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Ind. Eng. Chem. Res. 2006, 45, 3624-3632
SEPARATIONS Determination of the Quantitative Relationships between the Synthesis Conditions of Macroporous Poly(styrene-co-divinylbenzene) Microparticles and the Characteristics of Their Behavior as Adsorbents Using Bovine Serum Albumin as a Model Macromolecule Cristina Garcia-Diego and Jorge Cuellar* Department of Chemical Engineering, UniVersity of Salamanca, Plaza de los Caidos 1-5, 37008 Salamanca, Spain
The adsorption properties of macroporous polymeric microparticles are dependent on their porous structure, which, in turn, is dependent on the synthesis conditions of the adsorbent material. In the present work, we have investigated whether it is feasible to find a quantitative relationship between the synthesis conditions of macroporous poly(styrene-co-divinylbenzene) adsorbent microparticles and their behavior in the adsorption of a protein of intermediate size. For this purpose, 10 types of such microparticles were synthesized from reactive mixtures with different divinylbenzene (cross-linker) and n-heptane (diluent) contents, and the equilibrium isotherms and adsorption kinetics of a model protein (the bovine serum albumin (BSA) protein) were studied. The results allowed us to determine some quantitative relationships that, within the limits of this research, permit the synthesis of adsorbents with predetermined adsorption properties. Thus, the model obtained, relating the synthesis conditions of the adsorbents to the adsorption equilibrium, reveals that the higher the divinylbenzene concentration and the higher the diluent content, the higher the maximum adsorption capacity of the adsorbents for this protein. With regard to adsorption kinetics, the fastest adsorption was achieved with the lowest divinylbenzene concentration and the highest diluent concentration. 1. Introduction In many downstream processes in the biotechnological industry, the separation and purification of biomolecules is performed by adsorption.1-5 A critical step in the adsorption processes is the choice of the most suitable adsorbent for a specific separation problem. It is desirable that this adsorbent should have a high adsorption capacity, fast kinetics in the adsorption-desorption process, and good mechanical and chemical stability. In this respect, polymeric adsorbents have proved to be useful in the adsorption of biomacromolecules and, in particular, macroporous poly(styrene-co-divinylbenzene) microparticles have been widely used, either in derivatized form or not.6-18 Their chemical nature and cross-linking are responsible for their high physical stability, and, with regard to their porous structure, their large pores allow rapid passage of biomolecules to the interior of each microparticle, whereas the shorter and numerous pores of small diameter provide the surface area for adsorption to occur. Many studies that address the synthesis of macroporous poly(styrene-co-divinylbenzene) microparticles have been conducted to investigate the influence of the synthesis conditions in the structural characteristics of such microparticles.19-29 The qualitative influence of the structural characteristics of the microparticles in the adsorption capacity of different types of molecules has also been studied.8,30 However, to our knowledge, * To whom correspondence should be addressed. Phone: +34923294479. Fax: +34923294574. E-mail:
[email protected].
there are no works that have reported either qualitative or quantitative relationships between the adsorption capacity of an adsorbent and its synthesis conditions, nor have we found any information about research focused on the quantitative influence of the synthesis conditions of the microparticles in the adsorption kinetics. In an earlier work,31 we described a thorough investigation in which we synthesized 10 different types of macroporous poly(styrene-co-divinylbenzene) microparticles and established some quantitative relationships between the structural characteristics of the microparticles (Brunauer-Emmett-Teller (BET) specific surface area, and macropore, mesopore, and micropore volumes) and their synthesis conditions with a view to synthesizing adsorbents with predetermined pore properties. Now, as an extension of that research, we report the study of the equilibrium isotherms and adsorption kinetics of the bovine serum albumin (BSA) protein, which is used as a model protein of intermediate size (140.9 Å × 41.6 Å × 41.6 Å ),32 on the surface of the 10 adsorbents. The first objective of this work is to find a quantitative relationship between the capacity of the macroporous poly(styrene-co-divinylbenzene) microparticles to adsorb BSA and their synthesis conditions. A further goal is to investigate the quantitative influence of the synthesis conditions of the microparticles in the adsorption kinetics of BSA on these microparticles. In both cases, the final objective is to facilitate the synthesis of adsorbent microparticles with predetermined adsorption properties. These quantitative relationships should be considered as a reasonable starting point in the choice of the most appropriate adsorbent for a specific adsorption process,
10.1021/ie051292l CCC: $33.50 © 2006 American Chemical Society Published on Web 04/06/2006
Ind. Eng. Chem. Res., Vol. 45, No. 10, 2006 3625 Table 1. Synthesis Conditions, Average Microparticle Radius, and Porosity of the Adsorbents
synthesis conditions was obtained by multiple nonlinear regression analysis.
Fmb
% DVBa
2. Experimental Section
adsorbent number
real values
coded values
real values
coded values
Rp (cm)
1 2 3 4 5 6 7 8 9 10
30.0 55.0 55.0 30.0 42.5 25.0 25.0 55.0 55.0 40.0
-2/3 +1 +1 -2/3 +1/6 -1 -1 +1 +1 0
0.40 0.40 0.60 0.60 0.50 0.50 0.70 0.70 0.50 0.60
-1 -1 +1/3 +1/3 -1/3 -1/3 +1 +1 -1/3 +1/3
0.0076 0.0081 0.0110 0.0085 0.0072 0.0072 0.0075 0.0075 0.0094 0.0072
0.752 0.768 0.572 0.471 0.672 0.535 0.147 0.333 0.646 0.523
a The divinylbenzene concentration (% DVB, w/w) is the weight percentage of the divinylbenzene isomers in the monomeric mixture used in the synthesis of the microparticles. b The monomeric fraction (Fm, v/v) is the volume fraction of monomers in the organic phase used in the synthesis of the microparticles.
either using the adsorbent without any change, or after modifying its adsorption properties through hydrophilic derivatization 7 or by coating the surface of the microparticles with a hydrophilic polymer.14 To achieve these objectives, adsorption isotherms of BSA on the 10 adsorbents were obtained from batch experiments, which permits us to determine the maximum adsorption capacity of the protein on each adsorbent by fitting the double Langmuir adsorption model to the isotherm data. Based on these results, the protein adsorption capacities of the adsorbents were correlated with their synthesis conditions by multiple linear regression analysis. Moreover, the kinetics of the adsorption process was determined and a quantitative relationship between the adsorption kinetics of the protein on the adsorbents and their
2.1. Chemicals and Buffers. The BSA protein (96% purity) was obtained from Sigma (Madrid, Spain). Methanol (highperformance liquid chromatography (HPLC) grade) was supplied by Scharlau (Barcelona, Spain). Salt-phosphate buffer solutions were prepared from monobasic anhydrous sodium phosphate, dibasic anhydrous potassium phosphate, and ammonium sulfate, all of them of reagent grade and all obtained from Panreac (Barcelona, Spain). The salt-buffer solutions (pH 6.8) contained 0.05 mol/dm3 of phosphate and 1 mol/dm3 of ammonium sulfate. 2.2. Adsorbents. Ten types of macroporous poly(styreneco-divinylbenzene) adsorbent microparticles with different structural characteristics were used to study the characteristics of the adsorption of BSA. These adsorbents were synthesized at our laboratory31 through a suspension polymerization technique, using different proportions of divinylbenzene (crosslinker) and n-heptane (diluent of the organic phase), as shown in Table 1. To facilitate the interpretation of the results, these proportions were coded, assigning a value of -1 to the low level and a value of +1 to the high level of the range studied for each factor j, through the coding equation
xj )
ξj - ξj,center ξj,max - ξj,center
(1)
where xj, ξj, ξj,center, and ξj,max are the coded, the real, the center real and the maximum real values of the range studied for the factor j, respectively.
Figure 1. Scanning electron microscopy (SEM) photomicrographs of (a) the spherical morphology, (b) the external surface, and (c) the inner structure of the adsorbent 5. Also shown are the inner structures of (d) adsorbent 3, (e) adsorbent 4, (f) adsorbent 6, (g) adsorbent 7, (h) adsorbent 8, (i) adsorbent 9, and (j) adsorbent 10. Photomicrographs of adsorbents 1 and 2 can be found elsewhere.31
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An average value of the particle radius for each adsorbent, Rp (given in centimeters), was obtained by sieving the microparticles (see Table 1). In addition, the porosity of the adsorbents () was determined from their volume of pores obtained by nitrogen adsorption and mercury porosimetry.31 Scanning electron microscopy (SEM) microphotographs of the adsorbents are shown in Figure 1, where the spherical morphology and the macroporous structure of the microparticles can be observed. 2.3. Operating Procedure. The equilibrium isotherms and the kinetics of the adsorption of BSA on each type of adsorbent were obtained via a batch method, using the procedure described below. Samples of each adsorbent were placed in a column and washed first with 2 column volumes of methanol and then with more than 10 column volumes of buffer, to displace any residual methanol. Following this, the adsorbent microparticles were equilibrated overnight with the buffer solution, which was later removed. The dry-weight fraction of the wet adsorbents was determined from the weight loss of adsorbent samples at 110 °C for 24 h in an oven. To obtain the adsorption isotherms, amounts of wet adsorbent (0.5 g approximately) were weighed and placed in tubes containing 10 mL of the salt-buffer solution with a known initial concentration of BSA. The solution was allowed to equilibrate for 24 h in a rotator mixer, which was placed inside an incubator to maintain the temperature at 25 °C. After this time, the BSA concentration in the supernatant (Ce, expressed in units of mg/ mL) was determined with a UV-Vis spectrophotometer (Varian, model Cary 50) at 280 nm. The amount of BSA adsorbed per unit dry weight of adsorbent (qe, expressed in units of mg/g of dry adsorbent) was obtained from a mass balance. To study the kinetics of adsorption of BSA on each type of microparticle, a salt-buffer solution with an initial protein concentration of 5 mg/mL was used. A volume of 10 mL of this solution was poured into tubes that contained known amounts of wet adsorbent. Each tube was allowed to rotate at 25 °C for a previously fixed time, after which point the BSA concentration in the supernatant (CBSA, given in units of mg/ mL) was determined. From these results, the amount of BSA adsorbed at different times (qads, given in units of mg/g of dry adsorbent) was calculated. 3. Results and Discussion 3.1. Adsorption Isotherms. The adsorption isotherms of BSA on all the adsorbents are shown in Figure 2, where qe is plotted versus Ce. Different adsorption models (Langmuir, double Langmuir, Freundlich, Langmuir-Freundlich, Toth, etc.) were fitted to the adsorption equilibrium data for each adsorbent in an attempt to find the best description of the adsorption phenomenon. Some models fitted the adsorption data very well for some adsorbents, but not for others. No model emerged as the best in all cases; however, the double Langmuir model was determined to be the best for most of the adsorbents. It was also observed that the calculated maximum adsorption capacities from the different models were very similar to each other for each adsorbent, not differing by more than 5%. Taking this into account, together with the fact that the maximum adsorption capacities of BSA were being investigated with the aim of obtaining a quantitative relationship between these maximum adsorption capacities and the synthesis conditions of the microparticles, the double Langmuir model was deemed a good enough model for all adsorbents. The double Langmuir model can be expressed as
Figure 2. Curves of the adsorption isotherms of bovine serum albumin (BSA) on the adsorbents used. Data points represent experimental data, and solid lines were calculated from the double Langmuir model (eq 2).
qe )
qm1b1Ce qm2b2Ce + 1 + b1Ce 1 + b2Ce
(2)
where qmi is the maximum adsorption capacity of BSA on the adsorption sites of type i, and bi is a parameter related to the affinity between the adsorbate and the adsorption sites of type i. This model assumes the presence of two types of adsorption sites (i ) 1, 2), although this assumption should be understood, taking into account that, even though adsorption sites with many different affinities toward the adsorbate may exist, it is possible to group that multiplicity of sites into two levels, the bi parameter being the average value of the affinity for the adsorption sites grouped in each level. The fit of the double Langmuir equation to the adsorption equilibrium data was accomplished by multiple nonlinear regression techniques. The best fit was observed for adsorbent 10. Thus, considering that (i) the chemical nature of all the adsorbents was very similar and (ii), as a result, the type of adsorption sites should not differ very much from one adsorbent to another, the values of the b1 and b2 parameters, obtained from the fitting of the adsorption data for adsorbent 10 (b1 ) 23.12 and b2 ) 0.59), were considered valid and were used in the estimation of the values of qmi for all the adsorbents. The calculated values of qm1 and qm2, as well as the correlation coefficient for each fit (R2), are shown in Table 2. From the values of qm1 and qm2, the total monolayer capacity, or maximum adsorption capacity of the adsorbents (qm), was calculated as the sum of qm1 and qm2, and these values are also given in Table 2. The curves resulting from these fits are plotted in Figure 2. 3.2. Correlation between the Maximum Adsorption Capacity of the Microparticles and Their Synthesis Conditions. An initial step in attaining the objective of synthesizing microparticles with predetermined adsorption characteristics is to determine the quantitative relationship between the synthesis conditions of the adsorbents and their maximum adsorption capacity. For this purpose, the values of qm (Table 2) were subjected to multiple linear regression analysis,33,34 affording the following second-order response surface model, which permits calculation of the adsorption capacity of the adsorbents as a function of the values of the %DVB and Fm used in their synthesis:
qˆ m ) 215.8 + 46.3x% DVB - 88.2xFm - 47.9x% DVBxFm 18.5x% DVB2 - 104.1xFm2 (3)
Ind. Eng. Chem. Res., Vol. 45, No. 10, 2006 3627 Table 2. Adsorption Characteristics of the Microparticles adsorption equilibrium adsorption kinetics
qm qm1a qm2b adsorbent (mg/g dry (mg/g dry (mg/g dry number adsorbent) adsorbent) adsorbent) 1 2 3 4 5 6 7 8 9 10
134.4 271.0 192.3 150.4 244.8 145.4 8.8 0.0 280.4 168.2
R2
Ds (cm2/s)
R2
1.3 × 6.0 × 10-11 1.1 × 10-10 1.6 × 10-10 2.0 × 10-10 5.6 × 10-10 4.6 × 10-10
0.9739 0.9629 0.9553 0.9598 0.9497 0.9637 0.9468
10-10
62.0 90.4 73.2 78.3 244.4 123.3 4.1
72.4 180.6 119.1 72.1 0.4 22.1 4.7
0.9946 0.9885 0.9973 0.9950 0.9297 0.9960 0.9606
109.6 59.1
170.8 109.1
0.9892 6.7 × 10-11 0.9462 0.9996 5.3 × 10-11 0.9620
a Adsorption capacity of the sites of type 1, calculated with the parameter b1 ) 23.12. b Adsorption capacity of the sites of type 2, calculated with the parameter b2 ) 0.59.
Table 3. Analysis of Variance (ANOVA) for Significance of Regression of the Quadratic Model of qm, Given by eq 3a source of variation
sum of degrees of mean squares freedom square
regression residual error total a
84313 226 84539
5 4 9
F
tabulated F
P-value
16862 298.06 F0.05,5,4 ) 6.26
