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Functionalized Adsorbents for the Purification of Cephalosporin C and Deacetylcephalosporin C L. Fernando Bautista,† Jose´ L. Casillas, Mercedes Martı´nez, and Jose´ Aracil* Department of Chemical Engineering, UniVersidad Complutense de Madrid, 28040 Madrid, Spain
In the present work, the adsorption of cephalosporin C and deacetylcephalosporin C on six different adsorbents has been studied. Two of the adsorbents were commercial Amberlite XAD-2 and Amberlite XAD-4, and the other four were their corresponding brominated and bromoethylated derived resins, respectively. The research was carried out by means of pulse experiments in a high-performance liquid chromatography (HPLC) system, and the corresponding chromatographic responses were analyzed using the moment theory. The results showed that the chemically modified XAD-2 resins enhanced the adsorption constant for cephalosporin C. Specially, brominated XAD-2 resin showed an increase in KA of ∼3× with respect to the commercial adsorbent at 15 °C. In addition to the equilibrium and thermodynamic properties of the adsorption process, the effective pore diffusion coefficient, Dp, was also calculated. In all experimental conditions, the values of Dp were always larger for the modified resins, showing a consequent decrease in the internal mass transfer resistance of both cephalosporin C and deacetylcephalosporin C. 1. Introduction The commercialization of products obtained by biotechnological processes requires a coordinated coupling of unit operations in order to develop an efficient process. In most of these biological production processes, the main product of interest is generally synthesized along with other byproducts showing analogous chemical and physical properties. Thus, the need for more efficient downstream processing is a main objective to decrease the costs of those biotechnological products. Adsorption processes are widely used unit operations in downstream processing of many products obtained by fermentation. So, one of the ways to improve their efficiency is the rational design and development of adsorbents with increased selectivity and capacity. Cephalosporin C (CP-C), a β-lactamic antibiotic precursor obtained by aerobic fermentation from fungal strains of Cephalosporium acremonium, is the starting raw material for the synthesis of a wide-ranging spectrum of bactericidal antibiotics1. During the course of the previously described fermentation process, the acetyl ester group of the biologically active cephalosporin C is catalytically hydrolyzed, yielding deacetylcephalosporin C (dCP-C). At industrial scale, the purification of CP-C comprises a first filtration step to remove the cell mass, followed by acidification of the supernatant in order to precipitate proteins and remove penicillin N, and, finally, purification either by solvent extraction or by chromatographic/ adsorption techniques. However, the former lacks the high specificity shown by the latter, so that adsorption appears to be a better alternative, since a very high specificity operation must be carried out by considering the chemical similarities between CP-C and dCP-C. Early works reported different types of adsorbents for the isolation of CP-C, for example, activated carbons2, polystyrene-based macroreticular hydrophobic resins,3,4 or ion exchangers.5 More recently, divinylbenzenestyrene-based macroreticular hydrophobic resins, such as Am* To whom correspondence should be addressed. E-mail:
[email protected]. † Present address: Department of Chemical and Environmental Technology, Rey Juan Carlos University. 28024 Mo´stoles, Madrid, Spain.
berlite XAD-2 and XAD-46,7 or Amberlite XAD-1180,8 have been found to achieve better performance, although the efficiency and selectivity for the separation of CP-C and dCP-C, when tested, was not entirely satisfactory. In the present work, the assessment of performance and selectivity for the separation of CP-C and dCP-C using chemically modified Amberlite XAD-2 and XAD-4 resins was performed by pulse high-performance liquid chromatography (HPLC) experiments and further moment analysis of the chromatographic peak responses obtained. 2. Experimental Section 2.1. Adsorbates and Chemicals. Cephalosporin C and deacetylcephalosporin C were supplied by Glaxo Inc. (Uxbridge, Middlesex, U.K.) in the form of a concentrate in liquid solution from the fermentation broth containing cephalosporin C, deacetylcephalosporin C, and little amounts of deacetoxycephalosporin C. The two main components, i.e., CP-C and dCP-C, were individually isolated and purified by recrystallization in various water/acetone mixtures at 2 °C and, subsequently, dried under vacuum conditions at 20 °C for 24 h. Cephalosporin C thus obtained contained no significant amounts ( XAD-2-(CH2)2-Br > XAD-2 > XAD-4 > XAD-4-Br > XAD-4-(CH2)2-Br. For all Amberlite XAD-2 based resins, this series agrees with the results of the average pore size and polarity. However, for the XAD-4 family, the adsorption constant was larger for the commercial resin, showing higher hydrophobicity and smaller pore size than the two modified resins thereof. The different trend between the adsorption of CP-C on XAD-2 based and XAD-4 based adsorbents, respectively, could be due to a different incorporation of both functional groups (i.e., bromine and bromoethyl) within the XAD-4 matrix, reducing the number of effective adsorption sites with respect to the commercial resin. Every resin studied, both commercial and chemically modified, showed lower KA values for the adsorption of dCP-C than for that of CP-C. The extent of the adsorption of dCP-C on functionalized XAD-2 resins was again larger than the adsorption on commercial Amberlite XAD-2, although the differences were not very significant and the system did not show the large impact on the affinity for the adsorption of CP-C showed by the functionalization of the commercial adsorbent. For the
adsorption of dCP-C on XAD-4 type adsorbents, as for the adsorption of CP-C, a decrease in the capacity on both modified resins was again observed. In addition, the adsorption constant for the XAD-4/dCP-C system was larger than that for the XAD-2/CP-C system. The reason may lie in the smaller size of dCP-C. Since the chemical nature of both commercial resins was similar, the narrower pore size, and the consequent larger surface area, of Amberlite XAD-4 makes it possible to accommodate more dCP-C molecules inside the pores. So, the extent of the adsorption equilibrium can be considered to be driven by a complex combination of the balance of hydrophobic/hydrophilic characteristics of both adsorbates and adsorbents, the polarity of the adsorption surface, and steric features including the ratio between molecular size of the adsorbate and average pore size of the resin and the chemical nature of the ligands, functional groups, or spacer groups introduced in the modified resins. Assuming an Arrhrenius’ type dependence of KA with temperature, the heat of adsorption was calculated by using the well-known van’t Hoff equation (eq 5) and then the change of
Ind. Eng. Chem. Res., Vol. 45, No. 9, 2006 3233 Table 2. Summary of Equilibrium and Kinetic Parameters from Moment Analysis of the Adsorption of Cephalosporin C and Deacetylcephalosporin C on Amberlite XAD-2 and Chemically Modified Resins Thereof
Table 3. Summary of Equilibrium and Kinetic Parameters from Moment Analysis of the Adsorption of Cephalosporin C and Deacetylcephalosporin C on Amberlite XAD-4 and Chemically Modified Resins Thereof
entropy associated to the process was calculated by eq 6.
∂ ln KA ∆H0 ) ∂T RT2 ∆S0 )
∆H0 + R ln KA T
(5)
(6)
The above-mentioned complex simultaneous effects of both chemical and structural properties of adsorbates and adsorbents
were also exerted on the thermodynamics of the system. The equilibrium of adsorption was favored at decreasing temperatures, within the experimental range studied, as observed from the negative values of ∆H° (Tables 2 and 3). For each resin, the heat of adsorption was larger for the adsorption process involving CP-C, which has a higher molecular weight than dCPC. In all systems studied, the entropy change associated to the adsorption process was negative, as observed in other hydrophobic systems caused by the solvation of the hydrophobic surfaces exposed to the aqueous medium, which, in turn,
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produced an ordered arrangement of water molecules around those hydrophobic areas.15 The choice of a specific adsorbent for the further modeling and scaling up steps of the purification of CP-C and dCP-C must be based not only upon the high adsorption capacity and affinity for the target compound but also upon the comparative low adsorption of the contaminant molecule, i.e., dCP-C. This can be evaluated by use of the separation factor, R, whose definition is as follows,
R)
CFCF-C CFdCF-C
(7)
where CFCP-C and CFdCP-C are the capacity factors of CP-C and dCP-C, defined as expressed in eq 8.
CF ) µF
(8)
The larger the value of the separation factor, the more suitable the adsorbent and the operating conditions used are, to carry out the separation of both components in a single step. As observed in Table 4, the separation factors for all the XAD-4
Table 4. Average Separation Factor at Each Temperature for Cephalosporin C and Deacetylcephalosporin C separation factor, R resin
293 K
303 K
313 K
XAD-2 XAD-2-Br XAD-2-(CH2)2-Br XAD-4 XAD-4-Br XAD-4-(CH2)2-Br
2.3 4.7 2.2 1.2 1.4 1.1
1.9 3.7 2.2 1.1 1.2 1.1
1.8 2.8 2.3 1.0 1.1 1.0
based resins were close to 1 and they were not temperature sensitive. This means that both solutes were adsorbed to a similar extent with comparable efficiency, and using these resins it is virtually not possible to achieve a good separation between CP-C and dCP-C. On the other hand, XAD-2 adsorbents gave better results from the point of view of the separation factor obtained. Amberlite XAD-2 showed similar separation-factor values than XAD-2(CH2)2-Br, so both resins had a comparable ability to separate dCP-C from CP-C within the operating conditions studied. These two desired characteristics (i.e., high adsorption and separation
Figure 2. HETP plot for the adsorption of CP-C and dCP-C on Amberlite XAD-2 (a), XAD-2-Br (b), XAD-2-(CH2)2-Br (c), Amberlite XAD-4 (d), XAD-4-Br (e), and XAD-4-(CH2)2-Br (f). (Legends: ] CP-C at 15 °C, 4 CP-C at 20 °C, 0 CP-C at 25 °C, [ dCP-C at 15 °C, 2 dCP-C at 20 °C, 9 dCP-C at 25 °C, - - - linear fitting.)
Ind. Eng. Chem. Res., Vol. 45, No. 9, 2006 3235
capacity) were merged in XAD-2-Br, showing the larger adsorption constant and the larger value of the separation factor. Both features were significantly enhanced at decreasing temperature, since the affinity for CP-C increased faster than that for dCP-C as temperature fell down. According to eq 4, when mass transfer control takes place, HETP linearly increases with linear velocity of the mobile phase. So, the plot of HETP versus V should yield a linear relationship for which the slope is a function of both Dp and kf, as well as physicochemical characteristics already measured, such as bed and particle porosity, particle radius, and adsorption constant, and the intercept corresponds to the axial dispersion contribution to mass transfer. This term, 2DL/V, can also be independently evaluated from pulse experiments using a nonadsorbable tracer that diffuses freely (i.e., not showing internal or external diffusion control) along the column. Under these assumptions, HETP ) 2DL/V. For this purpose, pulses of 0.25 M NaNO3 were injected into each column at a different flow rate and temperature. The moment analysis was applied to the peak responses of the tracer. The values of HETP for the tracer were found to be independent of both temperature and linear velocity of the mobile phase. The external mass transfer coefficient can be estimated by a correlation adequate for the Reynolds number range used in the experiments carried out in the present work.16 Figure 2 shows plots of HETP as a function of the interstitial linear velocity for CP-C and dCP-C in all resins studied at different temperatures. As can be observed, the plots show the linear behavior expected from eq 4. The values of the pore diffusion coefficient were calculated from the corresponding slopes, and they are tabulated in Tables 2 and 3. As expected, the pore diffusivity increases with temperature for all systems studied. Concerning the adsorption of CP-C, Dp increased in the XAD-2 series according to the following order: XAD-2-Br > XAD-2-(CH2)2-Br > XAD-2. So, for the commercial resin, the internal mass transfer resistance was higher than that for the modified resins, which have larger average pore size, favoring the diffusion of the solute along the pores. However, pore size was not the only feature affecting pore diffusivity, since both modified resins showed similar values of pore diameter and pore volume (Table 1). So, the chemical modifications, somehow, must have affected the pore structure in a different manner, yielding different physicochemical characteristics. A similar effect was observed for dCP-C in the same resin series, except that the values of Dp on XAD-2-Br and XAD2-(CH2)2-Br were very similar to that for the case of CP-C. By comparing both solutes, the smaller molecule (i.e., dCP-C) showed larger values of pore diffusion coefficient, indicating a weaker resistance to diffuse inside the pores due to its smaller size. For the XAD-4 series, the expansion of the pore structure, caused by the chemical functionalization of the commercial resin, produced a decrease in the internal mass transfer resistance, too. In this case, the order of the Dp values for both CP-C and dCP-C was found to be as follows: XAD-4-(CH2)2Br > XAD-4-Br > XAD-4. 5. Conclusions The study of the adsorption of CP-C and dCP-C on six different adsorbents was performed by pulse perturbations in HPLC. The peak responses were analyzed by the moment analysis. Two of the adsorbents were the commercial hydrophobic Amberlite XAD-2 and Amberlite XAD-4. The other four
where prepared “in-house” by chemical functionalization of the above resins by introducing either bromine or bromoethyl functional group into the polymeric matrix. For the adsorption of CP-C, the results showed that the bromoethylated XAD-2 resin slightly increased the value of the adsorption constant while the brominated XAD-2 improved the adsorption capacity up to 3×, with respect to the commercial Amberlite XAD-2. However, for dCP-C, the chemical modification of commercial XAD-2 only yielded an enhancement of ∼30%. This selective affinity for CP-C of the XAD-2-Br resin resulted in an important improvement of the separation factor between CP-C and dCP-C, especially at 15 °C, which makes this adsorbent particularly appropriate for the purification of CP-C in solutions or broths containing dCP-C. The pore diffusion coefficients for CP-C and dCP-C were also calculated, showing an increase after the functionalization of both commercial resins due to the expansion of the pore structure caused by the chemical modification. This feature produced a decrease in the internal mass transfer resistance and a consequent enhancement of the kinetics of the adsorption process. Nomenclature C ) adsorbate concentration in the liquid phase (g/L) CF ) capacity factor (cm3), defined in eq 8 DL ) axial dispersion coefficient (m2/s) Dp ) pore diffusion coefficient (m2/s) F ) volumetric flow rate (cm3/min) KA ) linear adsorption constant k ) apparent adsorption constant kf ) external-film mass-transfer coefficient (m/s) L ) column length (m) R ) particle radius (m) t ) time (s) V ) interstitial velocity of the mobile phase (m/s) Greek Letters R ) separation factor, defined in eq 7 ) bed voidage p ) particle porosity µ ) first moment as defined by eq 1 σ2 ) second central moment as defined by eq 3 Literature Cited (1) Abraham, E. P.; Loder, P. B. Cephalosporins and penicillins: Chemistry and biology; Flynn, H. E., Ed.; Academic Press: New York, 1972. (2) Nara, K.; Ohta, K.; Katamoto, K.; Fukuda, H.; Mikozami, N. Method for separating cephalosporin C. U.S. Patent 3926973, 1975. (3) Voser, W. Isolation of hydrophilic fermentation products by adsorption chromatography. J. Chem. Technol. Biotechnol. 1982, 32, 109-118. (4) Pirotta, M. Amberlite ER-180sA new styrene divinylbenzene adsorbent specially designed for industrial chromatography and particularly for the extraction of cephalosporin C. Angew. Makromol. Chem. 1982, 109/ 110, 197-214. (5) McCormick, S. L.; Mack, H. Process for the recovery and purification of cephalosporin C. U.S. Patent 3467654, 1969. (6) Addo-Yobo, F.; Slater, N. K. H.; Kenney, C. N. Measurement of heats of adsorption of amino acids on Amberlite XAD-2 by an HPLC technique. Chem. Eng. J. 1988, 39, B9-B16. (7) Hicketier, M.; Buchholz, K. Investigations on cephalosporin C adsorption kinetics and equilibria. Appl. Microbiol. Biotechnol. 1990, 32, 680-685. (8) Hicketier, M.; Buchholz, K. Fluidized bed adsorption of cephalosporin C. J. Biotechnol. 2002, 93, 253-268.
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(9) Martı´nez, M.; Casillas, J. L.; Addo-Yobo, F.; Kenney, C. N.; Aracil, J. An HPLC technique for the study of adsorption of amino acids on functionalized resins. In Separations for Biotechnology 3; Pyle, D. L., Ed.; The Royal Society of Chemistry: Letchworth, U.K., 1994; pp 294-300. (10) Bautista, L. F.; Pinilla, J.; Aracil, J.; Martı´nez, M. Adsorption isotherms of Aspartame on commercial and chemically modified divinylbenzene-styrene resins at different temperatures. J. Chem. Eng. Data 2002, 47 (3), 620-627. (11) Ruthven, D. M. Principles of adsorption and adsorption processes; Wiley-Interscience: New York, 1984. (12) Miyabe, K.; Guiochon, G. The moment equations of chromatography for monolithic stationary phases. J. Phys. Chem. B 2002, 106 (34), 8898-8909. (13) Venkata Saritha, N.; Madras, G. Modeling the chromatographic response of inverse size-exclusion chromatography. Chem. Eng. Sci. 2001, 56 (23), 6511-6524.
(14) Yu, H. W.; Ching, C. B. Kinetic and equilibrium study of the enantioseparation of fluoxetine on a new β-cyclodextrin column by high performance liquid chromatography. Chromatographia 2001, 54 (11/12), 697-702. (15) Bautista, L. F.; Martı´nez, M.; Aracil, J. Adsorption equilibrium of R-amylase in aqueous solutions on ion-exchange and hydrophobic polymeric adsorbents. AIChE J. 1999, 45 (4), 761-769. (16) Hidajat, K.; Aracil, J.; Carberry, J. J.; Kenney, C. N. Laboratory catalytic studies: The role of transport phenomena. J. Catal. 1987, 105, 245-248.
ReceiVed for reView November 3, 2005 ReVised manuscript receiVed February 2, 2006 Accepted March 1, 2006 IE051221M
