TetraniethyI-cu-niethyl-glucoside 2,3,5,6-Tetrarnethyl-0-methyl-glucoside 2,3,5,6-Tetramethyl-a-methyl-glucoside 2,3,4-?'rirnethgl-glucosan-a l , 5 -p 1,6 2,3,4-Trirnethyl-p-methyI-glucopyranoside
2 32 2 80 3 41
3 4 4 5 6 7 8
2,3,6-Trimethyl-p-nieth3.l-glucofurant~side 2,3,6-Trime th3-1-ol-methgl-glucufuran~side
25
87 25
0 1%;,HCI Ag20/CHJ
Dir. nieth. BaO/CHaI 0 3%
10 3%
46 2 5 0 14%
4 67, 49 9%
29 4 5
35 2%
0 9%
1 7 7
61 01 2 2Cc
03 9 87
2,3,4-Trimethyl-a-me th3.1-gluropyranoside
2,3,6-Trimethyl-~-methyl-glucopyranoside 2,3,6-Trimethyl-ol-niethyl-gluropyranoside 2,3,5-Triniet~hgl-methyl-glucoside
,13,4,6 - t'etramethyl - o( - methyl - D fructoside, it is readily detected as may be seen from the occurrence of this substance in directly methylated fructose (see Table 11). I n addition to these main components, the keto-fructose-pentamethyl ether is present a t 3.47,. To verify that this material which appears with a relative retention value of 2.16 is actually the keto form, it was isolated by means of preparative gas chromatography ( 3 ) . Appearance of the carbonyl band a t 5.75 microns in the infrared spectrum was consistent with t,he assumption. Two bands in the chromatogram in the amount of approximately 4% could not be identified. It was csuppo.sed that these compounds were sugar anhydrides which are present in greater amounts in the case of glucose. The keto form was also present in the methyl ethers obtained by direct methylation where thp &isomers predominate (Table 11).
88
180" C.
=
3 35 6 87
10 84 14 42 15 00
Galactose. Formation of methylglycosides and methylation of Dgalactose in the usual manner gave relatively simple chromatograms (Figure 2 ) . Vnfortunately the two pyranosides were not resolved on conventional columns, and therefore these two substances are listed together in Table 111. These isomers, homever, were completely separated on capillary columns (Figure 3). The pentamethyl ether of the aldehyde form was clearly resolved and was present in amounts up to 0.7Yo. The isomeric composition of the ethers as formed under various conditions is given in Table 111. I t appears that with increasing concentration of hydrochloric acid and with decreasing temperatures the carbonyl sugars are present in greater amounts. The mixtures obtained by direct niethylation contained a,- much as 2y0 2,3,J,S,6-pentnmet hyl-aldo- L, - galac t ose.
1
2 3% 7 0%
From these observations there can be little doubt that the aldehydes are present in the equilibrium mixture of sugars in solution. The chromatograms of the directly methylated galactose (Table 111) lead to another interesting conclusion. One may observe two additional peaks with relative retention values of 3.02 and 5.30. The latter substance is the main component and is present to the extent of 64.2Yo. These two substances were identified as the p- and a-septanosides, sugars with seven-membered-ring atructurea. It has been predicted (9)that glycosides having a (1,6)-ring should have the same order of stability as furanosides. To date it was not possible to prove the existence of these rings in solution. The results were confirmed by synthesis of 2,3,4,5tetramethyl-D-galactosides (3, 9) yielding the same retention behavior on parked and on capillary columns. Glucose. The methylated methylglucosides yielded mixtures which exhibited only the normal pyranoses and furanoses. Table IT demon-
v) Y
6
B L
W L
a
er
8 L W
TIME
Figure 2 . Chromatogram of methylethers by glycosidification of D-galactose with 0.1 370 methanolic hydrochloric acid- and methylation 'with AgzO/ dirnethylformamide 2 - m . column with 20% polyglycol on kieselguhr; T = 180' C., standard: B = succinic acid ester. The numbering of peaks corresponds to numbering in Toble Ill
1454
ANALYTICAL CHEMISTRY
Start TtME
Figure 3. Separation of 2,3,4,6-tetramethyl-/3-methyl-~-galactoside ( 3 a ) and 2,3,4,6-tetramethyl-a-methyl-D-galactoside ( 3 b ) 50-m.-capillary, impregnated with polypropylene glycol. a r d succinic acid ester
T
= 180" C.
B = stand-
strates t h a t t h e p-glucosides predominate over the a-glucosides. As opposed to D-galactose no trace of aldehydes or septanosides occurs. The mixture obtained from direct methylation in D M F however contained 0.3y0 of the aldehyde with a relative retention of 2.32 (Table IV). I n addition, traces of the sugar anhydride, 2,3,5trimethyl - D - glucosane - a - [1,4] p - [1,6], and incompletely methylated compounds were present in these mixtures. I t has not been resolved whether this anhydride takes part in the glucose equilibrium in solution or is an artificial product formed by water elimination from incompletely methylated glucose. Arabinose. This case is analogous t o glucose in t h a t t h e methyl ethers formed from the methylglycoside contained only the furanosides and pyranosides. The mixture obtained by direct methylation was found to exhibit a peak with a relative retention of 2.50 indicating the presence of the aldo form (0.47,). CONCLUSION
I t is possible to determine the amounts of various ring forms and carbonyl forms of equilibrium mixtures of sugars in solution. The proportion of isomers changes under varying conditions. Investigations of this nature are very important in studies concerning the mechanism of mutarotation. S o t only should a-p-isomerization and transitions of furahoses and pyranoses be Considered, but, as in the case of D galactose, the presence of septanosides must be taken into account. CrPyranOse 4
r, bPVranoee
r r - F ~ m ~p i d o - f o r m 8-FUl’mO@ a-Septanoee p e-septanose
Table V.
Content of Different Ethers in Methylated D-Arabinose
2-m. column (0.4 mm.) with 2 0 7 , polyethylene glycol on kieselguhr (0.2-0.3 mm.). T = 150” C. Retention relative t o succinic acid ester
Relative retention Substance 2,3,5-Trimethyl-a-methyl-~-arabinoside 1 60 2,3,5-Trimethyl-p-methyl-~-arabinoside 2.04 2,3,4,5-Tetramethyl-al-n-arabinose 2 50 2,3,4-Trimethyl-/3-methyl-~-arabinoside 3 05 2,3,4-Tr~rnethyl-~-methyl-~-arabinoside 3 20
Gas chromatography is the first reliable method that demonstrates the existence of carbonyl forms of sugars in solution which, according to the above scheme, are important intermediates in transitions of the ring forms. By using the results of analytical investigations it is possible to devise simple syntheses of pure isomeric methyl ethers ( 3 ) . Tables I1 to V indicate the feasibility of obtaining very high yields of any desired component merely by varying the conditions under which the methylglycosides and permethyl ethers are formed. I n the past the synthesis of pure isomers was always a troublesome task which included many steps and, as in the case of carbonyl and septanose forms, was not always successful. Direct methylation and subsequent separation by preparative gas chromatography now produces the desired substances in a very short time. LITERATURE CITED
(1) Bayer, E., Survey on Application of
Gas Chromatography in Sugar Chemistr in E. Bayer, “Gas-Chromatograplie,” Springer-Verlag Berlin, 2. Auflage, 1962. (2)Bayer, E.,Hupe, K. P., Witsch, H. G., Angew. Chem. 73, 525 (1961).
Glyc. 0.013% HCI Ag*O/CHJ 61 1y0 26 5 7 , 6 3% 5 9%
Dir. perm. BaO/CH31 3 3% 34 1 % 0 47, 26 0% 36 6%
(3) Bayer, E.,Widder, R.,Liebigs Ann. Chem. In press., See also Ph.D. thesis, R . Widder, Cniversity of Tubingen, 1964. (4) Cox, E. G., Goodwin, T. H., Wagstaff, A. J., J . Chem. SOC. (London) 1935, 1495. ( 5 ) Henri, V., Schou, S., Hoppe Seylers, 2. Physiol. Cheni. 174, 295 (1928). (6) Kuhn, R., Trischmann, H. Low, I., Angew. Chem. 67, 32 (1926); 72, 805 (1960). ( 7 ) Kwiecinski, L., Marchlewski, L., Hoppe Seylers, 2. Physiol. Chem. 169, 300 (1939). (8) Levene, P. A , , Meyer, G. M., J . Biol. Chem. 69, 176 (1926); 74, 695 (1927). (9) Micheel, F., Suckfull, F., Chem. Ber. 66, 1957 (1933). (10) Overend, W. G., Peacocke, A. R., Smith, J. B., J . Chem. SOC.(London) 1961,3487. (11)Petuely, F., Meixner, IT., Chem. Ber. 86, 1255 (1935). (12)Sweeley, C.C., Bentley, R., Makita, M., Wells, W. W., J . A m . Chem. SOC. 85. 2497 (1963). (13)’Sweeley, C,’ C., Walker, B., ~ ~ N A L . CHEM.36, 8, 1461 (1964).
RECEIVEDfor review March 26, 1964. Accepted April 27, 1964. 2nd International Symposium on Advances in Gas Chromatography, University of Houston, Houston, Texas, March 23-26, 1964. We are indebted to the Deutsche Forschungsgemeinschaft for supporting this work.
Pressure Changes during Passage of a Solute through a Theoretical Plate in Gas Liquid Chromatography R. P. W. SCOTT Unilever Research laboratory, Sharanbrook, Bedford, England
b Peak distortion owing to solute partial pressure is considered theoretically and the elution curve equation, derived from the plate theory, is modified to account for the effect. A pressure curve, coincident with the elution curve, is confirmed experimentally using a novel pressure transducer. The effect of solute partial pressure is negligible on analytical packed columns for charges of less than 1 mg., but significantly distorts early peaks on preparative scale columns. The increase in exit flow from a column during the elution of a
peak is also considered and a simple method given for molecular weight determinations using an anemometer detector.
D
of a solute through a theoretical plate the column pressure will increase owing to the partial pressure of the solute. This change in column pressure will cause transfusion of the solute band away from the normal peak maximum due to the difference in volume flow of carrier gas on either side of the peak. To assess the significance of this pressure URING THE PASSAGE
effect on peak shape, column efficiency, and retention volume, the plate theory has to be modified and another elution curve equation derived. Such an equation would indicate conditions where this effect would cause errors in results or reduce column performance. To confirm the existence of a pressure pulse coincident with the elution curve, i t was necessary to employ a suitable pressure transducer that would continuously monitor the pressure a t a point in the column as a solute band passes ( I ) and it will be seen that this transducer acts as a detector n i t h a sensitivVOL. 36, NO. 8, JULY 1964
e
1455
