Analytical and Bioanalytical Chemistry

, Volume 411, Issue 15, pp 3321–3330 | Cite as

Comprehensive two-dimensional liquid chromatography for the characterization of acrylate-modified hyaluronic acid

  • Zanelle Viktor
  • Céline Farcet
  • Claudine Moire
  • Fabien Brothier
  • Helen PfukwaEmail author
  • Harald PaschEmail author
Research Paper


Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives.

Graphical abstract


Hyaluronic acid Liquid chromatography Comprehensive 2D-LC Chemical composition analysis Molar mass analysis 



The preparation of the HA and HAM samples by L’Oréal Laboratories is acknowledged.

Funding information

The authors received financial support from the L’Oréal Research and Innovation (Aulnay-sous-Bois, France). ZV received funding for her MSc project from the NRF.

Compliance with ethical standards

There was no research involving human participants and/or animals. The work has been submitted with the consent of all authors.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2019_1799_MOESM1_ESM.pdf (2.9 mb)
ESM 1 (PDF 2918 kb)


  1. 1.
    Liu L, Liu Y, Li J, Du G, Chen J. Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb Cell Factories. 2011;10:99–108.CrossRefGoogle Scholar
  2. 2.
    Chong BF, Blank LM, Mclaughlin R, Nielsen LK. Microbial hyaluronic acid production. Appl Microbiol Biotechnol. 2005;66:341–51.CrossRefPubMedGoogle Scholar
  3. 3.
    Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid (hyaluronan): a review. Vet Med (Praha). 2008;53:397–411.CrossRefGoogle Scholar
  4. 4.
    Sze JH, Brownlie JC, Love CA. Biotechnological production of hyaluronic acid: a mini review. 3 Biotech. 2016;6:1–9.CrossRefGoogle Scholar
  5. 5.
    Burdick JA, Prestwich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011;23:41–56.CrossRefGoogle Scholar
  6. 6.
    Schanté CE, Zuber G, Herlin C, Vandamme TF. Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications. Carbohydr Polym. 2011;85:469–89.CrossRefGoogle Scholar
  7. 7.
    Liu J, Willför S, Xu C. A review of bioactive plant polysaccharides: biological activities, functionalization, and biomedical applications. Bioact Carbohydrates Diet Fibre. 2015;5:31–61.CrossRefGoogle Scholar
  8. 8.
    Zong A, Cao H, Wang F. Anticancer polysaccharides from natural resources: a review of recent research. Carbohydr Polym. 2012;90:1395–410.CrossRefPubMedGoogle Scholar
  9. 9.
    Alkrad JA, Merstani Y, Neubert RHH. New approaches for quantifying hyaluronic acid in pharmaceutical semisolid formulations using HPLC and CZE. J Pharm Biomed Anal. 2002;30:913–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Čožíková D, Šílová T, Moravcová V, Šmejkalová D, Pepeliaev S, Velebný V, et al. Preparation and extensive characterization of hyaluronan with narrow molecular weight distribution. Carbohydr Polym. 2017;160:134–42.CrossRefPubMedGoogle Scholar
  11. 11.
    Oudshoorn MHM, Rissmann R, Bouwstra JA, Hennink WE. Synthesis of methacrylated hyaluronic acid with tailored degree of substitution. Polymer. 2007;48:1915–20.CrossRefGoogle Scholar
  12. 12.
    Finelli I, Chiessi E, Galesso D, Renier D, Paradossi G. Gel-like structure of a hexadecyl derivative of hyaluronic acid for the treatment of osteoarthritis. Macromol Biosci. 2009;9:646–53.CrossRefPubMedGoogle Scholar
  13. 13.
    Buffa R, Šedová P, Basarabová I, Bobula T, Procházková P, Vágnerová H, et al. A new unsaturated derivative of hyaluronic acid—synthesis, analysis and applications. Carbohydr Polym. 2017;163:247–53.CrossRefPubMedGoogle Scholar
  14. 14.
    Pravata L, Braud C, Boustta M, El Ghzaoui A, Tømmeraas K, Guillaumie F, et al. New amphiphilic lactic acid oligomer–hyaluronan conjugates: synthesis and physicochemical characterization. Biomacromolecules. 2008;9:340–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Šedová P, Buffa R, Kettou S, Huerta-Angeles G, Hermannová M, Leierová V, et al. Preparation of hyaluronan polyaldehyde—a precursor of biopolymer conjugates. Carbohydr Res. 2013;371:8–15.CrossRefPubMedGoogle Scholar
  16. 16.
    Palumbo FS, Pitarresi G, Mandracchia D, Tripodo G, Giammona G. New graft copolymers of hyaluronic acid and polylactic acid: synthesis and characterization. Carbohydr Polym. 2006;66:379–85.CrossRefGoogle Scholar
  17. 17.
    Vasi A-M, Popa MI, Butnaru M, Dodi G, Verestiuc L. Chemical functionalization of hyaluronic acid for drug delivery applications. Mater Sci Eng C. 2014;38:177–85.CrossRefGoogle Scholar
  18. 18.
    Pouyani T, Prestwich GD. Functionalized derivatives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials. Bioconjug Chem. 1994;5:339–47.CrossRefPubMedGoogle Scholar
  19. 19.
    Eenschooten C, Guillaumie F, Kontogeorgis GM, Stenby EH, Schwach-Abdellaoui K. Preparation and structural characterisation of novel and versatile amphiphilic octenyl succinic anhydride–modified hyaluronic acid derivatives. Carbohydr Polym. 2010;79:597–605.CrossRefGoogle Scholar
  20. 20.
    Zawko SA, Truong Q, Schmidt CE. Drug-binding hydrogels of hyaluronic acid functionalized with β-cyclodextrin. J Biomed Mater Res Part A. 2008;87A:1044–52.CrossRefGoogle Scholar
  21. 21.
    Botha C, Viktor Z, Moire C, Farcet C, Brothier F, Pfukwa H, et al. Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography. Anal Bioanal Chem. 2018;410:4259–73.CrossRefPubMedGoogle Scholar
  22. 22.
    Botha C, Kuntz J-F, Moire C, Farcet C, Pfukwa H, Pasch H. Molar mass analysis of hydrophobically modified hyaluronic acid by SEC-MALLS: facing the challenges of amphiphilic biomacromolecules. Macromol Chem Phys 2018;
  23. 23.
    Pasch H. Hyphenated separation techniques for complex polymers. Polym Chem. 2013;4:2628–50.CrossRefGoogle Scholar
  24. 24.
    Pasch H, Trathnigg B. HPLC of polymers. Berlin: Springer; 1997. p. 223.Google Scholar
  25. 25.
    Radke W, Falkenhagen J. Chapter 5. Liquid interaction chromatography of polymers. In: Fanali S, Haddad PR, Poole CF, Schoenmakers P, Lloyd D, editors. Liquid chromatography. Amsterdam: Elsevier; 2013. p. 93–129.CrossRefGoogle Scholar
  26. 26.
    Raust J-A, Brüll A, Moire C, Farcet C, Pasch H. Two-dimensional chromatography of complex polymers: 6. Method development for (meth)acrylate-based copolymers. J Chromatogr A. 2008;1203:207–16.CrossRefPubMedGoogle Scholar
  27. 27.
    Radke W. Polymer separations by liquid interaction chromatography: principles – prospects – limitations. J Chromatogr A. 2014;1335:62–79.CrossRefPubMedGoogle Scholar
  28. 28.
    Maiko K, Hehn M, Hiller W, Pasch H. Comprehensive two-dimensional liquid chromatography of stereoregular poly(methyl methacrylates) for tacticity and molar mass analysis. Anal Chem. 2013;85:9793–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Schoenmakers P, Aarnoutse P. Multi-dimensional separations of polymers. Anal Chem. 2014;86:6172–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Shakun M, Heinze T, Radke W. Characterization of sodium carboxymethyl cellulose by comprehensive two-dimensional liquid chromatography. Carbohydr Polym. 2015;130:77–86.CrossRefPubMedGoogle Scholar
  31. 31.
    Ghareeb HO, Radke W. Characterization of cellulose acetates according to DS and molar mass using two-dimensional chromatography. Carbohydr Polym. 2013;98:1430–7.CrossRefPubMedGoogle Scholar
  32. 32.
    Stern R, Kogan G, Jedrzejas MJ, Šoltés L. The many ways to cleave hyaluronan. Biotechnol Adv. 2007;25:537–57.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and Polymer ScienceUniversity of StellenboschStellenboschSouth Africa
  2. 2.L’Oréal Research and InnovationAulnay-sous-BoisFrance

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