Radiation Physics and Chemistry: Articleinfo
Radiation Physics and Chemistry: Articleinfo
Radiation Physics and Chemistry: Articleinfo
A R T I C L E I N F O A B S T R A C T
Keywords: Novel chromatographic materials for protein purification with high adsorption capacity and fouling resistance
PVA hydrogel are highly demanded to improve downstream processes. Here, we describe a novel adsorptive material based on
Radiation-induced polymerization reticulated polyurethane foam (rPUF) coated with a functional hydrogel layer. rPUF provides physical rigidity
Chromatography through its macroscopic structure, whereas the hydrogel layer provides capacity to adsorb proteins by specific
Protein adsorption
interactions. The hydrogel coating process was performed by the dip-coating method, using a polyvinyl alcohol
(PVA) solution. The PVA hydrogel was linked to the rPUF material by using a radiation-induced crosslinking
process in aqueous ethanol solution. The ethanol in the solvent mixture allowed a balance between PVA swelling
and PVA dissolution during the irradiation step. The resulting material showed higher thermal stability than the
non-irradiated one. In addition, a simultaneous radiation-induced grafting polymerization (SRIGP) was done by
simple addition of glycidyl methacrylate monomer into the irradiation solution. In a further step, sulfonic ligands
were included specifically in the hydrogel layer, which contained around 200% of PVA respect to the original
rPUF. Materials were characterized by FT-IR, thermogravimetric analysis, SEM microscopy and EDX analysis.
The cation-exchange rPUF material was functionally characterized by the Langmuir isotherm and a dynamic
adsorption experiment to analyze the chromatographic properties for protein purification processes.
⁎
Corresponding author.
http://dx.doi.org/10.1016/j.radphyschem.2017.08.006
Received 6 January 2017; Received in revised form 6 July 2017; Accepted 6 August 2017
Available online 09 August 2017
0969-806X/ © 2017 Elsevier Ltd. All rights reserved.
M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311
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M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311
calculated as the percentage of solvent absorption per dry material 2.3.5. Thermal gravimetric analysis (TGA)
weight after wetting the sample. Thermal analyses were performed with TA Instruments Q500 ther-
mobalance (TA Instruments, Delaware, USA) in rPUF samples.
Approximately 5 mg of each sample was heated using a linear heating
2.3.2. Scanning electron microscopy (SEM)
ramp (20 °C min−1) from 30 °C up to 600 °C with a continuous N2 flow
SEM pictures were captured with SEM-Carl Zeiss NTS-SUPRA40
at 60 mL min−1 to guarantee an inert atmosphere. Weight loss was
(Centro de Microscopías Avanzadas, Universidad de Buenos Aires
measured as a function of temperature, and derivative of weight change
(UBA), Buenos Aires, Argentina). All the samples were equilibrated in
with respect to temperature (DTG) was calculated from the original
phosphate buffer with 3 M KCl, rinsed with distilled water, dried in an
signal. The temperature necessary to reach 5% of weight loss (T95) was
oven at 55 °C up to constant weight, and examined using SEM at dif-
considered as the initial degradation point.
ferent magnifications. The samples were no more than 5–8 mm thick.
The samples were mounted on a circular metallic sample holder and
coated with 20–50 nm thick of gold by sputtering before being ana- 2.3.6. Protein adsorption equilibrium under static conditions
lyzed. The adsorptive static capacity was determined in batch mode by a
single experiment, where 0.16 g of modified material was equilibrated
2.3.3. Energy dispersive X-Ray (EDX) analysis with 10 mL Phosphate Buffer 20 mM pH 7 (Buffer A) for 2 h under
EDX spectra were captured with a microanalysis system Oxford agitation. After equilibration, rPUFs were saturated with 10 mL of
Instruments INCA Energy 250 including a Si(Li) detector of 10 mm2. Lysozyme solution 2 mg mL−1 in Buffer A for 3 h under agitation. The
The system is coupled to Carl Zeiss SMT Model Supra 40. Data were decrease in the optical density at 280 nm of the supernatants was re-
processed with INCA Energy and Thin Film ID of Oxford Instruments corded, and the equilibrium concentration and the amount of protein
programs (Centro de Microscopías Avanzadas, UBA). adsorbed to the material were calculated from absorbance measure-
All the requirements mentioned for SEM are also valid for EDX ments. A washing step with Buffer A was done. Desorption experiments
analysis. However, for accurate quantitative analysis of the elements in were performed in a similar way by changing the elution Buffer A to
the sample, the samples should be flat. For qualitative analysis, any Buffer A + 1 M NaCl (Buffer B).
shape of the sample is workable. Samples were coated with 20–50 nm
thick of carbon.
2.3.7. Protein adsorption equilibrium under dynamic conditions
Protein adsorption and elution under dynamic conditions were
2.3.4. Fourier transform infrared spectroscopy with attenuated total performed using an ÄktaPrime (GE Healthcare Life Science). A column
reflectance (FTIR-ATR) of 1 mL was prepared with 0.15 g of SP-rPUF. Before loading the
Dried samples were measured directly using an FTIR IR-Affinity column with the protein solution, it was equilibrated using Buffer A
instrument (Shimadzu Corporation, Japan), equipped with attenuated until the absorbance of the outlet stream had reached that of the inlet
total reflectance accessory, GladiATR diamond single reflection (PIKE stream. Lysozyme 2 mg mL−1 in Buffer A was pumped onto the column
Technologies, USA). Spectra were acquired by scanning the specimens at a defined flow rate and the outlet absorbance at 280 nm measured.
for 64 times in the wavenumber range from 4000 to 550 cm−1 with a After loading the sample, the column was washed with 10 column vo-
resolution of 4 cm−1. ATR and atmosphere correction were applied for lumes of Buffer A. The column was then eluted with Buffer B. The flow
all spectra. rate was 1420 cm/h for all the steps.
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In the last decade, the application of porous monolith materials to As mentioned in the Materials and Methods section, the preparation
affinity chromatography has been expanded to analytical applications of the material was divided into different steps (Fig. 1): (i) physical
(Pfaunmiller et al., 2013). These materials have interconnected chan- coating of a water-soluble polymer on the open-foam polymer; (ii)
nels which facilitate mass transport and reduce the diffusion process. crosslinking of both polymers; (iii) grafting of a reactive polymer; and
However, the inner surface of these materials is very low, which re- (iv) specific chemical functionalization of the hydrogel. Steps (ii) and
duces their binding capacity and consequently the industrial applica- (iii) were done simultaneously.
tions. Therefore, the development of materials which can increase this
property are welcome to expand the applications of these materials.
3.1.1. Foam coating
Here, we described the preparation of a novel protein adsorptive
The coating process was accomplished by the dip-coating technique
material based on a hydrogel-coated porous polymer. In particular, a
by using rPUF material as base polymer. Three different hydrophilic
commercial rPUF polymer was used as base material and a poly-
polymer solutions were analyzed for the coating process: agarose, HEC
methacrylate-grafted PVA hydrogel was prepared in situ by radiation-
and PVA.
induced process. Fig. 1 shows the scheme of the work proposed.
Agarose was selected because it is the base polymer of the com-
rPUF is a chemically inert industrial material with three-dimen-
mercial chromatography matrices used for industrial protein purifica-
sional structure, excellent mechanical properties (high strength and
tion. This polymer is a neutral polysaccharide which forms a highly
elasticity), commercial availability and low cost. It has a high porosity
hydrophilic hydrogel with a porous structure (Hjertén, 1964). HEC is a
(about 97%) and an open highly structured macrostructure (Fig. 1).
water-soluble polymer derived from cellulose. It is a nonionic polymer
Being an elastic material, the flexibility of rPUF also provides good
which has been used for membrane coating (Beeskow et al., 1995).
stability and resistance to compression. However, rPUF is generally
Finally, PVA is a widely used synthetic hydrophilic polymer with ex-
hydrophobic, with low compatibility with water soluble polymers.
cellent film-forming properties and good flexibility. In addition, it is
On the other hand, as mentioned in the introduction section, hy-
water-soluble and inexpensive (Hallensleben and Hallensleben, 2000).
drogels are important materials to prepare chromatographic matrices
The coating process was done onto ten rPUF pieces (cylinder
for protein purification and there are many very well established
shaped) by immersion in solutions of the hydrophilic polymers as de-
commercial products in this field.
scribed in Section 2.2.1. After drying the material at 55 °C to constant
Ionizing radiation is a powerful technique to prepare hydrogels in a
weight, the coating degree (CD %) was determined and plotted in Fig. 2.
single step without using initiators or crosslinkers, and reactions can be
The coating process using agarose and HEC showed a CD% lower
initiated at room temperature or sub-ambient temperature (Peppas and
than 20%w/v and 50%w/v respectively, whereas that using PVA
Merrill, 1976). Also, in the last decade, efforts have been made to study
yielded the highest performance, with a CD% about 100%, two-fold
super-absorbent hydrogels by radiation-induced crosslinking (Ibrahim
higher than that obtained with the HEC coating.
et al., 2007). One of the key factors for the success to prepare these
The coating process with PVA was performed at 85 °C to reduce the
hydrogels is the intermolecular radiation-induced crosslinking under
viscosity of polymer solutions and improve the yield. However, some of
low water concentration during the irradiation processes. As an ex-
the polymer leached during the drying process. To overcome this pro-
ample, PVA is degraded by ionizing radiation in dry state (Bhat et al.,
blem, a coagulation step was added before the drying step to increase
2005) and may result in a cross-linked appearance when it is irradiated
the coating yield. Therefore, 2-propanol was selected to coagulate the
under a specific aqueous phase condition (Wang et al., 2000). There-
coating layer of PVA, because it is well known that PVA has poor so-
fore, the irradiation conditions and sample preparation are critical is-
lubility in this solvent. In this way, after the immersion step in the
sues to obtain the desired material. To our acknowledge, water is the
polymer bath, samples were soaked in a solvent bath for 10 s and after
only solvent so far reported used to prepare PVA hydrogels by irra-
its removal, they were dried in an oven at 55 °C.
diation crosslinking methods of aqueous solutions of this polymer.
We also studied the influence of a lag time between the immersion
In addition to the crosslinking process, the radiation-induced
step in the polymer bath and that in the coagulation bath. For these
grafting process can be used to functionalize the material. Grafting
experiments, the coating condition used was that corresponding to the
copolymerization methods are able to generate surface and/or bulk
best condition previously achieved (PVA 10%w/v, pH 7 at 85 °C). Each
polymer modifications. There are several grafting techniques by ap-
experimental condition was prepared using a set of ten cylindrical rPUF
plying radiation technology (Uyama et al., 1998). A comprehensive
pieces. Fig. 3 shows the results of the CD% of samples prepared with
review has been published by Nasef and Güven (2012).
different lag times. A lag time of less than 2 h was able to improve the
The SRIGP procedure has been long applied for the preparation of
coating process by around 20%.
adsorptive membranes (Grasselli et al., 1999). It has been used to im-
mobilize different kinds of ligands onto microfiltration membranes and
applied to purify proteins by specific recognition. The following ligands
have been used: iminodiacetic acid (Grasselli et al., 1999), sulfonic
groups (Camperi et al., 1999), reactive dyes (Wolman et al., 2005),
amino groups (Trelles et al., 2010) and arsenic ligands (Carbajal et al.,
2009). In all these cases, the base materials were hydrophobic polymers
such as polyethylene or polysulfone, the irradiation solvent was an
ethanol/water mixture, and the main grafted monomer was GMA. Also,
in all cases, the grafted polymethacrylate polymer was localized onto
the surface of the base material.
In this work, we describe a similar procedure based on the SRIGP
method applied onto a water-soluble polymer coated in hydrogel-state
onto rPUF material. The epoxy reactive rings of the grafted polyGMA
will be further used to introduce cation exchange properties to the
material.
Fig. 2. Coating Degree (CD %) for the different water-soluble polymer formulations de-
scribed in Table 1.
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analysis.
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M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311
Fig. 5. TGA curves of non-irradiated rPUF material (upper left), non-irradiated PVA-coated rPUF material (upper right) and irradiated rPUF material (lower left) and irradiated PVA-
coated rPUF material respectively.
entry 4). This value is compatible with the presence of crosslinked compared with the spectrum of non-irradiated PVA. The characteristic
fractions. absorption peaks agree with previous reports (Bhat et al., 2005; Sugiura
To observe the effect of irradiation onto single materials, a PVA film et al., 2001). The spectrum of non-irradiated PVA showed a small signal
and rPUF were compared with the irradiated ones under these experi- in the carbonyl region (1700–1740 cm−1 range), in agreement with
mental conditions. Fig. 6 shows the great difference in the derivative of that previously described (Bhat et al., 2005) (Fig. 7a). No additional
TGA for both polymers irradiated in ethanol:water mixture. The curves increment in the carbonyl signal was found in irradiated samples, in-
of the derivative of TGA of rPUF material showed minor changes in dicating that the degradation process by scission of ketone moieties
both degradation zones, as described in the TGA curve (Fig. 5). generated by the keto-enol tautomerization of α-PVA radicals (Wang
Meanwhile, a shift of more than 130 °C was found in the degradation et al., 2000) is not a relevant process under these experimental condi-
zone of the PVA film. As reported in Table 2, the T95 in PVA-coated tions. In addition, no signals differences have been found between PVA
crosslinked rPUF increased to 248 °C, and this increase, in comparison and PVA hydrogels irradiated in water and ethanol:water mixture
with the non-irradiated one, could be attributed to the PVA crosslinking conditions (Fig. 7). Therefore, if PVA crosslinking occurs it will not
(entries 3 and 4). involve the hydroxyl groups, as it is proposed for other crosslinked PVA
The creation of covalent bonds by crosslinking requires an addi- hydrogels (Akhter et al., 1988; Mansur et al., 2008).
tional amount of energy which shifts to higher degradation tempera- The effect of the PVA concentration on the yield of radiation-in-
tures and could be correlated with an increase in its thermal stability. duced crosslinking deserves to be discussed. In this system, the PVA
Although it cannot be conclusive from TGA data, a crosslinking process hydrogel is irradiated at concentrations in the range of 30%w/v (see
involving both polymers is highly possible. above); therefore, according to the data reported by Wang et al. (2000)
Chemical modification onto PVA hydrogel irradiated under oxygen- for PVA hydrogels irradiated in water, the G value for intermolecular
free atmosphere was analyzed by FTIR. PVA films were irradiated at crosslinking is the maximum. However, this discussion does not include
10 kGy immersed in water and ethanol:water 1:1 v:v. the presence of ethanol in the medium. The presence of high amount of
The FTIR spectra of the dried PVA films are shown in Fig. 7 and ethanol in the irradiation solvent will produce at least two phenomena:
Table 2
TGA parameters corresponding to rPUF materials.
Sample T95 1st Max. Integration 1st 2nd Max. Integration 3rd Max. Integration 3rd
Decomp. Derivative zone Decomp. derivative 2nd zone Decomp. Derivative zone
zone (1st) zone zone (3rd)
Unit (°C) (°C) (% °C−1) (%) (°C) (% °C−1) (%) (°C) (% °C−1) (%)
rPUF 251.2 297.4 1.149 52.2 379.2 0.907 44.5 No – –
rPUF/irradiated 240.0 284.4 0.928 46.8 371.5 1.119 47.8 No – –
PVA-coated 212.4 282.5 1.022 39.5 349.8 0.444 54.7 No – –
rPUF
PVA coated 248.4 303.0 0.663 32.5 368.4 0.626 30.9 411.39 0.782 30.3
rPUF/
Irradiated
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Fig. 6. Derivative of TGA curves of non-irradiated rPUF material (upper left), non-irradiated PVA film (upper right) and irradiated rPUF material (lower left) and irradiated PVA film
(lower right) respectively.
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Fig. 10. GD % for PVA-coated rPUF samples irradiated in the presence of GMA 4% in
3.2.3. Energy-dispersive X-ray spectroscopy (EDX)
ethanol:water solution (1:1 v:v). Total irradiation dose: 10 kGy.
Grafting polymerization can be performed on the coating layer or in
the polymer bulk according to the solubility of the monomers and
solvents within the polymeric matrix. Using the same monomer (GMA)
and solvent mixture (ethanol/water), the grafting location could be
different according to the base material to be grafted. For example,
when using polyethylene and polysulfone membranes as base materials,
surface grafting is reached (Grasselli et al., 1999; Carbajal et al., 2003).
However, when using acrylamide-based cryogel as base material, the
polyGMA grafting occurs in the bulk of the polymer structure (Bibi
et al., 2011). In both cases, the SRIGP technique was performed under
the same experimental conditions.
To study the localization of the grafted polymer in the sample, an
elemental in situ X-ray microanalysis was performed (SEM-EDX).
Copper ions have characteristic energy values for the s electron dif-
ferent from other elements available in the sample. Therefore, a grafted
PVA-coated rPUF sample was labeled with copper ions. This was per-
formed by functionalization of polyGMA with iminodiacetic ligand, and
Fig. 11. GD % as a function of CD % for PVA-coated rPUF samples irradiated in the copper ions were chelated on it, as previously described (Grasselli et al.,
presence of GMA 4% in ethanol:water solution (1:1 v:v) at 10 kGy and 1 kGy/h. 1999). Two different regions of the SEM picture in Fig. 13 were selected
to perform the EDX analysis: one in the bulk of the rPUF material and
the other on the outer region (PVA coating).
Previous studies have shown that a homogenous grafting can be
It can be clearly seen that the presence of copper is delimited to the
achieved in polymers that are able to swell because grafting proceeds
surface region of the material, indicating that the PVA-hydrogel layer
by front mechanism, where it starts in layers close to the surface, and
was grafted with polyGMA.
then proceeds inward towards the core of the film by progressive
monomer diffusion through the swollen layers (Nasef, 2001). In this
3.3. Adsorptive properties
research, the swelling occurred onto the coated PVA-hydrogel. There-
fore, grafting was expected to occur in the hydrogel layer supported on
The functional rPUF material containing sulfonic ligands (SP-rPUF)
the rPUF. Section 3.2.3 shows the grafting localization by EDX (see
was characterized by standard adsorption experiments. A protein ad-
below).
sorption isotherm was performed under static conditions (Static
The swelling property of the GMA-grafted PVA-hydrogel coated
Binding Capacity, SBC) and a protein adsorption experiment was per-
rPUF was measured using distilled water at room temperature. The
formed under dynamic conditions. In both cases, we used Lysozyme as a
swelling degree was 80% considering the whole material, which means
model protein. SBC was tested in batch as described in Section 2.3.6,
approximately 150–200% swelling of the PVA hydrogel.
reaching 475 +/− 30 mg protein g−1 matrix (Fig. 14).
The PVA hydrogel reduced the pore diameter in a small fraction.
3.1.4. Chemical functionalization of PVA-coated rPUF Therefore, considering the adsorptive process under dynamic condi-
The GMA-grafted PVA-hydrogel coated rPUF material was further tions, no measurable backpressure was detected when the liquid flow
functionalized with sulfonic groups according to very well described through the foam filled the column.
add-on chemical reactions (Camperi et al., 1999). Ligand capacity was Dynamic adsorption/desorption assays were tested onto a 1-mL
calculated by quantification of sulfonic groups by elemental analysis, column by injection of a protein sample and further elution, changing
corresponding to 0.13 meq g−1 rPUF. to the elution buffer (Fig. 15), to test the potentiality of the SP-rPUF to
be used as an ion-exchange chromatographic matrix. It is important to
3.2. Material characterization remark that this adsorptive analysis was made under plug-flow condi-
tions.
3.2.1. Infrared spectroscopy analysis
The FTIR-ATR spectrum corresponding to the GMA-grafted material 4. Conclusion
showed comparatively higher carbonyl signal (1720 cm−1) than the
PVA-coated one, which was assigned to polyGMA. The characteristic A novel functional hydrogel-coated porous monolith material was
epoxy ring signal at 900 cm−1 was overlapped with the signals of the prepared based on commercial rPUF material. From three different
base material (data not shown). hydrophilic polymers analyzed for the coating process, PVA was the
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Fig. 12. SEM images of (a/b) virgin rPUF and (c/d) coated and grafted rPUF material, respectively.
60
best to obtain a high coating degree. An optimized coating method was Co source. The irradiation conditions involved the use of a solvent
also described considering the polymer concentration, temperature and mixture which balanced the PVA swelling, preventing the dissolution of
coagulating conditions. PVA during the irradiation step. In addition, ethanol acts as an sca-
PVA coating was stabilized by crosslinking by irradiation with a venger of the hydroxyl radicals generated by water radiolysis.
Fig. 13. SEM image of cross-section of adsorptive rPUF material (inner and coated zone). Spectra of elemental composition of the retro-dispersed electrons of rPUF-PVA-pGMA-IDA-Cu2+.
Spectrum 1: inner zone of the material. Spectrum 2: coated zone of the material.
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