Process Biochemistry 45 (2010) 13251329

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Optimization of single-column chromatographic separation of

fructooligosaccharides
Katarna Vankov, Milan Polakovic
Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering,
Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskho 9, 812 37 Bratislava, Slovak Republic

a r t i c l e i n f o a b s t r a c t

Article history: An experimental study aimed at the design and optimization of single-column separation of
Received 21 July 2009 fructooligosaccharides from mono- and disaccharides was carried out. AmberliteTM CR1320Ca, a cation-
Received in revised form 30 April 2010 exchange resin with a calcium ion as the functional group was packed in two laboratory columns of
Accepted 30 April 2010
different dimensions. Optimization parameters were the supercial velocity and the column load. In a
smaller column, the maximal yield of fructooligosaccharides of 86% and the selectivity of separation of
Keywords:
82% were achieved for the column load of 2.0% and supercial velocity of 5.0 105 m s1 . At the same
Fructooligosaccharides
process conditions in a larger column, the yield and selectivity increased to 90%.
Elution chromatography
Cation exchanger 2010 Elsevier Ltd. All rights reserved.
Poly(styrene-divinyl benzene) resin
Optimization
Process design

1. Introduction
Fructooligosaccharides (FOSs) represent a major class of fructan
oligomers. They have prebiotics effects therefore they support the
growth of bidobacteria in human colon [1,2]. Owing to their rela-
tively high sweetness, noncariogenicity and nondigestibility, they
have broad applications in diabetic, child and low-energy food [3,4].
Moreover, they stimulate adsorption of magnesium and calcium,
and decrease the total cholesterol, phospholipids and triglycerides
in serum [5,6]. Natural sources of FOSs are agricultural plants [7,8].
On industrial scale, they are produced from sucrose by the action
of enzyme fructosyltransferase [9,10]. Separation of FOSs is carried
out using simulated moving-bed chromatography. Unfortunately,
available literature provides information on the chromatographic
separation of FOSs from mono- and disaccharides only for analyt-
ical purposes [11,12]. On the other hand, there are a number of
papers about the process separation of glucose, fructose or sucrose
mixtures [1315].
Elution chromatography is a method that allows an effective
separation and purication of multicomponent mixtures with var-
ious adsorption afnities of their components to a chromatographic
adsorbent. In general, it provides a high yield and selectivity. In
spite of some disadvantages such as high product dilution, low pro-
ductivity and long separation time, elution chromatography has
Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52496920.
E-mail address: milan.polakovic@stuba.sk (M. Polakovic).
been extensively studied and applied in large-scale in the last two
decades [16]. Separation efciency of elution chromatography can
be determined by several factors.
The key point of the optimization of this separation process is
the choice of a suitable adsorbent. Cation-exchange resins have
been the most commonly used type of adsorbents for saccha-
ride separation in the last decade. Several authors have studied
the inuence of the resin ion-form type and the degree of cross-
linking on the distribution coefcients of saccharides [1720]. In
our previous study, we dealt with the characterization of single-
component adsorption equilibria of glucose, fructose, sucrose and
fructooligosaccharides on several commercial adsorbents [20]. A
cation-exchange resin AmberliteTM CR1320Ca was selected as the
best one. In a subsequent study, adsorption equilibria of binary
mixtures of these saccharides on AmberliteTM CR1320Ca were
investigated [19]. Another important parameter is temperature,
which inuences the viscosity of liquid phase, rate of saccharide
hydrolysis and adsorption intrinsic kinetics. The separation can
be carried out at an ambient temperature but an elevated tem-
perature improves the separation performance due to enhanced
mass transfer. At high temperatures, however, increased hydrolysis
of sucrose and fructooligosaccharides was observed and a tem-
perature of 60 C was therefore recommended [21]. The column
diameter and length as well as supercial velocity have an effect
on the height equivalent and on the number of theoretical plates.
Adsorbent capacity is decisive for the volume of resin and the col-
umn load. A key issue in the process chromatography is the scale-up
of a separation process from a smaller column to a larger one. Exist-

1359-5113/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.04.025
1326 K. Vankov, M. Polakovic / Process Biochemistry 45 (2010) 13251329

Table 1
Column characteristics.

Column 1 Column 2

Column type Superformance 150-16 XK26 Pharmacy

Diameter (mm) 16 26
Bed length (cm) 11.5 61.00
Volume of resin (cm3 ) 23.1 323.9
Bed voidage 0.384 0.009 0.392 0.014
Total bed porosity 0.794 0.012 0.805 0.021

ing studies show that it is rather difcult to preserve separation

efciency in scaling up chromatographic columns [22,23].
The aim of this work was to investigate the separation of
FOSs using preparative elution chromatography. For this purpose,
true process-size particles of cation-exchange resin AmberliteTM
CR1320Ca was used to separate FOSs from fructose, glucose and
sucrose in a xed-bed column at 60 C. Supercial velocity and load
Fig. 1. Column outlet concentration courses at the supercial velocity of
were varied in order to achieve optimal yield of FOSs and separa- 21.6 105 m s1 and column load of 1.85% for Column 1.  1F -fructofuranosyl
tion selectivity. Scale-up experiments were performed for optimal nystose, nystose,  kestose,  sucrose, and  glucose. For better distinc-
conditions. tion, the concentrations of low-content components (fructose, FOS hexamer and
heptamer) forming only 2.9% of total saccharides are not displayed.
2. Materials and methods
differential ow refractometer (Knauer, Berlin, Germany) at 25 C and fractions for
2.1. Materials analysis were collected in predened intervals.

Fructose, glucose and sucrose were obtained from Mikrochem s.r.o. (Pezi-
2.3. Analysis of saccharides
nok, Slovakia). Kestose, nystose and 1F -fructofuranosylnystose were products of
Wako Pure Chemical Industries (Osaka, Japan). Water, which was used to pre-
All saccharide concentrations were determined by HPLC using the column
pare saccharide solution and eluent, was redistilled and ltered through a 0.2 m
REZEX RSO-Oligosaccharide (Ag+ form, 200 mm 10 mm, Phenomenex, Torrance,
cellulose-nitrate lter. All other chemicals were of analytical grade and were
CA). The concentrations of higher FOSs, for which no standards were available,
obtained from readily available commercial sources.
were determined on the basis of specic refractivity of FOS standards which was
found to be approximately constant. The temperature of the analytical column was
2.2. Separation of saccharides maintained at 40 C using a thermostat (Jetstream Plus II, Thermotechnic Products,
Langenzersdorf, Austria). Redistilled water was used as eluent at the ow rate of
A resin with the functional group (SO3 )2 Ca2+ , AmberliteTM CR1320Ca (Rohm 0.3 cm3 min1 . An autosampler from Gilson (Middleton, WI) was used to inject 10 L
and Haas France s.a.s., France), was used. The particle diameter and ion-exchange samples which were detected by the differential ow reactometer Knauer at 25 C.
capacity were 320 m and 1.63 equiv. L1 , respectively. The water content of the
resin was determined by drying wet particles at 80 C until constant weight was
reached. The value of water mass fraction was 48.3% with a standard deviation 3. Results and discussion
of 0.7%. The resin slurry was decanted several times with redistilled water and
then packed into a column Superformance 150-16 (Gotec Labor Technik, Muhltal, The rst two series of separation experiments were carried out
Germany) or XK26 Pharmacy (Uppsala, Sweden). The basic characteristics of the
in the smaller column, Column 1; the column load and super-
columns are summarized in Table 1. The bed voidage was measured with a dextran
of the molecular weight of 2 000 000 (Polymer Standard Service, Mainz Germany), cial velocity were varied in each of the series. Fig. 1 shows the
which did not diffuse into the resin particles. The total bed porosity was deter- column outlet concentration courses for the supercial velocity of
mined from the retention volume of acetone that was injected in a form of a 20 vol.% 21.6 105 m s1 and the column load of 1.85%. The mean reten-
aqueous solution. tion times of individual saccharides decrease with their molecular
For the determination of the height equivalent to a theoretical plate (HETP),
weight but the column adsorption zones of the saccharides are
individual saccharide solutions were injected at the supercial velocity of
21.6 105 m s1 . HETP was then calculated from the rst and the second moment rather wide, which is evident from the overlap of the concentra-
of residence the time distribution using the following equations: tion courses. The goal of industrial separation is, however, not to
 obtain pure compounds but to recover a mixture of FOSs from other
tc(t) dt
= 0 (1) sugars. Fig. 2 therefore presents the total concentrations of the two
c(t) dt main groups of compounds. The possibility to obtain a product frac-
0
 2
tion containing only FOSs is evident. This would however be only
(t ) c(t) dt at the expense of a large loss of FOSs in a by-product fraction.
2
 = 0
 (2)
c(t) dt An optimal separation of this mixture thus requires a compro-
0
mise between the requirements for maximal recovery of FOSs and
 2
 minimal presence of lower saccharides in the product. For that
HETP = Lc (3)
 purpose, FOS yield Yt and selectivity St are dened as follows:
where  is the rst statistical moment,  2 is the second central moment, c(t) is the mF,t
Yt = (4)
column outlet concentration in time t, and Lc is the column length. mF0
The mixture of saccharides, which was used in the separation, contained (mass
fraction %): 2.1 of fructose, 21.4 of glucose, 14.8 of sucrose, 24.3 of 1-kestose, 28.8 mF,t
St = (5)
of 1-nystose, 7.8 of 1F -fructofuranosyl nystose, 0.7 of FOS hexamer and 0.1 of FOS mT,t
heptamer. The saccharide feed concentration was 700 g L1 . A column was rst equi-
librated with distilled water at the temperature of 60 C and the supercial velocity where mF,t and mT,t are the masses of FOSs and total saccharides
was set in the range of (2.1621.6) 105 m s1 by a HPLC pump Knauer (Berlin, recovered from the column outlet in time t and mF0 is the mass of
Germany). The mixture of saccharides was injected into the column through a six- FOSs in the feed.
port switching valve (Knauer, Berlin, Germany) at various amounts. Column load,
dened as the ratio of the total saccharide mass in the feed and dry resin mass in
Fig. 3 illustrates the calculated courses of Yt and St for the sep-
the column, ranged from 0.62% to 10.5%. Most experiments were made in duplicate. aration experiment presented in Figs. 1 and 2. While the FOS yield
The total saccharide concentration in the column outlet stream was monitored by a increased from 0% to 100%, the selectivity decreased from 100% to
 K. Vankov, M. Polakovic / Process Biochemistry 45 (2010) 13251329
Fig. 2. Column outlet concentration courses of total FOSs () and mono- and dis-
accharides () at the supercial velocity of 21.6 105 m s1 and column load of
1.85% for Column 1. The shaded area corresponds to the product fraction while the
non-shaded area the by-product fraction.
only 56%, which was the portion of FOSs in the feed. As optimal
values of FOS yield and selectivity were considered those obtained
for the cut time, which is the time at the cross-section of the frac-
tion concentration curves [24]. The cut time divides the collection
of the column outlet stream into two fractions (Fig. 2). The product
fraction contains mainly FOSs and the by-product fraction mainly
glucose and sucrose. In this particular case, the cut time was about
10 min and the FOS yield and selectivity had incidentally the same
value of about 80%. The FOS yield and selectivity values presented
in the further text are those obtained for the cut time.
Fig. 4 presents the results of separation in Column 1 for the con-
stant supercial velocity of 6.5 105 m s1 and the column load
varying from 0.5% to 20%. It is well known that the increase of load
decreases the effective length of the column because the center
of the feed zone is in larger distance from the inlet. Moreover, a
larger length difference between the fraction zone centers must
be achieved for full separation. The FOS yield was about 85% up to
the column load of 3% but it strongly decreased above this value.
The selectivity decreased gradually with the column load in this
interval due to accumulation of glucose, fructose and sucrose in
the product fraction. The decrease of FOS yield was partly reduced
by the increase of the cut time from about 33 min at 0.5% to about
45 min at 20% of the column load. The decrease is a consequence of
broadening of adsorption zones of individual components.
Fig. 3. Dependence of FOS yield () and selectivity () on time at the supercial
velocity of 21.6 105 m s1 and column load of 1.85% for Column 1.
1327
Fig. 4. Dependence of FOS yield (), selectivity () and cut time () on the column
load at the supercial velocity of 6.5 105 m s1 for Column 1.
A different picture was obtained for the inuence of
supercial velocity on separation of FOSs in the range of
(2.1621.6) 105 m s1 at the constant column load of 1.85%
(Fig. 5). Both the FOS yield and the selectivity varied relatively lit-
tle; the FOS yield from 77% to 86% and the selectivity from 78%
to 82%. While the selectivity was a decreasing function of the
supercial velocity, the FOS yield had a mild maximum at around
7.5 105 m s1 . It could be concluded that the FOS yield depen-
dence was an inverse function of the HETP dependence on the
supercial velocity. Fig. 5 further shows that the cut time was
almost an inverse function of the supercial velocity. This means
that the cut time was approximately proportional to the liquid
mean residence time.
In order to better map the region of optimum FOS yield, a second
set of experiments was designed. The range of the investigated col-
umn load was reduced to 0.510%, whereas the supercial velocity
range was the same as in the rst series. A second order polyno-
mial function was applied to the simultaneous description of the
experimental data from both series of experiments in the following
form:
y = b0 + b1 x1 + b2 x2 + b3 x1 2 + b4 x2 2 + b5 x1 x2 (6)
where y is either the FOS yield or selectivity, x1 is the column load,
x2 the supercial velocity, and bi . are the regression coefcients.
The polynomial function provided very close ts of the FOS yield
and selectivity values with the standard error of 0.6% and 0.1%,
respectively. The regression coefcients were also estimated with
Fig. 5. Dependence of FOS yield (), selectivity () and cut time () on the super-
cial velocity at the column load of 1.85% for Column 1.
1328 K. Vankov, M. Polakovic / Process Biochemistry 45 (2010) 13251329

Table 2 Table 3
Results of regression analysis of the dependence of FOS yield and selectivity on the HETP for individual saccharides in Columns 1 and 2 at the supercial velocity of
supercial velocity and column load for Column 1. 21.6 105 m s1 .

Coefcient FOS yield Selectivity Saccharide HETP 103 (m)

b0 86.8 0.2 84.3 0.1 Column 1 Column 2

b1 1.02 0.07 1.17 0.01
b2 (3.18 0.30) 104 (1.13 0.06) 104 Fructose 2.79 2.81
b3 (8.58 0.61) 102 (3.11 0.12) 102 Glucose 4.96 4.91
b4 (3.21 0.11) 108 (2.32 0.22) 107 Sucrose 6.47 6.39
b5 (2.24 0.21) 103 (1.92 0.40) 102 Kestose 7.13 7.10
1-Nystose 7.49 7.52
The values after the plus/minus sign denote the standard deviation of the regression 1F -Fructofuranosyl nystose 8.07 7.94
coefcient.

a good accuracy (Table 2). They were used to plot the dependences
of the FOS yield and selectivity on the supercial velocity and col-
umn load (Figs. 6 and 7). The gures conrm the above mentioned
observations of selectivity being a decreasing function of both fac-
tors whereas the FOS yield had a mild optimum located at the
values of the column load of 2.0% and the supercial velocity of
5.0 105 m s1 . The value of the FOS yield at the maximum was
85.6% and the selectivity was 81.5%.
In the next step, a scale-up of FOSs separation was carried out.
The volumetric aspects of the scale-up were limited by the dimen-
sions of Column 2. Consequently, the bed cross-section area and
Fig. 6. Dependence of FOS yield on the supercial velocity for Column 1 at column
load 0.62% (), 1.85% (), 5.56% () and 10.5% (). The curves represent the tted
values calculated from Eq. (6).
length were increased by the factors of 2.64 and 5.30 which cor-
responded to a 14-fold increase of the bed volume. Table 1 shows
that bed porosity was essentially the same in both cases. The ef-
ciency of bed packing was compared through HETP values at a
single supercial velocity value of 21.6 105 m s1 , which were
determined using the rst and second moments as described in
Materials and methods. Table 3 shows that the HETP values for each
saccharide were the same in both columns. The number of theo-
retical plates for Column 2 was thus 5.3 times larger than that of
Column 1. Therewith, a potential for improving the separation ef-
ciency in Column 2 was created. A set of experiments was designed
in the same intervals of the column load and supercial velocity as
for Column 1.
Results of the experiments are presented in Figs. 8 and 9. It is
evident that higher values of the FOS yield and selectivity were
achieved in the larger column. They were above 90% in the region
of process conditions optimal for Column 1. At the column load
of 2.0% and the supercial velocity of 5 105 m s1 , the FOS yield
was higher by 4.9% and selectivity by 8.8%. The trends of the column
load and supercial velocity dependences were qualitatively very
similar as for the smaller column. The effect of both factors was,
however, weaker for Column 2, which was best demonstrated on
the ts of the experimental data with Eq. 6. Accuracy of the ts was
again very good, with the standard error of 0.45% for the FOS yield
and 0.18% for the selectivity. Values of regression coefcients are
summarized in Table 4.
Higher values of the coefcient b0 than in the previous case con-
rm the higher FOS yield and selectivity in the region of optima.
On the other hand, several times lower values of other regression
coefcients prove that the effect of the column load and that of the
supercial velocity were weaker in this case.

Fig. 7. Dependence of selectivity on the supercial velocity for Column 1 at column Fig. 8. Dependence of FOS yield in cut time from supercial velocity in case of Col-
load 0.62% (), 1.85% (), 5.56% () and 10.5% (). The curves represent the tted umn 2 for column load 0.62% (), 5.56% () and 10.5% (). The curves represent the
values calculated from Eq. (6). tted values calculated from Eq. (6).
 K. Vankov, M. Polakovic / Process Biochemistry 45 (2010) 13251329 1329

Acknowledgement

This study was supported by the Science and Technology Assis-

tance Agency APVV (LPP-0234-06) and Slovak Grant Agency for
Science VEGA (Grant No. 1/0655/09).

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