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Preparation of Supermacroporous Composite Cryogel Embedded with SiO2 Nanoparticles
BIOTECHNOLOGY AND BIOENGINEERING Chinese Journal of Chemical Engineering, 18(4) 667ü671 (2010)
Preparation of Supermacroporous Composite Cryogel Embedded with SiO2 Nanoparticles* XU Panping (༘૬)1, YAO Yuchen (ྍုт)2, SHEN Shaochuan (ಋҎ)1, YUN Junxian (䋴ࢋບ)1 and YAO Kejian (ྍࢸ߆)1,** 1 2
State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, China School of Chemical Engineering, University of New South Wales, Sydney NSW 2052, Australia
Abstract Supermacroporous composite cryogels embedded with SiO2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide (AAm) and N,N-methylene-bis-acrylamide (MBAAm) containing SiO2 nanoparticles (mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08) under the freezing-temperature variation condition in glass columns. The properties of these composite cryogels were measured. The height equivalent to theoretical plate (HETP) of the cryogel beds at different liquid flow rates was determined by residence time distribution (RTD) using tracer pulse-response method. The composite cryogel matrix embedded with the mass fraction of SiO2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization. Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was carried out to evaluate the protein breakthrough and elution characteristics. The chromatography can be carried out at relatively high superficial velocity, i.e., 15 cm·min1, indicating the satisfactory mechanical strength due to the embedded nanoparticles. Keywords composite cryogel, SiO2 nanoparticle, protein chromatography
1
INTRODUCTION
Supermacroporous cryogel monolithic chromatography is a new method for separation and purification of biomolecules, such as proteins, plasmid DNA and viruses, from crude feedstocks [13]. Cryogel has interconnected pores with sizes of several microns to several hundreds of microns, which permit the solid particulates in the culture fluids to pass through freely without blockage. Different kinds of cryogels have been prepared successfully [211]. However, as the cryogels are relatively soft and flexible [12, 13], they may be compressed at high flow velocities. In recent years, polymer-nanoparticle composite materials have attracted the interest of researchers due to the synergistic and hybrid properties derived from several components [1418]. In previous studies, we prepared composite cryogels embedded with Fe3O4 nanoparticles to improve the binding capacity of protein [6, 8]. The cryogel bed embedded with Fe3O4 nanoparticles showed good properties such as mechanical strength, porosity, permeability, dispersion and chromatography, but it is unstable under acidic condition. In this study, a new composite cryogel embedded with SiO2 nanoparticles is developed because of the special properties of SiO2 [17, 1921]. Different mass ratios of SiO2 nanoparticles are used to mix with the monomer solution (AAm and MBAAm) and the cryogenic copolymerization is conducted under the freezing-temperature variation condition in glass columns. The properties of these cryogels are tested and the satisfactory matrix is grafted with AMPSA. The pro-
tein adsorption capacities of the cryogel are measured at different flow velocities. 2 2.1
EXPERIMENTAL Materials
Acrylamide (AAm, 99.9%) and lysozyme were supplied by Sangon (Shanghai, China). N,N-methylenebis-acrylamide (MBAAm, 99%) and 2-acrylamido-2methyl-1-propanesulfonic acid (AMPSA, 99%) were bought from Sigma-Aldrich (Steinheim, Germany). SiO2 nanoparticles (99.8%, BET: 380 m2·g1, particle size: 7 to 40 nm) were bought from Aladin (Shanghai, China). N,N,Nƍ,Nƍ-tetramethylethylenediamine (TEMED, 99%), ammonium persulfate (APS, 98%), and other chemicals were of analytical grade obtained from local sources. 2.2 Preparation of composite cryogel columns embedded with SiO2 nanoparticles The composite supermacroporous cryogels were prepared in 10 mm inner-diameter glass columns under the freezing temperature variation condition as reported previously [6]. The reactant mixture of monomers (AAm and MBAAm) and SiO2 nanoparticles were initiated by TEMED and APS. Monomers (4.6 g of AAm and 1 g of MBAAm) were dissolved in 74.3 ml of deionized water and different amounts of SiO2 nanoparticles (the mass ratios of nanoparticles to
Received 2010-01-13, accepted 2010-04-23. * Supported by the National Natural Science Foundation of China (20876145) and the Natural Science Foundation of Zhejiang Province (Y4080329). ** To whom correspondence should be addressed. E-mail: [email protected]
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AAm from 0.01 to 0.08) were added with sufficient stirring. Then, 0.028 g of TEMED and 0.0672 g of APS were added into the mixture, which was poured into eight glass columns (I. D. 10 mm, length 120 mm). After the reaction, the column was fitted with a commercial top adaptor and washed with 100 ml of 0.1 mol·L1 HCl and a mass of deionized water [5, 6, 8]. 2.3
Properties of cryogel columns
Properties of cryogels were measured with the method described previously [48]. Briefly, a pulse response technique was used to measure the residence time distribution (RTD). Acetone solution [3% (by volume)] was used as tracer (150 ȝl) and injected into the cryogel column. At the same time, the response signals were measured by UV spectrometer at 280 nm and recorded by a computer with an A/D transformer. The total theoretical plate number N was calculated by the retention time tR and the half-height peak width w1/2 from the RTD curves according to the following formula 2
§ t · N 5.54 ¨ R ¸ (1) © w1/ 2 ¹ The values of HETP were calculated by the total theoretical plate number and the monolith length L (HETP L/N) [48, 22, 23]. The porosity of cryogel monolith M was estimated by measuring the content of free water and the monolith volume of a given sample, similar to the procedure reported [24, 25]. The cryogel structure was measured by scanning electron microscope (SEM) (S-4700-ESEM, HITACHI, Japan). 2.4
In each run, lysozyme solution (1 mg·ml1 in 20 mmol·L1 phosphate buffer, pH 7.2) was applied to the column followed by washing with phosphate buffer. Elution was performed with 1.5 mol·L1 NaCl in phosphate buffer (20 mmol·L1, pH 7.2) and re-equilibration was performed with phosphate buffer. The lysozyme contents were measured at the optical density of 280 nm and the amounts of lysozyme by elution were used to calculate the protein binding capacities of the cryogel under the corresponding conditions. 3 3.1
RESULTS AND DISCUSSION Porosity and permeability of cryogels
Figure 1 displays the porosities M of the composite cryogel matrix with different mass ratios of SiO2 nanoparticles to AAm. The porosities of these cryogels are in the range from 79% to 89% and the effect of the amount of embedded SiO2 nanoparticles on the porosity is insignificant. The reason is that the volume fractions of nanoparticles within the cryogel matrix are small [about 1% to 8% (by volume) for the mass ratio of SiO2 nanoparticles to AAm increased from 0.01 to 0.08]. The porosities of these cryogels are slightly higher than those of classical polymeric monoliths or cryogels embedded with Fe3O4 nanoparticles, which are about 70% to 73% [6, 12]. The porosities of cryogels without embedded nanoparticles [15, 7, 29] are around 80%, which are close to the present composite cryogels. The cryogel structure at the mass ratio 0.02 of nanoparticles to AAm observed by SEM is shown in Fig. 2. The pores within the cryogel are inter-connected and with a few dead pores.
Graft composite cryogel
The cryogel matrix with satisfactory properties was used in the graft polymerization. K5[Cu(HIO6)2] solution (0.0562 mol·L1) was prepared according to references [2628]. The volume ratio of K5[Cu(HIO6)2] solution to NaOH (1 mol·L1) was 2Ή1, mixed as initiator and pumped through the given cryogel column at a constant flow velocity of 1.5 cm·min1. Then the cryogel was sealed and incubated at 48°C water bath for 45 min. Approximately 20 ml AMPSA monomer solution (2 mol·L1) was pumped into the cryogel bed. The graft polymerization was carried out at 48°C for 2 h. Finally, the cryogel was washed with 0.1 mol·L1 HCl and deionised water for further use. 2.5 Chromatography of lysozyme at different flow velocities Protein chromatography was carried out at the liquid velocities of 1, 5, 10 and 15 cm·min1 separately at room temperature. The chromatographic process was monitored using the same UV spectrometer at 280 nm.
Figure 1 Porosities of the composite cryogel matrix with different amounts of SiO2 nanoparticles
Figure 3 shows the relationship between the pressure drop across the column and the flow rate of the liquid in the cryogel columns with different mass amounts of SiO2 nanoparticles. The water permeability kw of the eight cryogels are calculated from the experimental data and summarized in Table 1. The cryogel with the mass ratio of 0.02 has better porous networks with interconnected pores than those with other mass ratios.
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Figure 2 SEM photograph of the composite cryogel embedded with SiO2 nanoparticles at mass ratio of 0.02
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Figure 4 HETP at various superficial liquid velocities in cryogel beds with different mass ratios of SiO2 nanoparticles mass ratio: ƶ 0.01; ƽ 0.02; Ƹ 0.03; ͩ 0.04; 0.05; 0.06; ƹ 0.07; 0.08
3.3 Protein chromatographic behavior in the grafted cryogel column
Figure 3 Flow rate as a function of pressure drop in the composite cryogel columns with different mass ratios of SiO2 nanoparticles (liquid viscosity ȝw 1×103 Pa·s) mass ratio: ƶ 0.01; ƽ 0.02; Ƹ 0.03; ͪ 0.04; 0.05; 0.06; ƺ 0.07; 0.08
Figure 5 shows the adsorption, washing, and elution profiles of lysozyme in 20 mmol·L1 phosphate buffer solution at liquid flow velocities of 1, 5, 10 and 15 cm·min1, where C and C0 are the concentrations of lysozyme in the effluent and the initial solution, respectively, and V is the loaded volume. The breakthrough curves are close and the adsorbed lysozyme molecules are eluted effectively by 1.5 mol·L1 NaCl in phosphate buffer (20 mmol·L1, pH 7.2). Fig. 6 shows the
Table 1 Permeability and length of the composite cryogel matrix with different mass ratios of SiO2 nanoparticles (column inner diameter: 10 mm) Mass ratios of SiO2 nanoparticles to AAm
kw×1011/m2
Cryogel length /mm
0.01
0.85
89
0.02
1.43
81
0.03
1.55
86
0.04
1.40
77
0.05
2.34
77
0.06
1.58
88
0.07
1.55
87
0.08
0.91
87
3.2
Figure 5 Breakthrough, wash, and elution profiles of 1 mg·ml1 lysozyme solution in 20 mmol·L1 phosphate for the grafted composite cryogel with 2 mol·L1 AMPSA at different liquid velocities (The column was washed with 1.5 mol·L1 NaCl in 20 mmol·L1 phosphate) liquid velocity/cm·min1:ƶ1;ƽ5; 10;Ƹ15
HETP of cryogel columns
The measurements of RTD were carried out to evaluate the HETP in cryogel columns at different flow velocities, as shown in Fig. 4. The values of HETP with low mass ratios of SiO2 nanoparticles (0.01 to 0.04) are in the range of 0.4 to 0.8 mm, while those with high ratios (0.05 to 0.08) are in the range of 1 to 2.3 mm. The values of HETP illustrate different axial dispersion and thus the separation efficiency [4]. The lowest value of HETP (0.05 cm) is at the mass ratio of 0.02, which was employed in the following graft polymerization.
Figure 6 Effect of liquid velocity on lysozyme binding capacity of the grafted cryogels with 2 mol·L1 AMPSA
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relationship between the flow velocity and binding capacity of the grafted composite cryogel. The lysozyme binding capacities are 1.21, 1.13, 1.02 and 0.98 mg·ml1 wet cryogel bed at flow velocities of 1, 5, 10 and 15 cm·min1, respectively. The SiO2 nanoparticles enhance the mechanical strength of cryogel, as the chromatographic process can be carried out at high flow velocities, e.g., 15 cm·min1. Although the protein binding capacity decreases slightly at high velocities, the property of SiO2 nanoparticles is inert and steady under acidic condition.
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CONCLUSIONS 8
The cryogel embedded with SiO2 nanoparticles can be prepared by cryo-copolymerization of acrylamidebased monomers in frozen state under the variation of freezing temperature. At the mass ratio of 0.02 of SiO2 nanoparticles to AAm, the composite matrix presents better properties, including high porosity, high permeability and weak axial dispersion in a wide range of liquid flow velocities. Due to the embedded adsorbent nanoparticles, the flow velocity for chromatography operation is improved. Furthermore, the cation exchange groups can be grafted to these crygels to get ion exchange cryogels for chromatography at high flow velocities. The new composite cryogel matrix may be applied to the separation of biomolecules in downstream processes.
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NOMENCLATURE 14 Dax HETP kw L N t
t tR UL u ȝw ȡw
V M
2 t
axial dispersion coefficient , m2·s1 height equivalent to theoretical plate, m water permeability of the cryogel bed, m2 length of cryogel bed, m theoretical plate number, dimensionless time, s mean residence time, s retention time, s superficial liquid velocity, cm·min1 interstitial flow velocity, m·s1 water viscosity, Pa·s water density, kg·m3 variance of RTD, m2 cryogel bed porosity
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Preparation of Supermacroporous Composite Cryogel Embedded with SiO2 Nanoparticles* XU Panping (༘૬)1, YAO Yuchen (ྍုт)2, SHEN Shaochuan (ಋҎ)1, YUN Junxian (䋴ࢋບ)1 and YAO Kejian (ྍࢸ߆)1,** 1 2
State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, China School of Chemical Engineering, University of New South Wales, Sydney NSW 2052, Australia
Abstract Supermacroporous composite cryogels embedded with SiO2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide (AAm) and N,N-methylene-bis-acrylamide (MBAAm) containing SiO2 nanoparticles (mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08) under the freezing-temperature variation condition in glass columns. The properties of these composite cryogels were measured. The height equivalent to theoretical plate (HETP) of the cryogel beds at different liquid flow rates was determined by residence time distribution (RTD) using tracer pulse-response method. The composite cryogel matrix embedded with the mass fraction of SiO2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization. Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was carried out to evaluate the protein breakthrough and elution characteristics. The chromatography can be carried out at relatively high superficial velocity, i.e., 15 cm·min1, indicating the satisfactory mechanical strength due to the embedded nanoparticles. Keywords composite cryogel, SiO2 nanoparticle, protein chromatography
1
INTRODUCTION
Supermacroporous cryogel monolithic chromatography is a new method for separation and purification of biomolecules, such as proteins, plasmid DNA and viruses, from crude feedstocks [13]. Cryogel has interconnected pores with sizes of several microns to several hundreds of microns, which permit the solid particulates in the culture fluids to pass through freely without blockage. Different kinds of cryogels have been prepared successfully [211]. However, as the cryogels are relatively soft and flexible [12, 13], they may be compressed at high flow velocities. In recent years, polymer-nanoparticle composite materials have attracted the interest of researchers due to the synergistic and hybrid properties derived from several components [1418]. In previous studies, we prepared composite cryogels embedded with Fe3O4 nanoparticles to improve the binding capacity of protein [6, 8]. The cryogel bed embedded with Fe3O4 nanoparticles showed good properties such as mechanical strength, porosity, permeability, dispersion and chromatography, but it is unstable under acidic condition. In this study, a new composite cryogel embedded with SiO2 nanoparticles is developed because of the special properties of SiO2 [17, 1921]. Different mass ratios of SiO2 nanoparticles are used to mix with the monomer solution (AAm and MBAAm) and the cryogenic copolymerization is conducted under the freezing-temperature variation condition in glass columns. The properties of these cryogels are tested and the satisfactory matrix is grafted with AMPSA. The pro-
tein adsorption capacities of the cryogel are measured at different flow velocities. 2 2.1
EXPERIMENTAL Materials
Acrylamide (AAm, 99.9%) and lysozyme were supplied by Sangon (Shanghai, China). N,N-methylenebis-acrylamide (MBAAm, 99%) and 2-acrylamido-2methyl-1-propanesulfonic acid (AMPSA, 99%) were bought from Sigma-Aldrich (Steinheim, Germany). SiO2 nanoparticles (99.8%, BET: 380 m2·g1, particle size: 7 to 40 nm) were bought from Aladin (Shanghai, China). N,N,Nƍ,Nƍ-tetramethylethylenediamine (TEMED, 99%), ammonium persulfate (APS, 98%), and other chemicals were of analytical grade obtained from local sources. 2.2 Preparation of composite cryogel columns embedded with SiO2 nanoparticles The composite supermacroporous cryogels were prepared in 10 mm inner-diameter glass columns under the freezing temperature variation condition as reported previously [6]. The reactant mixture of monomers (AAm and MBAAm) and SiO2 nanoparticles were initiated by TEMED and APS. Monomers (4.6 g of AAm and 1 g of MBAAm) were dissolved in 74.3 ml of deionized water and different amounts of SiO2 nanoparticles (the mass ratios of nanoparticles to
Received 2010-01-13, accepted 2010-04-23. * Supported by the National Natural Science Foundation of China (20876145) and the Natural Science Foundation of Zhejiang Province (Y4080329). ** To whom correspondence should be addressed. E-mail: [email protected]
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AAm from 0.01 to 0.08) were added with sufficient stirring. Then, 0.028 g of TEMED and 0.0672 g of APS were added into the mixture, which was poured into eight glass columns (I. D. 10 mm, length 120 mm). After the reaction, the column was fitted with a commercial top adaptor and washed with 100 ml of 0.1 mol·L1 HCl and a mass of deionized water [5, 6, 8]. 2.3
Properties of cryogel columns
Properties of cryogels were measured with the method described previously [48]. Briefly, a pulse response technique was used to measure the residence time distribution (RTD). Acetone solution [3% (by volume)] was used as tracer (150 ȝl) and injected into the cryogel column. At the same time, the response signals were measured by UV spectrometer at 280 nm and recorded by a computer with an A/D transformer. The total theoretical plate number N was calculated by the retention time tR and the half-height peak width w1/2 from the RTD curves according to the following formula 2
§ t · N 5.54 ¨ R ¸ (1) © w1/ 2 ¹ The values of HETP were calculated by the total theoretical plate number and the monolith length L (HETP L/N) [48, 22, 23]. The porosity of cryogel monolith M was estimated by measuring the content of free water and the monolith volume of a given sample, similar to the procedure reported [24, 25]. The cryogel structure was measured by scanning electron microscope (SEM) (S-4700-ESEM, HITACHI, Japan). 2.4
In each run, lysozyme solution (1 mg·ml1 in 20 mmol·L1 phosphate buffer, pH 7.2) was applied to the column followed by washing with phosphate buffer. Elution was performed with 1.5 mol·L1 NaCl in phosphate buffer (20 mmol·L1, pH 7.2) and re-equilibration was performed with phosphate buffer. The lysozyme contents were measured at the optical density of 280 nm and the amounts of lysozyme by elution were used to calculate the protein binding capacities of the cryogel under the corresponding conditions. 3 3.1
RESULTS AND DISCUSSION Porosity and permeability of cryogels
Figure 1 displays the porosities M of the composite cryogel matrix with different mass ratios of SiO2 nanoparticles to AAm. The porosities of these cryogels are in the range from 79% to 89% and the effect of the amount of embedded SiO2 nanoparticles on the porosity is insignificant. The reason is that the volume fractions of nanoparticles within the cryogel matrix are small [about 1% to 8% (by volume) for the mass ratio of SiO2 nanoparticles to AAm increased from 0.01 to 0.08]. The porosities of these cryogels are slightly higher than those of classical polymeric monoliths or cryogels embedded with Fe3O4 nanoparticles, which are about 70% to 73% [6, 12]. The porosities of cryogels without embedded nanoparticles [15, 7, 29] are around 80%, which are close to the present composite cryogels. The cryogel structure at the mass ratio 0.02 of nanoparticles to AAm observed by SEM is shown in Fig. 2. The pores within the cryogel are inter-connected and with a few dead pores.
Graft composite cryogel
The cryogel matrix with satisfactory properties was used in the graft polymerization. K5[Cu(HIO6)2] solution (0.0562 mol·L1) was prepared according to references [2628]. The volume ratio of K5[Cu(HIO6)2] solution to NaOH (1 mol·L1) was 2Ή1, mixed as initiator and pumped through the given cryogel column at a constant flow velocity of 1.5 cm·min1. Then the cryogel was sealed and incubated at 48°C water bath for 45 min. Approximately 20 ml AMPSA monomer solution (2 mol·L1) was pumped into the cryogel bed. The graft polymerization was carried out at 48°C for 2 h. Finally, the cryogel was washed with 0.1 mol·L1 HCl and deionised water for further use. 2.5 Chromatography of lysozyme at different flow velocities Protein chromatography was carried out at the liquid velocities of 1, 5, 10 and 15 cm·min1 separately at room temperature. The chromatographic process was monitored using the same UV spectrometer at 280 nm.
Figure 1 Porosities of the composite cryogel matrix with different amounts of SiO2 nanoparticles
Figure 3 shows the relationship between the pressure drop across the column and the flow rate of the liquid in the cryogel columns with different mass amounts of SiO2 nanoparticles. The water permeability kw of the eight cryogels are calculated from the experimental data and summarized in Table 1. The cryogel with the mass ratio of 0.02 has better porous networks with interconnected pores than those with other mass ratios.
Chin. J. Chem. Eng., Vol. 18, No. 4, August 2010
Figure 2 SEM photograph of the composite cryogel embedded with SiO2 nanoparticles at mass ratio of 0.02
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Figure 4 HETP at various superficial liquid velocities in cryogel beds with different mass ratios of SiO2 nanoparticles mass ratio: ƶ 0.01; ƽ 0.02; Ƹ 0.03; ͩ 0.04; 0.05; 0.06; ƹ 0.07; 0.08
3.3 Protein chromatographic behavior in the grafted cryogel column
Figure 3 Flow rate as a function of pressure drop in the composite cryogel columns with different mass ratios of SiO2 nanoparticles (liquid viscosity ȝw 1×103 Pa·s) mass ratio: ƶ 0.01; ƽ 0.02; Ƹ 0.03; ͪ 0.04; 0.05; 0.06; ƺ 0.07; 0.08
Figure 5 shows the adsorption, washing, and elution profiles of lysozyme in 20 mmol·L1 phosphate buffer solution at liquid flow velocities of 1, 5, 10 and 15 cm·min1, where C and C0 are the concentrations of lysozyme in the effluent and the initial solution, respectively, and V is the loaded volume. The breakthrough curves are close and the adsorbed lysozyme molecules are eluted effectively by 1.5 mol·L1 NaCl in phosphate buffer (20 mmol·L1, pH 7.2). Fig. 6 shows the
Table 1 Permeability and length of the composite cryogel matrix with different mass ratios of SiO2 nanoparticles (column inner diameter: 10 mm) Mass ratios of SiO2 nanoparticles to AAm
kw×1011/m2
Cryogel length /mm
0.01
0.85
89
0.02
1.43
81
0.03
1.55
86
0.04
1.40
77
0.05
2.34
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0.06
1.58
88
0.07
1.55
87
0.08
0.91
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Figure 5 Breakthrough, wash, and elution profiles of 1 mg·ml1 lysozyme solution in 20 mmol·L1 phosphate for the grafted composite cryogel with 2 mol·L1 AMPSA at different liquid velocities (The column was washed with 1.5 mol·L1 NaCl in 20 mmol·L1 phosphate) liquid velocity/cm·min1:ƶ1;ƽ5; 10;Ƹ15
HETP of cryogel columns
The measurements of RTD were carried out to evaluate the HETP in cryogel columns at different flow velocities, as shown in Fig. 4. The values of HETP with low mass ratios of SiO2 nanoparticles (0.01 to 0.04) are in the range of 0.4 to 0.8 mm, while those with high ratios (0.05 to 0.08) are in the range of 1 to 2.3 mm. The values of HETP illustrate different axial dispersion and thus the separation efficiency [4]. The lowest value of HETP (0.05 cm) is at the mass ratio of 0.02, which was employed in the following graft polymerization.
Figure 6 Effect of liquid velocity on lysozyme binding capacity of the grafted cryogels with 2 mol·L1 AMPSA
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relationship between the flow velocity and binding capacity of the grafted composite cryogel. The lysozyme binding capacities are 1.21, 1.13, 1.02 and 0.98 mg·ml1 wet cryogel bed at flow velocities of 1, 5, 10 and 15 cm·min1, respectively. The SiO2 nanoparticles enhance the mechanical strength of cryogel, as the chromatographic process can be carried out at high flow velocities, e.g., 15 cm·min1. Although the protein binding capacity decreases slightly at high velocities, the property of SiO2 nanoparticles is inert and steady under acidic condition.
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CONCLUSIONS 8
The cryogel embedded with SiO2 nanoparticles can be prepared by cryo-copolymerization of acrylamidebased monomers in frozen state under the variation of freezing temperature. At the mass ratio of 0.02 of SiO2 nanoparticles to AAm, the composite matrix presents better properties, including high porosity, high permeability and weak axial dispersion in a wide range of liquid flow velocities. Due to the embedded adsorbent nanoparticles, the flow velocity for chromatography operation is improved. Furthermore, the cation exchange groups can be grafted to these crygels to get ion exchange cryogels for chromatography at high flow velocities. The new composite cryogel matrix may be applied to the separation of biomolecules in downstream processes.
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NOMENCLATURE 14 Dax HETP kw L N t
t tR UL u ȝw ȡw
V M
2 t
axial dispersion coefficient , m2·s1 height equivalent to theoretical plate, m water permeability of the cryogel bed, m2 length of cryogel bed, m theoretical plate number, dimensionless time, s mean residence time, s retention time, s superficial liquid velocity, cm·min1 interstitial flow velocity, m·s1 water viscosity, Pa·s water density, kg·m3 variance of RTD, m2 cryogel bed porosity
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