Article

pubs.acs.org/Biomac

Structural Regime Identification in Ionotropic Alginate Gels:

Influence of the Cation Nature and Alginate Structure
Pierre Agulhon,† Mike Robitzer,† Laurent David,‡ and Françoise Quignard*,†
†
Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-UM2-ENSCM-UM1, Matériaux Avancés pour la Catalyse et la Santé,
ENSCM, 8 rue de l’Ecole Normale, 34296 Montpellier cedex 5, France
‡
Université de Lyon, Institut de Chimie de Lyon FR 3023, Université Claude Bernard Lyon 1, Ingénierie des Matériaux Polymères
CNRS UMR 5223, IMP@Lyon1, Ecole Polytechnique Universitaire de Lyon 1, 15 bd Latarjet, F-69622 Villeurbanne Cedex, France
*
S Supporting Information

ABSTRACT: The morphologies of several ionotropic alginate

hydrogels and aerogels were investigated by SAXS according
to the nature of the divalent metal cation (Mn2+, Co2+, Zn2+,
Cu2+) and the guluronic fraction of the alginate. All alginate
hydrogel and aerogel samples show isotropic small-angle X-ray
scattering. Gelation results from cooperative associations of
cations and chain segments and yields different nanostructures,
that is, nanofibrillar morphology or multiple junction
morphology, according to cation type and eventually
mannuronic/guluronic ratio. Therefore, Mn and Cu gels
present the same morphology whatever the guluronic ratio,
whereas Co and Zn gels yield different nanostructures. In the size range investigated by SAXS (∼10−200 Å), the structure of
aerogels obtained by CO2 supercritical drying is found to be inherited from the morphology of the parent hydrogel whatever the
initial structural regime.

■ INTRODUCTION
Alginates are natural polysaccharides produced by brown
guluronic content give gels with a higher strength than alginate
with low guluronic content. This was attributed to the stronger
algae,1 composed of (1−4)-linked uronate residues (pyranose affinity of the guluronic residues for divalent cations.13−15 The
rings with a carboxylic group in C-6) (Figure 1). β-D- effects of metal ion complexation on the morphology of the
Mannuronate and α-L-guluronate residues are present in resultant gels were evaluated by polarized optical microscopy
varying proportion, sequence, and molecular weight.2−4 The and electron microscopy.16−18 Alginates can also form acidic gel
axial orientation of the (1−4)-links of the guluronate residues by lowering the pH of a sodium alginate solution.5,19 Acidic
and equatorial in the case of mannuronate account for gels, like ionotropic gels, present a higher mechanical strength
variations of the organization and stability of the ionotropic for a high guluronic content. Small-angle X-ray scattering
gels formed by alginates with different mannuronic/guluronic (SAXS) suggested the formation of junction zones with a high
(M/G) ratios. degree of multiplicity. In the case of calcium or acidic
Alginates are able to form heat-stable strong gels with hydrogels, SAXS experiments were performed on gels obtained
divalent or trivalent cations (except Mg2+), whereas mono- by slow release of gelling agent.20,21 Draget et al. showed that
valent metal ions form soluble salts. This property is the basis the two gel systems could not be described by a simple
for applications of alginates in drug release systems5 and as monodisperse rod-like model and multiplicity of junction zone
supports in biocatalysis. Cations show different affinity for nature should be taken into account.22,23 Calcium induces
alginate. The interaction of the polyuronate chains with various gelation with polymer chains aggregated to form rod-like
divalent cations has been monitored by circular dichroism structures with cross-sectional radii of the order of 10 to 20
(c.d.).6−8 Spectral change when gelation is induced by Cu2+ Å.23,24 Acidic gels show larger aggregated structures (size ∼50
suggested a nonspecific binding mechanism, consistent with the Å). M and G sequences both contribute to cooperative
lack of selectivity of Cu2+ for different polyuronates.9,10 aggregation, and MG alternated structures favor solubility at all
The influence of the nature of the divalent cations on the pH values. In a previous study, we demonstrated the interest of
network elasticity was demonstrated by calorimetric and a structural study in the case of calcium−alginate hydrogels
dilatometric methods. The Young modulus of the gels followed systems to predict the morphology of the corresponding
the order Cd > Ba > Cu > Ca > Ni > Co > Mn. There is a
linear relation between the observed values of the Young Received: October 20, 2011
modulus and the logarithm of the association constant for Revised: December 13, 2011
divalent cations-alginate complexes.11,12 Alginates with high Published: December 15, 2011

© 2011 American Chemical Society 215 dx.doi.org/10.1021/bm201477g | Biomacromolecules 2012, 13, 215−220
Biomacromolecules Article

Figure 1. Structural units of alginate. M: mannuronate residue; G: guluronate residue.

aerogels. The SAXS patterns exhibited an asymptotic behavior formed by stepwise solvent exchange: the hydrogel microspheres were
at low-q values (in the experimental q range 7 × 10−3 to 2 × dehydrated by immersion in a series of successive ethanol−water baths
of increasing alcohol concentration (10, 30, 50, 70, 90, and 100%) for
10−2 Å−1) close to I(q) ≈ q−1, which was indicative of rod-like
15 min each. Ethanol was replaced by liquid CO2, which was
scattering objects with random orientation. The evolution of eliminated in a Polaron 3100 apparatus under supercritical conditions
the diameter of such rod-like objects was thus deduced from beyond 31 °C and 74 bar (typically 39 °C and 85 bar) leading to the
the maxima observed on Kratky plots, that is, I(q)·q2 versus q. aerogel spheres. The same procedure was followed for acidic gels using
The results were in perfect agreement qualitatively (rod-like a 1 mol·L−1 hydrochloric acid gelling solution. To avoid any
anisometry type of the scattering objects) and quantitatively morphological differences due to depressurization conditions, the 12
samples described were dried together.
(diameter of the rods) with direct SEM observations of the
Characterization. Nitrogen adsorption isotherms at −196 °C
morphology of aerogels and with the results of N2 adsorption were recorded on a Micromeritics Tristar apparatus. Prior to the
on the aerogel. This was evidence that under the experimental analysis, the aerogel samples were outgassed at 50 °C under dynamic
conditions chosen, the nanostructure of the aerogel depends on vacuum. The volume of the adsorbed monolayer was evaluated by the
the morphology of pre-existing rod-like objects within the gel, BET equation, and the surface area was calculated by assuming a N2
that is, that the structure of the aerogel provided a correct molecule to cover 0.162 nm2.
Scanning electron microscopy (SEM) pictures were recorded on a
image of the structure of the parent gel.25 Hitachi S-4800 microscope after platinum metallization.
Recent developments for cleaner sustainable chemistry are The SAXS data were collected on BM2-D2AM beamline at the
being driven by a shift from petrochemical-based feedstocks to European Synchrotron Radiation Facility (Grenoble, France) 9 days
biological materials. There is indeed a considerable interest in after the preparation of the samples. The data were collected at
exploiting natural polymer macrostructures, and, in particular, incident photon energy of 16 keV, the synchrotron working in 16-
those from polysaccharides, to create high-performance and bunch mode. A bidimensional detector (CCD camera from Ropper
environmentally friendly catalysts. Nevertheless, the introduc- Scientific) was used. The sample holders were metallic disks (diameter
tion of renewable resources in the production of catalyst 10 mm, thickness 2 mm) with a central hole where the microspheres
supports and adsorbents is only possible if the materials were placed. All data corrections were performed by the software
bm2img available on the D2AM beamline. The data were corrected for
intended to replace oil-derived or energy-intensive solids dark image (response of the camera with no incident beam), flat field
present adequate properties, such as high surface area, response (obtained after illumination of the camera with a fluorescent
appropriate surface chemistry and porosity, thermal and sample), and geometrical image distortion of the camera. Finally, the
chemical stability, and low cost. Algal polysaccharides, for the radial average around the image center (location of the center of the
economics of their growth and their ease of extraction, are incident beam) was performed for the q-range calibration standard
classed among the less-energy consuming alternatives to fossil (silver behenate) and the studied samples. The scattering data are
fuels.1,26 As far as remediation and heterogeneous catalysis are normalized by the transmitted intensity so that the scattering pattern
of the empty cell can be subtracted from all other scattering diagrams.

■
concerned, diffusion properties and high surface areas are
common requisites. Aerogel formulations of ionotropic alginate
gels fulfill the textural requisites,27 and metal transition cations RESULTS AND DISCUSSION
are of prime interest for catalytic purposes. In this work, the Dropwise addition of 2% w/w sodium alginate solution into the
morphologies of alginate hydrogels and aerogels are inves- various gelling media leads to hydrogel beads formation
tigated by SAXS, according to the nature of the divalent cation whatever the cation used. Alginate beads are mechanically
and the guluronic fraction of the alginate. The textural stable and the solvent exchange to alcogel or the supercritical
properties of the aerogels are discussed in relation to the drying does not change the macroscopic shape of the samples.
SAXS patterns. (See Figure 2.)

■ MATERIALS AND METHODS

Ionotropic Alginate Gels Synthesis. Alginate samples were
Metal loading of each system is investigated through
thermogravimetric studies of aerogel samples and allow
estimating the number of uronate units per cation (Supporting
supplied by FMC Biopolymer (Norway) and characterized by 1H Information, Table 2). Aerogel calcination under an air
NMR according to Grasdalen.28,29 According to their respective atmosphere leads to the corresponding oxide formation.30 At
guluronate content, alginates were designated as HG and LG for “high 900 °C, all of the organic part is eliminated, and the residual
G” and “low G”. The percents of guluronate unities in the alginate mass is only due to the oxide of the gelling cation. The number
backbone were 63 and 33, respectively (complete characterization
reported in Table 1 of the Supporting Information). of uronate units per cation is not very precise, but it allows
Hydrogel millimetric beads were obtained by dropwise addition of a some qualitative conclusions. Therefore, for each alginate and
2% sodium alginate solution (10 mL) into a gelling solution prepared each cation this ratio is close to 2, which is the theoritical value
with divalent cation chloride salt (0.1 mol·L−1, 10 mL). After for total complexation with divalent cations. Along with the
formation, the beads were left in gelling medium for 3 h. Alcogels were absence of sodium in the oxide (determined by EDX), this
216 dx.doi.org/10.1021/bm201477g | Biomacromolecules 2012, 13, 215−220
Biomacromolecules Article

range). The only difference concerns the relative intensity due

to the different contrast between the gel and the solvent (water
or air).
The SAXS patterns can be divided in two main ranges,
analyzing the intensity data according to
I(q) ≈ A /qα
In the q range 0.03 < q < 0.1 Å−1 (Figure 4), the slope of the
log−log plot, that is, the scattering exponent α, stays between

Figure 2. Aerogel beads of alginate HG (upper range) and alginate LG

(lower range) with various gelling conditions: Acidic, Mn2+, Co2+,
Zn2+, Cu2+, and Ca2+ from left to right, respectively.

result confirms that each uronate unit (M or G) participates in

the complexation when using an excess of gelling cation. The
slight deviation from 2 (1.9 ± 0.1) may be due to a little part of
acidic gelation31 with cation solution pH ranging from 4 to 6
(the pKa of guluronic and manuronic acids is close to 3.45), but
it is not clearly alginate depending.
Nanostructure of Hydrogels and Aerogels. All alginate
hydrogel and aerogel samples show isotropic small-angle X-ray
scattering. Plots of scattered intensities averaged along azimutal
angles versus the scattering vector q in log−log scale for
hydrogel and aerogel samples are displayed in Figure 3. For a
Figure 4. SAXS patterns of hydrogel samples obtained with alginate
HG (solid line) or alginate LG (dashed line) with different cations.
For clarity, the curves of each cation are arbitrary shifted up by
successive 100 factors, and the relative intensity for a given cation is
kept unchanged.

−4 and −5 for aerogel samples and ranges from −3 to −4 for

hydrogel samples. At lower q values (q < 0.03 Å−1), the
scattering exponent is lower and ranges from −1 to −4
according to the investigated system. The low-q scattering
exponents were determined by least-squares fits (Supporting
Information, Table 3). This analysis was kept even for acidic or
manganese hydrogels where the separation of the scattering
diagram into two well-apparent q ranges may not be obvious.
The scattering behavior observed in the low-q range
(Supporting Information, Table 3) enables us to evidence
two structural organizations in hydrogels. Mn-alginate systems
exhibit a high scattering exponent value α (above 2), whereas
Cu and Ca cations yield hydrogel structures with an α value
below 2. As discussed by Draget et al.,32 such behavior is
indicative of a fine fibrillar morphology when α is close to 1
(Ca-LG and Ca-HG; Cu-LG and Cu-HG; Zn-HG and Co-HG)
and a more complex morphology with junction nature
multiplicity when α is higher than 2 (H-HG and H-LG; Mn-
Figure 3. Small-angle X-ray scattering profiles for aerogel (solid lines) LG and Mn-HG; Zn-LG and Co-LG).
or hydrogel (dashed lines) samples obtained with alginate HG (on the The case of Zn and Co hydrogels systems has also to be
left) or alginate LG (on the right). For clarity, the curves of each
discriminated because the structural regime depends on the
cation are arbitrary shifted up by successive 10 000 factors, but the
relative intensity between an aerogel and the corresponding hydrogel type of alginate: High guluronic alginate (HG) induces the
is kept unchanged. formation of the fibrillar organization, whereas low guluronic
alginate (LG) induces the formation of junctions with multiple
given cation and a given alginate, the scattering pattern is little morphologies.
affected by the gel physical state: hydrogel or aerogel as As for previous studies, calcium and acidic gels seem to have
different behaviors. Acidic gels show a stronger initial slope
previously reported for calcium HG-alginate gel.25 Except for along with a smoother transition with the second part of the
alginate LG with manganese, aerogel shows the same scattering scattering pattern. For the investigated transition-metal cations
behavior type as the corresponding hydrogel (at least in low-q (Mn, Co, Zn, Ca, and Cu), we can classify each sample in two
217 dx.doi.org/10.1021/bm201477g | Biomacromolecules 2012, 13, 215−220
Biomacromolecules Article

Figure 5. Kratky plot of alginate HG hydrogel samples with various cations: (a) calcium-type with fibrillar morphology and (b) acidic-type with
multiple junction morphology.

categories whether its low-q slope is higher or lower than −2, aerogels. The main differences between metallic cation-alginate
that is, whether it displays the scattering behavior of acidic- systems would thus lie in the molecular organization within the
alginate hydrogel or calcium-alginate hydrogel. This classi- junction zones, where fibrillar or more complex junctions with
fication reveals in fact a third category comprising the samples multiple natures could be found.
behaving like calcium with alginate HG and like acidic gel with To gain more insight into the role of the metal cation-uronic
alginate LG. Therefore, with cobalt and zinc, the guluronic unit interactions, quantum chemical calculations on the model
fraction impacts the hydrogel morphology because scattering complex structures are in progress. Quantitative information
patterns with “calcium-type” shape or “acidic-type” shape with about the local structure of the complexes as a function of the
alginate HG or LG, respectively, are obtained. On the contrary, chemical nature of the cation and the uronic G or M unit are
with both alginates, manganese always shows an acidic expected. Indeed, in the case of Ca2+, molecular dynamics
behavior, whereas copper exhibits a calcium behavior. simulation revealed that changes in the ion-binding mode must
To go a step further in the investigation of the nanostructure play a rule in chain−chain association within junction zones
(i.e., secondary structure) of hydrogels, we tested in more detail and in the stiffening of the chains.33,34 This effect is expected to
the rod-like approximation by means of Kratky plots (i.e., be even more pronounced for transition-metal ions.
q2·I(q) vs q plots) as shown in Figure 5. The scattering Relation of the Textural Properties and the Nano-
behavior of a collection of randomly oriented rods should structure of Aerogel. SEM on the cross-section of aerogel
exhibit a maximum at q ≈ 1/Rc, where Rc is the mean gyration beads reveals a macroporous texture. Interconnected fibers
radius in the cross-section of the rods. The outer radius of the form a 3D network as shown in Figure 6. This morphology is
fibrils is then given by R0 ≈ Rc√2. The results for the diameter
of the fibrils d ≈ 2R0 = 2Rc√2 are gathered in Table S4 in the
Supporting Information.
In agreement with the published interpretation,32 the
differences in morphology result from the cooperative
interactions between the metallic cations and mannuronic
and guluronic sequences within the primary structure of
alginates. Complexation of cations do not depend on the
guluronic content of alginate, as shown by Table 2 of the
Supporting Information, but the structuration resulting from
cooperative associations of several macromolecular chains is
shown to depend on the nature of the cation or the structure of
the alginate chain. Copper cations seem to interact strongly
with both mannuronic and guluronic residues and induce a
comparable fibrillar morphology whatever the guluronic
content. The scattering exponents are significantly impacted
by alginate type in calcium-alginate hydrogels, and thus calcium
cations seem to induce chain associations into fibrils Figure 6. SEM picture of M-alginate HG and LG aerogel (same scale
for all images).
preferentially with guluronic residues. A clear transition in the
morphology is observed from HG to LG for cobalt and zinc
observed in each sample with slight differences in the network
hydrogels, which again should be due to a decrease in the
cooperative interaction effects for LG systems. Mn and acidic density and the roughness of the fibers.
hydrogels display the nonfibrillar morphology that could be due The fibrillar network of aerogel beads leads to high surface
to an absence of cooperative interchain associations for all areas as determined by the analysis of the N2 adsorption−
investigated alginate types. The morphology of aerogel samples, desorption isotherm at 77 K.35 The shape of adsorption−
as deduced by SAXS, was shown to be mainly inherited for the desorption isotherm of N2 at 77 K is typical of a type IV
parent morphology of corresponding hydrogels for calcium- isotherm in the classification of the IUPAC36 with a strong
alginate gels.25 In this work, we observed the same trends for a adsorption at low relative pressure and a hysteresis loop at very
variety of cations for two alginate systems. This resulted in high relative pressure. This behavior characterizes large
similar values of the power exponents α characterizing the mesopores and macropores. The application of the αS method
morphology type of hydrogels and their corresponding indicates that the solid does not present micropores. The broad
218 dx.doi.org/10.1021/bm201477g | Biomacromolecules 2012, 13, 215−220
Biomacromolecules Article

size distribution of the mesopores is centered around 30−40 strongly with both mannuronic and guluronic residues and
nm. induce a comparable fibrillar morphology whatever the
The range of surface area goes from 80 to 540 m2·g−1 (Figure guluronic content. With cobalt and zinc, the guluronic fraction
7). In the absence of microporosity, these surface areas can be impacts the hydrogel morphology. Mn and acidic hydrogels
display the nonfibrillar morphology that could be due to an
absence of cooperative interchain associations for all inves-
tigated alginate types. In the size range investigated by SAXS
(∼10−200 Å), the structure of aerogels is found to be inherited
from the morphology of the parent hydrogel, whatever the
initial structural regime (fibrillar junctions gels or gels with
multiple junction nature). The surface areas and thus the
overall performance of such materials as catalysts are in close
relation with the structural regime exhibited by aerogels.

■
*
ASSOCIATED CONTENT
S Supporting Information
Figure 7. Surface areas of aerogel of HG alginate (black bars) or LG Complete characterization of the alginates, mass analysis of
alginate (gray bars) with various cations. aerogel samples, the scattering exponent in the low-q
asymptotic regime as function of cation nature, alginate
attributed to the outer surface of the fibrils. Alginate HG structure, and hydrogel/aerogel physical state, and the fibril
generally produces samples with higher surface area compared diameter estimated through the maxima of Kratky plots are
with alginate LG. A difference around 100 m2·g−1 is observed reported. This material is available free of charge via the
except for acidic and copper gels where the values are very Internet at http://pubs.acs.org.

■
close. Moreover, the surface area seems to be strongly related
to the gelling agent giving a ranking which is independent of AUTHOR INFORMATION
the alginate if we exclude the case of acidic gelling.
Corresponding Author
In view of Figure 6 displaying fiber morphologies in all
*E-mail: [email protected]. Fax: (33) 4 67 16 34 70.

■
systems, the morphology studied by SAXS should be related to
a more local scale within fibers.
As far as the preparations of highly dispersed materials are ACKNOWLEDGMENTS
needed, for example, for adsorption, catalysis, and remediation We gratefully acknowledge Didier Cot (IEMM) for the
in liquid or gas phase applications, the higher surface areas are scanning electron microscopy. SAXS was performed at ESRF
obtained for the systems that display the fibrillar organization during experiment IN-728 from March 31, 2010 to April 1,
resulting from the higher intermacromolecular interaction and 2010. It is a pleasure to acknowledge the BM2-D2AM beamline
structuring. Figure 8 evidences the correlation between the staff and more specifically Dr. Cyrille Rochas for his invaluable
help during pre-experiment formalities, SAXS experiments, and
data treatment.

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