Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography
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.
KeywordsAmphiphilic 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.
- 30.Trathnigg B. Size-exclusion chromatography of polymers. In: Meyers RA, editor. Encyclopedia of analytical chemistry. Chichester: John Wiley & Sons, Ltd; 2000. pp. 8008–34.Google Scholar