Biochemical Engineering Journal 15 (2003) 185–192

Affinity adsorption and hydrodynamic behavior in

a tapered-bed of upward flow
Jianjun Li, Bolun Yang∗ , Guangxu Cheng
Department of Chemical Engineering, Xi’an Jiaotong University, Xi’an 710049, China
Received 8 August 2002; accepted after revision 28 November 2002

Abstract
Affinity chromatography operated in a tapered-bed with the continuous up-flow was researched. Purification of Lysozyme by Blue
Dextran Sepharose 4B was chosen as a model system. A series of experiments such as the batchwise adsorption, tapered columnwise
adsorption, elution and pressure drop determination were carried out to find out characteristics such as adsorption equilibrium, hydrodynamic
performance and adsorption efficiency for this kind of new affinity process. A mathematical model to describe this system was also
established and verified. Research results showed that the affinity purification by the tapered-bed with up-flow could be operated in three
regimes, i.e. the fixed-bed regime, fully fluidized-bed regime and partially fluidized-bed regime, which depended on the flow rate. The
adsorption efficiencies were the highest in the fixed-bed regime, and poor adsorption efficiencies were shown in the fully fluidized-bed
regime.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Affinity chromatography; Protein; Tapered-bed; Adsorption; Modeling; Hydrodynamic

1. Introduction tapered-bed can operate under a low-pressure drop and

Affinity chromatography is a useful technique in the
separation and purification of biologically active macro-
molecules. It can give a high purification degree in a single
step process. However, it is generally operated in a fixed-bed
mode by using a cylindrical column with the down flow
method. The pretreatment of the crude feed is necessary
because that the chromatography column is easily clogged.
Mechanically soft properties of the affinity adsorbent may
also preclude the high flow rate, and a longer operation
time will be necessary in this case. As a kind of improved
method, an expanded bed with a cylindrical column with
up-flow is suggested [1–3]. However, it will be difficult to
avoid high axial back mixing and adsorbent beads will be
lost easily. A combined process of batchwise adsorption
and columnwise elution is also investigated by the authors
[4]. However, the operation became discontinuous.
A tapered-bed with up-flow is proposed here to improve
the affinity chromatography process. Due to its variation
in the superficial velocity in the column, hydrodynamic
characteristics in the tapered column differ greatly from
the operation in the cylindrical column with up-flow. The
∗ Corresponding author. Tel.: +86-29-2668569; fax: +86-29-3237910.
E-mail address: blunyang@xjtu.edu.cn (B. Yang).
avoid over-expansion of the bed [5]. The tapered-bed has
been successfully used in immobilized enzyme reaction and
microbiological reaction [6,7]. In this work, the purification
of Lysozyme by using Blue Sepharose 4B is carried out,
and the relation between the fluidization properties and the
affinity separation efficiency is discussed.
2. Experiments
2.1. Materials
Lysozyme as a model protein was purchased from Sigma
Co., and Dextran 2000 and CNBr-activated Sepharose 4B
were purchased from Pharmacia Co. All other chemicals
used were purchased as analytical pure reagents from Xi’an
Chemical Reagent, China.
Solution of 0.4 M NaHCO3 –NaOH (pH = 9.5) is named
buffer A; solution of 0.02 M sodium phosphate buffer (pH =
7.4, containing 0.15 M NaCl) is named buffer B; solution
of 0.02 M sodium phosphate buffer (pH = 7.4, containing
0.15 M NaCl solution and 50% glycol) is named buffer C;
solution of 0.02 M sodium phosphate (pH = 7.4) is named
buffer D; solution of 0.02 M sodium phosphate (pH = 7.4,
containing 0.01 M NaCl) is named buffer E.

1369-703X/03/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S1369-703X(03)00002-0
186 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192

Nomenclature 2.2. Preparation of adsorbent

Cin concentration just inside the bed at the Blue Dextran and Sepharose 4B were linked directly.
inlet (kg/m3 ) CNBr-activated Sepharose 4B were swollen with the cold
Cout concentration just inside the bed at the 1 mM HCl in a conical flask and washed with the same so-
outlet from the bed (kg/m3 ) lution several times to remove impurities. Blue Dextran sus-
Cp concentration of protein in the particle pended in buffer A was shaken for 3 h and added to this
(kg/m3 ) conical flask, then put the conical flask in a refrigerator
C1 , C2 constant in the Ergun equation defined in shaken for 8 h at 277 K. Filtrating this reaction solution, the
Eq. (18) filter cake was washed with buffer A, distilled water, buffer
DB diameter of conical bed at position HB (m) B, buffer C and buffer B in turn, until the absorbance at
DL axial dispersion coefficient (m2 /s) 630 nm (A630 ) was less than 0.01 (pH = 7.2). The adsorbent
Dp intra-particle diffusion coefficient (m2 /s) (Blue Dextran Sepharose CL-4B) was then stored in buffer
DT diameter of conical bed at position HT (m) B and used in all the experiments. The particle diameter was
D0 bottom diameter of conical bed (m) a distribution of 45–165 ␮m and the average diameter was
D1 top diameter of conical bed (m) 100 ␮m.
Ef friction loss in terms of mechanical energy
change (m2 /s2 ) 2.3. Batch adsorption
g gravity (m/s2 )
h vertical position (m) It was found in the preparatory experiment that the ad-
HB height of lower surface of the fixed region sorption rate is independent of agitation speeds in the range
in partially fluidized-bed (m) of our experiment. This shows that the external diffusion re-
HT height of upper surface of the fixed region sistance is not significant, so the external diffusion can be
in partially fluidized-bed (m) ignored and the process is controlled by internal diffusion
Hm height of particle bed (m) and adsorption kinetics.
kf av average volumetric coefficient of mass A series of batchwise adsorption experiments in 278 K
transfer (s−1 ) were carried out to evaluate the intra-particle effective dif-
K adsorption equilibrium constant fusivity. Different initial concentrations of Lysozyme in the
P pressure (N/m2 ) solution of buffer D were prepared. The adsorption was car-
−PN net pressure drop (N/m2 ) ried out in a stirred tank and the variation in protein con-
q adsorbed amount (kg/m3 ) centration with time was recorded continuously.
qm maximum adsorbed amount (kg/m3 ) Adsorption was also carried out in the test-tubes with
r intra-particle radius of adsorbent (m) different initial concentration of Lysozyme solution under
R radius of adsorbent particle (m) 278 K to determine the adsorption equilibrium. The concen-
t time (s) tration of protein in the supernatant after 24 h was deter-
u superficial velocity of the fluidizing fluid mined by UV adsorption (280 nm). This value was used as
(m/s) the equilibrium concentration. Adsorption capacities were
u0 inlet velocity of the tapered-bed (m/s) calculated from the difference between initial and final con-
umff minimum velocity of full fluidization centrations.
(m/s)
umfp maximum velocity of partial fluidization 2.4. Continuous adsorption and elution
(m/s)
V volume of solution in batchwise (m3 ) Fig. 1 indicates the experimental apparatus constructed to
carry out the adsorption and elution operation. The inside
Greek symbols diameter of the inlet-tapered-bed is 0.7 cm and tapered angle
α constant in Langmuir equation is 3◦ . The column is equipped with a cooler solution jacket
ε voidage of the fluidized-bed and kept at 278 K. A straight column of 1 cm inter diam-
εf particle voidage eter was used for comparison which operated at the same
ε0 voidage of the fixed-bed condition with the tapered-bed.
θ angle of tapered-bed In all operations, the feed solution flows into column from
ρf fluid density (kg/m3 ) the bottom and flows out from the top of column, then into
ρp density of particle (kg/m3 ) the ultraviolet (UV) spectrophotometer for the detection of
ρs solid density (kg/m3 ) absorbance at 280 nm. At the adsorption stage, when the
Φs sphericity of the solid particle absorbance of the solution becomes constant and equal to
that of the feed solution, the column is washed with buffer
D and eluted with the buffer E in turn, until the absorbance
 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192 187

Fig. 2. Structural representation of tapered-bed.

Fig. 1. Schematic diagram of experimental apparatus. particles (internal diffusion). In the calculation of batch ad-
sorption, pore diffusion controlling was assumed. The tran-
reaches the baseline. The breakthrough and elution curves sient concentration profile was showed in Fig. 4.
are then obtained.
3.3. Adsorption in the tapered-bed
2.5. Determination of pressure drop
The concept for the tapered-bed is denoted in Fig. 2. Here,
The pressure drop in the system was measured by the the diameter of the bed increases with the height, and the
manometer connected with the bottom of the column by a superficial velocities thus are changed with the height of
three-way cock. In each experimental process, the flow rate column. From Fig. 2, the relationship of inlet and outlet of
of the feed was increased incrementally. When a stable state the taper bed can be written as
was established after each increment, records were taken


θ θ
from steady readings on the manometer to obtain the pres- R = (h + H0 ) tg = h tg + R0
sure drop. The resistance of the apparatus was eliminated 2 2

  
by subtracting the pressure drop in the empty column. For h θ h tg(θ/2)
= R0 1 + tg = R0 1 +
the comparison of pressure drop of tapered-bed and down R0 2 H0 tg(θ/2)


flow cylindrical column (fixed-bed), the pressure drops in h
the fixed-bed were also measured in the same operated con- = R0 1 + = R0 (1 + η) (2a)
H0
ditions.
where η = h/H0 , and the superficial velocity can be pre-
sented by follows:
3. Theory u0
u= (2b)
(1 + η)2
3.1. Adsorption isotherm
here, h is the height of the bed.
Langmuir correlation with two parameters, based on the Since the voidage is not uniform throughout the bed, the
assumption of monolayer adsorption, is used to describe the average voidage is used to simplify the calculation. A mass
adsorption system in many reports. However, the affinity balance in the inter-particle liquid over a section of the bed
action between some adsorbent and protein molecules is yields
not uniform; and the monolayer adsorption is not accurate ∂C ∂2 C ∂C 1 − ε̄B
in this case. In this work, the revised Langmuir equation = DL 2 − u − K f av (C − Cp ) (3)
∂t ∂h ∂h ε̄B
with three parameters is used to describe the equilibrium of
Lysozyme–Blue Dextran Sepharose 4B. The equation can The transport within the particle is represented as
be written as follows:  
∂Cp ∂q ∂ 2 Cp 2 ∂Cp
qm KCα εp + (1 − εp ) = εp Dp + (4)
q= (1) ∂t ∂t ∂r 2 r ∂r
1 + KCα

3.2. Batch adsorption Overall volumetric mass transfer resistance is represented

by the sum of the liquid film mass transfer and intra-particle
diffusion resistance
It was found in the preparatory experiment that the ex-
ternal diffusion could be ignored. Therefore adsorption is 1 1 R2
= + (5)
controlled by diffusion of the solute through the pores in the K f αv kf αv 15Dp (1 − ε̄B )
188 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192

The following two boundary conditions for the inlet and 1 − ε 0 ρf

C2 = 1.75 × (11b)
the outlet of the tapered-bed have been used: ε30 φs dp
∂Cin u0
= (Cin − C0 ) (6) The net pressure drop of the fixed-bed is
∂h DL
∂Cout D0 D0 (D02 + D0 D1 + D12 ) 2
=0 (7) (−PN ) = C1 Hm u 0 + C 2 Hm u0
∂h D1 3D13


4

These equations are solved numerically by the Crank– 1 u0 2 D0

+ − 1 ρf (12)
Nicoson method. 2 ε0 D1

3.4. Hydrodynamic characteristics of the tapered-bed The maximum (−PN ) can then be calculated by replacing
u0 with umpf in (12).
The flow regime in the tapered-bed is different from By equating the overall friction force to the overall effect
the fixed-bed with down flow. With an increase in the weight of the bed, the minimum velocity of partial fluidiza-
fluid velocities, three kinds of flow patterns can be found: tion (umpf ) can be obtained by the following equation:
fixed-bed regime, partially fluidized-bed regime and fully

fluidized-bed regime. These regimes can be distinguished D0
C1 umpf + C2 u2mpf − (1 − ε0 )(ρp − ρf )g
by the critical point in the variation of pressure drops with D1
the flow rate. The calculation method of the pressure drop  
D02 + D0 D1 + D12
and the critical velocities are delineated as: × =0 (13)
3D02
3.4.1. Fixed-bed regime
In this regime, the kinetic energy decreases owing to the 3.5. Partially fluidized-bed regime
reduction in the superficial velocity of the vertical direction
[6]. The macroscopic mechanical energy balance around the
In this regime, the tapered-bed may ideally be divided into
fluid flowing through the space between the top and bottom
two regions: the fluidized region at the bottom and the fixed
surfaces of the bed can be written as
region at the top. The total pressure drop (−PN ) decreases
P 1  u 2 with an expansion of the fluidization region.
0= +  + g h + Ef (8)
ρf 2 ε It is assumed that the particles in the fluidized region are
completely suspended. The total frictional force Fs exerted
The first term on the right-hand side of the equation repre-
on the particles can be written as
sents the static pressure change; the second term shows the
kinetic energy change; the third term shows the potential en-  HT
ergy change; the fourth term shows the friction loss. Here, Fs = A(C1 u + C2 u2 ) dh
the net pressure drop PN is defined by combining the effects HB
of static pressure P with the gravitational force ρf gh which = πr02 [C1 u0 (HT − HB )
acts in the opposite direction as follows: 
HT − H B
+ C2 u20 H02 (14)
PN = P + ρf gh (9) (H0 + HB )(H0 + HT )

From Eqs. (8) and (9), Eq. (10) can be obtained. The effective weight of the fixed-bed region in the upper
1  u 2
bed is
(−PN ) = Ef ρf +  ρf (10) 
2 ε HT
G= (1 − ε0 )(ρp − ρl )gA dh
This states that (−PN ) due to the combined effect of static HB
pressure with gravitational force is equal to the combination

(HT + H0 )3 − (HB + H0 )3
of the pressure drop due to the frictional loss Ef ρf and that = πr0 (1 − ε0 )(ρp − ρl )g
2

due to the kinetic change (1/2)(u/ε)2 ρf in the bed. 3H02

The well-known Ergun equation is adopted here to de- (15)
scribe the pressure gradient:
where Hf and HB represent the height of the upper surface
f(u) = C1 u + C2 u2 (11) and the under surface of fixed region, respectively.
where From the aforementioned assumption, the frictional
force on the particles should be balanced by their effec-
(1 − ε0 )2 µf tive weight in any control volume of the height dh in the
C1 = 150 (11a)
ε30 (φs dp )2 bed. The relationship between the flow rate of fluid and
 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192 189

voidage of the bed can be described by the Richardson–Zaki

equation.
u = ut εn (16)

where n is estimated from experiments.

From Eqs. (14)–(16), the values of HT and HB can be
calculated. Along with the increase of the flow rate the value
of HT − HB gets to zero. At this moment, the entry velocity
is the minimum velocity of full fluidization (umff ).
The magnitude of (−PN ) can be calculated by combin-
ing the net pressure drop of the two regions

D02
(−PN ) = C1 (HT − HB ) u0 + (1 − εf )(ρp − ρf )gHB Fig. 3. Adsorption isotherms.
DT DB
D4 (D2 + DB DT + DT2 )L20 2
+ C2 (HT − HB ) 0 B u0
3DT3 DB
3
 

2

1 2 1 D0 4 1
+ u0 − ρf (17)
2 ε02 DT εf

3.5.1. Fully fluidized-bed regime

When all particles in the bed are fluidized by increasing
the fluid velocity, the minimum velocity of full fluidization
(umff ) is obtained. In this regime, the particles in the bed are
completely suspended; the pressure drop through the bed
then can be expressed as

(−PN ) = (1 − εf )(ρs − ρf )gH


4

Fig. 4. Experimental and calculated results for the batchwise adsorption

1 u0 2 D0
+ − 1 ρf (18) (V = 1 × 10−4 m3 , the absorbent’s masses are 0.2106, 0.2108, and
2 εf D1 0.2104 g, respectively).

4. Results and discussions corresponding to each pattern is given in Fig. 5 (obtained by

increasing liquid velocity). It can be seen from Fig. 5 that the
4.1. Adsorption isotherms and batchwise adsorption results calculated compare favorably with the experimental
data.
It can be seen from Fig. 5 that due to the increase of the
Adsorption isotherm is shown in Fig. 3. It can be devel-
flow rate, the pressure drop of the bed rises rapidly. Before
oped as a Langmuir equation with three parameters.
104.3c1.513
q= × 0.971 (19)
1 + 104.3c1.513
The transient profile of the bulk liquid concentration in
the batchwise adsorption is shown in Fig. 4. Each mark
indicates experimental data. It can be seen from this figure
that the lower the initial concentration of protein solution
is, the steeper the curve becomes. Intra-particle effective
diffusivity is evaluated to be 5.3 × 10−11 m2 /s as a result of
comparison of the experimental data with calculated curves.

4.2. Pressure drop and critical velocities of fluidization

All experimental runs are performed at the temperature

of 278 K under the atmospheric pressure of 96.2 ± 00.5 kPa.
The variation of the net pressure drop of the tapered-bed Fig. 5. Effect of the superficial fluid velocity on pressure drop.
190 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192

the pressure drop reaches the maximum value, i.e. at a low

flow rate, the fluid simply passes upward through the bed
without disturbing the particles. At the maximum value of
pressure drop, the flow rate corresponds to the minimum
velocity of fluidization (umpf ). The tapered-bed is operated
in fixed-bed regime during this stage. At this velocity, an
almost empty cavity containing a small number of particles
is formed near the inlet. This cavity is unstable and swiftly
a fluidized region is formed in the place of the cavity. When
the flow rate increases continuously, the pressure drop of
bed decreases from the maximum and the flow regime is
accordingly changed to partially fluidized-bed regime. In this
regime, the fluidized region expanded continuously because Fig. 7. The elution curves of the tapered-bed.
the flow rate increases. Moreover, the particles in the center
of the bed are less than them outside the center. When the
cause the voidage of the tapered-bed increases with increas-
flow rate reaches the minimum velocity of full fluidization
ing flow rate, the tapered-bed has a much lower pressure
(umff ), the regime becomes a fully fluidized-bed one. In this
drop than the fixed-bed. The same reason, fluidized-bed has
regime the pressure drop remains almost constant.
the lowest pressure drop among three types of beds.
Fig. 9 shows the breakthrough curves of fixed-bed and
4.3. Breakthrough curve and elution curve tapered-bed under the same operation conditions, i.e. ini-
tial velocity was 0.10 × 10−3 m/s or 0.07 × 10−3 m/s that
Fig. 6 shows a typical breakthrough curve. The bed is between the Umpf and Umff . The initial concentration is
packed with 2.5 × 10−6 m3 of adsorbent. The inlet flow rate C0 = 0.4 kg/m3 . The calculated results were shown by solid
and the concentration of feed solution are 0.13 × 10−3 m/s lines. In the calculated of the fixed-bed, the pore diffusion
and 0.4 kg/m3 , respectively. The average voidage is deter- controlling and linear driving forces were assumed. By us-
mined as 0.412 from the ratio of the adsorbent volume to the ing well-mixed model, adsorption in the fluidized-bed was
total expanded volume of the tapered-bed. Therefore, a good also calculated for comparison with the other two opera-
agreement between calculation and experiment is obtained. tions. The breakthrough curve was slightly gentler for the
Fig. 7 shows the elution curve with the flow rate was tapered-bed than that for the fixed-bed because of larger bed
0.65×10−3 m/s. Lysozyme can be eluted from the adsorbent voidage. On the other hand, it is rather steeper than that for
in a short time. The maximum concentrated ratio C/C0 is the fluidized-bed.
about 3.3 times.
4.5. Effect of superficial velocity on the adsorption
4.4. Comparison of pressure drop and breakthrough performance
curves of fixed-bed, tapered-bed and fluidized-bed
The shape of the breakthrough curve is mainly affected by
As shown in Fig. 8, the pressure drop in down flow the equilibrium and the flow rate. As shown in Fig. 10, the
cylindrical column (fixed-bed), up-flow cylindrical column adsorption efficiency can be obtained for a given adsorption
(fluidized-bed) and up-flow tapered-bed was compared. In process when the breakthrough point is decided.
the comparison, the same volume of adsorbent was used in S1
η̄c = (20)
all case and the resistance of the apparatus was eliminated S1 + S 2
by subtracting the pressure drop of the empty column. Be-

Fig. 6. The breakthrough curves of the tapered-bed. Fig. 8. Comparison of pressure drops in different column.
 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192 191

Fig. 9. Comparison of breakthrough curves in different column.

(Recovery efficiency of the solute in feed solution)

S1
η̄q = (21)
S1 + S 3
(Utilization efficiency of the adsorbent bed capacity)
The values S1 , S2 and S3 indicate the areas of the shad-
owed portions denoted in Fig. 10. The value 1 − η̄c repre-
sents the quantity of the solute lost in the effluent. The value
1− η̄q represents the unused bed capacity. The breakthrough
concentration XBT corresponds to the process concentration.
These values, η̄c , η̄q and XBT , are important in the evalua-
tion of the performance of adsorption.
Fig. 11 manifests the influence of the flow rate on the
breakthrough curve. The inlet flow rates are 0.04 × 10−3 ,
0.13 × 10−3 and 0.2 × 10−3 m/s, respectively, and the initial Fig. 11. Comparison of breakthrough curves in three regimes.
concentration is C0 = 0.4 kg/m3 .
From Fig. 11, it can be seen that an increase in the in- Table 1
Adsorption efficiency in different flow regimes
let flow rates can affect the transient profiles for the break-
through curves. This demonstrates that the adsorption behav- Regime Fixed-bed Partially Fully fluidized-bed
regime fluidized-bed regime regime
ior in the tapered-bed is affected by the hydromantic of the
η̄c 0.958 0.942 0.420
column. These breakthrough curves are typical results rep-
η̄q 0.852 0.775 0.438
resenting the different flow regimes. When the breakthrough −PN (Pa) 61.8 60.5 58.5
point XBT is chosen as 0.25, the adsorption efficiency that
can be calculated based on the curves in Fig. 11 were shown
in Table 1. regime, the partially fluidized-bed regime is taken as the sec-
It is clear from Table 1 that the highest adsorption effi- ond place and the fully fluidized-bed regime is the lowest.
ciency can be obtained for the adsorption in the fixed-bed The same trend is also observed for the pressure drop. A
lower pressure drop in the partially fluidized-bed regime
than that of fixed-bed regime led shorter contact time and
less bed clogging. On the other hand, higher purification ef-
ficiency can be obtained than fully fluidized-bed regime led
higher process capability of column. Therefore, partial flu-
idization will be the best choice from the view of both the
adsorption efficiency and the pressure drop.

5. Conclusions

The tapered-bed was evaluated for affinity chromatogra-

phy. This operation indicates: a lower pressure drop than
Fig. 10. Schematic diagram of breakthrough curve. that of cylindrical fixed-bed without bed clogging and higher
192 J. Li et al. / Biochemical Engineering Journal 15 (2003) 185–192
purification efficiency than that of fluidized-bed can be ob-
tained.
Affinity purification by the tapered-bed with up-flow can
be operated in the three regimes, i.e. the fixed-bed regime;
fully fluidized-bed regime and partially fluidized-bed regime
based on the flow rate. The adsorption efficiency is the high-
est in the fixed-bed regime, and the poor adsorption effi-
ciency is shown in fully fluidized-bed regime.
Equations have been derived to predict the critical ve-
locity and the pressure drops in various flow regimes. By
purifying Lysozyme using Blue Dextran Sepharose 4B, the
tapered-bed with up-flow method is an efficient way to eval-
uate the affinity chromatography process.
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