Abstract
The separation of carbohydrate diastereomers by an ideal size-exclusion mechanism, i.e., in the absence of enthalpic contributions to the separation, can be considered one of the grand challenges in chromatography: Can a difference in the location of a single axial hydroxy group on a pyranose ring (e.g., the axial OH being located on carbon 2 versus on carbon 4 of the ring) sufficiently affect the solution conformational entropy of a monosaccharide in a manner which allows for members of a diastereomeric pair to be separated from each other by size-exclusion chromatography (SEC)? Previous attempts at answering this question, for aqueous solutions, have been thwarted by the mutarotation of sugars in water. Here, the matter is addressed by employing the non-mutarotating methyl-α-pyranosides of d-mannose and d-galactose. We show for the first time, using SEC columns, the entropically driven separation of members of this diastereomeric pair, at a resolution of 1.2–1.3 and with only a 0.4–1% change in solute distribution coefficient over a 25 °C range, thereby demonstrating the ideality of the separation. It is also shown how the newest generation of online viscometer allows for improved sensitivity, thereby extending the range of this so-called molar-mass-sensitive detector into the monomeric regime. Detector multidimensionality is showcased via the synergism of online viscometry and refractometry, which combine to measure the intrinsic viscosity and viscometric radius of the sugars continually across the elution profiles of each diastereomer, methyl-α-d-mannopyranoside and methyl-α-d-galactopyranoside.
Highlights
Among liquid-phase separation methods, size-exclusion chromatography (SEC) is virtually unique in its ability to separate analytes based on their solution conformational entropy, ΔS
Anomeric differences have been found responsible for α-glucose selectively binding sugar transport proteins over β-glucose [13], while diastereomeric configurational differences between the glucose, galactose, mannose, and talose carbohydrate moiety in C-linked antifreeze glycoproteins have been found to be essential to modulating recrystallization–inhibition activity [14]
We examine the methyl glycosides of α-dgalactose and α-d-mannose, the structures of which are shown in Scheme 1. (The equilibrium composition of galactose in water is given above; for mannose, non-glycosylated 30 °C aqueous solutions are composed of 32.8% 4C1 β-pyranose, 66.2% 4C1 α-pyranose, and a < 1% combination of α- and β-furanose isomers [6, 18].) As can be observed from Scheme 1, these two monosaccharides constitute a diastereomeric pair, the only difference between them being that in α-MeMan the axial hydroxy group is located on carbon 2 (C2), whereas on α-MeGal the axial OH is located at C 4. (The difference in the location of one hydroxy group, denoted by a red asterisk in Scheme 1, obviously corresponds to a two-stereocenter difference between these sugars.)
Summary
Among liquid-phase separation methods, size-exclusion chromatography (SEC) is virtually unique in its ability to separate analytes based on their solution conformational entropy, ΔS. The research presented aims to demonstrate that SEC separations performed under “ideal” conditions, i.e., in the virtually complete absence of enthalpic contributions to the separation, are both possible and can be employed for the near-baseline separation of monosaccharide diastereomers representing a single conformer in solution; and to further demonstrate that online viscometry is a sensitive means of detection in this monomeric regime.
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