DEVELOPMENT OF TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY
METHOD FOR ANALYSIS OF OLIGOSACCHARIDES FROM HYDROPHOBIC
HYALURONAN DERIVATIVES
Vido M.1,2, Hermannová M.2, Táborský P.1
1
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 753/5,
625 00 Brno, Czech Republic
2
Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic
(email: marekvido95@gmail.com)
Derivatives of hyaluronan with hydrophobic functional groups are promising
macromolecules for many possible biomedical applications.[1] For complete
characterization of novel derivatives not only essential parameters are needed,
such as a degree of substitution, molar mass distribution and polydispersity, but
also distribution of substituents along the molecule chain[2] is indispensable for
proper assessment of structure-property relationship of these polymers. A
method, that utilizes two-dimensional liquid chromatography hyphenated with
mass spectrometry for separation of oligosaccharides derived from hyaluronan
derivatives, was developed. Oligosaccharides can be obtained by enzymatic
degradation of polymeric derivatives with hyaluronidases (e.g. from
Streptococcus Pneumoniae)[3] and because of digestion they differ in a length (a
number of hyaluronan units) and in a number of bound substituents. Therefore,
the combination of size-exclusion chromatography in the first dimension and
reversed-phase LC in the second dimension was used to successfully separate
complicated mixtures of oligosaccharides by size and polarity at the same time.
Also, the active solvent modulation was used while connecting two modes of
separation which can suppress a “breakthrough” effect[4] and further help with
optimizing the separation.
lauroyl derivative of HA
enzyme
SpHyl
separated oligosaccharides
[1] A. Fallacara, E. Baldini, S. Manfredini, and S. Vertuani, “Hyaluronic Acid in the Third
Millennium,” Polymers, vol. 10, no. 7, p. 701, Jul. 2018, doi:
10.3390/polym10070701.
[2] P. Mischnick, “Analysis of the Substituent Distribution in Cellulose Ethers - Recent
Contributions,” in Cellulose Science and Technology, T. Rosenau, A. Potthast, and J.
Hell, Eds. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2018, pp. 143–173. doi:
10.1002/9781119217619.ch7.
[3] R. Stern, G. Kogan, M. J. Jedrzejas, and L. Šoltés, “The many ways to cleave
hyaluronan,” Biotechnology Advances, vol. 25, no. 6, pp. 537–557, Nov. 2007, doi:
10.1016/j.biotechadv.2007.07.001.
[4] B. W. J. Pirok, D. R. Stoll, and P. J. Schoenmakers, “Recent Developments in TwoDimensional
Liquid Chromatography: Fundamental Improvements for Practical
Applications,” Anal. Chem., vol. 91, no. 1, pp. 240–263, Jan. 2019, doi:
10.1021/acs.analchem.8b04841.