South African Journal of Chemical Engineering 45 (2023) 256–268

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South African Journal of Chemical Engineering

journal homepage: www.elsevier.com/locate/sajce

Mathematical model of astaxanthin purification process using the

low-pressure column chromatography method
Putri Restu Dewati a, Rochmadi b, Abdul Rohman c, Arief Budiman b, d, *
a
Chemical Engineering Department, Universitas Pembangunan Nasional Veteran Yogyakarta, Jl. SWK104, Yogyakarta 55283, Indonesia
b
Chemical Engineering Department, Universitas Gadjah Mada, Jl. Grafika 2, Yogyakarta 55284, Indonesia
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
d
Center of Excellence for Microalgae Biorefinery, Universitas Gadjah Mada, Sekip K1A, Yogyakarta, 55281, Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: Astaxanthin is an antioxidant compound that can be extracted from the microalgae Haematococcus pluvialis using
Astaxanthin the microwave-assisted extraction (MAE) method. Purification of the astaxanthin compound can be conducted
Mathematical model using a low-pressure column chromatography (LPCC) method. However, the mathematical model illustrating the
Low-pressure column chromatography
adsorption and desorption process using the LPCC method has not been built by the other authors yet. Therefore,
Purification
the purposes of this study were (1) to build the mathematical model of the purification process using the LPCC
method, and (2) to simulate the experimental data using the model to find the important kinetic constant values
that can be used in the industrial scale-up in the future. The purification process was carried out with silica gel as
a stationary phase and a mixture of n-hexane:acetone of 3:1 (v/v) as a mobile phase. The silica gel diameter was
varied to 0.2–0.5 mm and 0.063–0.2 mm, while the eluent velocity was varied to 1.85 mL/min and 3 mL/min.
The mathematical model having five kinetic constants of De1, De2, kc2, kc2.a, and H_A2 was successfully built. The
De1 and De2 (m2/s) were the effective diffusivities of astaxanthin in the intraparticle direction of the adsorbent
granule and in the axial direction of the column, respectively. The kc2 (m/s) was the mass transfer coefficient of
astaxanthin from the solution to the adsorbent. The kc2.a (/s) was the volumetric mass transfer coefficient of
astaxanthin from the solution to the adsorbent. The H_A2 (g silica gel/m3) was the Henry’s constant for astax­
anthin concentration at equilibrium at the liquid-solid interface. In the variation of silica gel diameter, the De2
value at the diameter of 0.2–0.5 mm was greater than that at 0.063–0.2 mm. However, the De1, kc2, kc2.a, and
H_A2 values were not affected by the different diameters. In the variation of eluent velocity, the kc2 and kc2.a
values at 3 mL/min were greater than those at 1.85 mL/min. However, the De1, De2, and H_A2 values were not
affected by the different eluent velocities.

1. Introduction
Astaxanthin is an antioxidant from the carotenoid group, which has a
higher antioxidant power than vitamins C and E (Krichnavaruk et al.,
2008; Wang et al., 2012; Lee et al., 2020). Astaxanthin (C40H52O4, 3,
3′-dihydroxy-β,β′-carotene-4,4′‑dione) is a compound having the
anti-cancer and anti-ageing activities and the ability to improve the
central nervous system, improve the heart function, and maintain the
eye health. In addition, astaxanthin can be used as a therapeutic drug for
patients with Coronavirus disease (Talukdar et al., 2020). Astaxanthin is
found in some organic materials such as fungi, microalgae, copepods,
krill, complex plants, snails, and some birds such as flamingos and quails
(Visioli and Artaria, 2017; Fassett and Coombes, 2011; Zhao et al.,
2019). One of the potential sources of astaxanthin is the microalgae
Haematococcus pluvialis (Dewati et al., 2021) because the cultivation
process for the microalgae does not require a large land area and its life
cycle is short enough (about 7–14 days) (Budiman et al., 2014). Astax­
anthin in the microalgae biomass is in the range of 0.5–5% dry cell
weight (Haque et al., 2016; Shang et al., 2016; Sun et al., 2016).
Astaxanthin from the microalgae is obtained through an extraction
process followed by a purification process. The purification process can
be carried out through several methods such as the gradient reversed-
phase high-performance liquid chromatography (Yuan and Chen,
1998), high-speed counter-current chromatography (Li and Chen, 2001;

* Corresponding author.
E-mail address: abudiman@ugm.ac.id (A. Budiman).

https://doi.org/10.1016/j.sajce.2023.06.004
Received 7 August 2022; Received in revised form 4 April 2023; Accepted 13 June 2023
Available online 14 June 2023
1026-9185/© 2023 The Authors. Published by Elsevier B.V. on behalf of South African Institution of Chemical Engineers. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P.R. Dewati et al. South African Journal of Chemical Engineering 45 (2023) 256–268

Du et al., 2016), and low-pressure column chromatography (Sun et al.,
2016; Hu et al., 2019). Table 1 shows the differences between the three
purification methods (Sethi et al., 2009; Sun et al., 2016; Khan and Liu,
2018).
Nowadays, many industries have used column chromatography in
their process like Novasep Company, Sartorius Company, etc. The three
purification methods using the column chromatography (shown in
Table 1) exist on industrial scales. In detail, compared to the other pu­
rification methods, the low-pressure column chromatography (LPCC)
method is more attractive (Table 1). The LPCC method is widely used in
the purification of synthetic chemicals and isolation of active com­
pounds from natural materials such as antifungals, antioxidants, and
antibacterials and is widely used for environmental waste analysis. The
basis of the separation process in the LPCC column is adsorption (Perry
and Green, 1999; Ismail and Nielsen, 2010; Horvath et al., 2014).
The microalgae extract, resulting from the extraction process of
microalgae, contains many compounds including astaxanthin. Further­
more, the purification process is used to separate the astaxanthin from
the other compounds. The purification process using the LPCC method
begins by inserting the microalgae extract into the LPCC column which
already contains a stationary phase. Then, the compounds of the
microalgae extract are adsorbed in the stationary phase. At the same
time, elution is carried out from the top of the column continuously with
a mobile phase, resulting in the desorption of the compounds from the
stationary phase. The desorption process occurs sequentially depending
on the affinity of each compound to the mobile phase, so there is a
separation of a specific compound from the microalgae extract.
Sun et al. (2015) carried out the extraction and purification of
astaxanthin from microalgae Haematococcus pluvialis using the LPCC
method with the LPCC column having a dimension of 500 mm × 40 mm,
silica gel as the stationary phase, and a mixture of n-hexane: dichloro­
methane: acetone = 5: 2.5: 1 (v/v/v) as the mobile phase. This study
resulted in trans-astaxanthin with a purity above 80%. Furthermore, a
Table 1
The differences between the three purification methods.
Parameters Purification methods
gradient reversed- high-speed counter- Low-pressure
phase high- current column
performance liquid chromatography chromatography
chromatography
study on the extraction and purification of astaxanthin from shrimp
shells has been conducted by Hu et al. (2019). The purification was
carried out using the LPCC method with silica gel as the filling material.
However, in the two studies, the development of a mathematical model
for the purification process in the LPCC column has not been conducted
yet. In addition, the experimental data, which were reported by the two
studies, were very limited.
Based on our best literature studies, a study on the astaxanthin pu­
rification process using the LPCC column has not been quantitatively
conducted by the previous authors yet. Therefore, this study focused on
the mathematical modelling of the astaxanthin purification process in
the LPCC column. The mathematical modelling can provide a better
understanding of the effect of affecting factors on the purification pro­
cess and the important kinetic constant values of the model are useful for
scaling up the purification process using the LPCC method in the future.
2. Mechanism and modeling of the adsorption-desorption
processes
Microalgae extract, containing astaxanthin and the other com­
pounds, was flowed into an LPCC column containing a stationary phase
(a pile of silica gel adsorbent granules called an adsorption bed). The
adsorption process occurred on the pore walls of the adsorbent granules,
hence astaxanthin in the LPCC column experienced the following pro­
cesses sequentially:
1) Mass transfer from the solution to the adsorbent surfaces.
2) Diffusion from the adsorbent surfaces into the adsorbent through the
pores.
3) Mass transfer from the solution in the pores to the pore walls.
4) Adsorption on the pore walls.
In the LPCC column, two mass transfer processes occurred, which
were the intraparticle mass transfer in the direction of the silica gel
granule and the axial mass transfer in the direction of the adsorption
bed.
The Fig. 1 shows a schematic of the axial and intraparticle mass
transfer process in the LPCC column.
2.1. Intraparticle mass transfer of astaxanthin

Pressure High There is an increase Low Intraparticle mass transfer of astaxanthin consisted of two processes
in pressure due to which were the mass transfer from the solution to the adsorbent surfaces
the rotation of the and the diffusion from the adsorbent surfaces into the adsorbent through
stationary phase
the pores.
Stationary Solid Liquid Solid
phase
Mobile phase Liquid Liquid Liquid 2.1.1. Mass transfer from the solution to the adsorbent surface
Advantages • It allows water to • There is no • Simple process. The Fig. 2 shows a schematic of the mass transfer of astaxanthin from
be used as a irreversible • Simple tools. the solution to the adsorbent surface with the assumptions below:
mobile phase so adsorptive loss. • Low installation
it is safer. • The sample is and operating
• High product easily adsorbed to costs. 1) There was a concentration gradient in the liquid film layer/solution.
purity. the stationary • High 2) There was a concentration gradient in the solid/adsorbent.
• Fast process. phase. selectivity.
• High yield. • High yield.
• High product • High product
purity. purity.
Disadvantages • High equipment, • The process in the • The process
operational, and column is takes a long
maintenance difficult. time.
costs. • Long separation • The number of
• Feedstock is in process. mobile phases
small quantities. • The search of the used is large
stationary and • There is an
mobile phases irreversible
takes a long time. adsorptive loss
• Usually for a
small feedstock
amount. Fig. 1. Schematic of (A) axial mass transfer and (B) intraparticle mass transfer.

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Fig. 2. Schematic of astaxanthin mass transfer process from the solution to the
adsorbent surfaces (NA2 = mass transfer of astaxanthin from the solution to the
adsorbent, CA2 = astaxanthin concentration in the solution, CA2* = astaxanthin
concentration at equilibrium at the liquid-solid interface, XA = astaxanthin
concentration in the adsorbent).

3) Equilibrium of the astaxanthin concentration at the liquid-solid

Fig. 3. Element volume in adsorbent granules (r = distance from the centre of
interface the sphere).
4) The adsorbent shape was spherical.

The mass transfer rate from the solution to the adsorbent surface can ∂ XA ∂ XA
be expressed through the Eq. (1). (r + Δr)2 De1 | − r2 De1 | (4)
∂r r+Δr ∂r r = r2 ∂XA
( ) Δr ∂t
*
NA2 = kc2 .a. CA2 − CA2 (1)

Where:
NA2 = mass transfer of astaxanthin from the solution to the If Δr → 0, so:
adsorbent per time per volume (g astaxanthin/(s.m3)). ( )
kc2 .a = volumetric mass transfer coefficient of astaxanthin from the ∂
r2 De1
∂XA
= r2
∂XA
(5)
solution to the adsorbent (/s). ∂r ∂r ∂t
CA2 = astaxanthin concentration in the solution (g astaxanthin/m3).
C*A2 = astaxanthin concentration at equilibrium at the liquid-solid ∂2 XA 2 ∂XA 1 ∂XA
+ = (6)
interface (g astaxanthin/m3). ∂r2 r ∂r De1 ∂t
The concentration ofC*A2 was approximated using the Henry equa­ Initial condition (at t = 0):
tion, shown in the Eq. (2)
XA (r, z, 0) = 0 (7)
*
CA2 = H A2.XA (2)
Boundary condition (at t>0):
Where:
1 At r = R
H_A2 = Henry’s constant for the equilibrium of astaxanthin
concentration at the liquid-solid interface (g adsorbent/m3) ∂XA ( )
− De1 *
(R, z, t) = kc2 CA2 − CA2 (8)
XA = astaxanthin concentration in the adsorbent (g astaxanthin/g ∂r
adsorbent).

2.1.2. Mass transfer from the adsorbent surface into the adsorbent 2 At r = 0
Astaxanthin in the solution diffused from the adsorbent surfaces into ∂XA
the adsorbent granules slowly. The schematic of the process can be (0, t) = 0 (9)
∂r
illustrated through the layer element volume as a function of the dis­
tance from the centre point of the adsorbent granules as shown in the
Fig. 3.
The astaxanthin mass balance equations on the element volume of Where:
the adsorbent granules can be described by the Eqs. 3 to 6 with the as­ XA = astaxanthin concentration in the adsorbent (g astaxanthin/g
sumptions below: adsorbent).
De1 = effective diffusivity of astaxanthin in the intraparticle
1) The system was isothermal. direction of the adsorbent (m2/s).
2) Silica gel was spherical with a uniform and fixed size. r= distance from the centre of the adsorbent (m).
3) The physical properties of liquids were constant. Δr = distance increment (m).
4) Astaxanthin degradation did not occur. t= time (s).
kc2 = mass transfer coefficient of astaxanthin from the solution to
rate of input − rate of output = rate of accumulation (3a)
the adsorbent (m/s).
[ ] [ ] CA2 = Astaxanthin concentration in the solution (g astaxanthin/m3).
∂XA ∂XA ∂XA
4π(r + Δr)2 De1 |r+Δr − 4π r2 De1 |r = 4πr2 Δr C*A2 = Astaxanthin concentration at equilibrium at the liquid-solid
∂r ∂r ∂t
interface (g astaxanthin/m3).
(3b)

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2.2. Axial mass transfer of astaxanthin
Axial mass transfer of astaxanthin occurred due to the flow of eluent
and due to the axial diffusion, described by the Eqs. (10) and 11
respectively.
Mass transfer due to the eluent flow = F.CA2
velocity,D is mass diffusivity (m2/s), isL is the travelled length of the
fluid. Based on the calculation, the Peclet number in this study was
0.357–4.19. The small Peclet number values indicated that the axial
dispersion in the model was necessary to consider.
The mass balance of astaxanthin in the solution can be described in
Eqs. 13 to 16 with the assumptions below:
(10)

∂CA2 1) The flow regime was a plug flow

Mass transfer due to the axial diffusion = − De2 . S . (11) 2) The system was isothermal.
∂z
3) Silica gel was spherical with a uniform and fixed size.
Where: 4) The physical properties of liquids were constant.
F= velocity of the inlet solution (m3/s). 5) Astaxanthin degradation did not occur.
CA2 = astaxanthin concentration in the solution (g astaxanthin/m3) 6) The eluent velocity into the LPCC column was constant.
De2 = effective diffusivity in the axial direction (m2/s). Rate of input − rate of output = rate of accumulation (13a)
S = cross-sectional area of the column (m2).
z= height of the column measured from the top end (m). [
∂CA2
] [
∂CA2
The schematic of the axial mass transfer in the LPCC column can be − De2 . S.
∂z z
| + F.CA2 |z − − De2 . S. |
∂z z+Δz
illustrated through the Fig. 4. The flow regime in a packed bed can be ] (13b)
( ) ∂CA2
known by the Reynolds number. The packed bed Reynolds number (Re*) + F.CA2 |z+Δz + kc2 .a. CA2 − *
CA2 .S.Δz = S.Δz.ε.
can be calculated using Eq. (12). The plug flow regime can be achieved ∂t
at Re* ≥ 10 (Wang et al., 2019b).
∂CA2 ∂CA2
| − |
* ρ.v.d ∂z z+Δz ∂z z
Re = (12)
μ Δz (14)
F CA2 |z+Δz − CA2 |z kc2 .a ( *
) ε ∂CA2
where: −
S.De2 Δz
−
De2
. CA2 − CA2 = .
De2 ∂t
Re* = Reynolds number
ρ= density of eluent flowing through the packed bed (kg/m3)
v= eluent velocity (m/s)
d= silica gel diameter (m) If Δz → 0, so:
µ= viscosity of eluent flowing through the packed bed (Pa.s) ( )
Based on the calculation, the packed bed Reynolds number was in the ∂ ∂CA2 F ∂CA2 kc2 .a ( ) ε ∂CA2
− . − . CA2 − *
CA2 = . (15)
range of 0.02–0.04. It means that the flow regime in the packed bed was ∂z ∂z S.De2 ∂z De2 De2 ∂t
still laminar. The main goal of this study was purification with selective
adsorption, therefore it needed a long contact time. Meanwhile, the ∂2 CA2 F ∂CA2 kc2 .a ( ) ε ∂CA2
− . − . CA2 − *
CA2 = . (16)
adsorbent amount (volume of silica bed) was limited. As consequence, ∂z2 S.De2 ∂z De2 De2 ∂t
the velocity of the mobile phase must be slow, resulting in the laminar Initial condition (t = 0):
flow. However, this study assumed that the flow regime was a plug flow
regime to simplify the calculations, but it still can be used to describe the CA2 (z, 0) = 0, at 0 < z ≤ L (17)
adsorption phenomena.
CA2 (0, 0) = CA20 (18)
Peclet number in the mass transfer is used to characterize fluid flows
with simultaneous momentum and mass diffusion convection processes. Boundary condition (z = 0):
u
Peclet number can be calculated using a formula ofD/L , whereu is flow The initial astaxanthin concentration was the same as the astax­
anthin concentration in the solution entering the column, as shown in
Eq. (19).
CA2 (0, t) = CA20 (19)

Where:
CA2 = astaxanthin concentration in the solution (g astaxanthin/m3).
C*A2 = astaxanthin concentration at equilibrium (g astaxanthin/m3).
De2 = effective diffusivity of astaxanthin in the axial direction of the
column (m2/s).
kc2 = mass transfer coefficient of astaxanthin from the solution to
the adsorbent (m/s).
a= area per volume of the column (/m)
ρb = bulk density of the adsorbent granules (g/m3).
ε= adsorbent porosity.
F= velocity of the inlet solution (m3/s).
S= cross-sectional area of the column (m2).
Z= height of the column measured from the top end (m).
Δz = height increment (m).
t= time (s).
Astaxanthin concentrations as a function of position and time can be
Fig. 4. Schematic of the axial mass transfer in the LPCC column (F = velocity of
the inlet solution, CA0 = initial concentration of the astaxanthin, Z = height of
predicted using a mathematical model containing two partial differen­
the column measured from the top end, L = height of the column). tial equations of Eqs. (6) and 16. By using the method of line solution
technique, the partial differential equations were converted into

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ordinary differential equations of a function of time (t) by discretizing 3. Astaxanthin accumulation in the silica gel
the dimensions of column length (z) and solid radius (r). This method
produced ordinary differential equations with the number of equations Astaxanthin accumulation in one granule of silica gel can be calcu­
as much as Nz(Nr+1), where Nz is the number of discretizations of the lated using the Eq. (22).
column length dimensions and Nr is the number of discretizations of the
∫r=R
solid radius. The completion of the two partial differential equations was
Astaxanthin accumulation = ρsilica gel .4.π.r2 .XA .dr (22)
conducted with the help of Matlab software. In detail, the flow of the
completion of the mathematical model can be seen in the Fig. 5. r=0

From the calculation with the help of Matlab software, the final
Where:
values of De1, De2, kc2.a, kc2, and H_A2 were determined when the Sum
ρsilica gel= density of the silica gel (g/cm3)
of Squared Error (SSE) between the experimental data and the calcu­
r= radius of the silica gel (cm)
lated data reached the minimum. The SSE was calculated using the Eq.
XA= astaxanthin concentration in the silica gel granules (g
(20).
astaxanthin/g silica gel)
SSE = (Experimental data − Calculated data)2 (20) Therefore, the total astaxanthin accumulation or the total remaining
astaxanthin in the all of silica gel granules can be calculated through the
Furthermore, to find out whether the proposed mathematical model Eq. (23).
is acceptable or not, the relative error was calculated using Eq. (21). If ⎛ r=r ⎞
the relative error is below 20%, the proposed mathematical model can ∫z=L
π 2⎝
∫
be accepted. Total astaxanthin accumulation = N Dc ρsilica gel .4.π.r .XA .dr⎠dz
2
4
⃒ ⃒ z=0 r=0
⃒Experimental data − Calculated data⃒
Relative error = ⃒⃒ ⃒ × 100%
⃒ (21) (23)
Experimental data
Where N is the number of silica gel granules in the adsorption bed per
volume bed. N can be calculated through the Eqs. (24)–26.

Fig. 5. The flow of the completion of the mathematical model using Matlab software.

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Total volume of particles in the bed / volume of one particle analysis was purchased from Sigma Aldrich, Singapore.
N = (24)
Volume of the bed
(π ) 4.2. Methods
/4
.D2c .L (1 − ϵ) .π.r3
N = 4 (π ) 3 (25) The Fig. 6 shows a series of purification tools with an LPCC column.
.D2 .L The preparation of the column chromatography for the purification
4 c
process was carried out in several steps. First, the cotton that has been
(1 − ϵ) shaped into a cone was inserted into the bottom end of the column to
N=
4 3 (26) prevent the silica gel being released from the column. After that, the
.π.r
3 column was installed in the upright position. Then, the adsorbent, that has
been soaked with the eluent for 24 h, was inserted together with the
Where:
eluent into the LPCC column. The adsorbent was poured into the column
N number of the particles per volume of adsorption bed (/cm3)
while gently tapping so that the porosity of the adsorbent in the column
Dc diameter of the column (cm)
was uniform. After all adsorbents and eluents were in the column, a
L height of the column (cm)
dispensing valve was opened to adjust the height of the eluent to be the
ϵ void fraction
same as the height of the adsorbent. The microalgae extract as much as 5
r radius of the silica gel (cm)
mL that contained 167 μg astaxanthin was placed right on top of the
Substituting the Eq. (26) to the Eq. (23) resulted in the Eq. (27).
adsorbent pile. Then, the valve of sample was opened. After all the
⎛ r=r ⎞
∫z=L ∫ microalgae extract entered the adsorbent, the eluent was added to a
(1 − ϵ) π 2 ⎝
Total astaxanthin accumulation = D ρsilica gel .4.π.r .XA .dr⎠dz
2 certain height. The eluent was continuously dripped at a constant veloc­
4 3 4 c ity. Before entering the column, the eluent was collected in an eluent
.π.r z=0 r=0
3
holding bottle. The addition of eluent was carried out in the eluent
(27)
holding bottle. Product samples as much as 5 mL were taken every 10 min.
4. Materials and methods
4.3. Astaxanthin compound analysis
4.1. Materials
Astaxanthin compound analysis was conducted using an HPLC tool
H. pluvialis biomass powder was obtained from Xi’an Saiyang with the brand of Shimadzu (Kyoto, Japan) LC-2010CHT with a pre­
BioTechnology Co., Ltd, China. Furthermore, the biomass powder was parative reversed-phase column C18 and detector of SPD-M10AVP at a
extracted using the microwave-assisted extraction (MAE) method with a wavelength of 475 nm. The mobile phase used was a mixture of 0.05%
solvent of acetone at 50 ◦ C for 10 min to get the microalgae extract. The trifluoroacetic acid: methanol of 3: 97 (v/v) at a column temperature of
microalgae extract contained an astaxanthin concentration of 33.41 mg/ 40 ◦ C. Astaxanthin was detected at a retention time of 4.2 min.
L. The LPCC column had a dimension of an outer diameter of 3 cm and a
height of 1 m (Fig. 6). Solvents of n-hexane and acetone with a ratio of 3:1 5. Results and discussion
(v/v) used as the eluent or the mobile phase were purchased from Merck.
Silica gel used as column filling material or stationary phase with diam­ 5.1. The effect of silica gel size and eluent flowrate in the purification
eter sizes of 0.063–0.2 mm and 0.2–0.5 mm was obtained from Merck. process
The pro analyst astaxanthin (with a purity of 98%, CAS 472–61–7) used as
a standard solution in High Performance Liquid Chromatography (HPLC) The purification process was conducted by varying the silica gel
diameter size and the eluent velocity. Two different silica gel diameter

Fig. 6. Astaxanthin purification device.

261
P.R. Dewati et al.
sizes of 0.063–0.2 mm and 0.2–0.5 mm. In this study, the average
diameter size was calculated using the ImageJ software. Some previous
studies also used ImageJ software to calculate the average particle
diameter size (Tajima and Kato, 2013; Rishi and Rana, 2015; Huling
et al., 2022). Silica gels with a size of 0.063–0.2 mm had an average
diameter of 0.18 mm, while those with a diameter size of 0.2–0.5 mm
had an average diameter of 0.35 mm. The Figs. 7 and 8 show the size
distribution for silica gel with diameter sizes of 0.063–0.2 mm and
0.2–0.5 mm, respectively.
The operating conditions in this purification process were variated
based on the Table 2. The difference between the Purifications 1 and 2
was the silica gel diameter size. Purification 1 and 2 used silica gel with a
diameter size of 0.2–0.5 and 0.063–0.2 mm, respectively. Table 3 shows
the Brunauer Emmett Teller (BET) test data for silica gel with a diameter
size of 0.063–0.2 mm and 0.2–0.5 mm. Furthermore, the difference
between the Purifications 2 and 3 was the eluent velocity which was
1.85 and 3 mL/min, respectively.
Fig. 7. Silica gel with a diameter size of 0.063–0.2 mm (63–200 µm): (A) photo of silica gel; (B) size distribution; (C) cumulative normal distribution.
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South African Journal of Chemical Engineering 45 (2023) 256–268
The Table 4 shows the experimental data of astaxanthin concentra­
tion as a function of time in the Purifications 1, 2, and 3. The Fig. 9
shows the differences in the astaxanthin concentration profile as a
function of time in the Purifications 1 and 2.
Based on the Table 4 and Fig. 9, at the beginning of the purification
process, the product did not contain astaxanthin. Then, the astaxanthin
concentration increased with the increase in the processing time. The
increase in the astaxanthin concentration indicated that the adsorption
and desorption processes occurred. Initially, the adsorption process only
occurred at the top of the column. Then, the adsorption process gradu­
ally decreased to the bottom of the column followed by the desorption
process rate starting from the top of the column. When the desorption
process was simultaneous at the top and bottom of the column, astax­
anthin concentration reached the peak on the curve. Astaxanthin con­
centration peaks of 0.65 mg/L and 0.77 mg/L were reached at the 80th
minute and 120th minute in the Purifications 1 and 2, respectively.
After reaching the peak, the astaxanthin concentration gradually
P.R. Dewati et al. South African Journal of Chemical Engineering 45 (2023) 256–268

Fig. 8. Silica gel with a diameter size of 0.2–0.5 mm (200–500 µm): (A) photo of silica gel; (B) size distribution; (C) cumulative normal distribution.

decreased and finally zero, indicating that the astaxanthin was no longer size of 0.2–0.5 mm, the astaxanthin needed 20 min to leave the column
released. This was due to the fact that during the purification process, no for the first time, while with that of 0.063–0.2 mm, the astaxanthin
microalgae extract entered again into the column, only the eluent was needed 60 min to leave the column for the first time. The length time of
continuously dripped at a constant velocity into the column so that the purification process and the length time of the first appearance of
desorption process of astaxanthin began to finish from the top of the astaxanthin indicated that silica gel with a diameter size of 0.063–0.2
column. These adsorption and desorption processes showed that the mm has superior sorption performance.
adsorption reaction of astaxanthin with silica gel adsorbent was physical The Fig. 10 shows the difference in the astaxanthin concentration
adsorption and reversible. profile as a function of time during the Purifications 2 and 3.
From the Table 4 and Fig. 9, it can be seen that the smaller the silica Based on the Table 4 and Fig. 10, the faster the eluent velocity, the
gel diameter size, the longer the time was needed for the purification faster the astaxanthin compound was obtained, in which the first time
process. For silica gel diameter size of 0.2–0.5 mm, the purification required to obtain the astaxanthin at an eluent velocity of 3 mL/min was
process needed 160 min, while for silica gel diameter size of 0.063–0.2 faster than that at 1.85 mL/min. The eluent velocity of 3 mL/min needed
mm, the purification process needed 190 min. With silica gel diameter
Table 3
Table 2 Results of the BET test for the different silica gel diameter size.
Operating conditions in the purification process. Parameter Size of 0.063–0.2 mma Size of 0.2–0.5 mmb
Parameter Purification 1 Purification 2 Purification 3 2
Surface area, m /g 480–540 181.415
Diameter of silica gel 0.2–0.5 mm 0.063–0.2 mm 0.063–0.2 mm Total pore volume, cm3/g 0.74–0.84 0.53
Mass of silica gel 60 g 60 g 60 g a
merckmillipore.com.
Velocity of eluent 1.85 mL/min 1.85 mL/min 3 mL/min b
Susanti, 2017.

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Table 4 of the column (De2, m2/s), the coefficient mass transfer of astaxanthin
Astaxanthin concentration out of the column (Purified astaxanthin from the solution to the adsorbent (kc2, m/s), the volumetric mass
concentration). transfer coefficient of astaxanthin from the solution to the adsorbent
t (min) Purified Astaxanthin Concentration (mg/L) (kc2.a, /s), and the Henry’s constant for the equilibrium of astaxanthin at
Purification 1 Purification 2 Purification 3 the liquid-solid interface (H_A2, g silica gel/ m3). Figs. 11, 12, and 13
0 0 0 0 show the simulation results of the mathematical modelling in the Puri­
10 0 0 0 fications 1, 2, and 3.
20 0.51 0 0
30 0.57 0 0.50
5.2.1. Purification 1
40 0.58 0 0.54
50 0.57 0 0.71 Fig. 11 shows the fitting between the experimental data and the
60 0.58 0.50 0.81 model data in the Purification 1.
70 0.63 0.50 0.60 By using the trapezoidal rule method, the total astaxanthin in the
80 0.65 0.51 0.51 product obtained from the Purification 1 was 140.60 μg. The relative
90 0.63 0.51 0.49
error of the simulation in the Fig. 11 was about 10.87% with an SSE of
100 0.56 0.55 0.35
110 0.53 0.57 0 0.0222. From the calculation, there was an astaxanthin compound
120 0.51 0.77 accumulated in the silica gel because the amount of astaxanthin com­
130 0.51 0.72 pound in the product was smaller than that in the feedstock (microalgae
140 0.47 0.65
extract). Based on the calculation, the amount of astaxanthin accumu­
150 0.30 0.57
160 0 0.52 lated in the silica gel was 26.45 μg or about 15.83% of the total astax­
170 0.48 anthin in the feedstock. In addition, the astaxanthin concentration in the
180 0.32 silica gel as functions of position and time can be predicted using the
190 0 mathematical model and the results are shown in the Fig. 12.
Based on the Fig. 12, the smaller the silica gel radius (R), the lower
30 min to get the first astaxanthin compound, while the eluent velocity the curve peaks of the astaxanthin concentration in the silica gel will be,
of 1.85 mL/min needed 60 min to get the first astaxanthin. By using the because the lower the astaxanthin amount was adsorbed in the silica gel.
eluent velocity of 3 mL/min, the purification process was completed in The astaxanthin adsorption process in silica gel started from the largest
110 min, while by using the eluent velocity of 1.85 mL/min, the puri­ R, then went to the smaller R, and then to the centre of the silica gel or at
fication process was completed in 190 min. R = 0. The adsorption process was followed by the desorption process.
At z = 0, the desorption process in the upper column immediately
started in the first minute, while at z = 20 the desorption process only
5.2. Application of the mathematical model to simulate the purification started at the minute 30th.
process in the LPCC column In the Fig. 12(a), at z = 2 cm, at the end time of the process, which was
minute 160th, all astaxanthin compound was completely desorbed in all
By using the mathematical model, the purification process can be silica gel radii, so in the LPCC column with z = 2 cm, there was no
predicted at various operating conditions, not only the operating con­ astaxanthin accumulation. While in the Fig. 12(b), at z = 20 cm, at the end
ditions studied in this study. From the completion of the mathematical time of the process, which was minute 160th, the XA did not reach 0 value
modelling of the purification process using Matlab software, the at all various silica gel radii. Therefore, from the Fig. 12, it can be
important kinetic constant values were found. They are the effective concluded that the silica gel in the LPCC column with a height (z) of 20 cm
diffusivity of astaxanthin in the intraparticle direction of the adsorbent still contained astaxanthin compounds. And at the other z values, there
(De1, m2/s), the effective diffusivity of astaxanthin in the axial direction may also be an accumulation of astaxanthin compounds in the silica gel.

Fig. 9. The effect of silica gel diameter size on the purification process with an eluent flowrate of 1.85 mL/minute and an astaxanthin concentration in the
microalgae extract of 33.4 mg/L.

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P.R. Dewati et al. South African Journal of Chemical Engineering 45 (2023) 256–268

Fig. 10. The effect of eluent velocity on the purification process with a diameter size of 0.063–0.2 mm and an astaxanthin concentration in the microalgae extract of
33.4 mg/L.

Fig. 11. Simulation results using the mathematical model for Purification 1 (a silica gel diameter size of 0.2–0.5 mm and an eluent velocity of 1.85 mL/min).

Fig. 12. Profiles of the astaxanthin concentration in the silica gel in the Purification 1 with (a) z = 2 cm, (b) z = 20 cm. Where z= height of the column and R-radius
of silica gel.

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P.R. Dewati et al. South African Journal of Chemical Engineering 45 (2023) 256–268

Fig. 13. Simulation results using the mathematical model for Purification 2 (a silica gel diameter size of 0.063–0.2 mm and an eluent velocity of 1.85 mL/min).

Fig. 14. Profiles of the astaxanthin concentration in the silica gel in the Purification 2 with (a) z = 2 cm, (b) z = 20 cm. Where z=height of column and R-radius of
silica gel.

Fig. 15. Simulation results using the mathematical model for Purification 3 (a silica gel diameter size of 0.063–0.2 mm and an eluent velocity of 3 mL/min).

5.2.2. Purification 2 product obtained from the Purification 2 was 132.37 μg. The relative
The Fig. 13 shows the results of the fitting between the experimental error of the simulation in the Fig. 13 was about 9.91% with an SSE of
data and model data in the Purification 2. 0.0121. Alike in the Purification 1, the astaxanthin was accumulated in
With the trapezoidal rule method, the total astaxanthin in the the silica gel in the Purification 2. The total amount of astaxanthin

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Fig. 16. Profiles of the astaxanthin concentration in the silica gel in Purification 3 with (a) z = 2 cm, (b) z = 20 cm. Where z=length of the column and R-radius of
silica gel.

accumulated in the silica gel in the Purification 2 was 34.41 μg or about zeaxanthin and lutein purification, the irreversible adsorption was 7.6%
20.60% of the total astaxanthin in the feedstock. The Fig. 14 shows the (Wang et al., 2019a). Therefore, by observing the astaxanthin concen­
profiles of the astaxanthin accumulated in the silica gel in the Purifi­ tration in the silica gel with the help of the mathematical model, it
cation 2. showed that the astaxanthin can be accumulated in the silica gel based
Based on the Figs. 11, 12, 13 and 14, at the end of the purification on the radius of silica gel. In an experimental analysis, a measurement of
process, the larger the diameter size of the silica gel, the more the astaxanthin concentration in solids along the silica gel radius is very
amount of astaxanthin came out of the column, and the less the amount difficult to be conducted, but it can be predicted through the mathe­
of astaxanthin that was still left in the silica gel. On the opposite side, the matical model. Thus, the mathematical model is very useful to give a
smaller the diameter size of the silica gel, the less the amount of astax­ better understanding about the purification mechanism process in the
anthin came out of the column, and the more the amount of astaxanthin chromatographic column.
that was still left in the silica gel. This indicated that the silica gel ad­ The kinetic constant values obtained from the simulation for the
sorbents with a large size were better used for the astaxanthin purifi­ Purifications 1, 2, and 3 using the mathematical model are shown in the
cation compared to the silica gel with a smaller size. Table 5.
To ensure that the kinetic constant values obtained from the simu­
5.2.3. Purification 3 lation were correct, a comparison between those values in this study and
The Fig. 15 shows the results of the fitting between the experimental the other studies was conducted. Gurgel et al. (2001) reported that the
data and the model data in the Purification 3. effective diffusivity constant of water into the silica gel at variations of
Based on the Fig. 15, by using the trapezoidal rule method, the total temperature, pressure and water concentration was in the range of 1.9 ×
astaxanthin in the product obtained from the Purification 3 was 135.30 10− 9 - 9.7 × 10− 9 m2/s. Ni and San (2002) reported that the diffusivity
μg. The relative error of the simulation in the Fig. 15 was about 6.52% coefficient of water vapour into the silica gel was in the range of 2 ×
with SSE = 0.0094. Alike in the Purifications 1 and 2, the Purification 3 10− 9 - 4 × 10− 11 m2/s. Meanwhile, the diffusivity coefficient of aerogel
showed that at minute 110th, there was no astaxanthin accumulation in silica and aerogel silica coated with a metal foam was in the range of
the LPCC column with z = 2 cm (Fig. 16). Furthermore, with z = 20 cm, 0.91 × 10− 10 - 13.79 × 10− 10 m2/s (Nawaz et al., 2014). In addition, at
there was astaxanthin accumulation in the silica gel (Fig. 16). The the adsorption of water vapour into the silica gel, the diffusivity value
amount of the remaining astaxanthin in the silica gel was 31.75 μg or was in the range of 1.4 × 10− 10 - 4.6 × 10− 10 m2/s and the mass transfer
about 19.01% of the total astaxanthin in the feedstock. The Fig. 16 rate coefficient value was in the range of 5 × 10− 3 - 6 × 10− 3 m/s
shows the profile of XA in the Purification 3. (Gwadera Kupiec, 2015).
Based on the simulation, after the purification process, astaxanthin Davarnejad et al. (2014) explained that the volumetric mass transfer
accumulation in the silica gel in the Purifications 1, 2, and 3 was constant in the extraction of β-carotene with supercritical fluid was
15.83%, 20.60%, and 19.01% respectively. These results were close to a affected by the pressure, time and temperature of extraction. The results
result reported by Hu et al. (2019) in which the astaxanthin purification showed that the value of the volumetric mass transfer constant was
process in the chromatographic column with adsorbents of silica gel 2.486 × 10− 2 /s at a pressure of 7.5 MPa, a temperature of 100 ◦ C, an
resulted in an irreversible adsorption of 14.05%. Meanwhile, for extraction time of 1 h, and 0.046 × 10− 2 /s at a pressure of 17.5 MPa, a
temperature of 120 ◦ C, an extraction time of 5 h. The optimum mass
transfer coefficient was 6.70 × 10− 3 /s obtained at a pressure of 17.7
Table 5 MPa, a temperature of 100.5 ◦ C and an extraction time of 3.9 h.
Kinetic constant values obtained from the simulation. Meanwhile, Jannah (2016) studied the ethanol purification with silica
Kinetic constants Purification 1 Purification 2 Purification 3 gel and stated that the volumetric mass transfer value was 3.8 × 10− 2 /s
, m2/s
De and the axial diffusivity coefficient was 1 × 10− 4 m2/s. Brandani and
1 1.09 × 10–8 1.09 × 10–8 1.09 × 10–8
,De
2
m2/s
2.80 × 10− 5 3.88 × 10− 6 3.88 × 10− 6 Mangano (2022) explained that the adsorption rate of water into silica
,kcm/s
2 5.90 × 10–5 1.09 × 10–5 1.68 × 10–4 gel (known as the average volumetric mass transfer coefficient) was 4.9
× 10− 3 /s at a temperature of 25 ◦ C.
kc /s
.a,
2 5.00 × 10–3 1.00 × 10–3 1.36 × 10–2
H_A2, g silica gel/m3
2.19 × 10− 1 2.19 × 10− 1 2.19 × 10− 1

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6. Conclusion
The purification process of astaxanthin from Haematococcus pluvialis
microalgae extract using the LPCC method was successfully simulated
by the proposed mathematical model having five kinetic constants
including the effective diffusivity of astaxanthin in the intraparticle di­
rection of the adsorbent (De1, m2/s), the effective diffusivity of astax­
anthin in the axial direction of the column (De2, m2/s), the mass transfer
coefficient of astaxanthin from the solution to the adsorbent (kc2, m/s),
the volumetric mass transfer coefficient of astaxanthin from the solution
to the adsorbent (kc2.a, 1/s), and the Henry’s constant for the equilib­
rium at the liquid-solid interface (H_A2, g silica gel/m3). In the variation
of the silica gel diameter size (0.2–0.5 mm and 0.063–0.2 mm), De2
value at the large diameter size of the silica gel was greater than that in
the smaller diameter size of the silica gel. However, the values of De1,
kc2, kc2.a and H_A2 were the same for both diameter sizes. Meanwhile,
for the variation of eluent velocity (1.85 mL/min and 3 mL/min), kc2
and kc2.a values at the large eluent velocity were greater than those at
the small eluent velocity. However, the values of De1, De2 and H_A2 were
the same for both eluent velocities. The largest irreversible adsorption
(20.60%) was obtained with a silica gel diameter size of 0.063–0.2 mm
and an eluent velocity of 1.85 mL/minute. The proposed model resulted
in relative error values of 6.52–10.87% which were below 20%.
Therefore, the model can be accepted. However, we realized that the
model still needs improvement to get a better prediction, especially at
the end of the experiment. Therefore, in the future, the mathematical
equations and boundary conditions can be modified to result in better
predictions.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors acknowledge the financial support in this research and
publication from the Ministry of Education, Culture, Research, and
Technology of the Republic of Indonesia.
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