Radiation Physics and Chemistry 141 (2017) 300–311

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Radiation Physics and Chemistry

journal homepage: www.elsevier.com/locate/radphyschem

Chromatographic matrix based on hydrogel-coated reticulated polyurethane MARK

foams, prepared by gamma irradiation
Mirna L. Sáncheza, Claudia Y. Giméneza, Juan F. Delgadob, Leandro J. Martíneza,
⁎
Mariano Grassellia,
a
Laboratorio de Materiales Biotecnológicos (LaMaBio), Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes-IMBICE-CONICET-CICPBA, Bernal,
Argentina
b
Laboratorio de Obtención, Modificación, Caracterización y Evaluación de Materiales (LOMCEM), Departamento de Ciencia y Tecnología, Universidad Nacional de
Quilmes, CONICET, Buenos Aires, Argentina

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.

1. Introduction From the early days of the radiation processing, radiation-induced

crosslinking has been identified as useful to prepare hydrogels
Modern bioprocesses depend heavily on the availability of materials (Alexander and Charlesby, 1957; Danno, 1958). At well-defined irra-
that allow obtaining competitive products in terms of quality and costs. diation doses, PVA hydrogels can be obtained from a PVA solution
Historically, hydrogels have been very important materials in the field (Peppas and Merrill, 1976). To introduce some functionality, PVA hy-
of biochemistry. Chromatographic matrices and electrophoresis gels are drogels have been grafted by radiation methods with different ligands
currently the most successful materials used in protein analysis and to prepare adsorptive materials to recover different ions. An example of
purification. In addition, hydrogels have expanded their applications this is the application of hydrogels in decontamination (Güven et al.,
into various biological areas, such as contact lens materials, orthopedic 1999).
applications, and devices for controlled release of drugs (Hyon et al., In the last two decades, one-piece porous polymeric materials (also
1994; Hoffman et al., 2002; Casolaro et al., 2006; Bae et al., 2006). called monoliths) have been applied to separate macromolecules
Different types of hydrogels can be prepared from natural polymers, (Krajnc et al., 2005). These materials add two advantages respect to
as well as from semisynthetic and synthetic polymers, most of which are hydrogels: (i) much higher mechanical resistance and (ii) an internal
obtained by crosslinking of their aqueous solutions. As an example, structure of interconnected channels which facilitate mass transport.
hydrogels have been obtained from polysaccharides and poly- Therefore, monolithic columns with these unique structures allow high
acrylamides (Porath and Flodin, 1959; Hjertén and Mosbach, 1962) and flow rates at low pressures without losing column efficiency; as a result,
chemical crosslinking has been performed to improve their mechanical a quick separation is achieved (Cabrera et al., 2000).
properties, which are still the main limitation. Porous monolithic materials can be prepared by means of a wide

⁎
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

variety of techniques (reviewed by Svec (2010)), including the use of Table 1

radiation-induced polymerization (Grasselli et al., 2001). As mentioned Polymer solutions and conditions of the coating process.
above, solid porous monoliths have good mechanical and hydro-
Polymer Concentration (w/v) pH Temperature (°C)
dynamic properties. However, they exhibit a very low adsorption ca-
pacity. This is associated with the fact that chromatographic ligands are Agarose 8 7 85
linked to a low specific surface area of these non-hydrogel-based ma- HEC (90 kDa) 1.4 7 R.T.
PVA (61 kDa) 10 7 85
terials.
One particular case is the porous monolithic material called cryogel.
This material is prepared by a polymerization reaction of water soluble
⎢ W − Wo ⎥
monomers in freezing state, where the pore structure is obtained by the CD% = 100 ∙ ⎢ 1 ⎥
phase separation of water in frozen state during the polymerization ⎣ W0 ⎦ (1)
process (Mattiasson et al., 2010). Therefore, the cryogel material is where W0 and W1 are the initial and final weights of the material, re-
formed by a hydrophilic crosslinked polymer; however, this material spectively.
has also low adsorption capacity for protein purification (Mattiasson
et al., 2010). A recent publication has described an improvement of the 2.2.2. Grafting/crosslinking process
adsorptive capacity of this material by simultaneous radiation-induced Radiation-induced crosslinking and grafting polymerization was
grafting polymerization (SRIGP) (Bibi et al., 2011). This improvement performed by irradiation of polymer samples with a gamma source.
is due to the grafting modification that occurs in the bulk of the cryogel Coated rPUF materials, soaked in an irradiation solution, were sealed in
material. SRIGP is a modification method based on ionizing radiations, a sample container. Then, 0.4 g of coated and dried material (10 cy-
which generate radicals onto the base polymer that are responsible for linders) was soaked in 100 mL of ethanol:water 1:1 v:v. To perform
chemical reactions. In this way, the grafting polymerization process simultaneous crosslinking and SRIGP, GMA or GMA and DMMA
occurs in the presence of methacrylate monomers onto a base polymer. monomers were added to the irradiation solution. The following con-
Here, we describe a novel approach to prepare porous monolithic ditions were studied: 2%v/v, 4%v/v, and 6%v/v of GMA, 5%v/v of
adsorptive materials, based on radiation-crosslinking and functionali- DMAA, 5%v/v of DMAA + 1%v/v of GMA, and 5%v/v of DMAA + 4%
zation of hydrophilic coating onto reticulated polyurethane foams v/v of GMA. Solvents were previously degassed for 15 min by nitrogen
(rPUFs). These materials were analyzed for chromatographic applica- gas bubbling.
tions to recover macromolecules such as proteins. The samples in the containers were irradiated in a 60Co irradiation
source (PISI semi-industrial source, Comisión Nacional de Energía
2. Materials and methods Atómica, Ezeiza, Argentina) with a 10 kGy irradiation dose at a dose
rate of 1 kGy h−1 (if not specified) and room temperature. Dosimetry
2.1. Materials was performed with Red Perspex type dosimeters. After irradiation,
materials were washed with ethanol:water 1:1 v:v and subsequently
rPUF from Eurofoam Deutschland GmbH was kindly donated by with 96% ethanol several times until all the homopolymer trapped in-
Prof. Marcelo Fernandez Lahore (Jacobs University). rPUF type Filtren side the porous structure was washed out. Materials were dried at 55 °C
TM 60, with a pore size of about 250 µm, was used as base material in an oven until constant weight. The grafting degree (GD%) was cal-
without purification. The material was cut into small cylinders of culated as the percentage of increase in weight, as follows (Eq. (2)):
0.7 cm in diameter and 2.8 cm in height.
PVA Mowiol 10–98, M.W. 61,000 (Sigma-Aldrich, catalog number ⎢ W − W1 ⎥
GD% = 100 ∙ ⎢ 2 ⎥
10852) hydrolysis grade 98–98.8%, Hydroxyethyl cellulose (HEC), ⎣ W1 ⎦ (2)
M.W. 720,000 (Sigma-Aldrich code number 9004-62-0) and Agarose
(Biodynamics, Molecular biology, low electroendosmosis grade) were where W1 and W2 are the weights of the coated and the grafted ma-
used. Glycidyl methacrylate (GMA), dimethyl acrylamide (DMAA) and terial, respectively.
Lysozyme were from Sigma Chemical Co. (USA). Acetone, sodium sul-
phite anhydrous, isopropanol, ethanol, and other chemical reagents 2.2.3. Hydrogel functionalization
were purchased from Anedra (Buenos Aires, Argentina). All chemicals Strong cation-exchange ligand was immobilized onto the hydrogel
used were analytical grade. based on the ring-opening reaction of the epoxy groups of polyGMA
with sodium sulphite (Camperi et al., 1999). Then, 0.2 g of grafted rPUF
2.2. Hydrogel coating preparation was incubated with 20 mL of a mixture of sodium sulphite/iso-
propanol/water (10:15:75 w/w/w) at 37 °C, overnight under agitation.
The preparation of the material was divided in four main steps: (i) After extensive washing with water, the remaining epoxy groups to
foam coating; (ii) crosslinking and (iii) grafting procedure and (iv) li- diol groups were hydrolyzed by incubation with a H2SO4 solution 0.5 M
gand functionalization reaction. at 80 °C for 2 h (Camperi et al., 1999). After extensive washing with
water, the cation-exchange rPUF (SP-rPUFs) were dried at 55 °C in an
2.2.1. Foam coating oven overnight.
rPUF was coated with different water-soluble polymer solutions. The sulfonic groups were quantified by Inductively Coupled Plasma
Each polymer solution was prepared taking into account the maximum Spectroscopy with Atomic Emission Spectroscopy (ICP-AES) (INCAPE-
dissolution of each polymer in water. The concentrations of each CENACA, Universidad Nacional de la Plata, La Plata, Buenos Aires,
polymer solution are listed in Table 1. Argentina).
rPUF cylinders were coated with hydrophilic polymer solutions by
the dip-immersion method. Pieces of the porous material were sub- 2.3. Material characterization
merged in a selected polymer solution at a given temperature for ten
seconds and further squeezed to remove the polymer in excess. 2.3.1. Swelling degree
Immediately, they were immersed in isopropanol at 85 °C for ten sec- Swelling experiments were performed by placing 0.04 g of the
onds, squeezed and then dried in an oven at 55 °C until constant weight. material in distilled water overnight. After the incubation, the material
The coating degree (CD%) was calculated as the percentage of increase was removed from the water, quickly and carefully dried with a filter
in dried weight, as follows (Eq. (1)): paper, and its weight gain measured. The swelling degree was

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Fig. 1. (a) Scheme of the coating process onto reticulated-poly-

urethane foam (rPUF).

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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3. Results and discussion 3.1. Preparation of the hydrogel-coated porous material

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.

3.1.2. Cross-linking processes

Since the PVA coating without any further treatment is non-stable in
aqueous media, a procedure is required to elude its leaching from the
PUF material. Ionizing radiation is a very powerful technique to modify
polymers. Polymer modification can be achieved by industrial sources
such as gamma rays and accelerated electrons. The straightforward
preparation of hydrogels by irradiation of aqueous solutions of PVA and
other polymers has long been demonstrated (Alexander and Charlesby,
1957; Peppas and Merrill, 1976). In this particular case, i.e. the irra-
diation with a gamma ray source, the high penetrability is capable of
generating reactive radicals through all the porous material and the
surrounding solvent inside its porous structure (considering rPUF
samples are irradiated in a solvent).
Fig. 3. Coating Degree (CD %) of rPUF coated with polyvinyl alcohol (PVA) 10%w/v The poor solubility of PVA in alcohols was used to choose etha-
followed by 2-propanol treatment. The X axis represents the lag times between both nol:water 1:1 v: v as irradiation solvent. Under this condition, the PVA
treatments. The broken line shows the CD % without 2-propanol treatment. is not dissolved and, at the same time, swells up to 300% (corre-
sponding to a PVA concentration of approximately 25% w/v).
3.1.1.1. Infrared spectroscopy analysis. Infrared spectroscopy is a To study the effect of the irradiation process onto the PVA-coated
powerful tool to study the chemical composition of polymers. In rPUF, this material was irradiated with 10 kGy in a 60Co source in
addition, the ATR module adds advantages that simplify the polymer aqueous/ethanol 1:1 v:v. This solvent condition keeps the PVA as a
analysis (direct analysis of opaque sample) and allow obtaining hydrogel in close contact with the rPUF surface. In addition, the solvent
additional information such as spatial resolution in case of flat surfaces. generates a number of different radicals by radiolysis in accordance to
As a consequence of the lower scale penetration of the IR signal the type of solvents (see below).
using the ATR accessory, only the first microns of the samples in close After irradiation, the samples were washed and dried as described in
contact with the crystal were analyzed. Therefore, FTIR-ATR is a Section 2.2.2 and their thermal stability and decomposition behavior
powerful technique to identify different functional groups onto coated analyzed by TGA.
samples; however, the intensity of the absorptive band is not indicative Fig. 5 and Table 2 show the TGA curves and thermal properties of
of the proportion of components (Theophanides, 1984). To analyze the rPUF, PVA-coated rPUF and the same samples irradiated and immersed
coating process, rPUFs coated with different polymers were pressed in ethanol:water mixture. The irradiated rPUF sample under the same
onto the diamond crystal and the FTIR spectra recorded. experimental conditions (irradiation solvent, dose and dose rate) is
All coated rPFU samples showed a CD% higher than 10%; however, shown as a comparison of the effects of ionizing radiation onto the base
no additional signals in the spectra in comparison to the base rPFU material.
material were observed in samples coated with agarose or HEC (data PUFs are block copolymers that owe their elastic properties to the
not shown). This could be due to the poor stability of these coated phase separation of hard and soft blocks. Hard blocks are rigid struc-
polymers, where the pressure of the arm onto the ATR crystal sweeps tures that are physically cross-linked, whereas soft blocks are stretch-
the coating layer from the rPUF. PVA-coated rPUF clearly showed the able chains which provide the polymer with elasticity. The thermal
presence of the hydrophilic polymer (Fig. 4c). For comparison, PUF and properties of these materials are governed by the weakest bond of the
PVA film spectra were plotted in the same Figure (Fig. 4a and b re- polymer chain, whereas the most frequent chain and the environment
spectively). Samples containing PVA showed an increase at 3300 cm−1 of the given groups are the dominant factors for overall thermal sta-
and 2900 cm−1 peaks, which correspond to the stretching of the -OH bility. In this way, two different decomposition zones were determined
and -CH2 groups respectively. in rPUF materials in agreement with previous observations
The rPUF material coated with PVA 10%w/v, pH 7.5, 85 °C (PVA- (Mohammadi et al., 2014). Soft and hard segments were formed during
coated rPUF) was selected for the following modifications and further the forming reaction of PU and are related to different functional
groups (Mequanint et al., 2002). While soft segments come from
polyols, hard ones are formed by the reaction between diisocyanate and
diol or diamine. Because each segment has different bond energy, they
are broken at different temperatures and can be differentiated in TGA
curves. A first zone in our rPUF sample extended from 210 °C to 330 °C,
whereas a second zone, next to the first, extended until 410 °C.
The thermal properties of the non-irradiated PVA-coated rPUF
sample also showed two degradation zones, but the second zone had a
maximum at a temperature lower than that of the rPUF sample (Table 2
entries 1 and 3). PVA degradation causes an intensive signal of weight
change (Strawhecker and Manias, 2000), which comes earlier and
overlaps with the degradation of both segments of rPUF (Mohammadi
et al., 2014).
The T95 value for the base material (rPUF) was determined at 251 °C
(Table 2), whereas, for the coated rPUF without any irradiation treat-
ment, this temperature decreased to 212 °C. This temperature of de-
composition could be related to the non-crosslinked PVA (Strawhecker
and Manias, 2000).
When the PVA-coated rPUF was irradiated under these experimental
Fig. 4. FTIR-ATR spectra of (a) base material rPUF; (b) PVA film (non-irradiated) (c) and conditions, the remaining weight after 400 °C was still higher than 20%
PVA-coated rPUF respectively.
(Fig. 5) and a new third maximum was observed at 411 °C (Table 2,

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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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M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311

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.

PVA-coated rPUF, the hypothesis of PVA-PVA intermolecular cross-

linking and intermolecular PVA-PU reactions is strongly supported.

3.1.3. SRIGP of PVA-coated rPUF

To add chemical functionality to the PVA-coated layer, a reactive
polyGMA was simultaneously grafted. This grafted polymer allowed
introducing different chemical ligands, such as sulfonic groups, to
customize the hydrophilic layer.
SRIGP is a versatile tool to provide tailor-made properties to a
polymer (Carbajal et al., 2003). This tool has been studied for decades
and still maintains an industrial interest due to its great flexibility to
prepare a complex variety of materials (Clough, 2001).
Ionizing radiation technology allows performing crosslinking and
Fig. 7. FTIR-ATR spectra of (a) non-irradiated PVA and (b) PVA film irradiated in water
and (c) irradiated in water:ethanol 1:1 mixture (Dose: 10 kGy). SRIGP simultaneously in one step onto PVA hydrogels (Bodugöz et al.,
1999). This second process can be done by including methacrylic
monomers in the irradiation solvent. Monomers dissolved in the solvent
(a) a solvent exchange in the PVA hydrogel (Takigawa et al., 1992) and
yield homopolymers and/or grafted polymers onto the base material. In
(b) different kinds of radicals generated by solvent radiolysis (Roux
the latter case, polymethacrylate will be linked to the swelled PVA
et al., 1973). The water exchange by ethanol causes a phase separation
hydrogel coated to the rPUF material.
as a consequence of the decrease in solvent power. This brings a het-
After the irradiation process including different monomers (GMA
erogeneity within the PVA gel structure where the PVA-rich phase has
and DMAA) and monomer concentrations (from 1% to 6%), the mate-
the polymer chains very close to each other (Takigawa et al., 1992).
rial was washed to remove the homopolymer. Then, it was dried and
Therefore, a high yield in the radiation-induced crosslinking process is
weighed to calculate the grafting degree (GD%), which corresponds to
expected. Considering the second phenomenon, water radiolysis yields
the percentage of increase in weight compared to the non-irradiated
a high number of hydroxyl radicals and hydrated electrons. However,
sample.
the presence of a high concentration of ethanol (> 8 M) reduced the
Fig. 8 shows a correlation between GD% and GMA concentration in
amount of HO. radicals and H2O2 to negligible values (Štefanić and
the irradiation solution considering the grafting of one or two mono-
LaVerne, 2002). Hydrogen- and carbon-based radicals, such as ethoxy
mers. When a simultaneous grafting is performed using low monomer
or hydroxyethyl radicals, are the main reactive species generated in the
concentration, the SRIGP continues until consuming all the monomer
solvent (Roux et al., 1973) and are able to react with the PVA hydrogel,
available; therefore, a linear correlation between these variables is
in all its volume and at the surface of the insoluble polymer (rPUF),
expected, as shown in Fig. 8. A similar behavior has been reported in
generating macro-radicals. Subsequently, these radicals will evolve
previous studies for polyethylene and polysulfone membranes grafted
mainly in crosslinking chemical reactions according to the solvent
under the same experimental conditions (Grasselli et al., 1999; Carbajal
composition and the heterogeneity of the sample in different regions of
et al., 2003).
the material. Considering the lack of carbonyl moieties, according to
In a previous study, the use of the second monomer DMAA onto
the FTIR data, and the increase in the thermal stability in the irradiated
SRIGP of polysulfone membranes allowed obtaining an adsorptive

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M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311

Fig. 8. Grafting Degree (GD %) of rPUF coated with

PVA 10% w/v after being irradiated in the presence
of (left) GMA in ethanol:water solution (1:1 v:v) and
(right) GMA with the addition of DMAA 5% in
ethanol:water solution (1:1 v:v).

membrane containing a surface grafted polymer with improved ad-

sorptive properties. The presence of the hydrophilic monomer DMAA
enhanced the adsorptive capacity as a consequence of improved hy-
drophilic properties of the grafted polymethacrylate layer (Wolman
et al., 2005). Therefore, in the present work, we also studied the
grafting procedure onto the PVA-coated rPUF using GMA/DMAA.
Considering the SRIGP process using GMA, GD% increased pro-
portionally to the monomer concentration. Also, the amount of homo-
polymer increased proportionally (data not shown); therefore, the
sample containing the initial monomer concentration of 6% showed a
high yield of homopolymerization, which was difficult to remove from
the foam. The best experimental condition to prepare grafted foam was
the sample prepared using the initial monomer concentration of 4% in
the sample.
Considering the copolymerization with DMAA, it showed a GD%
similar to that without DMAA but higher amount of homopolymer was Fig. 9. GD % for PVA-coated rPUF samples irradiated in the presence of GMA 4% in
developed. These results are different from those previously reported different solvents.
(Wolman et al., 2005); however, the base polymers used in both works
were different (polysulfone versus PVA-coated rPUF) and thus different
3.1.3.2. Irradiation dose and dose rate of the SRIGP process. The
grafting localization was reached (see below). Therefore, the SRIGP
irradiation dose and dose rate are important parameters of SRIGP,
process using only GMA monomer was selected for further functiona-
which could strongly affect the GD%. The former influences the amount
lization of the PVA-coated rPUF.
of radicals, while the latter has a strong impact on the survival of such
radicals and the termination of the formed graft growing chains.
Generally, higher irradiation doses lead to higher GD% values. This is
3.1.3.1. Solvents of the SRIGP process. In SRIGP processes, solvents have
due to the enhancement of free radical formation in the grafting system
an important role in bringing about the swelling of the polymer to be
and the involvement of more radicals in the grafting reaction.
grafted and hence enhancing the monomer accessibility to radical sites
Considering the low monomer concentration and the total dose of
(Nasef, 2001). Accordingly, the solvent is one of the essential elements
10 kGy used in this work, the SRIGP process evolves until it consumes
towards the success of SRIGP (Nasef, 2001). Generally, there are two
all the monomer available (Grasselli et al., 1999).
reasons for the specific effect of solvents on SRIGP: (i) the radiolytic
Considering the dose rate, higher rates yield equal or lower GD%
products from the solvent and its chain transfer constant, and (ii) the
values as a consequence of the low diffusion rates of monomers. In this
redistribution of the absorbed dose between the components of the
work, dose rates of 1 and 10 kGy h−1 were used with a total dose of
monomer/solvent system. It has been established that the nature of the
10 kGy (Fig. 10). It is important to mention here that when the si-
solvent not only determines the degree of grafting but also the location
multaneous irradiation technique is applied, the irradiation dose is a
of grafting (i.e. surface or bulk). If a poor-swelling solvent is used,
function of the grafting time, whereas when the pre-irradiation tech-
surface grafting most likely takes place due to the slowdown in
nique is applied, the irradiation dose and the grafting time are two
monomer diffusion. This is due to the low concentration of absorbed
independent parameters. Moreover, relatively lower doses and dose
monomer and low viscosity of poorly swollen polymer matrix.
rates are required for SRIGP to achieve high GD% values (Nasef, 2001).
However, if a good-swelling solvent is used, bulk grafting is highly
As expected, GD% was higher at the lower dose rate. This may be
favored and homogenous grafting is most likely obtained.
explained by the fact that at higher dose rates, the kinetics of free ra-
In the present work, isopropanol, ethanol and ethanol:water
dical formation is much higher than the diffusion of the monomer, fa-
(1:1 v:v) were used as solvents for the SRIGP process. Fig. 8 shows the
voring the production of homopolymer in the solvent. Thus, 1 kGy h−1
GD% obtained using 4% GMA dissolved these solvents.
was the dose rate selected for the SRIGP process.
As shown in Fig. 9, the maximum GD% was reached using the
ethanol:water mixture as a solvent. In addition, we also noticed that the
SRIGP process carried out with isopropanol and absolute ethanol 3.1.3.3. Dependence between CD%and GD%. Considering that the
changed the integrity of the rPUF, reducing the flexibility of the ma- grafting process occurs in the PVA-coated hydrogel layer, GD% is
terial. In addition, alcohols prevent PVA swelling. Therefore, the high expected to be directly correlated with CD%. Therefore, three PVA-
molar proportion of water in the solvent increased the swelling of PVA coated rPUF samples containing different PVA amounts were modified
and reduced the swelling properties of PU, yielding higher GD%. by SRIGP using 4% GMA. As shown in Fig. 11, the amount of grafting
had a direct correlation with CD % in the range studied.

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M.L. Sánchez et al. Radiation Physics and Chemistry 141 (2017) 300–311

3.2.2. Scanning electron microscopy (SEM)

rPUF modifications were analyzed by observing scanning electron
micrographs. SEM images of untreated and modified rPUF (coated/
grafted rPUF) are shown in Fig. 12a/b and c/d, respectively.
A rough structure, which can be assigned to the modification pro-
cess, was recognized onto the foam surface. Also, the modified rPUF (c/
d) indicated no bulk deterioration compared to the untreated sample
and a thicker structure was also noticed. Similarly, no pore blockage
was detected.
The b/d images show the cross-section of the polymer of rPUF in
addition to the surface. It is possible to differentiate the morphology of
the internal body of rPUF in both cases. However, a contrast can be seen
between the smooth surface of the untreated rPUF and the rough sur-
face of the modified rPUF.

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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