Non-linear effects in quantitative 2D NMR of polysaccharides: pitfalls and how to avoid them.
Quantitative 2D NMR is a powerful analytical tool which is widely used to determine the concentration of small molecules in complex samples. Due to the site-specific response of the 2D NMR signal, the determination of absolute concentrations requires the use of a calibration or standard addition approach, where the analyte acts as its own reference. Standard addition methods, where the targeted sample is gradually spiked with known amounts of the targeted analyte, are particularly well-suited for quantitative 2D NMR of small molecules. This paper explores the potential of such quantitative 2D NMR approaches for the quantitative analysis of a high molecular weight polysaccharide. The results highlight that the standard addition method leads to a strong under-estimation of the target concentration, whatever the 2D NMR pulse sequence. Diffusion measurements show that a change in the macromolecular organization of the studied polysaccharide is the most probable hypothesis to explain the non-linear evolution of the 2D NMR signal with concentration. In spite of this non-linearity--the detailed explanation of which is out of the scope of this paper--we demonstrate that accurate quantitative results can still be obtained provided that an external calibration is performed with a wide range of concentrations surrounding the target value. This study opens the way to a number of studies where 2D NMR is needed for the quantitative analysis of macromolecules.
EBSI Team, Chimie et Interdisciplinarité: Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes, CNRS, UMR 6230, LUNAM Université, France; Spectromaîtrise, CAPACITÉS SAS, 26 Bd Vincent Gâche, 44200 Nantes, France.
Estelle
E
Martineau
EBSI Team, Chimie et Interdisciplinarité: Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes, CNRS, UMR 6230, LUNAM Université, France; Spectromaîtrise, CAPACITÉS SAS, 26 Bd Vincent Gâche, 44200 Nantes, France.
Kamel
K
El Khantache
Sanofi Pasteur, Analytical Research Department - CBB, Campus Mérieux - 1541, Avenue Marcel Mérieux 69280 Marcy l'étoile, France.
Marion
M
Pupier
Sanofi Pasteur, Analytical Research Department - CBB, Campus Mérieux - 1541, Avenue Marcel Mérieux 69280 Marcy l'étoile, France.
Patricia
P
Sepulcri
EBSI Team, Chimie et Interdisciplinarité: Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes, CNRS, UMR 6230, LUNAM Université, France.
Serge
S
Akoka
EBSI Team, Chimie et Interdisciplinarité: Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes, CNRS, UMR 6230, LUNAM Université, France; Institut Universitaire de France, 1 rue Descartes, 75005 Paris Cedex 5, France. Electronic address: patrick.giraudeau@univ-nantes.fr.
Patrick
P
Giraudeau
D002138:Calibration; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008970:Molecular Weight; D017711:Nonlinear Dynamics; D011134:Polysaccharides / Q000737:chemistry*
D011134:Polysaccharides
2D NMR; Nuclear magnetic resonance; Polysaccharides; Quantitative analysis; Standard additions