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S0009-2509(96)00289-8
1. INTRODUCTION
The downstream processing of biochemical compounds as well as the industrial effluents treatment for
pollution control use adsorbents onto which the target molecules are adsorbed. These molecules are then
recovered or discarded by means of a desorbing agent.
For the proper design of an industrial process, it is
important to be able to predict the extent of adsorption on the substrates used for that purpose. In order
to achieve this goal, we must find a kinetic model
describing the phenomenon. This requires the knowledge of transport and equilibrium parameters that
are closely related to external mass transport towards
the adsorbing particles, intraparticle diffusion of the
target molecules through the pores of the adsorbent,
and the adsorption process of the molecules at the
adsorption sites at the surface of the pore wails.
This is a rather difficult problem that is often solved
numerically (Arve and Liapis, 1987; Casillas et al.,
1993; Firouztale et al., 1992; Horstmann and Chase,
1989). In the case of the adsorption in a closed stirred
tank only very simple situations give rise to analytical
solutions that are easy to use (Chase, 1984; McKay
et al., 1983; Somerset al., 1989).
Ruthven (1984) reviews in great detail the mathematical solutions available for a wide range of
4489
4490
J. MEMBREZ
et al.
2. THEORETICAL BASIS AND MODEL
2.1. Generalities
We assume that the spherical particles, once swollen with the solvent, all have the same geometrical
radius, R. The porosity, ,, is the ratio of the volume of
liquid contained in a particle accessible to the protein,
Vvoid, to its total volume, Vp.
Vvoid
=
- -
(I)
v~
(2)
~qi
Pp -ff
~Cli
(0 2 ctl
77 = m,
2 Oc,'~
-;
(3)
(4)
(5)
and finally in terms of ci
(1 + p ~ _ ) Oc,
/02c,
2 ac,"l
(6)
4491
Use of the Laplace transform technique for simple kinetic parameters evaluation
The amount of protein per unit volume of swollen
resin obeys a diffusion equation
Oc, D f 02 c, 2 0_f2~
\ T;-:: + -r Or]
T; =
where D = 1 +
Dt
ppK/e"
(7)
Vp
rrro :o c, 2 Oe,"/
(8)
Vp
where Cbu~k(t) is the amount of protein per unit volume of free solvent (bulk concentration), k I is the
liquid film mass transfer coefficient, kz is the modified
liquid film mass transfer coefficient, and De is the
effective diffusion coefficient.
At equilibrium, there is no more driving force for
diffusion since the liquid concentration of the solute is
the same throughout the system.
The amount of protein present at time t inside one
particle, npart(t), whether adsorbed or in the liquid of
the pores, is given by
npart(t) = ~00(t)dt.
ni(t) =
n
Vbu]k
Npar, ~ ~(t)dt
Vbulk Jo
(14)
(9)
[-0c~(r, t)]
4nR2 Dt rLOci(R,t)]
Or J,=R = 4nR2k,[eCb,lk(t)- ei(R,t)]
(15)
nbulk (t)
(I)(t) Cbulk(t)
Vbulk
C i (R,
~,
t)
(I)(t)
4rcR2kte.
(10)
The flux at the boundary of a particle, per geometrical unit area of particle, is controlled by mass transfer
and assumed to be proportional to the difference in
protein concentration in the bulk of the solution and
in the pores at the edge of the particles. At the onset of
the adsorption, no protein is present in the particles.
This leads to
= kt[Cbulk(t) -- cu(R,t)]
e=
or
O, L ~ / r(r,: R'I l
(13)
rl)(t)dt
DzI~l,=R
Npartnpart(t) = Npart ~0
n = nbulk(t) +
Vp
(12)
k: [~Cbulk(t) --
ci(R ,
t)]
c~(r,O) = 0
with k: = kt" e
and De = Dl"
(16)
(11)
6(R)
R er'/77-~ - e-',/;7"6
7 e-~--~--_ e ~
(17)
4492
J.
MEMBREZet al.
4~R2kt
e~ (18)
s~oo
(19)
n
Cbulk =
~([
Vbulk
A= R
Bi
[e-2A(1-Bi+A)]-I
at
kl R
Dl
D~
4rcR2 Npart
- - ,
+Bi+A
(20)
Vbulk
?bulk -
where
(23)
s~O
[Cbulk(t = 0) -- a -- b]
d+ s
(22)
AND
METHODS
4493
Use of the Laplace transform technique for simple kinetic parameters evaluation
Table 1. Characteristics of HSA (van der Scheer, 1978)
Concentration in human plasma (kg/m 3)
Calculated molecular mass (kg/kmol)
Diffusion coefficient in pure water (25C) (m2/s)
Isoelectric point (-)
Extinction coefficient at 280 nm (m2/kg)
35-40
66,248
6.9x 10-1~ calculated (Young and Carroad, 1980)
4.5- 5.0
0.53
Table 2. Physical properties of Blue Sepharose CL-6B (*molecule used to determine porosity)
Particle
mean
diameter
(/~m)
93 + 1
Internal
bead
porosity (-)
*(t-D-glucose)
Internal
bead
porosity (-)
*(human
albumin)
External
bed
porosity (-)
*(dextran
T2000)
Archimedes
number (-)
(20C)
Papp
(kg/m 3)
/gp
(kg/m 3)
0.95
0.55
0.38
2.0+0.1
1257
72
(24)
(25)
Relim= PUlim
(26)
Stirringpowersupply
proteinInjectio~
gearp u m ~ _ _ ~ ~
II
Com[ter
//-q
"''r
4494
J. MEMBREZ
of 0.60 x 10- 6 m3/s. At the beginning of the experiments, 0.9 g of dry Blue Sepharose CL-6B (Npart
3 l0 T) was placed in the tank and equilibrated
with 1.50 x 10 -4 m 3 Tris buffer under soft stirring (50
rotations per minute) during 30 min. Agitation is then
stopped and the adsorbent beads settle at the bottom
of the vessel. Absorbance acquisition is switched on
and 1 ml of a 20 kg/m 3 HSA solution (0.05 M Tris
pH = 7, 0.1 M KC1) is gently added in the liquid.
After approximately 10s the stirring is abruptly
switched on (330rpm). Bulk-phase concentration
reaches its maximum (which can be predicted by mass
balance) and the adsorption process begins. Data acquisition is stopped when equilibrium is attained. The
adsorbent particles showed no sign of damage due to
agitation, even after periods of 10 h.
4.5. Adsorption equilibrium study
In a typical adsorption kinetics experiment, equilibrium is reached after 5000 s and the corresponding
value of absorbance is recorded. Mass balance on
HSA gives the value of the adsorbed amount of protein (expressed as the weight of adsorbed HSA per
unit weight of dry adsorbent). Subsequently, 1 ml of
20 kg/m 3 HSA solution is again added to the system.
When equilibrium is reached again, the value of absorbance is noted and q, the adsorbed quantity of
HSA, is calculated. The same procedure is repeated
until adsorbent is saturated. The resulting adsorption
isotherms were found to fit well to the Langmuir-type
expression as given in eq. (27).
5. RESULTSAND DISCUSSION
5.1. Adsorption isotherms
The adsorption isotherm is determined to estimate
the concentration range for which a linear adsorption
isotherm can be assumed and to estimate the adsorption equilibrium constants by independent measurements. The result can be used to serve as a control of
the fitted kinetic parameters by comparison or to reduce the fitting kinetic parameters to two (De and k~).
A typical result for adsorption isotherm measurement is shown in Fig. 2. The set of equilibrium data
was fitted to the Langmuir model [eq. (27)] by nonlinear least-squares regression.
qm
q(eq)
0.120.100.08"o
0.06~
~JD 0.04 ~
.YJ
0.02r~
0.00 ~
~ + 3 .
8%
Kd= 0.259 lkg/m3l:~9.4 %
0.0 . . . . . . .
(27)
al.
Cbulk (eq)
Kd + Cbulk(eq)'
et
0'.2 . . . . . . .
014 . . . . . . .
0'.6 . . . . . . .
o'.s
c ~k (eq) [kg/m3]
Symbol
Value
q,
Kd
K = q,,/Ka
0.156 (kg/kg)
0.260 (kg/m 3)
0.600 (ma/kg)
3.8
9.4
13.2
Use of the Laplace transform technique for simple kinetic parameters evaluation
0.16-]
0.14 -~
0.12 /t
n
0.10-[
a=
b=
d=
f=
2.83.02
2Ale..02
2..~'-03
2.57e.4)2
:k'0.12 %
:kl.66 %
+i.82 %
:L1.06 %
0.08 - ~
Z2=
6.72e-04
IT
0.06 -[~,
I~
o preteln concentration
_ _ double exponential fit
o.o2
0
10 7
--"=
-10 " ~
'
2000
-?--S--:
--,
1000
'
,-
30@0
1000
'
4000
i
3OOO
2OO0
'
I
4OOO
'
[s]
Time
0.16-
Results
0.14 - D
0.12 ~_ D e
kf
_ f "~ -
0.~0.04- ~
--
0.02
"l
-S
,o
13
-4
'
'
-2
-1
,.~_~./
o.oo~ - r -
-5
-4
-3
-2
""':
i
-1
los(s)
e-St Cbulk(t) dt
Cbulk =
4495
(31)
Cbulk~ ~
e -~''
Cbulk(h)At.
(28)
i=0
If At is taken as the time between two measurements (5 s) the preceding equation becomes
Cbulk "~ ~
Cbulk(t =
0)At
i=O
4496
et al.
J. MEMBREZ
Protein (kg/kmol)
Adsorbent
IgG
150,000
Insulin
5600
BSA
66,300
Sepharose
HSA
66,248
kI (m/s)
4.0
10 - 6
Amberchrome
CG-71MS
Sepharose FF
5.0 10-6
Blue Sepharose
CL-6B
4.2 x 10-6
5.0
10 - 6
Table 5. Values of De found in the literature compared to the results of this study
Reference
De 10x1
Protein
Matrix
Method
0.96
1.5
Bovine albumin
Bovine albumin
Sepharose CL-4B
Sepharose CL-6B
Moment analysis
--
(Experimental)
(Correlated)
This work
2.2
Human albumin
Sepharose CL-6B
(Experimental)
(m2/s)
0.12~ 0.10-~
~_ 0.0s"--~
" o.oo~ 0.04-
(32)
0.02-
the
dCbulk
dt
0.00-
kl
= D-'-~[eCb,lk -- Ci (R, t)]
-*
soo
Numerical simulation
Experimental data
to~oo
Is~oo
2o~oo
Time Is]
(33)
~rr=o =0
Oc~
0.14-
kf
(34)
Furthermore, c~(r, t = O) = O.
We used the kinetic parameters for pore diffusion
and film mass transfer that we found by the Laplace
transform technique. The equations are treated according to the method of lines fully described in the
paper of Horstmann (1989). The governing partial
differential equations for film and pore diffusion are
solved using the program SimuSolv (The Dow Chemical Company, Midland, Michigan, U.S.A.) running on
a Sun Spare station 10. A program defining the input
parameters and setting up the discretized differential
equations is written in a special ACSL language (Advanced Continuous Simulation Language). Among
the many integration methods available, a fourthorder fixed-step Runge-Kutta algorithm has been
chosen. Point concentrations were transformed into
a five-component time-dependent vector, due to the
discretization of the space variable. The result of the
numerical computation is compared to the experimental data (Fig. 5). It confirms that the kinetic parameters found by the Laplace transform technique
provide a good agreement with the experimentally
measured time dependence.
6. CONCLUSIONS
Kinetic parameters for the adsorption of a protein
on porous beads can be obtained via a Laplace transform method. The adsorption of HSA on Blue
Sepharose CL-6B has been chosen as a model system.
The method uses the Laplace transform technique to
solve the partial differential equations system that
describes the phenomenon. In the range where a linear adsorption isotherm is valid, by fitting the Laplace
Use of the Laplace transform technique for simple kinetic parameters evaluation
transform of the exact solution to the Laplace transform of the data, the kinetic parameters obtained by
least-squares fitting are in good agreement with the
values found in the literature for film mass transfer
coefficients and diffusion coefficient in porous adsorbent beads. In order to assert the validity of the values
found by this method, a numerical simulation with the
obtained model parameters was compared with the
experimental results and showed a good agreement,
The main advantage of the Laplace transform technique is that the time-consuming numerical approaches are not needed. The parameters are rapidly
and easily found via two algebraic fits, one in the time
domain followed by another in the Laplace domain,
The quality of the results is largely determined by the
quality of the fit in the time domain. It may be possible to further improve the results by selecting other
satisfactory ad hoc functions to fit the experimental
data in the time domain.
Future work will focus on the application of this
method to other adsorbents and on the use of other
functions that may improve the result in other situations where the four-parameter double exponential
does not provide a satisfactory description of the data.
4497
nbu,k
(t)
Greek letters
NOMENCLATURE
a
at
A
Ar
number
[ = P(Papp - P)gd 3/
/tz], dimensionless
double exponential coefficient, kg/m 3
b
Bi
Biot number ( = ktR/Dt = kyR/De), dimensionless
Cbulk(t) protein concentration in the bulk of the
solution, k g / m 3
Cbulk (eq) protein concentration in the bulk of the
solution at equilibrium, kg/m 3
protein amount per unit volume of swollen
ci (r, t)
particle, kg/m 3
cu (r, t) protein amount per unit volume of liquid
inside a particle, kg/m 3
double exponential coefficient, s d
mean diameter of the particles, m
dp
modified diffusion coefficient { = Ot/[1 +
D
(p,K /e)]}, m2/s
effective diffusion coefficient ( = O~. ~), mZ/s
De
diffusion coefficient in the pores of the adDl
sorbent, m2/s
double exponential coefficient, s- 1
f
acceleration due to gravity, m2/s
g
modified mass transfer coefficient, m/s
kt
mass transfer coefficient, m/s
k:
proportionality constant for linear adsorpK
tion isotherm, mS/kg
Langmuir equilibrium constant, kg/m s
Kd
At
e
p
papp
pp
p
(t)
Superscript
9~
Boyer, P. M. and Hsu, J. T., 1992a, Effects of ligand concentration on protein adsorption in dye ligand adsorbents.
Chem. Engng Sci. 47, 241-251.
Boyer, P. M. and Hsu, J. T., 1992b, Experimental studies in
restricted protein diffusion in agarose matrix. A.I.Ch.E.J.
38, 259-272.
Casillas, J. L., Martinez, M., Addo-Yobo, F. and Aracil, J.,
1993, Modelling of the adsorption of cephalosporin C on
modified resins in a stirred tank. Chem. Engng J. 52,
B71-B75.
Chase, H. A., 1984, Prediction of the performance of preparative affinity chromatography. J. Chromatogr. 297,
179-202.
4498
J. MEMBREZet al.
Ruthven, D. M., 1984, Principles on Adsorption and Adsorption Processes. Wiley, New York.
Skidmore, G. L., Horstmann, B. J. and Chase, H. A., 1990,
Modelling single-component protein adsorption to the
cation exchanger S Sepharose FF. J. Chromatogr. 498,
113-128.
Somers, W., Van't Riet, K., Rozie, H., Rombouts, F. M. and
Visser, J., 1989, Isolation and purification of endo-polygalacturonase by affinity chromatography in a fluidized
bed reactor. Chem. Engng J. Biochem. Enon # J. 40,
B7-B19.
van der Scheer, A. T., 1978, Adsorption of plasma proteins:
adsorption behaviour on apolar surfaces and effect on
colloid stability. Dissertation, University of Twente.
Young, M. E. and Carroad, P. A., 1980, Estimation of diffusion coefficients of proteins. Biotechnol. Bioe~ng 22,
947-955.