Analytical and Bioanalytical Chemistry

, Volume 410, Issue 18, pp 4259–4273 | Cite as

Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography

  • Carlo Botha
  • Zanelle Viktor
  • Claudine Moire
  • Céline Farcet
  • Fabien Brothier
  • Helen Pfukwa
  • Harald Pasch
Paper in Forefront


Amphiphilic hyaluronic acid (HA), synthesised by modifying HA to varying extents with acrylate groups, was successfully separated according to degree of substitution (DS) using solvent gradient high performance liquid chromatography (HPLC). Two HPLC methods based on the amphiphilic structure of the HA were developed. In the first approach, normal phase gradient HPLC was explored, and separation was based on the interactions of HA’s polar hydroxyl groups with a polar cyano stationary phase. In the second approach, separation was based on the interaction of the hydrophobic acrylate moieties with a non-polar C8 stationary phase (reversed phase gradient HPLC). The separation was optimised by using an electrolyte in the sample solvent to suppress non-covalent interactions and improve the selectivity of the developed method. The photolytic stability of the modified and unmodified HA was also investigated in order to optimise the sample preparation procedure. Furthermore, an alternative method to NMR spectroscopy was developed for determining the DS of HA.

Graphical abstract


Amphiphilic biopolymers Hyaluronic acid Solvent gradient HPLC Degree of substitution Glycosaminoglycans 



The financial support of this work by L’Oréal Research & Innovation (Aulnay-Sous-Bois, France) is gratefully acknowledged. Bertrand Lion, Julien Portal and Franck Hernandez from L’Oréal laboratories are acknowledged for providing the modified HAs and information related to their synthesis process.

Compliance with ethical standards

The authors declare no conflicts of interest.

Supplementary material

216_2018_1123_MOESM1_ESM.pdf (350 kb)
ESM 1 (PDF 349 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Carlo Botha
    • 1
  • Zanelle Viktor
    • 1
  • Claudine Moire
    • 2
  • Céline Farcet
    • 2
  • Fabien Brothier
    • 2
  • Helen Pfukwa
    • 1
  • Harald Pasch
    • 1
  1. 1.Department of Chemistry and Polymer ScienceStellenbosch UniversityMatielandSouth Africa
  2. 2.L’Oréal Research and InnovationAulnay-Sous-BoisFrance

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