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Preparation of an anion exchanger based on TiO2-densified cellulose beads for expanded bed adsorption
REACTIVE & FUNCTIONAL POLYMERS
Reactive & Functional Polymers 62 (2005) 169–177
www.elsevier.com/locate/react
Preparation of an anion exchanger based on TiO2-densified cellulose beads for expanded bed adsorption Yin-Lin Lei a
a,b
, Dong-Qiang Lin a, Shan-Jing Yao
a,b,*
, Zi-Qiang Zhu
a
Department of Chemical and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, PR China b Ningbo Institute of Technology of Zhejiang University, Ningbo 315100, PR China Received January 2004; received in revised form July 2004; accepted August 2004 Available online 22 December 2004
Abstract A novel TiO2-densified cellulose beads as the matrix used in expanded beds were exploited. The matrix was activated by epichlorohydrin and then coupled with diethylamine to function as an anion exchanger for the adsorption of proteins. Several factors influenced the efficiency of activation, such as the amount of epichlorohydrin, the concentration of NaOH, and the contents of TiO2 and cellulose in the matrix, were investigated. The optimal procedure for activation was found to be: mixing 20 g matrix beads in 40 mL 2.5 mol/L NaOH solution containing 8 mL epichlorohydrin for 8 h at 25 C. The coupling efficiency of diethylamine with the activated matrix is close to 90% under the given conditions. The prepared adsorbent has favorable expanded bed characteristics and an accordant protein breakthrough behavior both in packed bed and in expanded bed, by possessing a dynamic adsorption capacity of 42.6 mg BSA/mL adsorbent at 10% breakthrough. The results demonstrated that the custom-made adsorbent can be applied to the expanded bed adsorption of proteins. 2004 Published by Elsevier B.V. Keywords: Expanded bed adsorption; Matrix; Cellulose beads; Activation; Epichlorohydrin
1. Introduction Among the various industrial chromatographic separation techniques developed in recent years, expanded bed adsorption (EBA) has been success*
Corresponding author. Tel.: +86 571 8795 1982; fax: +86 571 8795 1015. E-mail address: [email protected] (S.-J. Yao). 1381-5148/$ - see front matter 2004 Published by Elsevier B.V. doi:10.1016/j.reactfunctpolym.2004.08.004
fully introduced to primary capture of target protein in the biotechnology industry, wherein several important bio-pharmaceuticals are being produced using the EBA technique [1,2]. Compared with other chromatographic processes, EBA provides a significant advantage because it enables proteins to be recovered directly from unclarified cultivations of microorganisms or homogenates of disrupted cells, without the need for prior removal
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Notation BSA C C0 E EBA DEAHP GMA
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
bovine serum albumin effluent concentration initial concentration expansion degree expanded bed adsorption diethylaminohydroxypropyl glycidyl methacrylate
of suspended solids [3]. A successful and robust EBA process depends on three key parameters: adsorbent matrix, ligand chemistry applied to the matrix, and the design of the columns. The matrix must possess a quite higher density than the feed stock to allow acceptable flow rates during the operation, and an appropriate size distribution to form a stable classification of adsorbent particles and thus a reduced back mixing in the column [4]. Cellulose, one of the most abundant and lowcost natural polymers, has been successfully manufactured in porous bead form [5–7], and widely used as column packing material for liquid chromatography and as matrix for further derivatization for ion exchange or affinity chromatography [8–10]. Recently, Lali et al. [11–13] prepared a rigid spherical macroporous cellulose matrix for the expanded bed affinity purification of enzymes, which had a mean pore size up to 3 lm and a bulk density of 1.6 g/cm3. The purpose of the present work is to use another cheap industrial material, superfine TiO2 powder, as a densifier which is embedded in regenerated cellulose beads to produce a novel composite matrix. After activation by epichlorohydrin and coupling with diethylamine, the matrix is derived to function as an anion exchanger for the EBA of proteins. The hydroxyl groups of cellulose beads can be converted by the usual chemical reactions applied to other hydrophilic matrices, e.g., fibrous cellulose and agarose. The reagents used for activation include sodium periodate [9], epichlorohydrin [14], 1,4-butanediol diglycidyl ether [15], and Cl-COONB [16]. Among these activation methods, oxidation with periodate leads to severe damage on the cellulose beads and is not recommended,
whereas epoxy activation methods are extensively used to couple diverse ligands or small molecules which contain a nucleophilic primary or secondary amine, a sulphydryl group or, less commonly, a hydroxyl group [17–19]. Epichlorohydrin is the usual reagent used for the cross-linking or activation of hydroxyl-containing polymers [20], and is therefore employed to activate the cellulose-based matrix in our study. Cellulose may be crystalline in several polymorphic forms, whereas regenerated cellulose usually exists in the thermodynamically stable cellulose II form. The differentiation of cellulose into two distinct regions, the amorphous zone and the crystalline zone, is probably a simplified description of the submicroscopic morphology [21]. In general, chemical reactions cannot take place easily in the crystalline regions of cellulose. Swelling in a compatible solvent may be needed for the adequate utilization of hydroxyl groups in cellulose-based matrix. On the other hand, embedding of TiO2 particles into regenerated cellulose should affect the semicrystalline structure of the matrix, which may bring an unacquainted accessibility of activation reaction. In order to further elucidate this kind of effect, several factors influencing the activation reaction, such as the amount of epichlorohydrin, the concentration of NaOH, and the contents of TiO2 and cellulose in matrix, would be investigated systematically in the present work.
2. Experimental 2.1. Materials Degreasing cotton with a degree of polymerization 1620 was ordered from Huamei Biotech Ltd. (Shanghai, China). Rutile TiO2 powder with a density of 4.2 g/cm3 and a size of 300 nm was provided by Huachang Polymer Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma (MO, USA). All other reagents were of analytical reagent grade and purchased from local suppliers.
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
2.2. Preparation of composite matrix The cellulose xanthate viscose was prepared by reacting 45 g alkali-treated and aged degreasing cotton with 20 mL CS2 and then dissolving into 6 wt% NaOH solution. The composite matrix based on TiO2-densified cellulose beads was prepared through the method of water-in-oil suspension thermal regeneration, described by the former paper [22]. Briefly, a given mass of TiO2 powder was mixed thoroughly with 100 g viscose (containing 6–9% cellulose). The mixture was dispersed in a solution of 200 mL chlorobenzene and 400 mL pump oil in a 1 l flask with agitation at 350 rpm for 0.5 h. The suspension was heated to 95 C for 1 h, then cooled down and filtered. The produced particles were washed with methanol. The decomposition of cellulose xanthate was completed by immersing particles in a solution of 50 mL acetic acid and 200 mL ethanol. Finally, about 30 mL beads with a diameter of 100–300 lm were obtained by washing with water and sieving. The composite matrix was nominated as Cell-Ti.
171
dorf, Germany. About 2 mL glass beads (0.3 mm diameter) were added to improve flow distribution at the column inlet. A movable top adapter was employed to adjust the position of liquid outlet just above the solid–liquid interface. A peristaltic pump (Lange Ltd., Baoding, China) was used for fluid supply. All operations were performed at 25 C with a settled bed height of 15.0 ± 0.2 cm. 2.5. Protein adsorption tests BSA was used as a model adsorbate in the present work for protein adsorption, with 50 mM Tris–HCl buffer (pH 7.5) as the fluid phase. A XK16/20 column (Amersham Biosciences, Sweden) was employed for packed bed adsorption with a settled bed height of 10.0 ± 0.2 cm, while the expanded bed mentioned above was adopted for EBA. The protein solution was continuously injected by the pump, and the BSA concentration at column outlet was monitored by a UV detector (Amersham Biosciences, Uppsala). All operations were carried out at 25 C.
2.3. Preparation of anion exchanger 2.6. Assays Cell-Ti was activated by epichlorohydrin, and then attached to diethylamine to produce an anion exchanger with diethylaminohydroxypropyl (DEAHP) weak base groups. The specific experimental conditions for activation reaction would be studied in Section 3.1. The activated beads (30 mL) were extracted with acetone in a Soxhlet apparatus for 4 h to remove low molecular weight polymers, and added into a solution of 90 mL dioxane and 90 mL diethylamine. Then, the mixture was heated to the set temperature and stirred for 12 h. The product was washed with 500 mL water and stored in 20% ethanol. The anion exchanger was designated Cell-Ti DEAHP. 2.4. Expanded bed operation A home made column (1000 · 20 mm ID) was used for expanded bed operation, which was a kind gift from Heinrich-Heine Universita¨t Du¨ssel-
The content of epoxy groups in the activated matrix was determined by the method described by Sundberg and Porath [15], and the content of DEAHP functional groups was analyzed with respect to the nitrogen content, by the quantitative elementary analysis with a 1106 elemental autoanalysis apparatus (Carlo-Erba, Italy). The ion-exchange capacity for small ions was determined by the HCl–NaOH titration. The infrared spectra were recorded on a Nicolet 170SX Fourier transform infrared spectrometer (Hitachi, Japan). SEMs were performed with an X-650 scanning electron microscope (Hitachi, Japan). Before operating SEM, samples were dehydrated by stepwise transfer into water–ethanol mixtures, followed by ethanol and finally diethyl ether. The diethyl ether was removed at room temperature. The degree of expansion E was measured as H/H0, where H was the expanded bed height and H0 was the sedimented bed height.
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3. Results and discussion 3.1. Factors on epoxy-activation For epoxy-activated reaction, the following parameters were studied in the present work during the activation of cellulose matrix: reaction time, concentration of NaOH, amount of epichlorohydrin, contents of TiO2 and cellulose. 3.1.1. Reaction temperature and time Activation of hydroxyl-containing polymers by epichlorohydrin is generally carried out at room temperature, because high temperature results in not only the reduction of reaction time but the loss of epoxy groups due to hydrolysis [15]. Fig. 1 shows the available epoxy groups in activated Cell-Ti and cellulose beads as a function of reaction time. As a result, the amount of activated epoxy group ascended to the maximum value after the reaction proceeded for 7–8 h. Compared to the case of cellulose beads, the activated Cell-Ti possesses more epoxy groups (up to 215 lmol/mL). This may be attributed to the addition of TiO2 superfine particles in the matrix, which looses the network structure of Cell-Ti. SEM images of both
matrices testified to this presumption, as exhibited in Fig. 2(a) and (b). From the practical viewpoint, TiO2 superfine particles played not only a role of densifier to impart an increased density to the composite matrix, but also a role of loose filler to facilitate the activation. 3.1.2. Concentration of NaOH Cross-linking and activation occur simultaneously when epichlorohydrin reacts with hydroxylcontaining polymers. Fig. 3 investigated the content of epoxy groups in the activated Cell-Ti beads as a function of NaOH concentration. The maximum content was found at a starting concentration of 3.0 mol/L NaOH. With increasing NaOH concentration, more hydrogen-bonds between the chains of regenerated cellulose had been broken, bringing more hydroxyl groups to be activated by epichlorohydrin. But at NaOH concentrations above 3.0 mol/L, chemical cross-linking through epichlorohydrin and the hydrolysis of epoxy groups tended to take place, which leaded to the obvious decrease of the content of epoxy groups with an increase of NaOH concentration. Similar results had been observed by Kong et al. [23]. Consequently, the optimal NaOH concentration for the activation reaction is 2.5–3.0 mol/L.
Epoxy group (µmol/mL)
250
3.1.3. Amount of epichlorohydrin Fig. 4 shows the content of available epoxy groups as a function of the amount of epichlorohydrin used, which indicates that excessive epichlorohydrin should be added into the reaction system in order to acquire a favorable activation result. According to the conclusion drawn by Kong et al. [23], when the mole ratio of epichlorohydrin to NaOH was less than 1.0, a majority of epichlorohydrin would be consumed by the cross-linking reaction rather than by activation.
Cellulose Beads Cell-Ti Beads
200
150
100
50
0 0
1
2
3
4
5
6
7
8
9
Reaction time (h) Fig. 1. Epoxy group content as a function of reaction time. Experiments were performed at 25 C, with 20 g Cell-Ti or cellulose beads in 40 mL of 2.5 mol/L NaOH solution containing 10 mL epichlorohydrin. The weight ratio of TiO2 to viscose was 15/100, and the content of cellulose in viscose was 7.5 wt%.
3.1.4. Content of TiO2 In the present work, TiO2 is used to densify the cellulose-based beads. With the weight ratio of TiO2 to viscose increased from 5/100 to 30/100, the wet density of composite beads increased accordingly from 1.12 to 1.35 g/cm3. It is declared
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
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Fig. 2. SEM images of matrices: (a) cellulose beads and (b) Cell-Ti beads.
once more that the embedding of TiO2 particles benefits the activation, by holding a high content of 164 lmol epoxy groups per mL beads even at a TiO2/viscose ratio of 30/100 (shown in Fig. 5).
3.1.5. Content of cellulose Fig. 6 exhibits the epoxy group contents at a series of cellulose contents in the starting viscose. The result indicates that the increase of cellulose content helps to gain more available epoxy groups. However, it is noticeable that too high cellulose content would make the matrix beads more compact, consequently preventing macromolecular adsorbates from permeating through. Here, the content of cellulose in viscose is chosen at 7.5 wt%.
3.2. Coupling with diethylamine At present, several cellulose-based anion exchangers having aminoethyl (AE), diethylaminoethyl (DEAE) and triethylaminoethyl (TEAE) groups as the ligands are commercially available [24,25]. These products are usually prepared by reacting halogenated amines with alkali-activated hydroxyls of cellulose. Here, diethylamine was coupled to epoxy-activated cellulose-based matrix for producing an anion exchanger with DEAHP groups, exhibiting a weak basicity similar to that DEAE groups [26,27]. The authors have studied the reaction of amine with the copolymer of glycidyl methacrylate
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Epoxy group (µmol/mL)
Epoxy group (µmol/mL)
300 250 200 150 100
200
150
100
50
50 0 0.00
0 0
1
2
3
4
5
6
0.05
0.10
0.15
0.20
0.25
0.30
0.35
TiO2/Viscose ( g/g )
NaOH concentration (mol/L) Fig. 3. Epoxy group content as a function of NaOH concentration. Experiments were performed at 25 C for 8 h, with 20 g Cell-Ti beads in 40 mL NaOH solution containing 8 mL epichlorohydrin. The weight ratio of TiO2 to viscose was 15/100, and the content of cellulose in viscose was 7.5 wt%.
Fig. 5. Epoxy group content as a function of TiO2/viscose. The experimental conditions were the same as those in Fig. 3, expect the ratio of TiO2/vicose varied.
250
Cellulose in viscose
Epoxy group (µmol/mL)
9.0% 200
150
100
8.0%
7.5%
6.0% 50
0
0 0
0.5
1.0
1.5
2.0
2.5
3.0
Epichlorohydrin/NaOH (mol/mol) Fig. 4. Epoxy group content as a function of epichlorohydrin amount. The experimental conditions were the same as those in Fig. 3, expect the amount of epichlorohydrin varied.
(GMA), illustrating some restrictive factors in the heterogeneous system [28]. Here for the coupling of diethylamine with activated Cell-Ti beads, Fig. 7 shows the coupling efficiency as a function of reaction time at 25 and 50 C, respectively. The result indicates that prolonging time and/or raising temperature tended to in-
50
100
150
200
250
300
Epoxy group ( µmol/mL) Fig. 6. Epoxy group content as a function of cellulose content in viscose. The experimental conditions were the same as those in Fig. 3, expect the content of cellulose in viscose varied.
crease coupling efficiency. After carrying out, for 12 h at 50 C, the efficiency was especially close to 90%. In view of the boiling point of diethylamine at about 56 C, a favorable coupling condition was determined as follows: rotating 30 mL activated matrix in a solution of 90 mL dioxane and 90 mL diethylamine for at least 12 h at 50 C.
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
ence of epoxy groups, which disappear in that of Cell-Ti DEAHP. The infrared spectra validate the efficiency in the different steps of Cell-Ti DEAHP preparation.
100 o
25 C o 55 C
Coupling efficiency
80
175
60
3.4. Expanded bed characteristics
40
20
0 0
2
4
6
8
10
12
Reaction time (h) Fig. 7. Coupling efficiency as a function of reaction time.
It is important for the EBA adsorbent to expand effectively in the column. Fig. 9 hereby measured the expanded bed characteristics of Cell-Ti DEAHP prepared, showing that the adsorbent could be expanded almost linearly in serial of mobile phases. The detailed examination for the hydrodynamic properties of Cell-Ti beads in an expanded bed has been made in the former paper [29].
3.3. Infrared spectra
3.5. Protein breakthrough capacities
Fig. 8 displays the infrared spectra of cellulose, Cell-Ti, activated Cell-Ti and Cell-Ti DEAHP. In Fig. 8, the adsorption peak of Cell-Ti at 682 cm 1 is due to the contribution of the stretching vibration of Ti–O. The peaks of activated Cell-Ti at 1244, 937 and 852 cm 1 account for the exist-
First, the ion-exchange capacity of Cell-Ti DEAHP was determined to be 0.2 mmol Cl /mL adsorbent. The BSA breakthrough capacity of Cell-Ti DEAHP was then measured in an expanded bed, with a comparison to that in a packed bed. As shown in Fig. 10, protein breakthrough behavior or dynamic adsorption efficiency in expanded bed accords with that in packed bed, except for a small advance in breakthrough time, which demonstrated that Cell-Ti DEAHP prepared in the present work might be used as an
3.5 in 20% glycerol in 10% glycerol in water
Expansion degree, E
3.0
2.5
2.0
1.5
1.0 0
100
200
300
400
500
600
700
Flow rate (cm/h) Fig. 8. Infrared spectra at various preparation stages.
Fig. 9. Expanded bed characteristics of Cell-Ti DEAHP.
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From this viewpoint, the study in this paper is a heuristic attempt. Another effort which embeds more dense stainless steel particles into regenerated cellulose beads is under way.
1.00 in packed bed in expanded bed
C/C0
0.75
0.50
Acknowledgements 0.25
0.00 0
10
20
30
40
50
60
70
80
Load BSA (mg/mL adsorbent) Fig. 10. Breakthrough curves in expanded and packed beds. The initial concentration of BSA is 1.0 mg/mL.
efficient anion exchanger for EBA. At the breakthrough of 10% of the initial concentration, the adsorption capacity was calculated to be 42.6 mg BSA/mL adsorbent.
4. Conclusions A novel anion exchanger for expanded bed adsorption was exploited, based on TiO2-densified cellulose beads as matrix. The activation of TiO2densified cellulose matrix by epichlorohydrin was investigated in details. The optimal procedure was obtained as follows: mixing 20 g matrix beads in 40 mL NaOH solution containing 8 mL epichlorohydrin for 8 h at 25 C. The coupling efficiency of diethylamine with activated matrix is close to 90% under the given conditions. The prepared adsorbent of Cell-Ti DEAHP has favorable expanded bed characteristics and an accordant protein breakthrough behavior with the dynamic adsorption capacity of 42.6 mg BSA/mL adsorbent at 10% breakthrough. It was demonstrated that when applying the composite TiO2-densified cellulose beads to EBA it is possible to take advantage of the regular spherical shape, high porosity, hydrophilicity, chemical reactivity and mechanical strength of individual particles. We believe that the results achieved in the field of EBA will promote greater enthusiasm for the large-scale manufacture of cellulose-based matrices in the next few years.
The authors wish to thank Prof. M.-R. Kula (Institute of Enzyme Technology, Heinrich-Heine Universita¨t Du¨sseldorf) for presenting the expanded bed column, and the National Natural Science Foundation of China for the financial support (No. 20206029).
References [1] R. Hjorth, Trends Biotechnol. 15 (1997) 230. [2] F.B. Anspach, D. Curbelo, R. Hartmann, G. Garke, W.D. Deckwer, J. Chromatogr. A 865 (1999) 129. [3] D.K. McCormick, Bio/Technology 11 (1993) 1059. [4] J. Tho¨mmes, Adv. Biochem. Eng. 58 (1997) 185. [5] J. Pesˇka, J. Sˇtamberg, Z. Blae`e, US Patent, 4,055,510, 1977. [6] J. Sˇtamberg, J. Pesˇka, H. Dautzenberg, B. Philipp, in: T.C.J. Gribnau, J. Visser, R.J.F. Nivard (Eds.), Affinity Chromatography and Related Techniques, Analytical Chemistry Symposia Series, vol. 9, Elsevier, Amsterdam, 1982, p. 131. [7] S. Kuga, J. Chromatogr. 195 (1980) 221. [8] J. Sˇtamberg, Sep. Purif. Meth. 17 (1988) 155. [9] H.-F. Boeden, K. Pommerening, M. Becker, C. Rupprich, M. Holtzhauer, F. Loth, R. Muller, D. Bertram, J. Chromatogr. 552 (1991) 389. [10] P. Gemeiner, M. Polakovic, D. Mislovicova, V. Stefuca, J. Chromatogr. B 715 (1998) 245. [11] A. Pai, S. Gondkar, S. Sundaram, A. Lali, Bioseparation 8 (1999) 131. [12] A. Pai, S. Gondkar, A. Lali, J. Chromatogr. A 867 (2000) 113. [13] N. Amritkar, M. Kamat, A. Lali, Proc. Biochem. 39 (2004) 565. [14] I. Matsumoto, Y. Mizuno, N. Seno, J. Biochem. 85 (1979) 1091. [15] L. Sundberg, J. Porath, J. Chromatogr. 90 (1974) 87. [16] W. Buttner, M. Becker, C. Rupprich, H.-F. Boeden, P. Henklein, F. Loth, H. Dautzenderg, Biotechnol. Bioeng. 33 (1989) 26. [17] G.H. Hermanson, A.K. Mallia, P.K. Smith (Eds.), Immobilized Affinity Ligand Techniques, Academic Press, San Diego, 1992, p. 11. [18] F. Sˇvec, H. Hrudkova´, D. Hora´k, J. Ka´lal, Angew. Makromol. Chem. 63 (1977) 23.
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177 [19] J.B. Wheatley, D.E. Schmidt Jr., J. Chromatogr. A 849 (1999) 1. [20] J. Nagasawa, T. Suehiro, A. Yamauchi, J. Appl. Biochem. 7 (1985) 296. [21] L. Westman, T. Lindstro¨m, J. Appl. Polym. Sci. 26 (1981) 2519. [22] Y.-L. Lei, D.-Q. Lin, S.-J. Yao, Z.-Z. Liu, J. Appl. Polym. Sci. 90 (2003) 2848. [23] D.-L. Kong, J. Dai, C.-Z. Chen, Y.-T. Yu, Chem. J. Chin. Univ. 21 (2000) 1848, in Chinese.
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[24] G.A. Tishchenko, M. Bleha, J. Sˇkvor, T. Bosˇtik, J. Chromatogr. B 706 (1998) 157. [25] T.M. Mohamed, M.A. Mohamed, S.A. Mohamed, A.S. Fahmy, Biores. Technol. 68 (1999) 215. [26] J. Hradil, F. Sˇvec, Enzyme Microb. Technol. 3 (1981) 336. [27] Y.-H. Yu, Y. Sun, J. Chromatogr. A 855 (1999) 129. [28] Y.-L. Lei, Z.-Z. Liu, Q.-F. Liu, X.-Y. Wu, React. Funct. Polym. 48 (2001) 159. [29] Y.-L. Lei, D.-Q. Lin, S.-J. Yao, Z.-Z. Liu, Z.-Q. Zhu, Chin. J. Chem. Eng. 11 (2003) 141.
Reactive & Functional Polymers 62 (2005) 169–177
www.elsevier.com/locate/react
Preparation of an anion exchanger based on TiO2-densified cellulose beads for expanded bed adsorption Yin-Lin Lei a
a,b
, Dong-Qiang Lin a, Shan-Jing Yao
a,b,*
, Zi-Qiang Zhu
a
Department of Chemical and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, PR China b Ningbo Institute of Technology of Zhejiang University, Ningbo 315100, PR China Received January 2004; received in revised form July 2004; accepted August 2004 Available online 22 December 2004
Abstract A novel TiO2-densified cellulose beads as the matrix used in expanded beds were exploited. The matrix was activated by epichlorohydrin and then coupled with diethylamine to function as an anion exchanger for the adsorption of proteins. Several factors influenced the efficiency of activation, such as the amount of epichlorohydrin, the concentration of NaOH, and the contents of TiO2 and cellulose in the matrix, were investigated. The optimal procedure for activation was found to be: mixing 20 g matrix beads in 40 mL 2.5 mol/L NaOH solution containing 8 mL epichlorohydrin for 8 h at 25 C. The coupling efficiency of diethylamine with the activated matrix is close to 90% under the given conditions. The prepared adsorbent has favorable expanded bed characteristics and an accordant protein breakthrough behavior both in packed bed and in expanded bed, by possessing a dynamic adsorption capacity of 42.6 mg BSA/mL adsorbent at 10% breakthrough. The results demonstrated that the custom-made adsorbent can be applied to the expanded bed adsorption of proteins. 2004 Published by Elsevier B.V. Keywords: Expanded bed adsorption; Matrix; Cellulose beads; Activation; Epichlorohydrin
1. Introduction Among the various industrial chromatographic separation techniques developed in recent years, expanded bed adsorption (EBA) has been success*
Corresponding author. Tel.: +86 571 8795 1982; fax: +86 571 8795 1015. E-mail address: [email protected] (S.-J. Yao). 1381-5148/$ - see front matter 2004 Published by Elsevier B.V. doi:10.1016/j.reactfunctpolym.2004.08.004
fully introduced to primary capture of target protein in the biotechnology industry, wherein several important bio-pharmaceuticals are being produced using the EBA technique [1,2]. Compared with other chromatographic processes, EBA provides a significant advantage because it enables proteins to be recovered directly from unclarified cultivations of microorganisms or homogenates of disrupted cells, without the need for prior removal
170
Notation BSA C C0 E EBA DEAHP GMA
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
bovine serum albumin effluent concentration initial concentration expansion degree expanded bed adsorption diethylaminohydroxypropyl glycidyl methacrylate
of suspended solids [3]. A successful and robust EBA process depends on three key parameters: adsorbent matrix, ligand chemistry applied to the matrix, and the design of the columns. The matrix must possess a quite higher density than the feed stock to allow acceptable flow rates during the operation, and an appropriate size distribution to form a stable classification of adsorbent particles and thus a reduced back mixing in the column [4]. Cellulose, one of the most abundant and lowcost natural polymers, has been successfully manufactured in porous bead form [5–7], and widely used as column packing material for liquid chromatography and as matrix for further derivatization for ion exchange or affinity chromatography [8–10]. Recently, Lali et al. [11–13] prepared a rigid spherical macroporous cellulose matrix for the expanded bed affinity purification of enzymes, which had a mean pore size up to 3 lm and a bulk density of 1.6 g/cm3. The purpose of the present work is to use another cheap industrial material, superfine TiO2 powder, as a densifier which is embedded in regenerated cellulose beads to produce a novel composite matrix. After activation by epichlorohydrin and coupling with diethylamine, the matrix is derived to function as an anion exchanger for the EBA of proteins. The hydroxyl groups of cellulose beads can be converted by the usual chemical reactions applied to other hydrophilic matrices, e.g., fibrous cellulose and agarose. The reagents used for activation include sodium periodate [9], epichlorohydrin [14], 1,4-butanediol diglycidyl ether [15], and Cl-COONB [16]. Among these activation methods, oxidation with periodate leads to severe damage on the cellulose beads and is not recommended,
whereas epoxy activation methods are extensively used to couple diverse ligands or small molecules which contain a nucleophilic primary or secondary amine, a sulphydryl group or, less commonly, a hydroxyl group [17–19]. Epichlorohydrin is the usual reagent used for the cross-linking or activation of hydroxyl-containing polymers [20], and is therefore employed to activate the cellulose-based matrix in our study. Cellulose may be crystalline in several polymorphic forms, whereas regenerated cellulose usually exists in the thermodynamically stable cellulose II form. The differentiation of cellulose into two distinct regions, the amorphous zone and the crystalline zone, is probably a simplified description of the submicroscopic morphology [21]. In general, chemical reactions cannot take place easily in the crystalline regions of cellulose. Swelling in a compatible solvent may be needed for the adequate utilization of hydroxyl groups in cellulose-based matrix. On the other hand, embedding of TiO2 particles into regenerated cellulose should affect the semicrystalline structure of the matrix, which may bring an unacquainted accessibility of activation reaction. In order to further elucidate this kind of effect, several factors influencing the activation reaction, such as the amount of epichlorohydrin, the concentration of NaOH, and the contents of TiO2 and cellulose in matrix, would be investigated systematically in the present work.
2. Experimental 2.1. Materials Degreasing cotton with a degree of polymerization 1620 was ordered from Huamei Biotech Ltd. (Shanghai, China). Rutile TiO2 powder with a density of 4.2 g/cm3 and a size of 300 nm was provided by Huachang Polymer Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma (MO, USA). All other reagents were of analytical reagent grade and purchased from local suppliers.
Y.-L. Lei et al. / Reactive & Functional Polymers 62 (2005) 169–177
2.2. Preparation of composite matrix The cellulose xanthate viscose was prepared by reacting 45 g alkali-treated and aged degreasing cotton with 20 mL CS2 and then dissolving into 6 wt% NaOH solution. The composite matrix based on TiO2-densified cellulose beads was prepared through the method of water-in-oil suspension thermal regeneration, described by the former paper [22]. Briefly, a given mass of TiO2 powder was mixed thoroughly with 100 g viscose (containing 6–9% cellulose). The mixture was dispersed in a solution of 200 mL chlorobenzene and 400 mL pump oil in a 1 l flask with agitation at 350 rpm for 0.5 h. The suspension was heated to 95 C for 1 h, then cooled down and filtered. The produced particles were washed with methanol. The decomposition of cellulose xanthate was completed by immersing particles in a solution of 50 mL acetic acid and 200 mL ethanol. Finally, about 30 mL beads with a diameter of 100–300 lm were obtained by washing with water and sieving. The composite matrix was nominated as Cell-Ti.
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dorf, Germany. About 2 mL glass beads (0.3 mm diameter) were added to improve flow distribution at the column inlet. A movable top adapter was employed to adjust the position of liquid outlet just above the solid–liquid interface. A peristaltic pump (Lange Ltd., Baoding, China) was used for fluid supply. All operations were performed at 25 C with a settled bed height of 15.0 ± 0.2 cm. 2.5. Protein adsorption tests BSA was used as a model adsorbate in the present work for protein adsorption, with 50 mM Tris–HCl buffer (pH 7.5) as the fluid phase. A XK16/20 column (Amersham Biosciences, Sweden) was employed for packed bed adsorption with a settled bed height of 10.0 ± 0.2 cm, while the expanded bed mentioned above was adopted for EBA. The protein solution was continuously injected by the pump, and the BSA concentration at column outlet was monitored by a UV detector (Amersham Biosciences, Uppsala). All operations were carried out at 25 C.
2.3. Preparation of anion exchanger 2.6. Assays Cell-Ti was activated by epichlorohydrin, and then attached to diethylamine to produce an anion exchanger with diethylaminohydroxypropyl (DEAHP) weak base groups. The specific experimental conditions for activation reaction would be studied in Section 3.1. The activated beads (30 mL) were extracted with acetone in a Soxhlet apparatus for 4 h to remove low molecular weight polymers, and added into a solution of 90 mL dioxane and 90 mL diethylamine. Then, the mixture was heated to the set temperature and stirred for 12 h. The product was washed with 500 mL water and stored in 20% ethanol. The anion exchanger was designated Cell-Ti DEAHP. 2.4. Expanded bed operation A home made column (1000 · 20 mm ID) was used for expanded bed operation, which was a kind gift from Heinrich-Heine Universita¨t Du¨ssel-
The content of epoxy groups in the activated matrix was determined by the method described by Sundberg and Porath [15], and the content of DEAHP functional groups was analyzed with respect to the nitrogen content, by the quantitative elementary analysis with a 1106 elemental autoanalysis apparatus (Carlo-Erba, Italy). The ion-exchange capacity for small ions was determined by the HCl–NaOH titration. The infrared spectra were recorded on a Nicolet 170SX Fourier transform infrared spectrometer (Hitachi, Japan). SEMs were performed with an X-650 scanning electron microscope (Hitachi, Japan). Before operating SEM, samples were dehydrated by stepwise transfer into water–ethanol mixtures, followed by ethanol and finally diethyl ether. The diethyl ether was removed at room temperature. The degree of expansion E was measured as H/H0, where H was the expanded bed height and H0 was the sedimented bed height.
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3. Results and discussion 3.1. Factors on epoxy-activation For epoxy-activated reaction, the following parameters were studied in the present work during the activation of cellulose matrix: reaction time, concentration of NaOH, amount of epichlorohydrin, contents of TiO2 and cellulose. 3.1.1. Reaction temperature and time Activation of hydroxyl-containing polymers by epichlorohydrin is generally carried out at room temperature, because high temperature results in not only the reduction of reaction time but the loss of epoxy groups due to hydrolysis [15]. Fig. 1 shows the available epoxy groups in activated Cell-Ti and cellulose beads as a function of reaction time. As a result, the amount of activated epoxy group ascended to the maximum value after the reaction proceeded for 7–8 h. Compared to the case of cellulose beads, the activated Cell-Ti possesses more epoxy groups (up to 215 lmol/mL). This may be attributed to the addition of TiO2 superfine particles in the matrix, which looses the network structure of Cell-Ti. SEM images of both
matrices testified to this presumption, as exhibited in Fig. 2(a) and (b). From the practical viewpoint, TiO2 superfine particles played not only a role of densifier to impart an increased density to the composite matrix, but also a role of loose filler to facilitate the activation. 3.1.2. Concentration of NaOH Cross-linking and activation occur simultaneously when epichlorohydrin reacts with hydroxylcontaining polymers. Fig. 3 investigated the content of epoxy groups in the activated Cell-Ti beads as a function of NaOH concentration. The maximum content was found at a starting concentration of 3.0 mol/L NaOH. With increasing NaOH concentration, more hydrogen-bonds between the chains of regenerated cellulose had been broken, bringing more hydroxyl groups to be activated by epichlorohydrin. But at NaOH concentrations above 3.0 mol/L, chemical cross-linking through epichlorohydrin and the hydrolysis of epoxy groups tended to take place, which leaded to the obvious decrease of the content of epoxy groups with an increase of NaOH concentration. Similar results had been observed by Kong et al. [23]. Consequently, the optimal NaOH concentration for the activation reaction is 2.5–3.0 mol/L.
Epoxy group (µmol/mL)
250
3.1.3. Amount of epichlorohydrin Fig. 4 shows the content of available epoxy groups as a function of the amount of epichlorohydrin used, which indicates that excessive epichlorohydrin should be added into the reaction system in order to acquire a favorable activation result. According to the conclusion drawn by Kong et al. [23], when the mole ratio of epichlorohydrin to NaOH was less than 1.0, a majority of epichlorohydrin would be consumed by the cross-linking reaction rather than by activation.
Cellulose Beads Cell-Ti Beads
200
150
100
50
0 0
1
2
3
4
5
6
7
8
9
Reaction time (h) Fig. 1. Epoxy group content as a function of reaction time. Experiments were performed at 25 C, with 20 g Cell-Ti or cellulose beads in 40 mL of 2.5 mol/L NaOH solution containing 10 mL epichlorohydrin. The weight ratio of TiO2 to viscose was 15/100, and the content of cellulose in viscose was 7.5 wt%.
3.1.4. Content of TiO2 In the present work, TiO2 is used to densify the cellulose-based beads. With the weight ratio of TiO2 to viscose increased from 5/100 to 30/100, the wet density of composite beads increased accordingly from 1.12 to 1.35 g/cm3. It is declared
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Fig. 2. SEM images of matrices: (a) cellulose beads and (b) Cell-Ti beads.
once more that the embedding of TiO2 particles benefits the activation, by holding a high content of 164 lmol epoxy groups per mL beads even at a TiO2/viscose ratio of 30/100 (shown in Fig. 5).
3.1.5. Content of cellulose Fig. 6 exhibits the epoxy group contents at a series of cellulose contents in the starting viscose. The result indicates that the increase of cellulose content helps to gain more available epoxy groups. However, it is noticeable that too high cellulose content would make the matrix beads more compact, consequently preventing macromolecular adsorbates from permeating through. Here, the content of cellulose in viscose is chosen at 7.5 wt%.
3.2. Coupling with diethylamine At present, several cellulose-based anion exchangers having aminoethyl (AE), diethylaminoethyl (DEAE) and triethylaminoethyl (TEAE) groups as the ligands are commercially available [24,25]. These products are usually prepared by reacting halogenated amines with alkali-activated hydroxyls of cellulose. Here, diethylamine was coupled to epoxy-activated cellulose-based matrix for producing an anion exchanger with DEAHP groups, exhibiting a weak basicity similar to that DEAE groups [26,27]. The authors have studied the reaction of amine with the copolymer of glycidyl methacrylate
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Epoxy group (µmol/mL)
Epoxy group (µmol/mL)
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2
3
4
5
6
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0.15
0.20
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0.30
0.35
TiO2/Viscose ( g/g )
NaOH concentration (mol/L) Fig. 3. Epoxy group content as a function of NaOH concentration. Experiments were performed at 25 C for 8 h, with 20 g Cell-Ti beads in 40 mL NaOH solution containing 8 mL epichlorohydrin. The weight ratio of TiO2 to viscose was 15/100, and the content of cellulose in viscose was 7.5 wt%.
Fig. 5. Epoxy group content as a function of TiO2/viscose. The experimental conditions were the same as those in Fig. 3, expect the ratio of TiO2/vicose varied.
250
Cellulose in viscose
Epoxy group (µmol/mL)
9.0% 200
150
100
8.0%
7.5%
6.0% 50
0
0 0
0.5
1.0
1.5
2.0
2.5
3.0
Epichlorohydrin/NaOH (mol/mol) Fig. 4. Epoxy group content as a function of epichlorohydrin amount. The experimental conditions were the same as those in Fig. 3, expect the amount of epichlorohydrin varied.
(GMA), illustrating some restrictive factors in the heterogeneous system [28]. Here for the coupling of diethylamine with activated Cell-Ti beads, Fig. 7 shows the coupling efficiency as a function of reaction time at 25 and 50 C, respectively. The result indicates that prolonging time and/or raising temperature tended to in-
50
100
150
200
250
300
Epoxy group ( µmol/mL) Fig. 6. Epoxy group content as a function of cellulose content in viscose. The experimental conditions were the same as those in Fig. 3, expect the content of cellulose in viscose varied.
crease coupling efficiency. After carrying out, for 12 h at 50 C, the efficiency was especially close to 90%. In view of the boiling point of diethylamine at about 56 C, a favorable coupling condition was determined as follows: rotating 30 mL activated matrix in a solution of 90 mL dioxane and 90 mL diethylamine for at least 12 h at 50 C.
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ence of epoxy groups, which disappear in that of Cell-Ti DEAHP. The infrared spectra validate the efficiency in the different steps of Cell-Ti DEAHP preparation.
100 o
25 C o 55 C
Coupling efficiency
80
175
60
3.4. Expanded bed characteristics
40
20
0 0
2
4
6
8
10
12
Reaction time (h) Fig. 7. Coupling efficiency as a function of reaction time.
It is important for the EBA adsorbent to expand effectively in the column. Fig. 9 hereby measured the expanded bed characteristics of Cell-Ti DEAHP prepared, showing that the adsorbent could be expanded almost linearly in serial of mobile phases. The detailed examination for the hydrodynamic properties of Cell-Ti beads in an expanded bed has been made in the former paper [29].
3.3. Infrared spectra
3.5. Protein breakthrough capacities
Fig. 8 displays the infrared spectra of cellulose, Cell-Ti, activated Cell-Ti and Cell-Ti DEAHP. In Fig. 8, the adsorption peak of Cell-Ti at 682 cm 1 is due to the contribution of the stretching vibration of Ti–O. The peaks of activated Cell-Ti at 1244, 937 and 852 cm 1 account for the exist-
First, the ion-exchange capacity of Cell-Ti DEAHP was determined to be 0.2 mmol Cl /mL adsorbent. The BSA breakthrough capacity of Cell-Ti DEAHP was then measured in an expanded bed, with a comparison to that in a packed bed. As shown in Fig. 10, protein breakthrough behavior or dynamic adsorption efficiency in expanded bed accords with that in packed bed, except for a small advance in breakthrough time, which demonstrated that Cell-Ti DEAHP prepared in the present work might be used as an
3.5 in 20% glycerol in 10% glycerol in water
Expansion degree, E
3.0
2.5
2.0
1.5
1.0 0
100
200
300
400
500
600
700
Flow rate (cm/h) Fig. 8. Infrared spectra at various preparation stages.
Fig. 9. Expanded bed characteristics of Cell-Ti DEAHP.
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From this viewpoint, the study in this paper is a heuristic attempt. Another effort which embeds more dense stainless steel particles into regenerated cellulose beads is under way.
1.00 in packed bed in expanded bed
C/C0
0.75
0.50
Acknowledgements 0.25
0.00 0
10
20
30
40
50
60
70
80
Load BSA (mg/mL adsorbent) Fig. 10. Breakthrough curves in expanded and packed beds. The initial concentration of BSA is 1.0 mg/mL.
efficient anion exchanger for EBA. At the breakthrough of 10% of the initial concentration, the adsorption capacity was calculated to be 42.6 mg BSA/mL adsorbent.
4. Conclusions A novel anion exchanger for expanded bed adsorption was exploited, based on TiO2-densified cellulose beads as matrix. The activation of TiO2densified cellulose matrix by epichlorohydrin was investigated in details. The optimal procedure was obtained as follows: mixing 20 g matrix beads in 40 mL NaOH solution containing 8 mL epichlorohydrin for 8 h at 25 C. The coupling efficiency of diethylamine with activated matrix is close to 90% under the given conditions. The prepared adsorbent of Cell-Ti DEAHP has favorable expanded bed characteristics and an accordant protein breakthrough behavior with the dynamic adsorption capacity of 42.6 mg BSA/mL adsorbent at 10% breakthrough. It was demonstrated that when applying the composite TiO2-densified cellulose beads to EBA it is possible to take advantage of the regular spherical shape, high porosity, hydrophilicity, chemical reactivity and mechanical strength of individual particles. We believe that the results achieved in the field of EBA will promote greater enthusiasm for the large-scale manufacture of cellulose-based matrices in the next few years.
The authors wish to thank Prof. M.-R. Kula (Institute of Enzyme Technology, Heinrich-Heine Universita¨t Du¨sseldorf) for presenting the expanded bed column, and the National Natural Science Foundation of China for the financial support (No. 20206029).
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