2
This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution 4.0 International License. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung 4.0 Lizenz. 24 TH. GHEORGHIU AND K. OETTE The Effect of Stereoisomeric Structure on Retention Time in Gas Liquid Chromatography of Per-O-trimethylsilyl Derivatives of Pentoses and Hexoses TH. GHEORGHIU and K. OETTE Department of Medicine, University of Cologne, West-Germany (Z. Naturforsch. 26 b, 24—25 [1971] ; eingegangen am 15. August 1970) The analysis of relationships between structure and retention time in gas liquid chromato- graphy of pentoses and hexoses leads to the formulation of following rule: the per-O-trimethyl- silyl derivatives of aldoses stereoisomers with conformational stability, identical ring form and the same number of substituted OH groups are longer retained on polar columns when the equatorial substituents are more numerous and are located nearer to the anomeric carbon. Gas liquid chromatography provides an excellent mean for the analysis of sugars and its derivatives. Thus, not only the different pentoses or hexoses re- sulting from the variable disposition of OH groups at C-2,3 (respectively C-2,3,4), but also their a and ß anomers and pyranose-furanose forms are partly or completely separated. These separation charac- teristics may disturb quantitative estimations in complex sugar mixtures of biological origin e. g. hydrolysates of mucosubstances from body fluids, especially because of the chromatographic inter- ference of some of the anomers present in aqueous solutions (GHEORGHIU et a l . 1-3 ) . The analysis of relationships between isomeric structure and the cor- responding retention time is therefore of peculiar interest. Based on the observation of KLEIN and BARTER 4 , BISHOP 5 postulated that for polymethyl ethers of methyl glycopyranosides the anomer in which the C-l methoxy group is placed in trans position to a C-2 substituent or in eis to an unsubstituted OH group at C-2 has the lower retention time. SWEELEY et al. 6 showed that this generalization can not be extended to per-O-trimethylsilyl derivatives of pentoses and hexoses; in this case, when the sugar Reprints request to TH. GHEORGHIU M. D., Med. Univer- sitätsklinik Köln, Haus 3 a, D-5000 Köln-Lindenthal, Josef- Stelzmann-Str. 9. 1 TH. GHEORGHIU, H. FROTZ, K. OETTE, R. PHLIPPEN, H. J. KLEIN, and M. WINTERFELD, Verh. dtsch. Ges. inn. Med. 75, 619 [1969]. 2 TH. GHEORGHIU, H. FROTZ, H. J. KLEIN, and R. PHLIPPEN, Verh. dtsch. Ges. Verdauungs- und Stoffwechselkrankh. [1969], in: R. AMMON and U. RITTER, Aktuelle Berichte aus dem Gebiet der Verdauungs- und Stoffwechselkrankhei- ten, Thieme-Verlag, Stuttgart 1961, p. 95. 3 TH. GHEORGHIU, H. J. KLEIN, H. FROTZ, and K. OETTE, Abstracts of the 4th World Congress of Gastroenterologv, p.115 [1970]. appears normally in one of the chair forms CL or 1C and does not manifest important conformational instability, the anomer with the equatorial OH group at C-L discloses the longest retention time. In 2 previous papers 7 ' 8 we presented results in gas chromatographic analysis of sugar-containing compounds of biological fluids and their hydrolysis. Our observations suggest a more general formula- tion of the relationship between retention time and structure, appliable — for the pentoses and hexoses examined — not only to anomers, but also to their other stereoisomers. One has to consider in addition to the equatorial position of the glycosidic O-tri- methylsilyl substituent all other trimethylsilyl groups located equatorially. It appears to be a rule, using polar columns *, that retention times of isomeric aldoses with a stable identical ring form (and with the same number of substituted OH groups) increase when the groups with equatorial position are more numerous and/or when they are located nearer to C-l (Table 1). Table 1 also shows that the effect caused by the number of equatorial substituents is generally by far greater than that of their position relatively to 4 E. KLEIN and C. J. BARTER, Textile Res. J. 31, 486 [1961]. 5 C. T. BISHOP, Methods biochem. Analysis 10, 1 [1962]. 6 C. C. SWEELEY, R. BENTLEY, M. MAKITA, and W. W. WELLS, J. Amer. diem. Soc. 85, 2497 [1963]. 7 TH. GHEORGHIU and K. OETTE, J. Chromatogr. [Amster- d a m ] 4 8 , 4 3 0 [1970]. 8 TH. GHEORGHIU, K. OETTE, and V. BAUMANN, Z. Natur- forsch. 25 b, 829 [1970]. * Various stationary phases (e. g. butane-l,4-diol-succinate, ethylene glycoladipate or ethylene glycolsuccinate poly- esters) were tested (for details see 1. c. 7 ).

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Page 1: The Effect of Stereoisomeric Structure on Retention Time ...zfn.mpdl.mpg.de/data/Reihe_B/26/ZNB-1971-26b-0024.pdf · The Effect of Stereoisomeric Structure on Retention Time in Gas

This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

2 4 TH. GHEORGHIU AND K. OETTE

The Effect of Stereoisomeric Structure on Retention Time in Gas Liquid Chromatography of Per-O-trimethylsilyl Derivatives

of Pentoses and Hexoses T H . GHEORGHIU a n d K . OETTE

Department of Medicine, University of Cologne, West-Germany

(Z. Naturforsch. 26 b, 24—25 [1971] ; eingegangen am 15. August 1970)

The analysis of relationships between structure and retention time in gas liquid chromato-graphy of pentoses and hexoses leads to the formulation of following rule: the per-O-trimethyl-silyl derivatives of aldoses stereoisomers with conformational stability, identical ring form and the same number of substituted OH groups are longer retained on polar columns when the equatorial substituents are more numerous and are located nearer to the anomeric carbon.

Gas liquid chromatography provides an excellent mean for the analysis of sugars and its derivatives. Thus, not only the different pentoses or hexoses re-sulting from the variable disposition of OH groups at C - 2 , 3 (respectively C - 2 , 3 , 4 ) , but also their a and ß anomers and pyranose-furanose forms are partly or completely separated. These separation charac-teristics may disturb quantitative estimations in complex sugar mixtures of biological origin e. g. hydrolysates of mucosubstances from body fluids, especially because of the chromatographic inter-ference of some of the anomers present in aqueous solutions (GHEORGHIU et a l . 1 - 3 ) . The analysis of relationships between isomeric structure and the cor-responding retention time is therefore of peculiar interest.

Based on the observation of KLEIN and BARTER 4 , BISHOP 5 postulated that for polymethyl ethers of methyl glycopyranosides the anomer in which the C-l methoxy group is placed in trans position to a C-2 substituent or in eis to an unsubstituted OH group at C - 2 has the lower retention time. SWEELEY et al. 6 showed that this generalization can not be extended to per-O-trimethylsilyl derivatives of pentoses and hexoses; in this case, when the sugar

Reprints request to TH. GHEORGHIU M. D., Med. Univer-sitätsklinik Köln, Haus 3 a, D-5000 Köln-Lindenthal, Josef-Stelzmann-Str. 9.

1 TH. GHEORGHIU, H . FROTZ, K . OETTE, R . PHLIPPEN, H . J. KLEIN, and M . WINTERFELD, Verh . dtsch. Ges . inn. M e d . 75, 619 [1969].

2 T H . GHEORGHIU, H . FROTZ, H . J. KLEIN, and R . PHLIPPEN, Verh. dtsch. Ges. Verdauungs- und Stoffwechselkrankh. [1969], in: R. AMMON and U. RITTER, Aktuelle Berichte aus dem Gebiet der Verdauungs- und Stoffwechselkrankhei-ten, Thieme-Verlag, Stuttgart 1961, p. 95.

3 TH. GHEORGHIU, H . J. KLEIN, H . FROTZ, and K . OETTE, Abstracts of the 4th World Congress of Gastroenterologv, p .115 [1970].

appears normally in one of the chair forms CL or 1C and does not manifest important conformational instability, the anomer with the equatorial OH group at C-L discloses the longest retention time.

In 2 previous papers 7 ' 8 we presented results in gas chromatographic analysis of sugar-containing compounds of biological fluids and their hydrolysis. Our observations suggest a more general formula-tion of the relationship between retention time and structure, appliable — for the pentoses and hexoses examined — not only to anomers, but also to their other stereoisomers. One has to consider in addition to the equatorial position of the glycosidic O-tri-methylsilyl substituent all other trimethylsilyl groups located equatorially. It appears to be a rule, using polar columns *, that retention times of isomeric aldoses with a stable identical ring form (and with the same number of substituted OH groups) increase when the groups with equatorial position are more numerous and/or when they are located nearer to C-l (Table 1).

Table 1 also shows that the effect caused by the number of equatorial substituents is generally by far greater than that of their position relatively to

4 E . KLEIN and C. J. BARTER, Texti le Res . J. 3 1 , 4 8 6 [ 1 9 6 1 ] . 5 C. T. BISHOP, Methods biochem. Analysis 10, 1 [1962]. 6 C . C . SWEELEY, R . BENTLEY, M . MAKITA, and W . W . WELLS,

J. A m e r . d i e m . Soc. 8 5 , 2 4 9 7 [ 1 9 6 3 ] . 7 TH. GHEORGHIU and K . OETTE, J. Chromatogr . [Amster -

d a m ] 4 8 , 4 3 0 [ 1 9 7 0 ] . 8 TH. GHEORGHIU, K . OETTE, and V . BAUMANN, Z . Natur-

forsch. 2 5 b , 8 2 9 [ 1 9 7 0 ] . * Various stationary phases (e. g. butane-l,4-diol-succinate,

ethylene glycoladipate or ethylene glycolsuccinate poly-esters) were tested (for details see 1. c. 7 ) .

Page 2: The Effect of Stereoisomeric Structure on Retention Time ...zfn.mpdl.mpg.de/data/Reihe_B/26/ZNB-1971-26b-0024.pdf · The Effect of Stereoisomeric Structure on Retention Time in Gas

STEREOISOMERIC STRUCTURE AND RETENTION TIME IN GASCHROMATOGRAPHY 2 5

Sugar Conformation E q u a t o r i a l O - T M S g r o u p s Relative retention timesb

of the ring Number P o s i t i o n C i C2 C3 C4

(Methyl)pentoses a-L-arabinose 1C (?) 1

a-L-fucose 1C 2 /3-L-arabinose 1C (?) 2

a-D-xylose C I 3 /3-L-fueose 1C 3

ß-T>-xylose C I 4

Hexoses a-D-mannose C I 2 a-D-galactose C I 2

a-D-glucose C I 3 /J-D-mannose C I 3 /S-D-galactose C I 3

ß-D-glucose C I 4

+ 1.00c 0.40

+ + 1.20 0.48 + + 1.32 0.53

+ + + 1.65 0.66 + + + 1.67 0.67

+ + + + 2.32 0.93

+ + 1.00d

+ + 1.39

+ + + 1.42 + + + 1.69 + + + 1.83

+ + + + 2.78

Table 1. The effect of isomeric structure on retention time in gas chromatographic analysis of per-O-trimethylsilyl derivati-ves of some monoses a.

a Packard Chromatograph with flame ionization detector; coiled glass column (185 x 5 mm inside diameter) packed with polyethylene glycolsuccinate 2.5% on Chromosorb G; Argon 99.99%, 100 cm3/min., 20 p.s.i.g.; temperatures: column 120 °C, detector 210 °C, inlet 250 °C, outlet 220 °C (for details see 1. c. 7) . b The retention times are related to a-L-arabinose as well at to a-D-mannose. c Absolute retention time at 120 °C = 3.14 min. d Absolute retention time at 120 °C = 7.75 min.

C- l ; the retention time differences between classes with 1, 2, 3 or 4 such groups are increasing non-linearly. The respective figures point to the influence of other known conformational factors too: a supplementary equatorial substituent may have a very different action depending on the general mole-cular constellation (e. g. between a-galactose and a-glucose this difference is minimal as compared to that between /^-galactose and /^-glucose).

The oxydation of an OH group, its replacement or any change in cyclic structure of sugars may of course greatly disturb these relationships. Galactose derivatives for example are eluated from Polyethy-len glycol succinate columns in the following order: 6-deoxy (y, a, ß) y peak (aldofuranose?) —>-aldopyranose (a, ß) 2-deoxy-2-amino (y,a,ß) —>• hexuronic acid (as methyl ester) —> hexuronic acid lactone —> 2-deoxy-2-acetamido (y, a, ß).