(374f) Optimizing the Separation of Oleanolic and Ursolic Acids by Simulated Moving Bed Chromatography using a C30 Adsorbent

Optimizing the separation of
oleanolic and ursolic acids by simulated moving bed chromatography using a C30
adsorbent

Ivo S.
Azenha¹, José P.S. Aniceto^1,*, Adélio Mendes²,
Carlos M. Silva¹

¹ CICECO, Department of Chemistry,
University of Aveiro, 3810-193 Aveiro, Portugal;

² LEPABE-Faculdade de Engenharia,
Universidade do Porto, 4200-465 Porto, Portugal

*joseaniceto@ua.pt

1. Introduction

Eucalyptus globulus
is a predominant species in the Portuguese forest [1] and a vital resource for
the pulp and paper industry. The bark residues are usually burned for energy
production without any further valorization. Recently, the bark has been
identified as a source of triterpenic acids (TTAs) such as oleanolic and
ursolic acids [2]. These compounds are known to possess a wide spectrum of
bioactivities, including anti-oxidative, antitumoral, anti-inflammatory,
anti-hyperlipidemic, and anti-microbial effects [3]. Thus, under the scope of
the biorefinery concept, E. globulus bark is a potential candidate to
extract high-value compounds such as TTAs. However, their separation after
extraction is challenging as oleanolic and ursolic acids are two structurally
related isomers and occur simultaneously in the same natural matrix.

Simulated moving bed
(SMB) chromatography is a continuous adsorption technique, which appears as an
efficient alternative to batch elution chromatography. The SMB continuous
countercurrent mode of operation maximizes the mass transfer driving force,
providing improved productivity and reduced solvent consumption [4]. Thus, an
SMB process may be a potential candidate for TTAs separation.

2.
Methods

A series of impulse
experiments were conducted to select appropriate mobile phases for the
separation of oleanolic and ursolic acids. Experimental breakthrough curves
were measured in a custom laboratorial installation. Breakthrough of pure
components were conducted to determine equilibrium and mass transport
parameters, by fitting a chromatographic model to the experimental data. The
model considers an axial dispersion plug flow pattern with the internal and
external mass transfer resistances lumped into a global linear driving force
coefficient. Parameters were validated through the successful prediction of
breakthrough assays of binary mixtures.

3. Results and
discussion

From a series of
several impulse tests methanol/water 95/5 (%, v/v) emerged as the most
favorable mobile phase to conduct their continuous separation by SMB providing
a value of selectivity of 1.08. The C30 column demonstrated a remarkable
separation capacity for the triterpenic acids, enabling simultaneously higher
selectivities and faster analysis times, when compared with previous results
using a C18 packing material and the same mobile phase [5]. Equilibrium and mass
transport parameters obtained from breakthrough experiments of pure acids were then
successfully validated for the prediction of breakthrough experiments of binary
mixtures of oleanolic and ursolic acids with errors (AARD) of 5.2 % and 11.9%,
respectively. These parameters were then used to design the SMB separation of a
representative mixture of oleanolic and ursolic acids from a natural extract of
E. globulus.

A classical SMB scheme
was simulated and optimized with optimal operating conditions determined using
a Design of Experiments – Response Surface Methodology (DoE-RSM) approach
previously presented by the authors [5]. This methodology allows SMB
optimization with a low number of simulation runs, resulting in a small
computational load. Purity requirements were defined while the productivity was
maximized. Rigorous phenomenological simulation results, of which the
concentration profile in the SMB at cyclic steady state is presented in Figure
1, demonstrated that the SMB is possible to attain purities levels of 99.9 %,
for both extract and raffinate outlets, and productivities of 1.705
kg/(m3adsorbent day) with 2-2-2-2 configuration. This is a significant
achievement as previous results with a C18 stationary phase showed that
purities of 99.4 % and 99.1 % for ursolic and oleanolic acids, respectively,
were achievable at the expense of using three columns per section (3-3-3-3),
and consequently at the expense of extremely low productivities [6]. The work
presented here with the C30 column represents important improvements towards
the successful chromatographic separation of these triterpenic acids.

Figure 1.  Simulation
results of SMB operation at cyclic steady state for the isolation of oleanolic
(blue line) from ursolic acid (orange line). E – eluent; F – feed; X – extract;
R – raffinate.

4. Conclusions

The separation of
oleanolic and ursolic acids by SMB was enhanced by applying C30 columns and a
methanol/water 95/5 (%, v/v) mobile phase. The SMB unit with two columns per
section, which was optimized combining the design of experiments and response
surface methodologies with phenomenological computer simulations, attained
purities of 99.9 %. This represents a significant improvement in terms of
purity and productivity when compared with previous results obtained with C18
columns, which required a 3-3-3-3 configuration to attain purities of 99.4 %.

Keywords: chromatography, simulated moving
bed, triterpenic acids

Acknowledgements

CICECO (FCT Ref. UID/CTM/50011/2019);
Multibiorefinery project (POCI-01-0145-FEDER-016403); LEPABE -
POCI-01-0145-FEDER; I.S. Azenha thanks PhD grant SFRH/BD/126509/2016.

References

[1] J.S. Uva, Instituto da
Conservação da Natureza e das Florestas (2013) p. 3.

[2] M.M.R. de Melo, E.L.G. Oliveira,
A.J.D. Silvestre, C.M. Silva, J. Supercrit. Fluid., 70 (2012), pp. 137-145.

[3] J. Liu, J. Ethnopharmacol. 49
(1995) 57–68.

[4] J.P.S. Aniceto, C.M. Silva, Sep.
Purif. Rev., 44 (2013), pp. 41-73.

[5] J.P.S. Aniceto, S.P. Cardoso,
C.M. Silva, Comput. Chem. Eng. 90 (2016) 161–170.

[6] J.P.S. Aniceto, I.S. Azenha,
F.M.J. Domingues, A. Mendes, C.M. Silva, Sep. Purif. Technol. 192 (2018)
401–411.