Fast and
Accurate Quantitation of Glucans in Complex
Mixtures by Optimized Heteronuclear NMR Spectroscopy
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Abstract
Nuclear magnetic resonance (NMR)
spectroscopy is a widely used
technique for mixture analysis, but it has shortcomings in resolving
carbohydrate mixtures due to the narrow chemical shift range of glycans
in general and fragments of homopolymers in particular. Here, we suggest
a protocol toward fast spectroscopic glycan mixture analysis. We show
that a plethora of oligosaccharides comprising only α-glucopyranosyl
residues can be resolved into distinct quantifiable signals with NMR
experiments that are substantially faster than chromatographic runs.
Conceptually, the approach fully exploits the narrow line widths of
glycans (ν<sub>1/2</sub> < 3 Hz) in the <sup>13</sup>C spectral
dimension while disregarding superfluous spectral information in compound
identification and quantitation. The acetal (H<sub>1</sub>C<sub>1</sub>) groups suffice to spectroscopically resolve ∼20 different
starch fragments in optimized <sup>1</sup>H–<sup>13</sup>C
NMR with a narrow <sup>13</sup>C spectral width (3 ppm) that allows
sampling the indirect <sup>13</sup>C dimension at high resolution
within 15 min. Rapid quantitations by high-resolution NMR data are
achieved for glycans at concentrations as low as 10 μg/mL. For
validation, comparisons were made with quantitations obtained by more
time-consuming chromatographic methods and yielded coefficients of
determination (<i>R</i><sup>2</sup>) above 0.99