Exact determination of the degree of substitution of high molar mass hyaluronan by controlling the conformation in solution.

•Quantitative 1D-NMR analysis of high molecular weight HA derivatives is feasible.•Variation of ionic strength is a powerful tool to enhance quality of NMR spectra.•Full proton mobility can be achieved by controlling conformation in solution.•A correct DS can be determined below the concentration of chain overlap point.•Proton mobility of the reference peak should be checked with an internal standard.