Appl Microbiol Biotechnol (1990) 32:680-685

Applied Microbiology Biotechnology © Springer-Verlag 1990

Investigations on cephalosporin C adsorption kinetics and equilibria* M. Hicketier I and K. Buchholz 2 ~ Effem, Postfach 1280, D-2810 Verden, FRG 2 Sugar Institute, Technische Universit~it, Langer Kamp 5, D-3300 Braunschweig, FRG Received 13 January 1989/Accepted 18 August 1989

Summary. T h e k i n e t i c s a n d e q u i l i b r i a o f c e p h a l o s p o r i n C adsorption on different commercial adsorbents were investigated. Adsorption isotherms could be analysed a c c o r d i n g to t h e B r u n a u e r , E m m e t t a n d T e l l e r t h e o r y . F o r the i n t e r p r e t a t i o n o f a d s o r p t i o n kinetics it was nece s s a r y to d e v e l o p a m o r e c o m p l e x m o d e l c o m p r i s i n g b o t h a r a p i d a n d a s l o w e r step. A n i n t e g r a t e d a p p r o a c h c o m b i n i n g k i n e t i c s a n d e q u i l i b r i a a l l o w e d for s i m u l a t i o n o f t h e e x p e r i m e n t a l d a t a a n d can b e u s e d as a b a s i s for p r e d i c t i n g t e c h n i c a l a p p r o a c h e s such as f l u i d i z e d bed technology.

Introduction R e c o v e r y a n d p u r i f i c a t i o n o f b i o p r o d u c t s r e q u i r e cont i n u o u s r e s e a r c h a n d d e v e l o p m e n t for o p t i m i z e d a n d e c o n o m i c o p e r a t i o n ( A t k i n s o n 1983). A d s o r p t i o n p r o c esses r a n g e a m o n g t h e m o s t often u s e d p r i n c i p l e s , e.g. for t h e r e c o v e r y o f a n t i b i o t i c s (Voser 1977; Belter 1985). H o w e v e r few results have b e e n p u b l i s h e d o n b a sic p h e n o m e n a ; o n l y s i m p l i f i e d m o d e l s h a v e b e e n a p p l i e d to d e s c r i b e t h e a d s o r p t i o n o f a n t i b i o t i c s w i t h o u t d i s c r i m i n a t i n g the d i f f e r e n t m a s s t r a n s f e r steps a n d a d s o r p t i o n p h e n o m e n a i n v o l v e d (Belter 1985; P r a s a d et al. 1980). W e h a v e i n v e s t i g a t e d the kinetics a n d i s o t h e r m s o f cephalosporin C (CPC) adsorption with different materials, b o t h i o n - e x c h a n g e a n d n o n - i o n i c resins. F u r t h e r more, we h a v e p e r f o r m e d e x p e r i m e n t s for a p p l y i n g f l u i d i z e d - b e d t e c h n o l o g y for a d s o r p t i o n o f C P C . S o m e p r e l i m i n a r y results were p u b l i s h e d e a r l i e r ( H i c k e t i e r et al. 1985) a n d a d e t a i l e d c o m m u n i c a t i o n is in p r e p a r a tion.

* Dedicated to Professor F. Wagner on the occasion of his 60th birthday Offprint requests to: K. Buchholz

Materials and methods Resins. Three non-specific, polystyrene-type, macroporous adsorbents were used: Diaion HP 20 (Mitsubishi Kasei, Tokyo, Japan), Amberlite XAD4 and Amberlite XAD 1180 (Rohm and Haas, Philadelphia, Pa, USA), and one ion-exchanger IRA 68 (Rohm and Haas). The resins are characterized as shown in Table 1.

Adsorption experiments. The optimum conditions for adsorption of purified Na-CPC (gift from Hoechst, Frankfurt, FRG) on non-specific adsorbents were in the pH range 2.5-3.0 in glycine/ HC1 buffer (Ionic strength, I = 0.05). Prior to use the resins were wetted with methanol and then degassed. Afterwards the methanol was displaced by the before mentioned buffer in order to condition the resins. For the ion-exchanger IRA 68 optimum results have been achieved with acetic acid/NaOH buffer (I=0.02), pH 3.6, as a solvent for CPC and conditioning the ion-exchanger in the same buffer. For measuring the adsorption isotherms definite amounts of adsorbents (0.3-1.0 g) were added to 5 ml CPC solution of different concentrations (10-25 g/l) respectively in a 25-ml conical flask and then shaken at 200 rpm at 28 ° C. After reaching the equilibrium loading (1.75-2.5 h) the concentration of the solution, partly after appropriate dilution, was analysed by measuring the extinction at 258 nm versus water. The amount adsorbed corresponded to the difference in concentration between the initial and the equilibrium concentration. Solutions were prepared with before mentioned buffer systems, i.e. glycine/HC1 buffer (pH 2.5; I = 0.05) and acetic acid/ NaOH buffer (pH 3.6; I = 0.015) for the use of non-specific adsorbents and ion-exchanger respectively. Emphasis was given to pH control of the solution at the beginning and end of experiments. In order to investigate the adsorption kinetics, 100 ml of 0.5 g/1 CPC were stirred into a thermostatted vessel which contained frit at the bottom. The solution was kept at 20 °C, the pH was adjusted with 1 N H2SO4 to 2.5 and a definite amount of adsorbent (2-3 g) was added. With a velocity of 18.7 ml/min the solution was pumped out of the vessel via a flow-through cuvette (1 mm light path). Extinction was measured at 258 nm versus water, so the CPC concentration could be monitored directly as the calibration plot within the used range is linear (UV absorbance 1-~0.73 g/1 (Fig. 1). The minimal stirring speed (680 rpm with all resins) and maximal stirring speed (100 rpm with XAD 4 and HP20; 130 rpm with IRA 68) were used to observe the influence of stirrer speed on mass transfer.

681 Table 1. Characteristics of adsorbents Specific surface area (mZ/g)

Pore volume

HP 20 XAD 4 XAD 1180

718 750 650

IRA 68

Exchange capacity 1.6 m e q / m l (wet) 5.6 m e q / m l (dry) 80 g CaCO3/1 (wet)

Product

(ml/g)

Mean pore diameter (nm)

Bead size distribution (mm)

Mean particle diameter (mm)

1.16 0.96 1.7

-5.0 14.0

0.15-0.55 0.30-0.65 a 0.15-0.80

0.35 0.51 0.44

Gel structure

0.45-0.96

0.69

Bead size after wet sieving

Theory. According to the multilayer model of Brunauer, Emmett and Teller (BET model) the amount adsorbed as a function of equilibrium solution concentration in an adsorption experiment can be described by Eq. 1, which takes the adsorption of more than one layer of molecules into consideration.

Cad - -

C~4

=

a. CL[1-(n+l)b".C~L+n.b'~+l.C'] +1] ( l - b . C ~ ) [ l + ( a - b ) C ~ - a ' b " ' C ~ +1]

d)

where n = n u m b e r of layers including the monolayer; a=ko/kl, equilibrium coefficient (relation of adsorption and desorption for the monolayer); b=kl/k2, equilibrium coefficient for all further layers (with b~ = b2 = b 3 . . . ) ; C L = concentration of the adsorbate in solution; CM= amount adsorbed related to total weight of adsorbent for a complete covering of the surface with a monolayer. The parameters a, b and C~t in the BET equation are to be determined. An estimate for a follows from the initial slope of the isotherms. An optimization of adaptation of a, b and C~t with given n was carried out by a simplex method for function minimization, modified by Nelder and Mead (1965). This optimization was carried out for the isotherms of CPC with XAD 1180, XAD 4, HP 20 and IRA 68 by an HP 9845 minicomputer (Hewlett Packard, Fort Collins, Colo, USA). Attempts to simulate the observed kinetics with known models (Helffrich 1959; Buchholz 1979; Borchert and Buchholz 1984) were unsuccessful. Therefore an empirical approach was chosen by analysing experimental data similar to a reaction and determining apparent orders or reaction. Thus a first order reaction would give a linear correlation of experimental data according to Eq. 2:

(2)

In (CLo-- C~ / C L -- C=) = f ( t )

whereas a higher order would obey Eq. 3: 1/(eL - c =)x-1

=f(t)

(3)

CLO= concentration of adsorbate at the time 0; c= = concentration of adsorbate in equilibrium;f(t)= function of time. The first order approach proved to be valuable when two adsorption processes with different kinetics were assumed. Then the driving force for adsorption must be the difference in the actual solution concentration and that concentration which would correspond to equilibrium (CL). The latter can be calculated from the BET model and its parameters calculated from experimental results. The kinetics of CPC uptake from solution can then be described phenomenologically by:

- d CL" VL=d(Mad)=ka3" ACl " VA "F1 .dt + ka4. AC2- VA"F2"dt

(4)

where CL = CPC concentration in solution; VL= solution volume; Mad = amount of adsorbed CPC; ka3, ka4 = kinetic coefficients ; ACI, ACz=driving concentration differences (see above text) in two different particle fractions; VA= particle volume; F1, F2 = particle fractions (c.f. results). An approximate solution of Eq. 4 was performed by a set difference equation approach where d(Mad)~-AMad and dt=At, At'~t (t=time of experiment). An overall simulation model for CPC adsorption can then be derived based on adsorption isotherm and on adsorption kinetics according to Fig. 2. The simulation procedure was as follows: calculation of A M a d , and AMad2 for an appropriate short At from kinetics according to Eq. 4; from the respective sum ZAM~o, and ZAMao~ the concentrations of adsorbed CPC (C~d, and C~d~) can be calcu-

""1

ration o

J

~

ke3.&C1.At.VA.F1

/ CL~

"f

~ k cir.. AC 2 • At • VA . F2 ~

i©1 Pump

ooo oo Photometer

Fig. 1. Experimental set-up for measurement of adsorption kinetics

BET

= AMid1

\

\ ~ (TAMod~)

C~-(rAM~) ~ . &Mad2

~, 0L2

" {~AMod2)

/

BET Fig. 2. Overall simulation model for cephalosporin C (CPC) adsorption; for parameter definitions, see Materials and methods, Theory

682 n=3

105

~o~__~_ o o~/~-"~ "~n=2

140

o

120 90

o

75,

~ 100

~o

~

~

2 1

80

o

~ 60 ,~

60 ~5 40 g

3O

20

~5

0

O0 5

CL[g/I]

10

Fig. 3. Adaption of experimental data for XAD 4 according to Eq. 1: -, ~moothed curve adapted manually to experimental data (©); -, calculated with reference to smoothed experimental curve; n = 1: Monolayer, n = 2: double layer, n = 3: three layers

lated with the volume of adsorbent; fromthese the corresponding (hypothetical) equilibrium solution concentrations (Cgl and Co2) can be calculated according to the isotherm; the (hypothetical) concentration difference, as driving force for the adsorption rate, can then be obtained (AC1= CL - C~I); these are substituted in Eq. 4 in order to calculate the kinetics of the following adsorption step (time, segment At, ÷1).

Results

Adsorption isotherms The a d s o r p t i o n isotherms o f C P C o n the n o n - s p e c i f i c m a c r o p o r o u s a d s o r b e n t s X A D 4 a n d H P 20 are s h o w n in Figs. 3 a n d 4 a c c o r d i n g to the c o r r e l a t i o n Cad vs CL. It is i m p o r t a n t to n o t e that e q u i l i b r i u m results are very sensitive to c h a n g e in i o n i c strength, w h i c h was b e e n kept c o n s t a n t t h r o u g h o u t the e x p e r i m e n t s . As the interp r e t a t i o n s o f these plots with the s i m p l e r f o r m u l a s of F r e u n d l i c h a n d L a n g m u i r were u n s a t i s f a c t o r y , a description o f all m e a s u r e d i s o t h e r m s was p e r f o r m e d with the B E T model. F o r o p t i m i s a t i o n of the three p a r a m e t e r s a, b a n d CM f o u r c o m b i n a t i o n s have to be estimated. I n accord a n c e with the before m e n t i o n e d s i m p l e x m e t h o d the set o f p a r a m e t e r s m u s t be c h a n g e d u n t i l the d e v i a t i o n of the c a l c u l a t e d curve from the e x p e r i m e n t a l d a t a is minimal. The c o o r d i n a t e s CL a n d Cad for the a d s o r b e n t s X A D 4, HP20 a n d I R A 68 were t a k e n from the s m o o t h e d curve a d a p t e d m a n u a l l y to m e a s u r e d points.

~

1~3

1~5 2'0 CL[g/I]

2'5

3'0

Fig. 4. Results with expanded concentration range and HP20. At about 12 g/1 CPC a change in ionic strength was made. 1: Monolayer, 2: double layer, 3: three layers, dots: experimental data

F o r H P 20, n = 2, a n a d d i t i o n a l c a l c u l a t i o n was carried o u t u s i n g the e x p e r i m e n t a l d a t a as c o o r d i n a t e s ; for X A D 1180 o n l y the e x p e r i m e n t a l d a t a were used. T h e n u m b e r o f layers, n, was set at 1, 2 or 3. The results of the o p t i m i z a t i o n of the c o o r d i n a t e s are s u m m a r i z e d in T a b l e 2.

Table 2. Results from optimized adaptation of the isotherm coefficients by means of the simplex method Number of layer (n)

Parameters a

b

Correlation factor CM

HP 20; coordinates taken from smoothed measuring curve 1 0.36 -85.0 0.284 2 0.72 0.06 53.8 0.988 3 0.22 0.00002 102.7 0.072 experimental data as coordinates 2 0.63 0.05 57.3 0.212 XAD 4; coordinates taken from smoothed measuring curve 1 0.88 -98.3 0.014 2 0.70 0.05 80.3 0.244 3 0.74 0.03 83.7 0.272 XAD 1180; experimental data as coordinates 1 0.22 -82.3 2 0.42 0.03 54.0 3 0.63 0.03 45.9

0.068 0.089 0.112

IRA 68; coordinates taken from smoothed measuring curve 1 0.05 -190.7 2.315 2 O.11 0.23 46.2 0.203 3 0.43 0.15 26.7 0.080 For parameter definitions, see Materials and methods, Theory

3'5

683

The highest value for the correlation factor when using HP20, and therefore the best adaptation was achieved with a calculation for n = 2. When the original experimental data were used for calculation, the deviation from the calculated curve was always more significant than with the coordinates taken from the smoothed experimental curve. Therefore the correlation factor is always bigger in these latter cases, but the achieved results for a, b and C~4 are not very different. Concerning XAD 4 it was difficult to select the best choice for n. Following the plot o f the isotherms with the calculated parameters (Fig. 3), the curve with n = 2 layers seemed to be better. For XAD 1180 the best adaptation was achieved with n = 3 layers, when using the measurements. In the case of the ion-exchanger IRA68 the best adaptation was obtained with n = 1 layer. A maximum specific loading of 190 g C P C / 1 IRA68 with n = 1 can be determined: therefore that is the value for C~t. The adaptation with n = l corresponded to the form of Langmuir's isotherm. The achieved result, i.e. the best adaptation for n = 1, is evident for an ion-exchanger. Following a comment from a manufacturer, the normal concentration of CPC in production should be about 16 g/l, so the isotherms describe a suitable range. The experimental conditions mentioned in some publications (Voser and Weiss 1980) suggest expanding the experimental range to 30 g CPC/1 for C/~. Such measurements were also carried out (Fig. 4). In the high concentration range adaptation was obviously better with n = 3 for H P 20.

a)

-1.0 --8 -2.0 £.3

'._, ~ -3.0 _t'-

-4.0

-5.0

0.5

d 0.3

1~0

10

210

~ 310

I

410

50

I

o

60

710

t [mini

0

b)

-1.0

-q~-2.0 ~ ¼ ~ -3.0

'..........:

--

"%,%°..°

-4.0 ~ . . . .

-5.0

CPC on IRA68

~

1~

1~

~

~

--~.~'~

2~ 2~5 t [mini

3~0

3~

4'0

l~ig. 6. Adsorption kinetics of CPC with HP 20 (a) and IRA 68 (la)

The kinetics of CPC adsorption was analysed in a stirred vessel by measurement of the UV absorbance in the fluid phase. Figure 5 shows typical results; those with other adsorbents were similar in principle. From two experiments with a different stirrer speed it could be concluded that external mass transfer played a minor role and could therefore be neglected. Attempts to analyse the experimental results with known kinetic approaches (c. f. Helffrich 1959; Buch-

0.1

I

-6.00

-6.00 Adsorption kinetics

CPC on HP20

2~0

3~0

//

6~0

7~0

t [min] Fig. 5. Adsorption of CPC on HP20 as a function of time at two stirrer speeds

on a logarithmic scale (In CL-- C~ as a function of t; stirrer speed 680 rpm)

holz 1979; Borchert and Buchholz 1984) were not successful. Therefore a first order reaction was taken as an attempt to simulate the data (Fig. 6). Obviously no model based on one single transport and adsorption mechanism could simulate the experimental results. It was concluded that a more complex mechanism must be involved, and that the heterogeneous nature of the adsorbents could be the reason for this. In a second step, the differences AC from the experimental data for CL and the line extrapolated to zero time (Fig. 6: time interval 0 to approx. 10 min) were again treated according to a first order approach, giving a good linear concentration for that part of the adsorption process (Fig. 7). Thus the overall adsorption can be described as a combined process with two distinct mechanisms, a rapid and a slow one. Both can be phenomenologically interpreted as first order reactions. The quick first step may be correlated with adsorption in the easily accessible macropores of the particles, and the slow second step to adsorption in micropores of the microspheres which make up the individual macroscopic spheres.

684 0.6

0 -1.0

0.5

-2.0 0.4

U -3.0

Investigations on cephalosporin C adsorption kinetics and equilibria.

The kinetics and equilibria of cephalosporin C adsorption on different commercial adsorbents were investigated. Adsorption isotherms could be analysed...
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