Polym. Bull. (2015) 72:195–218 DOI 10.1007/s00289-014-1267-2 ORIGINAL PAPER
Effect of the surface parameters on the interaction of epoxy polymer supports with a lipase enzyme Aitana Tamayo • Antonio Aires-Trapote • Fausto Rubio • Maria J. Hernaiz • Angel Rumbero Juan Rubio
•
Received: 27 January 2014 / Revised: 5 September 2014 / Accepted: 22 October 2014 / Published online: 31 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Four porous polymeric supports prepared from epoxy monomers have been analyzed with respect to their catalytic behavior and characterized by means of infrared spectroscopy (FTIR-ATR), nitrogen adsorption and Inverse Gas Chromatography at Infinite Dilution (IGC-ID). The Pseudomonas stutzeri lipase has been used to determine the adsorption and desorption rates on each support. It has been found that the specific surface area increases with the number of epoxy groups and the pore volume increases as well. The surface energies are also related with the number of epoxy groups in the monomer and show a decrease in the dispersive component of the surface energy, cdS, from 104.87 to 71.12 mJ m-2, but an increase in the polar or acid–base characteristics. Regarding the adsorption–desorption rates of the enzyme lipase, it occurs in two stages being the short-time processes controlled by the dispersive surface energy of the polymer, but the desorption rate is opposite to the base-to-acid surface energy ratio of the polymer indicating that for long-time processes the enzyme is attached to the polymer surface mainly by acid– base interactions. Keywords Inverse gas chromatography Epoxy polymer Surface energy Interaction parameters
A. Tamayo (&) F. Rubio J. Rubio Ceramics and Glass Institute, CSIC, C/Kelsen 5, Campus De Cantoblanco, 28049 Madrid, Spain e-mail:
[email protected] A. Aires-Trapote A. Rumbero Department of Organic Chemistry, Faculty of Science, Autonoma University of Madrid, Av. Francisco Toma´s y Valiente 7, 28049 Madrid, Spain M. J. Hernaiz Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, Complutense University of Madrid, Pz/Ramo´n y Cajal s/n., 28040, Madrid, Spain
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Introduction Protein separation and purification processes are key technologies playing an important role in the biotechnology industry for studying the biological properties of the enzymes. These processes are also required to understand the structure of the proteins and enzymes and to analyze the interactions occurring with other adjacent molecules [1]. In this sense, probably liquid chromatography is the most versatile technique among all the different routes for protein separation and purification since it can be used for the purification of many different biomolecules [2]. In this methodology, particle packed columns are traditionally used as the chromatographic stationary phase, although membranes and monoliths have gained importance in the recent years. This stationary phase in the chromatographic column should be carefully selected to ensure the optimal processing conditions, a procedure that, in most of the cases, is quite expensive and time consuming due to the variations in both texture and chemical structure of supports and ligands [3]. Moreover, protein immobilization can be also used as a rapid method for separation and purification of crude proteins. Although there are hundreds of immobilization protocols [4–8], new designs and developments would permit the enhancement of the enzyme properties during immobilization and the efficiency of the associated processes. As an example, the immobilization via adsorption requires strong hydrophobic or electrostatic interactions between the enzyme and support [2, 8–12], but when using hydrophobic supports, there is a strong dependence on the van der Waals interactions between both protein uncharged patches and the support surface. This hydrophobic interactions become critical in enzyme immobilization and are difficult to control, since there are many factors affecting this hydrophobic interaction, being the most common the hydrophobic degree of the support, the type of the salt employed and its concentration, pH of the medium and temperature or the number of hydrophobic amino acid side chains on the protein surface such as valine, tryptophan, phenylalanine, and leucine [8, 12–14]. The design of novel biocompatible materials suitable for protein separation and purification became one of the top priority tendencies in modern chemistry. Particularly, advances in biopolymer science are responsible for accelerated development of many specific areas in biotechnology since most of the advanced materials with biotechnological importance are related with polymeric systems. In this sense, there are some specific requirements for the designing of these new polymeric biomaterials that are related with their biocompatibility and non-toxicity, stability, controlled biodegradability, adequate mechanical strength, porous microstructure, etc. When using biological polymeric nano- and microparticles, the physic-chemical parameters of the surface of the enzyme and the particle become significant since the interactions will take place on the active sites distributed all over the surface [1]. For this reason, the optimized application of these particles requires a given porosity which should permit the penetration of biological particles of specified size into the bulk material and thus, the exploitation of all the sites existing on the surface [15]. Although the vast majority of current porous polymers are based on styrene– divinylbenzene copolymers, other monomers including acrylates, methacrylates,
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vinylpyridines, vinylpyrrolidone, vinyl acetate and epoxy-activated supports have also been developed in the last few years [16]. Epoxy-activated supports seem to be the almost-ideal systems for carrying out very easy protocols for enzyme immobilization because of their stability and physic-chemical properties [4, 6, 7, 17, 18]. Epoxy groups are very stable at neutral pH even in wet conditions and can be stored for long periods. This fact is an useful advantage for the preparation of the epoxy supports which can be performed in different places from where enzymes would be immobilized and it is also important for the commercialization in large scale [19]. As described in the literature, [17–23], immobilization on the polymeric epoxy supports proceeds via a two-step immobilization mechanism: a rapid preliminary physical adsorption of the protein on the polymer surface and a subsequent covalent bonding of the active epoxy groups present in the support with the nucleophile residues placed on the surface of the adsorbed protein, such as amino, thiol or hydroxyl groups. The design of the polymer supports used for immobilization of biological macromolecules like enzymes should contain a certain distribution of meso- and/or macropores well connected and accessible to the biomolecules. Macroporous polymer particles support the diffusion of large enzyme molecules within the pores and the open microstructure facilitates the enzyme circulation towards the reactive epoxy groups of the polymer [1]. Moreover, when a given enzyme is bonded to a specific support the expression of such enzyme is not only dependent on diffusional factors, but on the enzyme–surface support interactions. Therefore, in polymers with high enzyme binding, the percent expression can be very low due to the increase of hydrophobic interactions between enzyme and polymer surface [24]. Different works in literature describe the influence of several reaction parameters on the pore size distributions (PSD) and the specific surface areas (SSA) in epoxy copolymers [25, 26]. For epoxy polymers, an important factor is the surface density of epoxy groups (SDEG), i.e., the number of epoxy groups per unit of surface area. Both SDEG and SSA of the porous polymer particle available for enzyme bonding decide the extent of enzyme immobilization [27, 28]. The allyl glycidyl ether monomer (AGE) is an interesting molecule that can be easily modified with various functional groups for synthesis of epoxy multi-activated polymers. This bifunctional molecule contains a terminal epoxy group and an acrylic functionality and, when AGE is polymerized the epoxy groups on the polymer surface become useful for both the introduction of amino or hydroxyl groups or for immobilization of different enzymes [27, 28]. Moreover, the monomer can be used to prepare new different species containing two and three epoxy groups and new porous polymers by incorporating different crosslink agents [29]. Inverse gas chromatography at infinite dilution (IGC-ID) is a highly sensitive and versatile technique to determine the surface energy parameters of different solids: Polymers [30], clays, glasses and ceramics [31], hybrids [32], etc. have been characterized by ICG-ID. This technique is based on the analysis of the retention time of different organic probes when pass through a chromatographic column in which the stationary phase is the solid material that is subjected to the characterization. This well-established method is the most suitable to obtain the
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dispersive (cdS) and specific (acid–base) surface characteristics of solid surfaces. In several papers, we have described the methodology to obtain such properties for polymer-derived hybrid materials and ceramics containing Si, Ti and B heteroatoms within the material network [32–36] and, in the present work IGC-ID has been used for characterizing the surface properties of the prepared polymeric materials. The use of non-specific organic probes such as n-alkanes leads to the determination of the dispersive component of the surface free energy (cdS), in this case of the epoxy polymer. The injection into the chromatographic column of organic vapors with polar properties allows the determination of the acid–base character of the polymeric substrate. In the present study, novel hydrophobic porous polymeric supports functionalized with epoxy groups have been prepared by solution polymerization of different allyl monomers using a hydrophobic crosslinker in the presence of a free-radical initiator. The effects of the epoxy groups, the SSA of the prepared materials, PSD and surface energies have been thoughtfully analyzed. These polymeric supports were then used for the immobilization of Pseudomonas stutzeri lipase via adsorption. The rate of enzyme adsorption and desorption has been related with the support surface properties. This lipase adsorption methodology is of general application for most lipases that combined with the characterization of the surface properties of the supports becomes a very promising method for an enhanced immobilization that will allow a larger selectivity in both adsorption and purification processes.
Experimental Materials Allyl glycidyl ether (AGE), divinylbenzene (DVB), 2,2-bis[(allyloxy)methyl]propane-1,3-diol (BAPD), 2,20 -azobis-isobutyronitrile (AIBN), glycerol diglycidyl ether and pentaerythritol were obtained from Sigma-Aldrich. Lipase from Pseudomonas stutzeri, lipase TLÒ was obtained from Meito & Sangyo. All chemicals were of analytical grade from Sigma-Aldrich. Figure 1 shows the corresponding monomers and crosslinkers used for the preparation of the polymers object of this study. Polymer preparation Porous polymers were synthesized by solution polymerization as described elsewhere [29]. Three epoxy-containing monomers (Allyl glycidyl ether, named AGE, pentaerythritol diallyl ether, labeled as APBOD and pentaerythritol triglycidyl allyl ether, named here AOOMPMO) and two different crosslinking agents (DVB and BAPD) were used in the synthesis of the polymeric materials. Cyclohexanol was employed as pore-generating solvent (porogen) (60 % respect the reaction mixture) and AIBN was used as free-radical initiator. The relative
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Fig. 1 Structure of the monomers and crosslinkers used for the synthesis of the polymeric supports. Monomers: AGE: Allyl glycidyl ether, APBOD: Pentaerythritol diallyl ether, AOOMPMO: Pentaerythritol triglycidyl allyl ether. Crosslinkers: DVB: divinylbenzene, BAPD: 2,2bis[(allyloxy)methyl]propane-1,3-diol
Table 1 Relative amount of reactants used for the synthesis of the studied polymers
Polymer name
Monomer
Crosslinker (%)
CLD
Acronym
(%)
DVB
BAPD
PAGE
AGE
70
30
0
77
PAPB
APBOD
70
30
0
74
PAOO
AOOMPMO
60
40
0
86
PAGE-BP
AGE
50
25
25
90
amount of each component used for the synthesis of the polymers is given in Table 1. The elemental analysis has been carried out in elemental analyzers Leco CS200, for determining the C amount, LECO TC-500, to obtain the O amount and LECO RC-412 for H. After reaction, polymers were ground in an agate mortar, vacuum filtered in a Buchner funnel and filtering paper and washed in a first instance with acetone and then rinsed with both water and methanol. Finally, the obtained polymers were dried under vacuum in an oven at 70 °C for 24 h. The powders were also sieved to obtain particles ranged between 100 and 200 lm to fill the chromatographic columns. Enzyme adsorption method Dried polymer (0.1 g) was suspended in 10.0 ml of 1 M sodium phosphate buffer (pH 7.0), containing 0.05 mg/ml of enzyme. Biomolecule adsorption testing was carried out by employing Lipase from Pseudomonas stutzeri under batch conditions in polyethylene cylindrical reactors at 25 °C with gentle stirring (200 rpm). The adsorption time was analyzed by taking aliquots of the supernatant solution to monitor the remaining amount of free protein. The percentage of adsorbed enzyme
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was determined by the difference between the initial amount of protein in the solution (0.05 mg/ml) and in the aliquots. Enzyme release method In vitro release of the enzyme retained on the polymeric supports was also performed under batch conditions in polyethylene cylindrical reactors at 25 °C with gentle stirring (200 rpm). Dried polymer (0.1 g) with a certain amount of enzyme adsorbed was suspended in 10.0 ml of 0.01 M sodium phosphate buffer (pH 7.0). Releasing time was analyzed by taking aliquots of the supernatant solution to monitor the amount of free protein. The percentage of released enzyme was determined by the difference between the initial amount of protein in the solution and in the aliquots. Protein concentration was determined using Bradford’s method (ref) following the manufacturer’s protocol (Bio-rad) and bovine serum albumin as standard Protein determination Protein concentration was determined using Bradford’s method [37] following the manufacturer’s protocol (Bio-Rad) and bovine serum albumin as standard. Analytical methods for the surface characterization of the obtained polymers 1.
2.
Infrared spectroscopy Attenuated Total Infrared Reflectance spectra (ATRFTIR) of the dried powders were collected in a FTIR spectrometer (Perkin Elmer model 1760x) using the MIRacleTM attenuated total reflectance accessory equipped with a single reflection diamond plate. Eight scans were used to obtain each spectrum and background was subtracted in all the cases. Nitrogen Adsorption. Specific surface areas, pore volumes and surface fractal dimensions. N2 adsorption–desorption isotherms at 77 K were obtained using a Micromeritics TriStar 3000 model (Micromeritics Instrument Corporation, Norcross, GA). Prior to all measurements, samples were outgassed overnight in a continuous nitrogen flow in the SmartPrep degasser unit (Micromeritics) to remove traces of gases or water adsorbed on the polymer surface. From the N2 isotherms SSA, PSD and surface fractal dimensions (Ds) have been calculated. SSA were determined using the Brunauer, Emmett and Teller equation [38]. PSD, in the range of 2–100 nm, were calculated with the method of Barrett, Joyner and Halenda (BJH) [39]. Mesopore surface areas (SMES), mesopore volumes (VMES) and average pore diameters (APD) were also determined by assuming cylindrical pores.
The Ds of a solid is used for characterizing the roughness of a given surface and can take on any non-integer value between 2 and 3. For a smooth surface, the topological dimension Ds = 2, however, rougher surfaces can reach the maximum value of Ds = 3. For solid materials, Ds can be determined from N2 isotherms by
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employing the well-established method based in the frenkel–Halsey–Hill (FHH) equation [40]: h iq V= ¼ ln RT ln P0= ð1Þ Vm P Here, V and Vm represent the volume adsorbed at pressure P and on the monolayer, respectively, R is the gas constant, T is the experimental temperature, P/ P0 is the partial pressure and q is related to the Ds of the sample. In the procedure, it is established that when N2 adsorption–desorption isotherms are used for obtaining Ds, there must be taken into account the presence of two adsorption regimes [41] and the magnitude of the parameter q is used to distinguish them [42]: the van der Waals (vdW) regime and capillary condensation (CC). If the vdW regime is dominant, i.e., the attraction between the solid and adsorbed film replicates the surface roughness, and Ds follows: q ¼ ð3 Ds Þ=3
ð2Þ
In the CC regime, the adsorption takes place in small pores where the liquid/ vapor interface becomes unstable and the adsorbed film collapses to fill the pore at some critical pressure. When the CC regime is dominant, the liquid/gas surface tension tends to reduce the interface area, and in these cases Ds is calculated from: q ¼ ð3 Ds Þ
ð3Þ
In general, for nitrogen adsorption isotherms, Eq. (2) should be used at low relative pressures, i.e., when vdW forces are dominant, while Eq. (3) should be used at high pressures, i.e., where CC forces prevail. However, in most of the cases, the exact threshold between vdW and CC is not clear. For this reason, Neimark proposed a new method based on thermodynamic considerations [43]: S P=P0 ¼ log acðDs 2Þ ð4Þ Here, S(P/P0) is the area of the solid/liquid interface, and ac is the main radius of curvature of this interface that can be determined from the Kelvin equation [38] ð5Þ ac ¼ 2rvm =RT ln P0=P where r and vm are the surface tension and the molar volume of the liquid nitrogen, respectively. S(P/P0) can be calculated according to the Kiselev equation by integrating the isotherm from the current value, V(P/P0), to the maximal value, Vmax, close to P/P0 = 1 [44]: i RT ZVmax h S P=P0 ¼ V P=P0 ln P0=P dV r
ð6Þ
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Inverse Gas Chromatography at Infinite Dilution (IGC-ID). Dispersive and Acid–Base Surface Energies of polymer surface
Here, cdS can be obtained from the adsorption of n-alkanes (non-polar probes) with increasing chain length using the following equation: . DGAðCH2 Þ 2 cds ¼ 1 4c ð7Þ ðCH2 Þ NaðCH2 Þ where N is the Avogadro constant, aðCH2 Þ is the area occupied by a methylene (–CH2–) group (0.06 nm2), DGAðCH2 Þ is the adsorption free energy of a –CH2– group, and cðCH2 Þ is the surface energy of a solid entirely made of –CH2– groups. DGAðCH2 Þ is calculated from the slope of the straight line obtained by plotting the retention volumes, VN, of a series of n-alkane probes versus their chain lengths. On the other hand, acid and base surface-specific characteristics, kA and kB, respectively, can be obtained from the interaction with organic probes containing acid, amphoteric and base properties. When the VN of these probes are plotted versus a given probe property such as the molecular induction polarizability (a0), all n-alkane probes fall on a straight line whereas the VN of the specific probes are located in the plot above this line. The difference in ordinates between any specific probe and the n-alkane line corresponds to the specific free energy of the specific probe (-DGsp). When -DGsp is obtained at different temperatures it can be calculated the specific enthalpy DHAsp , which is related with the kA and kB values with the following equation [45]: DHAsp ¼ kA DN þ kB AN
ð8Þ
Here, DN and AN* are the Gutmann’s donor and acceptor numbers of the specific probes [45]. DN and AN* values of different organic probes used in this work are given in Table 2 [46, 47]. kA and kB reflect the ability of the surface to act as electron acceptor (acid) and donor (base), respectively. IGC-ID measurements were carried out using a gas chromatograph (Perkin Elmer, Autosystem) fitted with a flame ionization detector (FID). 50 cm long and 2 mm of internal diameter tubes were filled with the polymeric particles sized and sieved between 0.1 and 0.3 mm. The flow rate of the carrier gas (He) was fixed to 20 cm3 min-1. Columns were conditioned at 100 °C overnight and the signal of the FID was analyzed using a GC integrator (Perkin Elmer Nelson). For surface energy characterization, n-alkanes from n-pentane to n-decane were used as non-polar probes and benzene, chloroform, acetone, ethyl acetate, diethyl ether and tetrahydrofuran as polar probes. The retention volume, VN, was obtained by injecting a small quantity of probe using a gas-micro-syringe (0.1 ll) Hamilton. The instrument response to the organic vapors passing through the FID detector is given in mV and, for our instrument, the optimum intensity of the peaks to be considered reproducible is of 1 mV. At least five injections per probe and temperature have
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Table 2 Properties of chromatographic probes AN* (kJ mol-1)
DN (kJ mol-1)
(hmL)1/2ao1049 (C3/2m2V-1/2)
Probe
Abbreviation
Character
n-Pentane
C5
Neutral
0
0
n-Hexane
C6
Neutral
0
0
9.2
n-Heptane
C7
Neutral
0
0
10.3
8.1
n-Octane
C8
Neutral
0
0
11.4
n-Nonane
C9
Neutral
0
0
12.6
Chloroform
CL
Acid
22.6
Benzene
BZ
Acid
0.7
0
7.8
0.4
8.6
Acetone
AC
Amphoteric
10.5
71.1
5.8
Ethyl acetate
EA
Amphoteric
6.3
71.5
7.9
Diethyl ether
DE
Base
5.9
80.6
7.3
Tetrahydrofuran
THF
Base
2.1
83.7
6.8
been taken to obtain elution peaks of 1 mV and the calculations include all the values of the retention volumes measured.
Results and discussion ATR-FTIR The ATR-FTIR spectra of the studied polymers are shown in Fig. 2. The similarity of the spectra is an indicative of the analogous polymerization reaction taking place in every monomer. The presence of epoxy groups can be detected by a small peak at 1,256 cm-1 and a medium one at 903 cm-1 corresponding to the symmetric breathing and symmetric bending vibrations of the C–O–C ring, respectively [48]. The details shown in the upper left corner in Fig. 2 represent the ATR intensity of the 903 cm-1 band where it is observed that the absorbance increases with the number of epoxy groups in the monomer, from PAGE to PAOO. These ATR-IR absorbances of the epoxy band only show a direct dependence with the number of epoxy groups on the monomer, but no relationship has been found with respect to the crosslink density values (CLD) collected in Table 1. The CLD values have been determined from the chemical composition of the polymers, taking into account that only the epoxy group contains O atoms. The relative amount of monomer and crosslinker contained in the solid substrate is calculated from the molecular weight of the components and the reaction yield. In the case of the PAGE-BP polymer, it was also necessary to determine the epoxidation degree of the polymer, which was estimated through titration to distinguish between the nonepoxidized and epoxidized oxygen. The fact that the CLD values are not reflected in the ATR-IR spectra implies that not all the epoxy groups are available to react in the surface since the ATR-IR technique is a surface technique whereas the CLD values
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Fig. 2 ATR spectra of polymer samples in the 600–1,700 cm-1 (the inserted box represents the spectra in the range 650–950 cm-1)
have been determined taking into account both the bulk and the surface of the polymer substrate. Nitrogen Adsorption: specific surface areas, pore volumes and surface fractal dimensions Figure 3a shows the nitrogen adsorption–desorption isotherms obtained for the prepared polymeric samples. These isotherms can be classified in accordance with the IUPAC classification as type IV [49]. In all the cases, it is observed a hysteresis loop indicating the presence of mesopores (pore diameter between 2 and 50 nm). The hysteresis loops are of type H1 which are associated with porous materials formed of agglomerates or compacts of approximately uniform spheres in a fairly regular array. Such hysteresis loops are located at partial pressures higher than 0.8 that correspond to large mesopores. In Fig. 3a, it is also observed that as the total adsorbed volume (VTOT) increases with the number of epoxy groups in the monomer except for the polymer prepared in the presence of the two crosslinking agents, PAGE-BP, which possesses the larger amount of nitrogen adsorbed at the maximum partial pressure. Most part of the VTOT corresponds to the hysteresis loop suggesting the major contribution to the surface area and pore volume of each polymer is attributed to mesopores. The evaluation of this porosity is assessed through the t-curve which is a plot of the adsorbed volume against the statistical thickness of the nitrogen film adsorbed on the polymer surface. For polymer materials, the most common approach to obtain
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Fig. 3 a N2 adsorption–desorption isotherms of studied polymers. b v–t plots
the statistical thickness of the nitrogen layer is the one proposed by Harkins and Jura [50], which is applicable to different adsorbents and adsorbates. The application of the v–t plot method (Fig. 3b) throws straight lines in all the cases, with a small upward depletion typical of mesoporous materials [38]. For each polymer, SSA, SMES, VTOT and VMES are given in Table 3. Table 3 also reflects the average pore diameters (APD) values calculated from the relationship 4VTOT/SBET [38] that acquire slightly lower values than the corresponding to the maximum (dP-max) of the pore size distributions (PSD) shown in Fig. 4. It can be observed that the average pore volume (APV) increases in accordance with the number of epoxy groups in the monomer, except for the polymer sample prepared with two crosslinking reagents. Fitting of the PSD to a theoretical curve gives a value of the full width at half maximum (FWHM) that also increases with the number of epoxy groups, being this distribution broader when the
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Table 3 Surface area and pore analysis of studied polymers Polymer name
SSA SMES (m2/g)
VTOT VMES (cm3/g)
APD (4V/ SBET) (nm)
dP-max (max. distrib)
FWHM (nm)
PAGE
96.8
87.8
0.577
0.546
23.87
37.4
12.75
PAPB
137.9
137.5
0.831
0.865
24.11
37.9
17.98
PAOO
207.7
212.9
1.179
1.221
22.70
43.5
33.62
PAGE-BP
333.2
321.7
1.703
1.602
20.44
45.1
29.34
Fig. 4 Pore size distributions of studied polymers (lines are only for guiding the eye)
two crosslinkers are used with respect to the curves corresponding to the same monomer with one crosslinking agent. N2 adsorption isotherms have been also used for calculating the surface fractal dimension, Ds, of the polymers and hence the geometrical complexity of their surface or roughness. The surface fractal dimension provides information about the physical structure of the particle surface since it acts at a scale smaller than the primary-particle diameter. The Ds is strongly related to effects of adhesion and surface forces on the primary particles whereas the mass fractal dimension would be useful to estimate possible particle aggregation. Moreover, it is well known that any rough surface can be considered as a smooth surface that has been creased or, alternatively, a surface with a distribution of pores with varying radii. The origin of rough surfaces may be then classified in roughness due to the presence of uniform pores, and roughness due to the presence of a broad pore size distribution [51]. In terms of fractality, a high Ds value is attributed to a large mesopore size distribution with higher contribution of the small pores than the big ones. The PSD shown in Fig. 4 present their maxima centered in the middle of the mesoporous range, concluding that this last consideration of a pore distribution is not satisfied and then the Ds values obtained for the studied polymers should correspond to surface roughness, but not to a distribution of pores.
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Fig. 5 FHH plots for different polymers
Figure 5 shows the FHH plots obtained from the application of Eq. 1. Similarly to other sorts of materials, all the curves show the presence of two linear ranges indicating the co-existence of the two above-mentioned regimes: vdW and CC [41, 52, 53]. The first linear region appears at X-axes values between 1 and -0.5 attributed to a N2 surface coverage corresponding to 0.5–2 adsorbed monolayers. In this region, the vdW regime occurs. At the same time, the second linear region appears between -0.5 and -3 and corresponds to 2–10 N2 adsorbed monolayers, or the CC regime. For both linear regions, the Ds dimensions have been calculated in accordance with Eqs. (2) and (3), being the obtained values collected in Table 4. In Table 4, there are also given the ranges of length scales, n, where the fractal behavior is observed and computed from the number of adsorbed layers, n = (N/ Nm)1/(3-Ds), and the N2 layer thickness of 0.35 nm. The N/Nm range of the length scale is computed from the Ds value. If the Ds value is close to 3, the N/Nm tends to infinite and does not coincide with the P/P0 values in which the FHH value is applicable. It is noticed that the calculated length scales are always three to four times larger than the N2 molecular size. The application of the FFH method provides invalid values of Ds when applied at low relative pressure values since a completely smooth surface possesses Ds = 2 and thus, any value lower than 2 does not have any physical meaning. With this method, the Ds must be calculated only in the CC regime (Eq. 3) giving as a result Ds values very similar to those obtained by applying the Neimark. The calculated Ds values suggest that the polymers possess rough surfaces with geometrical irregularities. From the Ds values obtained by the Neirmark equation, there is no evidence that the different number of epoxy group in the monomers would affect the surface roughness of the polymer. However, the small increase of Ds when the two crosslinkers have been used indicates that the surface of this polymer is rougher than the others.
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PAGE-BP
PAOO
PAPB
PAGE
Polymer
vdW
CC
-0.60
CC
-0.48
vdW
-0.67
CC
-0.51
vdW
-0.89
CC
-0.41
vdW
-0.64
Regime
-0.43
Slope
FHH
2.39
1.57
2.32
1.47
2.11
1.78
2.36
1.72
Ds
5.37–32.54
1.19–2.03
5.03–27.87
1.07–1.91
3.51–17.13
1.29–1.97
2.74–23.91
1.12–1.90
Application range of N/Nm
1.88–11.39
0.42–0.71
1.76–9.76
0.37–0.67
1.23–6.00
0.45–0.69
0.96–8.37
0.39–0.67
Length scale (nm)
2.38
2.24
2.31
2.20
Ds
Neimark
4.35–10.71
7.71–22.33
3.20–13.87
4.89–29–68
Application range of N/Nm
Table 4 Fractal Dimensions of studied polymers calculated through the application of the FHH and Neimark methods
1.52–3.75
2.70–7.82
1.91–16.18
1.71–10.39
Length scale (nm)
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Inverse gas chromatography at infinite dilution (IGC-ID): dispersive and acid–base surface energies of solid surfaces Both dispersive and acid–base surface properties of the prepared polymers have been determined from IGC-ID measurements. Dispersive properties have been obtained from the variation of VN of different n-alkanes. Figure 6a shows the results corresponding for the PAGE polymer at different measurement temperatures and Fig. 6b for the four polymers at 50 °C. In all cases, experimental data can be wellfitted with straight lines with correlation coefficients higher than 0.999. From the slope of these lines, the free energy of adsorption of one methylene group DGAðCH2 Þ is obtained and then, the dispersive component of the surface energy, cdS, can be calculated from Eq. (7). cdS values are given in Table 5. At 50 °C, the dispersive component of the surface energy, cdS, shows a decrease from 104.87 to 71.12 mJ m-2, and, as observed in Table 5, the cdS value decreases with the measurement temperature. This behavior is in accordance with the variation of the intermolecular interactions with the temperature increase. The change of cdS with temperature represents the ability of the surface to reversibly change its dispersive properties. According to Eq. (9), plot of cdS versus T can be linearly fitted, being the slope the contribution of the dispersive entropy (sdS) and Yintercept the dispersive enthalpy (hdS): cdS ¼ hdS TsdS
ð9Þ
The influence of the temperature in the surface properties of the studied polymers can be quantified using the relative temperature gradient (Eq. 10) of cdS expressed as percentage (Table 5): d ð10Þ sS cd S The calculated values of the temperature gradient of cdS are very similar for all polymers except for PAOO, indicating the similar behavior of the surface with the temperature even though their surface energies are quite different. The polymer PAOO, whose monomer contains three epoxy groups, possesses a high—sdS/cdS value indicating that the surface energy is highly affected by temperature changes. Table 5 also shows that cdS values decrease with the number of epoxy groups in the monomer, indicating a possible effect of such epoxy groups in the respective acid– base properties. Acid–base surface properties have been obtained using both n-alkanes and specific vapor probes. Since the enthalpy, DHAsp , is related with the acid and base constants according with Eq. (8), the determination of DHAsp values for the specific probes is carried out by knowing their respective DGsp A of at least at three different temperatures [54]: sp SP DGSP A ¼ DHA TDSA
ð11Þ
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Fig. 6 Variation of VN versus carbon number. a PAGE polymer at different temperatures. b Different polymers at 50 °C
Table 5 Dispersive component of the surface free energy of the studied polymers Polymer name
cdS (mJ m-2) 50 °C
60 °C
hdS (mJ m-2)
sdS (mJ m-2 °C)
sdS/cdS * 100 (°C-1)
70 °C
PAGE
104.87
93.67
92.99
298.63
-0.60
-0.57
PAPB
77.85
75.83
70.44
95.83
-0.35
-0.45
PAOO
71.12
69.40
56.72
105.35
-0.66
-0.92
PAGE-BP
81.87
67.21
72.64
107.81
-0.57
-0.69
Moreover, it is well recognized that DGsp A values can be obtained by plotting VN versus the molecular induction polarizability or (hmL)1/2ao, as it is shown in Fig. 7a,
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b. The DGdA of the n-alkanes can be fitted with a straight line whereas the difference in ordinates represents the specific free energy of the probe DGsp A . By sp calculating DGsp A at least at three temperatures and applying Eq. (11), DHA has been obtained. After that, from Eq. (8), the acid–base surface, kA and kB, properties have been obtained by plotting DHAsp versus DN/AN* (Fig. 8). In all the cases, correlation coefficients between 0.95 and 0.99 have been obtained. Table 6 gives the corresponding kA and kB values for the prepared polymers. Results of Table 6 show that the studied polymers possess amphoteric surface character because they present both acid and base sites on the surface. Both kA and kB increase with the number of epoxy groups in the polymer. However, the base surface energy is more affected by the monomer functionality than the acid one, and thus the ratio kB/kA increases with the number of epoxy groups in the monomer. Table 6 also shows that the acid–base polarity (kA ? kB) increases with the number of epoxy groups in the monomer contrary to the observed variation in cdS, so as the polarity increases the dispersive energy decreases. Enzyme adsorption–desorption The enzyme adsorption and desorption methods have been carried out with different saline concentrations. In the adsorption, the buffer must contain a high saline concentration to enhance the hydrophobic interactions, since all the polymers posses a high hydrophobic character. Desorption of the enzyme must be carried out in a low salt concentration and, therefore, the ionic interactions must compete with the hydrophobic interactions and thus, the enzyme is released. We have tested both the enzymatic adsorption–desorption processes using the Pseudomonas stutzeri enzyme. Figure 9a shows the corresponding enzyme adsorption retention rate (the amount of enzyme retained per time unit: dc/dt) on the studied polymers whereas Fig. 9b represents the amount of enzyme that is desorbed as a function of the retention time. Here, it is observed that the retention rate is very similar for all polymers in spite of their different SSA and porosities. Curves of Fig. 9 have been fitted with straight lines for different time ranges, and Table 7 gives their corresponding slopes and correlation coefficients. In the case of enzyme adsorption, fitting was carried out for two time regions being the first of linear type and the second of logarithmic type. From the slope of the curves presented in Fig. 9 at the initial stages of the reaction, it can be stated that the enzyme adsorption rate increases in the order: PAOO \ PAPB \ PAGE-BP \ PAGE, but when the adsorption time increases, the % of enzyme that is adsorbed on the surface of the polymer can be correlated with the retention time in a logarithmic scale (Table 7). In this case, the logarithmic rate increases in the order PAGE-BP \ PAGE \ PAPB \ PAOO. On the other hand, the enzyme retained on the polymer supports can be recovered with 0.01 N sodium phosphate buffer solution being the corresponding enzyme desorption curves shown in Fig. 9b. It can be appreciated that the curves are practically overlapped although at 50 h the PAOO polymer has desorbed 90 % of the adsorbed enzyme, PAPB has desorbed 95 % and the desorption is almost complete in both the PAGE-BP and PAGE polymers. At 80 h
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Fig. 7 Variation of VN versus (hmL)1/2ao for the PAOO polymer at a 333 K, b 343 K
desorption is complete in all the polymers so we can state that desorption rates follow the order PAOO \ PAPB \ PAGE-BP = PAGE. Since all of the polymer supports are functionalized with the same epoxide group, the differences found in the adsorption and desorption curves suggest that the surface properties of each polymer are affecting the interaction with the lipase enzyme. Thus, the results obtained from the adsorption–desorption curves can be correlated with the polymer surface characteristics. As observed by different authors [24, 27, 28] SSP, VTOT, VMES and APD are related to CLD values, but they do not depend on the number of epoxy groups in the monomer. The reaction conditions (type of monomer, crosslinker, porogen, temperature, etc.) are the main factors affecting the polymer physical surface properties. Moreover, the obtained values of Ds collected in Table 4 indicate that
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Fig. 8 Calculation of kA and kB from DHA and AN* and DN
Table 6 Acid and base constants derived from the polar component of the surface free energy of the studied polymers
kA (kJ/mol)
kB
kA ? kB (kJ/mol)
kB/kA
PAGE
0.23
0.13
0.36
0.54
PAPB
0.28
0.38
0.66
1.35
PAOO
0.42
0.79
1.21
1.80
PAGE-BP
0.44
0.44
0.88
1.00
Polymer name
the surfaces of the prepared polymers present similar roughness independent on the used monomer. At the same time, polymer surface energies characterized by cdS, kA and kB are strongly related to the IR absorbances of the epoxy groups which depend on the number of this functional group in the monomer, but no evidence of relationship with SSA, VTOT, VMES, APD or CLD values has been found. In general, for all studied polymers cdS decreases and kB/kA increases with the epoxy IR absorbance indicating the polar nature of epoxy groups present on the surface of the prepared polymers. However, well-fitted correlations of the adsorption or desorption rates of the lipase enzyme with the surface energies, especially with cdS and kB/kA, have been found. For the adsorption of enzyme, the variation of the slope dc/dt follows the same trend than cdS. This result indicates that the first adsorption of the enzyme is controlled by the non-dispersive interactions on the polymer surface. At higher times, the adsorption of the enzyme is probably controlled by the kB/kA ratio, i.e., by the surface polar characteristics, since the slopes dc/dt and the mentioned ratio vary in a similar manner. In the case of enzyme desorption, the slopes show a variation in the opposite to the kB/kA ratio. This result indicates that after adsorption the enzyme
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Fig. 9 Mass percent of enzyme on the polymer. a Adsorbed. b Desorbed
Table 7 Slope (a) of the of enzyme adsorption and desorption curves calculated for different polymers Polymer name
Enzyme adsorption
Enzyme desorption
% = at ? b (0 \ t \ 60)
% = alog(t) ? b (45 \ t \ 300)
% = at ? b (0 \ t \ 60)
PAGE
0.99
85.55
1.99
PAPB
0.62
87.62
1.93
PAOO
0.59
91.01
1.87
PAGE-BP
0.83
81.63
1.99
is attached to the polymer surface by polar forces mainly characterized by the acid– base surface properties and that the enzyme desorption occurs slowly when such acid–base interactions are stronger. These results are in accordance with those of
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Thudi et al. [55] who reported a decrease of enzyme activity attributed to polar interactions of the ester groups present on the polymer with the protein surface.
Conclusions The present study has explored the effect of polymer surface properties prepared from three different monomers and two different crosslinking agents, on the adsorption and desorption rates of the enzyme Pseudomonas stutzeri lipase. These rates have been analyzed in terms of the number of epoxy groups, SSA, VTOT, VMES, APD, Ds, cdS and kB/kA energies. SSA, VTOT, VMES, and APD also increase with the number of epoxy groups of the monomer and mainly in the case of the use of two crosslinking agents. However, the availability of these groups to interact with the enzyme is dependent on the monomer used, and not on the crosslink density. The prepared polymer supports present similar fractal surfaces with low fractal dimension and cdS and kB/kA surface energies increase with the number of epoxy groups of the monomer. The kB surface constant is well related with the number of epoxy groups of the monomer and with the absorbance of the epoxy group determined by ATR. A largest amount of epoxy groups in the polymer matrix, as determined through the crosslink density analysis, does not imply that all of them are available to react with the enzyme. The experiments have revealed that the adsorption rates of the enzyme occur in two stages, being the first one controlled by the cdS of the polymer and the second one is controlled by the base-to-acid surface energy ratio of the polymer. On the contrary, the desorption rates are conducted by the acid–base interactions, which are also responsible for directing the long-time adsorption rates in the enzyme. These analyses conclude that in the study of the adsorption or desorption rates of enzymatic solutions, the determination of the number of active groups in the monomer or the surface topography (in terms of specific surface area, pore volume and so on) is not sufficient enough to define the interactions between the enzyme and the polymeric supports. In our study, we have found that the dc/dt is mainly influenced by cdS and kB/kA surface interactions rather than for the polymer surface topography characteristics or the number of epoxy groups in the monomer.
References 1. Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme immobilisation. Chem Soc Rev 38(2):453–468. doi:10.1039/b711564b 2. Villeneuve P, Muderhwa JM, Graille J, Haas MJ (2000) Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J Mol Catal B Enzym 9(4–6):113–148. doi:10.1016/s1381-1177(99)00107-1 3. Ka´nˇavova´ N, Kosior A, Antosˇova´ M, Faber R, Polakovicˇ M (2012) Application of a micromembrane chromatography module to the examination of protein adsorption equilibrium. J Sep Sci 35(22):3177–3183. doi:10.1002/jssc.201200396 4. Bickerstaff GF (1996) Methods in biotechnology—immobilization of enzymes and cells. Humana Press, New Jersey
123
216
Polym. Bull. (2015) 72:195–218
5. Chibata I, Tosa T, Sato T (1986) Biocatalysis—immobilized cells and enzymes. J Mol Catal 37(1):1–24. doi:10.1016/0304-5102(86)85134-3 6. Hartmeier W (1985) Immobilized biocatalyst—From simple to complex systems. Trends Biotechnol 3(6):149–153. doi:10.1016/0167-7799(85)90104-0 7. Katchalski-Katzir E (1993) Immobilized enzymes—learning from past successes and failures. Trends Biotechnol 11(11):471–478. doi:10.1016/0167-7799(93)90080-s 8. Cheetham PSJ (1993) The use of biotransformations for the production of flavors and fragrances. Trends Biotechnol 11(11):478–488. doi:10.1016/0167-7799(93)90081-j 9. de Oliveira PC, Alves GM, de Castro HF (2000) Immobilisation studies and catalytic properties of microbial lipase onto styrene-divinylbenzene copolymer. Biochem Eng J 5(1):63–71. doi:10.1016/ S1369-703X(99)00061-3 10. Basri M, Yunus W, Yoong WS, Ampon K, Razak CNA, Salleh AB (1996) Immobilization of lipase from Candida rugosa on synthetic polymer beads for use in the synthesis of fatty esters. J Chem Technol Biotechnol 66(2):169–173. doi:10.1002/(sici)1097-4660(199606)66:2 11. Arica MY, Halicigil C, Alaeddinoglu G, Denizli A (1999) Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GA-derived poly(HEMA EGDMA) microspheres: partial purification and characterization. Process Biochem 34(4):375–381. doi:10. 1016/S0032-9592(98)00104-6 12. Arica MY, Testereci HN, Denizli A (1998) Dye-ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins. J Chromatogr A 799(1–2):83–91. doi:10.1016/s0021-9673(97)01079-0 13. Balcao VM, Paiva AL, Malcata FX (1996) Bioreactors with immobilized lipases: State of the art. Enzym Microb Technol 18(6):392–416. doi:10.1016/0141-0229(95)00125-5 14. Arica MY (2000) Epoxy-derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system. J Appl Polym Sci 77(9):2000–2008. doi:10.1002/1097-4628(20000829)77:9 15. Lozinsky VI (2008) Polymeric cryogels as a new family of macroporous and supermacroporous materials for biotechnological purposes. Russ Chem Bull 57(5):1015–1032. doi:10.1007/s11172-0080131-7 16. Arshady R (1991) Beaded polymer suports and gels. 1. Manufacturing techniques. J Chromatogr 586(2):181–197. doi:10.1016/0021-9673(91)85124-x 17. Wheatley JB, Schmidt DE (1999) Salt-induced immobilization of affinity ligands onto epoxideactivated supports. J Chromatogr A 849(1):1–12. doi:10.1016/s0021-9673(99)00484-7 18. Katchalski-Katzir E, Kraemer DM (2000) Eupergit (R) C, a carrier for immobilization of enzymes of industrial potential. J Mol Catal B Enzym 10(1–3):157–176. doi:10.1016/s1381-1177(00)00124-7 19. Mateo C, Torres R, Fernandez-Lorente G, Ortiz C, Fuentes M, Hidalgo A, Lopez-Gallego F, Abian O, Palomo JM, Betancor L, Pessela BCC, Guisan JM, Fernandez-Lafuente R (2003) Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules 4(3):772–777. doi:10.1021/bm0257661 20. Hannibal-Friedrich O, Chun M, Sernetz M (1980) Immobilization of beta-galactosidase, albumin and gamma-globulin on epoxy-activated acrylic beads. Biotechnol Bioeng 22(1):157–175. doi:10.1002/ bit.260220112 21. Smalla K, Turkova J, Coupek J, Hermann P (1988) Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxymethyl methacrylate with ethylene dimethacrylate. Biotechnol Appl Biochem 10(1):21–31. doi:10.1111/j.1470-8744.1988.tb00003.x 22. Melander WR, Corradini D, Horvath C (1984) Salt-mediated retention of proteins in hydrophobicinteraction chromatography—applications of solvophobic theory. J Chromatogr 317:67–85. doi:10. 1016/s0021-9673(01)91648-6 23. Mateo C, Fernandez-Lorente G, Abian O, Fernandez-Lafuente R, Guisan JM (2000) Multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 1(4):739–745. doi:10.1021/bm000071q 24. Kotha A, Raman RC, Ponrathnam S, Kumar KK, Shewale JG (1996) Beaded reactive polymers 2. Immobilisation of penicillin G acylase on glycidyl methacrylate divinyl benzene copolymers of differing pore size and its distribution. React Funct Polym 28(3):235–242. doi:10.1016/13815148(95)00089-5
123
Polym. Bull. (2015) 72:195–218
217
25. Kotha A, Raman RC, Ponrathnam S, Shewale JG (1996) Beaded reactive polymers 1. Effect of synthesis variables on pore size and its distribution in beaded glycidyl methacrylate-divinyl benzene copolymers. React Funct Polym 28(3):227–233. doi:10.1016/1381-5148(95)00081-X 26. Vaidya BK, Karale AJ, Suthar HK, Ingavle G, Pathak TS, Ponrathnam S, Nene S (2007) Immobilization of mushroom polyphenol oxidase on poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous beaded copolymers. React Funct Polym 67(10):905–915. doi:10.1016/j. reactfunetpolym.2007.05.015 27. Yasuda M, Kobayashi M, Kotani T, Kawahara K, Nikaido H, Ueda A, Ogino H, Ishikawa H (2002) Synthesis of amphiphilic polymer particles by seed polymerization and their application for lipase immobilization. Macromol Chem Phys 203(2):284–293. doi:10.1002/1521-3935(20020101)203:2 28. Vaidya BK, Ingavle GC, Ponrathnam S, Kulkarni BD, Nene SN (2008) Immobilization of Candida rugosa lipase on poly(allyl glycidyl ether-co-thylene glycol dimethacrylate) macroporous polymer particles. Bioresour Technol 99(9):3623–3629. doi:10.1016/j.biortech.2007.07-035 29. Aires-Trapote A (2012) Disen˜o y sı´ntesis de soportes polime´ricos porosos para la obtencio´n de nuevos biocatalizadores y su aplicacio´n en procesos de quı´mica sostenible. Universidad Autonoma de Madrid, Madrid 30. Ansari DM, Price GJ (2004) Correlation of mechanical properties of clay filled polyamide mouldings with chromatographically measured surface energies. Polymer 45(11):3663–3670. doi:10.1016/j. polymer.2004.03.045 ˇ erovic´ LS, Milonjic´ SK, Hourlier-Bahloul D (2005) Adsorption of n-alkanes on silicon nitride 31. C nanopowder. J Am Ceram Soc 88(7):1875–1878. doi:10.1111/j.1551-2916.2005.00371.x 32. Pen˜a-Alonso R, Tamayo A, Rubio F, Rubio J (2005) Influence of boron concentration on the surface properties of TEOS-PDMS hybrid materials. J Sol Gel Sci Technol 36(1):113–124. doi:10.1007/ s10971-005-2498-3 33. Martos C, Rubio F, Rubio J, Oteo JL (2001) Surface Energy of Silica-TEOS-PDMS Ormosils. J Sol Gel Sci Technol 20(2):197–210. doi:10.1023/a:1008759708396 34. Pena-Alonso R, Tellez L, Rubio J, Rubio F (2006) Surface chemical and physical properties of TEOS-TBOT-PDMS hybrid materials. J Sol Gel Sci Technol 38(2):133–145. doi:10.1007/s10971006-7116-6 35. Tamayo A, Tellez L, Pena-Alonso R, Rubio F, Rubio J (2009) Surface changes during pyrolytic conversion of hybrid materials to oxycarbide glasses. J Mater Sci 44(21):5743–5753. doi:10.1007/ s10853-009-3805-0 36. Tamayo A, Tellez L, Rubio J, Rubio F, Oteo JL (2010) Effect of reaction conditions on surface properties of TEOS-TBOT-PDMS hybrid materials. J Sol Gel Sci Technol 55(1):94–104. doi:10. 1007/s10971-010-2220-y 37. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72(1–2):248–254. doi:10.1006/abio.1976. 9999 38. Gregg SJ, Sing KSW (1982) Adsorption, surface area and porosity. Academic Press, London 39. Barrett EP, Joyner LG, Halenda PP (1951) The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms. J Am Chem Soc 73(1):373–380. doi:10.1021/ja01145a126 40. Avnir D, Farin D, Pfeifer P (1985) Surface geometric irregularity of particulate materials: the fractal approach. J Colloid Interface Sci 103(1):112–123. doi:10.1016/0021-9797(85)90082-7 41. Rubio F, Rubio J, Oteo JL (1997) Effect of heating on the surface fractal dimensions of ZrO2. J Mater Sci Lett 16(1):49–52. doi:10.1023/a:1018592532337 42. Ismail IMK, Pfeifer P (1994) Fractal analysis and surface roughness of non porous carbon fibers and carbon blacks. Langmuir 10(5):1532–1538. doi:10.1021/la00017a035 43. Neimark AV, Unger KK (1993) Method of discrimination of surface fractality. J Colloid Interface Sci 158(2):412–419. doi:10.1006/jcis.1993.1273 44. Jaroniec M (1995) Evaluation of the fractal dimension from a single adsorption isotherm. Langmuir 11(6):2316–2317. doi:10.1021/la00006a076 45. Gutmann V (1978) The donor–acceptor approach to molecular interactions. Plenum Press, New York 46. Riddle FL, Fowkes FM (1990) Spectral shifts in acid-base chemistry. 1. van der Waals contributions to acceptor numbers. J Am Chem Soc 112(9):3259–3264. doi:10.1021/ja00165a001 47. van Asten A, van Veenendaal N, Koster S (2000) Surface characterization of industrial fibers with inverse gas chromatography. J Chromatogr A 888(1–2):175–196. doi:10.1016/s0021-9673(00)004878
123
218
Polym. Bull. (2015) 72:195–218
48. Conley RT (1972) Infrared spectroscopy. Allyn and Bacon, Boston 49. Sing KSW, Everett DH, Haul RAW, Moscou L, Pierotti RA, Rouquerol J, Siemieniewska T (1985) Reporting physisorption data for gas solid systems with special reference to the determination of surface area and porosity (Recommendations 1984). Pure Appl Chem 57(4):603–619. doi:10.1351/ pac198557040603 50. Harkins WD, Jura G (1944) Surfaces of solids XIII A vapor adsorption method for the determination of the area of a solid without the assumption of a molecular area, and the areas occupied by nitrogen and other molecules on the surface of a solid. J Am Chem Soc 66:1366–1373. doi:10.1021/ ja01236a048 51. Lee CK, Tsay CS (1998) Surface fractal dimensions of alumina and aluminum borate from nitrogen isotherms. J Phys Chem B 102(21):4123–4130. doi:10.1021/jp9803485 52. El Shafei GMS, Philip CA, Moussa NA (2004) Fractal analysis of hydroxyapatite from nitrogen isotherms. J Colloid Interface Sci 277(2):410–416. doi:10.1016/j.jcis.2004.05.002 53. Fu J, He Q, Liu B, Zhang J, Hu B (2010) Study in adsorption behavior of polymer on molecular sieves by surface and pore properties. Colloids Surf A 369(1–3):113–120. doi:10.1016/j.colsurfa. 2010.08.011 54. Sun CH, Berg JC (2003) A review of the different techniques for solid surface acid-base characterization. Adv Colloid Interface Sci 105:151–175. doi:10.1016/s0001-8686(03)00066-6 55. Thudi L, Jasti LS, Swarnalatha Y, Fadnavis NW, Mulani K, Deokar S, Ponrathnam S (2012) Enzyme immobilization on epoxy supports in reverse micellar media: Prevention of enzyme denaturation. J Mol Catal B Enzym 74(1–2):54–62. doi:10.1016/j.molcatb.2011.08.014
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