Characterization tools and techniques of hydrogels

CHAPTER 16

Characterization tools and techniques of hydrogels Sayan Ganguly1, Poushali Das2 and Narayan Ch. Das1,2,* 1 2

Rubber Technology Centre, Indian Institute of Technology, Kharagpur, India School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur, India

Abbreviations , r02 . μ-CT 3D BC C60 CCD CDs Cn CNTs DUT FH GQDs IPN l LCST OCTSM PAAm PEG PNIPAM PTFE PVA SA SANS SAXS SR SWCNTs USANS VPT θ ξ


root mean square distance between two consecutive cross-linking points microcomputed tomography three-dimensional bacterial cellulose buckminsterfullerene charge-coupled device carbon dots characteristics ratio proposed by Flory carbon nanotubes device under test fluorescent hydrogel graphene quantum dots interpenetrating polymeric network bond length lower critical solution temperature optical coherence tomography-based spherical microindentation poly(acrylamide) poly(ethylene glycol) poly(N-isopropylacrylamide) poly(tetrafluoroethylene) poly(vinyl alcohol) sodium alginate small-angle neutron scattering small-angle X-ray scattering swelling ratio single-walled carbon nanotubes ultra small-angle neutron scattering volume phase transition bond angle distance between two consecutive cross-linking points

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00016-1 © 2020 Elsevier Inc. All rights reserved.

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482 Chapter 16

16.1 Introduction Hydrogels are a three-dimensional (3D) cross-linked, insoluble mass consisting of hydrophilic polymeric chains which are quite susceptible to volume alteration against external stimuli. The properties of hydrogels are always of concern to scientists because of their mode and area of applications. The hydrophilicity of hydrogels appears to be due to their polar pendant groups inherited from precursor monomers. Cross-linking of hydrogels is classified in various aspects; most commonly they are covalent, ionic, and physical hydrogels [1]. Experimentally, hydrogels can be evaluated by their flow behavior against external pressure. This flow property of materials is termed rheology. As a matter of fact, it has already been proved that a dilute solution of polymer in water acts with Newtonian flowability, whereas in a gelled system the flow property is slightly different. From a rheological point of view hydrogels are basically a combination of elastic and viscous attributes. Hence, to assess a hydrogel; their flow behavior analysis is a common characterization. The most prominent use of hydrogels is in the biomaterial field because hydrogels mimic the soft tissues and muscles of animals. For the last 30 years, a large number of hydrophilic polymers have also served in this area. Natural polymers like alginate, starch, agarose, gelatin, collagen, carrageenan, and other polysaccharides had been explored. In synthetic hydrogel fabrication, the first hydrogel was reported by Wichterle and Lim in 1960 [2]. With commercial success in soft materials research, hydrogels are the most promising candidate and can be regulated by means of external stimuli, namely, pH, temperature, ionic strength, electric pulse, light, solvent, enzymes, and a saline environment [36]. In 1980, Lim and Sun developed calcium ion cross-linked alginate hydrogel beads for microcapsule formation [7]. Yannas et al. first fabricated burn dressing/healing hydrogel skins from collagen [8]. The research results based on improvement of hydrogel technology can be sorted into several areas [9]. First-generation hydrogels basically emphasized the cross-linking methods. The initial research was directed at the improvement of water uptake behavior, desirable mechanical properties, and swelled gel dimensional integrity. After that, in the 1970s, research was channelized to the stimuli-responsive behaviors and the mode of applications. In this phase, most hydrogels were loaded with small molecules which were subjected to leaching out from the gel matrix by means of their external stimuli response. In this domain, drug delivery, fertilizer release, and other dye releases had immense significance [10]. The third-generation hydrogels were comprised of stereo-complex formation-assisted gelation. These are poly(ethylene glycol) (PEG)PLA physical interactions [11,12].

16.2 Hydrogels: microstructureproperty relationship Before going on to the main segment of this discussion, one basic query has to be answered. Why do we need to uncover the microstructure and hydrogel properties before

Characterization tools and techniques of hydrogels 483 discussing the characterization of hydrogels? The answer can be given as to evaluate the practical life span and usability of hydrogels. There are various kinds of hydrogels, based on their origin, synthesis methods, mode of application, type of polymeric network, and cross-linking methods. The most common hydrogels are chemical cross-linked hydrogels. The hydrogel network, which is liable to cover the whole 3D network of hydrogel, can be categorized as synthetic polymeric, natural, and a combination of these [13]. The most common synthetic polymers used to fabricate hydrogels are PEG or polyethylene oxide, polyacrylamide (PAAm), polysaccharides, poly(hydroxyethyl methacrylate), poly(vinyl alcohol) (PVA), etc. [14,15]. Natural polymer-based hydrogels are normally fabricated from collagen [16], silk [17], gelatin [18], and polysaccharides [19]. The most common commercially available polysaccharides are agar [20], alginate [21], carrageenan [22], starch [5,23], and psyllium [24,25]. For chemically cross-linked hydrogels these polymers are used as graftable polymers. This mechanism of fabricating hybrid hydrogels forms IPN-type hydrogels. In the case of physical hydrogels, the most significant distinction with chemical hydrogels is the physical entanglement of macromolecular chains. Fig. 16.1 illustrates the hypothetical formation of chemical and physical hydrogels for chemical gels and covalent anchoring at the specific intersection points of macromolecular chains, where physical hydrogels consist of entanglement or interactions throughout a domain not in a specific point. For alginate-based hydrogels ionic cross-linking is a common phenomenon.

Figure 16.1 (A) Ideal cross-linked 3D assembly with tetra-functional linkages. (B) Nonideal cross-linked network including chain ends and loops. (C) Ideal cross-linked double network gel. (D) Physically entangled network. (E) Helix formation in network. (F) Alginate-like network with ionic linkages between adjacent chains. 3D, Three-dimensional.

484 Chapter 16 Cross-linking points and the density of cross-linking points in a hydrogel matrix impact significantly on the mechanical viscoelastic or flow property of the hydrogels. At a molecular level hydrogels are also considered as porous materials. The pores present in hydrogel house water molecules when they are subjected immersed in aqueous media. The porous morphology of hydrogels is an outcome of internal cross-linking. Porosity in the hydrogel matrix is directly related to the water uptake behavior of hydrogels, called swelling behavior, this can be carried out dynamically or in equilibrium. Moreover, the gradual leaching of water molecules from hydrogel matrix is called a desoiling or poroelastic relaxation experiment. The swelling ratio (SR) or swelling coefficient is defined as the ratio of weight of swollen gel to dry gel. SR 5

Vswollen Vdry

(16.1)

The swelling ratio has utmost significance to define various properties which are critical in the gel matrix, viz. mechanical toughness and transport properties. The swelling ratio differs on the type of polymers and the hydrophilicity of the monomers taken for fabricating hydrogels. For example, poly(HEMA) shows for relatively low swelling whereas hydrogels made of acrylic acid monomer has immense soiling ratio. SR 5

Vpolymer 1 5 Q Vpolymer 1 Vwater;initial 1 Vwater;imbibed

(16.2)

As shown in Eq. (16.2) water uptake is dependent on two condition, the initial water to be imbibed and the surplus volume of water penetration during swelling. The microstructural attributes of the hydrogel matrix can also be evaluated by another scaling parameter called cross-link density. Fig. 16.2 is a graphical representation of the macromolecular change among the junction points. The average molecular weight between two adjacent cross-linking points is Mc, which is related to the SR by the following relation.

Figure 16.2 Microstructural parameters of hydrogels.

Characterization tools and techniques of hydrogels 485 This relation is applicable for moderate to high swelling (superabsorbent) hydrogels (SR or Q . 10); 3=5

SR or Q 5 βM c

(16.3)

where β is a constant related to the specific volume of the polymer, the main interaction parameter between polymer and solvent molecules and the molar volume of water. Again the SR is related to the 3D network mesh size, sometimes called the pore diameter, that is, the distance between two consecutive cross-linking points (ξ) in the spatial (3D) network. ξ 5 Q1=3 ðr 20 Þ1=2

(16.4)

where ,r02 . is the root mean square distance between two consecutive cross-linking points. ,r02 . can be written as, ðr 20 Þ1=2 5 lðCn Nb Þ2

(16.5)

where l is the bond length, Nb stands for the number of bonds, and Cn implies the characteristics ratio proposed by Flory. Cn generally lies in the range of 510 which corroborates deviations from ideality for freely rotating chains in the hydrogel mass. An approximation to evaluate the value of Cn for freely movable macromolecular chains is: CN D

1 1 cosθ 1 2 cosθ

(16.6)

where θ is the bond angle between consecutive segments. The mesh size (ξ) is basically a result of the gelation chemistry associated with the cross-link density, temperature, environmental pH, and other external stimuli. The pore size of a hydrogel also influences its mechanical and structural properties and adsorbent nature. As the mechanical properties of hydrogel are quite poor compared to other viscoelastic polymer composites, to improve their structural properties become the primary requirement to commercialize a hydrogel system. Generally, in hydrogel internal microstructure, the dimensional stability of any hydrogel system is related to the cross-link density. A highly dense network hydrogel with high crossing density provides high gel strength and good dimensional stability but the water uptake behavior is sacrificed due to high cross-link density. Thus, depending on the mode of application, the water uptake behavior and cross-link density are optimized. For physical hydrogels the macromolecular chain entanglement occurs due to hydrophobic interactions between the blocks present in the polymer. In the domain of physical hydrogels, the major two classes are thermoreversible and rheo-reversible.

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16.3 Mechanical characterizations of hydrogels 16.3.1 Uniaxial tensile testing As hydrogels are soft materials they lack mechanical robustness. The mechanical properties have significance in evaluating the extent of failure and longevity of hydrogels. In recent times, the most common method to evaluate the mechanical properties of hydrogels was by uniaxial tensile experimentation, which is sometimes called strip extensiometry. These methods are widely accepted by the scientific community to determine the mechanical toughness and ultimate tensile strength of various hydrogels. Moreover, uniaxial tensile testing infers a mechanical toughening mechanism for anisotropic filler-embedded nanocomposite hydrogels. Uniaxial tensile testing for soft/light samples is generally carried out by holding the terminals of specimens between two grips to ensure there is no slippage. The terminals of the hydrogels are physically covered by rough surfaced papers. Fig. 16.3 shows pictorial illustrations of several conventional techniques applied to carry out the mechanical testing of soft materials. In the case of extensiometry, two major classifications are available, one is strip extensiometry and the other is ring extensiometry. These experimentations result in a typical stressstrain plot for hydrogels. From the stressstrain plot one can obtain the finite values of Young’s modulus, stress, and ultimate tensile stress [26]. In addition to this extensiometry, the viscoelastic nature of hydrogels also can be determined. In terms of rheology these are called the creep test and stress relaxation experiment. Though this is a destructive experiment, it has immense significance due to nontime-consuming data collection and multidimensional explanations related to microstructure and morphology. For biological samples, hydrogels are a major concern because of their flexibility, noncytotoxicity, tuneable mechanical properties, and tissue mimetic features.

Figure 16.3 (A) Uniaxial extensiometry; (B) ring extensiometry; (C) compression test; (D) hydraulic bulging test; (E) indentation test.

Characterization tools and techniques of hydrogels 487

16.3.2 Compressive testing The compressive toughness testing of hydrogel is another destructive experiment for hydrogels to evaluate their mechanical properties. This testing is generally carried out by placing the hydrogel specimen between parallel plates followed by vertical pressing. A pictorial illustration of compressive experimentation has been given in Fig. 16.3C. The platens used for compressive experimentation are normally nonporous and smooth as per their surface morphology. But sometimes a single porous platen also has been utilized for multiphase gel materials. For multiphase materials, if there is any leaching out of fluids from the gel matrix, the porous platen will resolve that situation by permeation. The application of external pressure with respect to strain gives the compressive stressstrain plot, which infers the typical load-bearing and compressive fracture toughness of soft materials. Moreover, from such a data acquisition technique several theoretical model fittings also have been implemented. Comparing the versatility and popularity points of view between extensiometry and compression, the majority of research uses compressive experimentations. This is due to the easy sample preparation and unconditional restrictions over the specimen geometry. In general, extensiometry demands more precise sample dimensions and as good as possible sample surface smoothness; whereas in the case of compressive stress measurement there are no such restrictions as this testing requires only flat surfaces for even distribution of external compressive stress throughout the gel matrix. Such compressive mode-enabled testing also has significance over the judgment of nanocomposite hydrogels. Nanocomposite hydrogels are a special class of hydrogel where nano-inclusions have been entrapped inside the hydrogel matrix in order to improve the hydrogel properties in a synergistic fashion. Most popularly, nano-reinforcement incorporated hydrogels have been nurtured because of their load-bearing and fatigue performances. Recently, in situ nanoclay-based biomimetic adhesive type polymerizable monomers have been used to enable the fabrication of tough hydrogels [27]. The extent of stress transfer and load-bearing response of hydrogels reflects their compressive cyclic loadingunload plots.

16.3.3 Bulge experiment Prior to rupture, tough hydrogels go through a process called necking. In this domain, the strain hardening phenomenon has marked theoretical inference. Strain hardening can be assessed by a hydraulic bulge experiment for hydrogel sheets under biaxial tensile force. In this test both the stress and strain can be evaluated, whereas for typical tensile testing the experiment is restricted to uniform strain. A bulge test of hydrogels reflects their plastic flow behavior. The sample architecture for a bulge test is circular in shape (diaphragmlike), tightly held at the outer fringe of the sample holder, followed by uniform stretching via an external force applied laterally. One restriction to the specimen preparation is that

488 Chapter 16 the thickness to bulge diameter ratio should be small enough to eradicate the bending effect of hydrogels during clamping. In the early years, researchers used this test for thin metal films [28], but, in 1987, polyimide film was first experimented with via a hydraulic bulge test [29]. The displacement after the external force is measured by a charge couple detectors (CCD) camera laser probe. The typical formula for interpretation of external applied pressure and displacement relation can be written as: P5

C1 σ 0 t C2 Et h 1 4 h3 2 a a

(16.7)

where σ0, t, and a correspond to the residual stress, film thickness, and membrane halflength, respectively. For half-length assumption, the radius is taken into consideration, whereas, for rectangular specimens, the half-length is considered as the shortest length/ dimension of the rectangle. h is the extent of deflection after application of stress. E is Young’s modulus of the hydrogel and C1, C2 are the constants depending upon the finite element analysis and Poisson’s ratio, respectively. Mitchell et al. first accumulated the C1 and C2 values calculated by various researchers. The brief data for the calculated constant values have been tabulated in Table 16.1. Maier-Schneider et al. evaluated the values of the constants by utilizing a minimization strategy [30]. In general, two typical types of loading configurations have been standardized in bulge testing; “deflection into the orifice” and “deflection away from the orifice”. This test was first popularized by Xiang et al. in 2005 when they proposed mathematical discussions of rectangular hydrogel membrane [31]. Moreover, they also provided information regarding the elasticity, hysteresis, and toughness of the membrane sheets. Thus it could be inferred that besides uniaxial tensile testing, the bulge/deflection experiment also has great significance in hydrogel characterizations to evaluate the tissue mimetic nature of soft materials. Table 16.1: Calculated C1 and C2 values obtained from pressuredisplacement models Bulge geometry

C1

C2(1 2 υ)a

Circular

4.0 4.0 4.0 3.04 3.04 3.41 3.39 3.45 1.55 2.0

2.67 2.67(1.026 1 0.233υ)21 (7 2 υ)/3 1.473(1 2 0.272υ) 1.473(1 2 0.272υ) 1.37(1.075 2 0.292υ) (0.8 1 0.062υ)3 1.994(1 2 0.271υ) [30/(1 1 υ)][0.035 2 (16/(800 2 89υ))] 8/[6(1 1 υ)]

Square

Rectangular a

υ 5 Poisson’s ratio.

Characterization tools and techniques of hydrogels 489

16.3.4 Indentation test The indentation test is another crucial mechanical restructure characterization of soft materials. It is actually a single-point compression test where the surface deflection has been quantified (Fig. 16.3E). In this experiment, a force displacement transducer is connected to an indentation probe. This arrangement results in a forcedisplacement plot from which the elastic modulus of the materials (here hydrogel) can be calculated. One thing that should be mentioned is that indentation tip/probe geometry has utmost importance in assessing material rigidity. Recently, many scientific modifications via software precision have made this technique more attractive to the academic and research community. The data obtained after indentation are normally the critical stress of indentation, amount/depth of indentation, stress relaxation, and sample rigidity. These data have been acquired also in nanosized domains [32]. The mode of applications of such testing has been implemented in gel coating and soft materials attached to a hard surface. The indentation test shows superiority over conventional mechanical characterizations in terms of very low time consumption, online, and real-time data acquisition. The localized failure behavior is ideally evaluated from this experiment. However the disadvantage to this set up is it is not as desirable for cell-assembled hydrogels. This is due to the restriction of this experiment to a sterile environment. To overcome this limitation, a modified version of indentation has been implemented where sterile environment-based testing can be carried out without any difficulties. The name of the modified version is long-focal microscopy-based spherical microindentation which was adopted to carry forward the research based on online 3D cell-impregnated hydrogels. This technique was first proposed by Liu and Ju to calculate the mechanical properties of egg shell membranes [33]. Hydrogel thickness for this testing has to be kept below 1 mm. In this instrument, there are two parts, that is, the sample holder consisting of a spherical pin pointed indenter and the image acquisition set up. The hydrogel sample is attached radially by means of a circular clamping system with an inner diameter of 20 mm. The hydrogel is point-indented by a 4-mm PTFE sphere, 316L stainless steel, and 440 stainless steel sphere. These probe materials were chosen after initial assessment of hardness of the gel surface. The hydrogel to indenter diameter ratio was kept constant at 5.0 throughout the process. For 3D cell-impregnated hydrogels, the whole system is kept in sterile phosphate buffer saline medium at 37 C, 5% CO2. This environment severely impacts the mechanical properties of hydrogels. The image-capturing device consists of a long-distance objective microscope and computer-controlled CCD camera. The displacement of gel membrane is measured by a computer-controlled stage micrometer. The viscoelasticity of the specimens was evaluated indirectly by deformation of the hydrogels. In addition, another type of indentation method also has been adopted, called optical coherence tomography-based spherical microindentation (OCTSM). The theory behind this

490 Chapter 16 method is Hertz contact theory, which is performed by means of a spherical ball. OCTSM is a noninvasive technique followed by backscattering of light passing through the specimen. In brief, the setup consists of two typical light beams; one passed through the specimen and the other passes without any scattering as a reference beam. The backscattering of light is detected by a photodetector which develops a photograph of the cross-sectional microstructure. The depth of penetration/indentation is reflected over the sample thickness and geometry of the specimen. To be precise, indentation is a type of localized compression test which records the load to depth with time as a triparameter compromisation. The obtained value from indentation is the reduced modulus or ER, which can be written as, ER 5

E 1 2 γ2

(16.8)

where γ is Poisson’s ratio. Indentation for soft materials is classified as macroscale indentation (“mm” scale) and micro/nanoscale indentation (“μm” or “nm” scale). In recent years, nanoscale indentation has become a very promising strategy to investigate the mechanical properties of hydrogels [34].

16.4 Rheology 16.4.1 Viscoelasticity and microstructure Viscoelasticity deals with the flow and deformational behavior of materials. The term is adopted from the Greek work “rheos” meaning “the river” or “flowing.” This technique has been used by various industries related to paints, adhesives, paper, packaging, cosmetics, foodstuffs, pharmaceutics, polymer product making, surface technology, and even in the glass/ceramic and metal industries. The basic term “viscoelastic” consists of two subwords indicating viscous dominating over elastic. This implies the time-dependent deformational behavior of materials. The viscoelasticity of a material is highly dependent on the external force applied to the material of interest. The mechanical response of hydrogels normally shows time-dependent exponential curves which have close resemblance to elastomers. Hydrogel consists of a huge amount of water which acts as a plasticizer for hydrogels. Soft hydrogels with elastomer-like behavior are welcomed nowadays for applying in soft artificial tissues, muscle mimetic devices, tissue actuations, artificial skin, and mussel mimetic electronic membranes [3537]. When external shear is applied to a hydrogel specimen there are several consecutive phenomena resulting which can be summarized as an initial acceptance of external force, initiating mobility in macrochains followed by stress dissipation of polymer chains over time resulting in a gradual decaying/sigmoidal curve.

Characterization tools and techniques of hydrogels 491 There are some specific characteristics to such chain mobility and deformations which are categorized as “creep” and “stress relaxation.”

16.4.2 Creep behavior Creep is time-dependent deformational change or strain (γ) monitored in a constant stress (σ). Fig. 16.4A and B show a typical creep curve for viscoelastic fluids. The creep compliance, J(t) is the ratio of shear strain to applied shear stress. If creep compliance has been measured over a very short or long period of time, it shows negligible time dependency. At a very small time scale the polymer molecular chain mobility is much higher which implies an unrelaxed state of the system, as designated by Junrelaxaed in Fig. 16.4C. In contrast, at a longer time scale the molecular reorganization is better, and has been termed as the relaxed state of the system, Jrelaxed. For the assessment of product performance in long-term applications, creep study is highly significant. The data obtained from creep study reflect the materials’ recovery after being subjected to a fixed load. Ganguly et al. performed a creep study for nanocomposite hydrogels where the impregnated filler was anisotropic clay tactoids [25]. Clay is a highly abundant ceramic material. It has several applications besides hydrogels, such as solid support for catalyst, rheology modifier, and air impermeable coating [3843]. In their work they showed enhancement in elasticity after incorporation of clay nanofillers into a hydrogel matrix. Clay-incorporated hydrogels

Figure 16.4 Model plots for creep (AC) and stress relaxation (DF) of viscoelastic materials.

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Figure 16.5 Creep (left) and strain recovery (right) of clay-reinforced nanocomposite hydrogels [25]. r 2018. Reproduced with permission from Elsevier.

showed better elasticity and minimization of residual strain after a creep experiment as shown in Fig. 16.5. In another experiment, ionic cross-linked PVA hydrogels were also tested in a creep experiment [44]. They showed that PVA with Fe31 cross-linked hydrogels generally had 23% viscoelastic deformation. In low cross-linked systems, molecular deformation is quite easy because of the low level of elasticity. However, in the case of high cross-linked systems, the spring back action is much better, implying a better viscoelastic response. For high cross-linking systems the macromolecular chains are deformed under an external load. When external forces are applied, the slippage of polymeric chains is inhibited due to the interchain attachment (either physical bonding or chemical bonding). Thus, this experiment can be done to discover the longevity of soft materials under constant load. Viscoelasticity is therefore affected by cross-linking in the gel matrix as well as fillerpolymer chain attachment (physisorption).

16.4.3 Stress relaxation Stress relaxation is another time-dependent phenomenon where a finite amount of strain has been kept constant. This experiment is depicted graphically in Fig. 16.4DF. When a material is subjected to deform up to a certain deformation, there is a molecular chain relaxation inside the matrix due to exponential stress dissipation. Such stress dissipation contracts the specimen after a finite time and the remaining part which is not returned back to the exact initial position is called the residual strain. If this deformation and sparing back action happen instantaneously, then the phenomenon is called “elastic.” But in the case of hydrogels, the spring back action is time-dependent and the time decay of stress has a

Characterization tools and techniques of hydrogels 493 resemblance to the molecular relaxation. In Fig. 16.4F the shear modulus G(t) has been illustrated in relaxed and unrelaxed states. If one tallies the creep compliance and the stress relaxation modulus, a reciprocal relationship is seen.

16.4.4 Dynamic mechanical behavior The quantitative estimation of viscoelastic and rheological properties is best evaluated by means of dynamic mechanical testing. This is a frequency-based experiment. This oscillatory test gives the measure of complex shear modulus, which is given in Eq. (16.9):


σ G 5 G 1 iG 5
γ


0

00

(16.9)

where G0 and Gv correspond to the elastic (storage or real part) and viscous (loss or imaginary part) shear modulus, respectively. G
is the complex shear modulus. In the dynamic mode, that is, the oscillatory mode, the complex strain/deformation can be evaluated as


γ 5 γ0 expðiωtÞ

(16.10)

were ω and γ 0 indicate the angular frequency and initial strain value, respectively. If the Maxwell model is taken into consideration, then the viscoelasticity can be implemented by calculating the first derivative of strain rate and the equation will be:








dγ 1 dσ σ 5 1 G dt dt Gθ

(16.11)

where θ is the characteristic time constant and is closely related to η/G. η is the viscosity of the polymer system. After calculating this equation, the linear form of the equation after integration is


σ Gω2 θ2 ωθG 1i
5 2 2 γ 1 1 ω2 θ2 11ω θ

(16.12)

or


σ 5 σ0 expðiωt 1 δÞ

(16.13)

where δ is the angular phase lag having a maximum amplitude of σ0. Now, after comparing the aforementioned equations, it can be summed up that 0

G5

Gω2 θ2 1 1 ω2 θ2

(16.14)

494 Chapter 16 and 00

G 5

Gωθ 1 1 ω 2 θ2

(16.15)

Eqs. (16.7) and (16.8) are characteristic equations for calculating the elastic (storage) and viscous (loss) modulus for the specimens, respectively. Another thing also can be evaluated from the analysis, which is a damping parameter or dissipation factor, or to be precise “tan δ.” “Tan δ” is the ratio of viscous to elastic modulus. Tan δ can be numerically written as tanδ 5

G00 1 5 0 ωθ G

(16.16)

“tan δ” is a measure of dissipation energy after the application of oscillatory forces to a polymer matrix. This dissipation energy is solely in terms of heat. In the case of rheology, low temperature and high frequency are related in experimental conditions. At low temperature the hydrogels have a rigid type of system, rather than “glassy.” However, after increasing the temperature the molecular chains’ mobility becomes reduced, so that the system shows a glassy to rubbery transition. This results in a gradual decrement in elastic modulus and an increment in viscous modulus. Similarly, at high frequency, the polymer chains are incapable of reorienting, thus there is no chain relaxation. Improper chain relaxation hinders the segmental mobility resulting in enhancement of the elastic modulus. However, at a relatively lower frequency, the relaxation time required for polymer chains is enough that the stress dissipation is facile in this case. This implies a comparative lowering of the elastic modulus as lower frequency which is related to the dynamic mechanical behavior at high temperature. The damping parameter is the measure of how much energy has been dissipated during oscillatory testing. As “tan δ” is proportional to the loss modulus of the polymer system, this means the damping character of the specimen will be greater if “tan δ” increases. From a typical “tan δ” plot the glass transition temperature (Tg) can be evaluated. In terms of “tan δ,” the glass transition temperature can be defined as that temperature where the damping factor shows an apex value in the plot. Moreover, several other temperature-assisted transitions also can be defined by this analysis. The most significant transition that occurred at the highest temperature is called the “α-transition.” Other subsidiary temperature-based transitions also take place during this experiment, and are called “β-transitions.” “β-transitions” have relatively lower temperature transitions with respect to “α-transitions.” If the inherent cross-linking is uniform in nature, then the “α-transitions” are more prominent and the broad peak of Tg can be observed. The rheological testing of hydrogels is carried out normally in a swollen state, as in the swollen state the hydrogels behave like soft materials with sufficient rubber-like flexibility. In addition to the dynamic mechanical system, equipment for such cases includes the

Characterization tools and techniques of hydrogels 495

Figure 16.6 Rheological properties of gelatin hydrogel (Gel-H), gelatin/BC composite hydrogel (Gel/BC-H), and magnetic composite hydrogel (Mag-H) [45]. r 2018. Reproduced with permission from Elsevier.

parallel plate rheometer. Herein, researchers can tune the desired rheological conditions; such as strain sweep, temperature sweep, and viscosity evaluation. The distinguishable difference between elastic and loss moduli implies the gel state of the materials. Gelatin and bacterial cellulose (BC)-based hydrogels have been evaluated to assess their elastic and loss moduli [45]. They showed that a very small amount of BC did not significantly improve the elasticity of hydrogel (Fig. 16.6). Dextrin-based hydrogels have been tested previously and showed a tan δ vs. time plot depicting the “gel point” [46]. The gel point is the particular point where a gradual decrement in tan δ curves in various frequencies converge to meet. Thus, for hydrogels, synthesis parameters like gel time estimation are significant because they have a direct relationship to the gel strength. Moreover, another trustworthy method to estimate the gel point is “cross-over point evaluation.” The crossover point is the point where elastic and loss moduli alter their values/trends. That means in a pre-gel state, the loss modulus is dominant over the elastic one, whereas after gelation the trend shows the opposite. This implies a comparative enhancement in elasticity after gelation cannot be ruled out. Cross-over point estimation has been estimated in several literatures [47,48]. Nanofiller-based semi-IPN hydrogels also showed similar rheological behaviors. Clay and graphene are two of the most used fillers in the hydrogel field. When anisotropic fillers are incorporated inside hydrogels, the gel rheology showed much better thixotropic characteristics. Clay-based hydrogels with high gel strength have been described elsewhere [49,50]. Graphene-reinforced hydrogels also show high gel strength during oscillatory shear force experiments [51]. The gel strength is a quantitative parameter to estimate the gel dimensional stability of hydrogels in dynamic/oscillatory conditions. Graphene is a unique filler which has immense ability to adhere polymer chains by means of physisorption. This enables delayed network rupturing with increasing graphene content. Fig. 16.7 is an illustrated experiment of this work.

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Figure 16.7 (A) Rheological behavior of the hydrogel in a frequency sweep experiment. (B) Rheological behavior of the hydrogel in a shear stress sweep experiment. (C) Elastic modulus to loss modulus ratio versus graphene concentration in hydrogel (corresponds to the gel strength or elasticity of the nanocomposite hydrogels). (D) Dependency of the effective concentration of elastic chains with graphene concentration. The plot shows enhancement of cross-linking points and effective concentration of chains with increasing graphene content [51]. r 2018. Reproduced with permission from Elsevier.

For double-network hydrogels, the flow property was also calculated by Liu et al. [52]. Double-network hydrogels are another special class of hydrogel where two intertwined polymeric networks are cross-linked. Generally, double-network hydrogels are also called full-interpenetrating polymer networks (full-IPN). PVA/PAAm and sodium alginate (SA) have been gelled together and rheologically characterized. Single-network hydrogels are greatly inferior to double-network hydrogels. Fluorescent injectable hydrogels were developed by another research group with a self-healable feature characterized by a rheological experiment [53].

Characterization tools and techniques of hydrogels 497

34°C G',G''×10a(Pa)

P-3: a=4

P-2: a=2

26°C

P-1: a=0 1E–3

10

21°C 20

30 40 Temperature (°C)

50

60

Figure 16.8 Temperature dependence of storage and loss modulus and their crossover. Crossover point designates the gel time [54]. r 2007. Reproduced with permission from Elsevier.

In addition to gel time, gel temperature also can be estimated from rheology assessment. Gel temperature is the critical temperature where the gelation initiates or takes place. As per the flow behavior of materials, elastic modulus corresponds to the gel state, whereas for liquid state, loss modulus is the most dominant factor. Tang et al. showed how to evaluate the gel temperature from a rheological study [54]. As per their study (Fig. 16.8), initially the pre-gel state showed both elastic modulus and loss modulus values, where elastic modulus is less dominant. In fluid form, the polymer chains are more labile to flow after application of external shear forces. However, when temperature was applied to the system, the initiator disintegrated to fragments followed by a free radical gelation process. As the polymer/monomer mix is thermally insulating in nature, the gain of activation energy from external temperature would be slightly delayed. Thus, for thermal or redox initiators, there will be an induction period for initiators to propagate gelation. As per the revealed data, it is clear that the gel temperature has a direct relation to the added polymer phase. In that report, the authors added chitosan as the polymer phase. With increasing polymer phase the gel temperature was increased. This is an outcome of intermolecular H-bonding. Initially hydrophilic polymer itself has a tendency to self-assemble over a long range. When they poured it into protic media, a competitive H-bond formation took place between the solvent front and polymer chains. Thus to form an intertwined microstructure, the intermolecular microstructure has to scission before homogeneous gelation, resulting in delayed gelation. Higher elastic modulus implies better gel strength. Sometimes a typical hydrophobic association between the polymer chains cannot be ruled out. Similar types of mechanistic approach have already been proposed elsewhere [55].

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16.5 Small-angle X-ray scattering technique X-ray was first discovered by Wilhelm Conrad Rontgen and earned him the Nobel Prize in physics. In 1920 P. Krishnamurti reported Small-angle X-ray scattering (SAXS) on amorphous materials which were basically colloidal systems [56]. After that, there was not no really significant research into SAXS. The main difficulty in measuring the scattering of X-rays after bombardment with samples is the distinguishable intensity measurement between background X-ray and scattered beams. Since then, several designs have been created to improve the collimation system. Collimation is related to the beam size and slit/pinhole tuning. There are various designs of collimators. The major ones are the pinhole (slit) collimation system, Kratky collimation system, and BonseHart channel cut collimation system (for very small angles). Synchrotron X-ray has a very high photon count, resulting in a high-intensity 1D plot. Furthermore, the tunability of X-ray wavelength across a drift close to the K or L edge of a component has made SAXS a practicable approach for structure and morphology evaluations of a particular system in the presence of other interacting materials [57,58]. The interactions between X-rays and the present particulate materials are briefly depicted in Fig. 16.9. SAXS can be utilized to assess a large variety of materials in which at least

Figure 16.9 Illustration of typical nanoprobes and their range of analysis. SAS, Small-angle scattering; USAS, ultrasmall-angle scattering.

Characterization tools and techniques of hydrogels 499 biphasic systems have been mostly nurtured. In the case of multiphase systems, a variation in electron density affects the drastic scattering intensities of the specimens. In the case of laboratory X-ray sources, the incident photon count (or flux) lies in the range of 103108 photons/s. However, in synchrotron-based systems, the flux generally lies around 10111013 photons/s. There are some specific beamlines with superiority in photon flux at around 1014 photons/s, like BL19LXU in the Spring-8 synchrotron. A similar type of flux is also found in SACLA, Japan, the European XFEL, and LCLS in the United States. Another very important aspect in this situation is background noice and parasitic scattering. Parasitic scattering is unwanted scattering which is intensity loss for SAXS evaluation. To be more precise, the SAXS detector has a step-scanning Geiger counter and photographic film simulators. Nowadays, most laboratories have replaced this type of detectors with 2D gasfilled wire detectors [59]. CCDs are another class of detector besides these 2D gas phase detectors [60]. The MYTHEN detector is a better and problem-free detector but is less cost-effective, which follows direct photon-counting irrespective of other factors [61]. Other modified 2D detectors are the PILATUS [62], EIGER [63], Medipix. and PIXcel detectors [64]. Ionic cross-linked hydrogels can be evaluated by SAXS. When alginate has been crosslinked by Ca21 ions, the junction points are more prominent as per Guinier approximation which was better assessed under a cross-sectioned system. The cross-sectional radius of gyration (Rg) can be evaluated by Eq. (16.17) I ðqÞ  expðR2g q2 =2Þ

(16.17)

Kim et al. performed analysis based on such ionic cross-linked alginated hydrogel beads where they proposed that ionic cross-linked hydrogels are stiff and rigid due to their extensive network formation inside the alginate matrix [65]. Ionic cross-linking among alginate chains are proposed as an “egg-box” model formation. Thus, in the case of ionotropic gelation, SAXS has great significance in assessing the network system, which has a direct relationship on the water uptake behavior. SAXS also can be compared with the flow feature rather than the rheological behavior. A stiffer network results in higher elastic modulus, that is, high gel strength (ratio of elastic to loss shear modulus). In another work, biocompatible PEG hydrogels were evaluated under SAXS study [66]. As per this study, PEG cross-linked by diacrylamide has an immense effect on their junction points which was carried out in in situ SAXS measurement. The in situ SAXS experiment is shown in Fig. 16.10. As per this figure, it can be seen that initially there was no such strong peak at the precursor solution, but after a few seconds, a rising peak was obtained. This peak is called the correlation peak, and has been attributed to the formation of a gel state. In addition, the peak shift was also seen in a higher q range, which implied a smaller length scale. The strong correlation peak is also attributed to the 3D network-like

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Figure 16.10 SAXS curves showing in situ polymerization of a PEG-diacrylate precursor solution [66]. r 2010. SAXS, Small-angle X-ray scattering. Reproduced with permission from the American Chemical Society.

morphology. In the case of a dense network system, the juncture points behave as scattering centers. In the case of a swollen network system, a similar kind of experiment was also carried out. When PEG was cross-linked in the presence of diacrylate, the macrochains of the PEG network were restricted to a perfect conformation, whereas, after water uptake, the PEG chains were more liable to rearrange themselves inside the gel matrix. Moreover, the cross-link and degree of polymerization also can be estimated by this SAXS study. Nanocomposite hydrogels with clay as a reinforcement are another area of superabsorbent hydrogels. In this context, polyacrylate (PAA) and nanoclay have been blended homogeneously in order to integrate their structures and properties [67]. In the case of a high clay concentration, interparticle repulsive interaction is more dominant for moderatemolecular-weight PAA. SAXS also gives an idea of the size and distribution of pores inside a porous hydrogel matrix. For semi-IPN type nanocomposites the gel strength, mechanical robustness, and filler dispersion are evaluated and discussed elsewhere [49]. In Ref. [49], nanoclay was in situ gelled by polymethacrylic acid and SA. Such semi-IPN hydrogels become more mechanically robust when anisotropic nanofillers are as reinforcement. As per the SAXS study, the filler distribution showed an improvement in scattering intensity. As nanoclay has an immense surface area, it has the opportunity to anchor to the hydrophilic polymer chains inside the hydrogel matrix. Nanocomposite hydrogels have a dual contribution in the scattering phenomenon; one is their mass fractal and the other is their surface fractals. Two such physical terms are quite significant in SAXS study.

Characterization tools and techniques of hydrogels 501 After obeying the simple power law equation, the SAXS spectra infer the combination of surface and mass fractals for nanocomposite hydrogels. As per the rule of Guinier and Porod, the determining factor is the power law exponent. The value of the power law exponent can give an idea of the microstructure and special configuration of hydrophilic network systems. In the case of in situ hydrogels, the nanoclay itself has relatively small hydrodynamic size or radius of gyration. The radius of gyration is the quantitative parameter of SAXS spectra which can imply the particle agglomeration character. Inside nanoclay confined in hydrogels, the agglomerated feature is more prominent due to the physisorption of polymer chains inside the gel matrix. A high agglomeration of such hydrophilic clusters enhances the degree of agglomeration, resulting in inferior mechanical and rheological properties. The most significant model related to this system is the HurdSchmidt model [68,69].

16.6 Small-angle neutron scattering The neutron is a subatomic particle with a mass of 1839 times that of an electron (1.674928 3 10227 kg), a magnetic moment of 29.6491783 3 10227 J/T, and a 15- minute lifetime. As per the wave particle duality, a neutron has both a particle as well as a wavelike nature. Thus it has a proneness to reflect, scatter, refract, and diffract after interacting with other particles/matter. First, it should be taking into consideration that during smallangle neutron scattering (SANS) experimentation, particle and neutron collisions occur in two ways; nuclear scattering and interactions with unpaired electrons. Interactions with unpaired electrons are corroborated as an enhancement in the magnetic moment. This is called magnetic scattering. The most common assumption is elastic scattering of neutrons after particle-to-neutron collisions. A brief illustration has been depicted in Fig. 16.11 for SANS set up. When a neutron collides with the nucleus, the scattering is dependent upon the interaction potential between the neutron and nucleus, which has a very small domain (10215 m).

Figure 16.11 Instrument and component design for typical SANS facility. SANS, Small-angle neutron scattering.

502 Chapter 16 This is a much shorter distance than a neutron wave (10210 m), resulting in the nucleus scattering. The spherically symmetrical scattered wave function will be: b ψi 5 2 e2ikz r

(16.18)

where z and k represent the distance from the nucleus and wave, respectively. b is the scattering length of nucleus and r is the distance between the neutron and nucleus. The “ 2 ” sign is due to the repulsive force of interactions. The scattering length is significantly dependent over the isotopes. As an example, 1H and 2H (or D) have some discrepancy in their scattering lengths. The scattering length of normal hydrogen is drastically distinguishable from D. Thus hydrogen replacement during a SANS experiment is a commonly adopted technique for analysis, and is called “contrast variation.” Scattering cross-section is another term for SANS experiments. Scattering cross-section is a hypothetical nomenclature corroborating how “big” the nucleus seems to the incident neutron. It is evaluated as the ratio of the neutron count after scattering per unit time to neutron flux. SANS analysis is related to the particles present in a phase or density difference between the phases. In this context, contrast variation is another technique for complex systems. Fig. 16.12 is a schematic representation of the contrast variation technique adopted by various hypotheses. PVA hydrogels are the most practiced area of neutron scattering analysis. As the degree of hydration of PVA affects the crystallinity, thus a great amount of anisotropy can be observed in this system. SANS is an ideal nondestructive tool to investigate the anisotropic character inside PVA gels where the structural alterations are monitored at a scale of 100 nm [70]. Ultra-SANS (USANS) was also performed to estimate the wide range of crystallinity in PVA hydrogels. The thermally induced anisotropy can be corroborated by a combination of SANS and USANS, as reported elsewhere [71,72].

Figure 16.12 Contrast variation measurement for SANS study. SANS, Small-angle neutron scattering.

Characterization tools and techniques of hydrogels 503 Volume phase transition (VPT) is another area of hydrogels (thermoreversible hydrogels), where macromolecular chains are prone to arrange themselves to withstand the gelled structure. In this context, poly(N-isopropylacrylamide) (PNIPAM) hydrogels are the most significant. PNIPAM hydrogels show discriminating temperature-responsive characteristics in a roughly ambient temperature window [7375]. Fig. 16.13 is the SANS intensity versus scattering vector plot for PINIPAM hydrogels at various temperatures [75]. The figure shows swelling and collapsing of hydrogels under temperature alterations. Such VPT was also observed in another report [76]. As shown in these curves, there was a distinct peak in the low q region. This gave the idea of concentration variation of macromolecular chains. The homogeneity in hydrogel systems can be evaluated here after assessing the peak. In the case of a swollen system the hydrogel is quite phase separated, in the order of a few nanometers. The peak is the proper indication of microscopic gel domains acting as the scatterer in a SANS experiment. Water molecules imbibed into hydrogel matrices work as spacer molecules. Thus, from a microscopic point of view, the hydrogel in a swollen state is an inhomogeneous system which has been scattered by incident neutrons. Similar phenomena can be attributed to the presence of various stimuli including pH, temperature, and other small molecules [77,78].

Figure 16.13 SANS spectra of PNIPAM hydrogel in a wide temperature window with temperature reversible behavior [75]. r 2011. PNIPAM, poly(N-isopropylacrylamide). Reproduced with permission from The Society of Polymer Science, Japan (SPSJ), Nature-Springer.

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16.7 Fluorescent behaviors of hydrogels Very recently, fluorescent hydrogel (FH) has drawn attention in soft materials research. FHs are special hydrogels which are most easily fabricate after the addition of fluorescent substances inside a hydrogel matrix. FHs have a wide range of applications in tissue engineering and cell differentiation works. Their inherent fluorescence character means they are illuminated from inside of the body resulting in significant in vivo applications [79]. In addition, they also have important characteristics in the enhancement of toughness and rubber-like elasticity of hydrogels. Polymer hydrogels are normally very soft and sometimes inferior in terms of their applicability. The best and most cost-effective way to prepare such hydrogels is to incorporate fluorescent carbon dot (CD)/graphene quantum dot-reinforced hydrogels. Usually carbon is known as a black material, and, until some recent research, it was difficult for it to be soluble in water and it unveiled a high luminescence property. However, only the classical bulk carbon was associated with the black material because when its size was reduced to the nanometer scale its physical and chemical properties were drastically changed from the macroscopic material [8084]. The best known carbon nanomaterial could make several shapes including buckminsterfullerene (C60) [82], graphene [85], CNTs [83,86], nanodiamonds [81], carbon nanofibers [80,87], and CDs [8890], which were recently discovered. Similar to its widespread famous older cousins like fullerene, CNTs, and graphene, the most recent form of nano-carbon, the CDs, is motivating intensive research efforts in its own right. CDs were discovered accidently during the purification of SWCNTs fabricated from arc-discharge soot by Xu et al. [91]. CDs are a new-fangled family member of carbon nano-structured materials that contain quasispherical, discrete nanoparticles with sizes less than 10 nm [92,93]. The size and surface chemistry are important for the fluorescent carbon materials as the surface of the nanosized carbon material is comprised of sp2 and sp3 carbons, oxygen, and nitrogen chemical groups, and postmodified functional groups (Fig. 16.14). CDs mainly include carbon nanodots (CNDs), graphene quantum dots (GQDs), and polymer dots (PDs) [94]. GQDs [95] are anisotropic and hold one or a few layers of graphene and linked functional groups on their edges. The lateral dimensions of GQDs are larger than their height. CNDs are spherical in shape and categorized into carbon nanoparticles without crystal lattice, and carbon quantum dots a with crystal lattice. PDs are aggregated or cross-linked polymers derived from monomers or linear polymers. Due to the variety of CDs, there are many fabrication approaches for CDs, mainly divided into two groups: the top-down and bottom-up techniques. The top-down method is realized by cleaving or breaking down carbonaceous materials by means of a chemical, physical, or electrochemical approach. The latter involves carbonization of small molecules, pyrolysis, or stepwise chemical fusion of small organic molecules. Scientists are making efforts in this field due to the superior

Characterization tools and techniques of hydrogels 505

Figure 16.14 Three categories of fluorescent CDs are GQDs, CNDs, and PDs [94]. r 2015. CDs, Carbon dots; CNDs, carbon nanodots; GQDs, graphene quantum dots; PDs, polymer dots. Reproduced with permission from Springer.

optical properties of CDs in comparison with other carbonaceous materials, as well as their cost-effectiveness, ease of synthesis, large-scale production, tunable surface properties, biocompatibility, etc. [92,9699]. This variety of features empowers them in numerous applications such as sensing, bio-labeling, catalysis, anticounterfeiting, and as drug carriers and energy devices [51,99104]. These CDs combine a number of auspicious attributes of semiconductor quantum dots in terms of photoluminescence dependent on excitation wavelength and size, long-time photostability, and ease of bioconjugation while not having the problem of intrinsic toxicity or elemental scarcity and unnecessary harsh, tedious, expensive, or ineffective synthesis steps [105,106]. Ruiz-Palomero et al. reported fluorescent nanocellulose-based hydrogels for laccase-sensing applications [107]. The improvement in FH physical features is multidimensional. The main characterizations which make FHs significantly distinguishable are their mechanical, thermal, and optical properties. Most commonly hydrogels are a cross-linked polymer mass which is insoluble in solvents. These cross-links are either chemical or physical. However, when CDs are incorporated into gels, two types of cross-linking can be seen: chemical and physical. Some researches however have been carried out where chemical and physical cross-linking were attributed in hydrogels to improve their excellent toughness. PAAm and CD-based hydrogel with 650% elongation has been reported elsewhere [108]. Such hydrogels are superior in terms of their stress dissipation. Similar work was also reported by Zhu et al. associated with a self-healing characteristic [109]. The optical properties of FHs are primarily their UV-visible spectra. An agar-CD composite was prepared and evaluated

506 Chapter 16 by UV spectra. They reported a sharp peak for FHs in which approximately 75% of the total fluorescent behavior came from CDs [110]. Incorporation of CDs in ionic liquid is another way to drastically improve their luminescent property and gel strength [111]. Such hydrogels are desirable in their sensing applications, biomedical diagnosis, and controlledrelease behavior. Controlled release is a diffusion-based phenomenon. In these ionic liquidbased hydrogels small molecular imbibition can be tuned by adjusting their molecular interactions. Similar FHs with diffusion-based drug-release behavior were also reported by other researchers [112]. The temperature-responsive behavior of thermoresponsive hydrogels was reported in another research work [113]. The optical property of FHs was evaluated in this work. As normal, CDs are almost indifferent in their photoluminescent (PL) behavior when temperature has been kept as a function. But surprisingly, an anomalous trend has been noticed when thermoresponsive FH was tested against temperature variation. The PL behavior of CD-based PINIPAM hydrogels showed a downward trend with an increment in temperature. This decrement in PL intensity has been accounted for by an enhancement in scattering points in PINIPAM hydrogels just above the LCST. FHs were also reported by the addition of fluorescent molecules inside the gel matrix. Chitosan, PVA, and 9anthraldehyde were reported as a gelled mass which was tested under FL microscopy [114]. Again these data showed similar results as the gel phase acts as an incident laser-scattering center. Here also the sol phase or pregel state showed better FL behavior than the gel state. This result could also be indirect proof of gelation. In another reported work, the authors prepared hyperbranched poly(amidoamine) and oxidized alginate-based hydrogels which were injectable and self-healable in nature [53].

16.8 Microcomputed tomography Microcomputed tomography (μ-CT) is an extra transmission image technique. In this instrument, incident X-ray beams are coming from an X-ray generator and traverses through assemble followed by recording of the transmitted are scattered beams to the detector. In the detector we obtain images called radiographs. The specimen for analysis can be rotated or tilted to several degrees. The mode of rotation can lie in the range of 180 or 360 generating a series of projection images. The projection images are processed using software to evaluate the internal structure, morphology, microstructure, and void-filler inclusions present in the specimen. This technique can result in horizontal and vertical assessment of the projected sample. In brief, the micro-CT process can be elaborated/ performed in four consecutive steps. First is the extra generation, second the transmission of X-ray beams throughout the sample, third is the desired rotation of the sample to acquire a series of projection images, and lastly reconstruction of the projected images or a radiograph by computer software. Fig. 16.15 illustrates graphically the micro-CT process.

Characterization tools and techniques of hydrogels 507

Figure 16.15 Micro-CT graphical illustration. CT, Computed tomography.

Figure 16.16 Basic anatomy of an X-ray source used in micro-CT. CT, Computed tomography.

First we discuss the X-ray source. For CT systems, fixed beams are generated by directing electrons produced in a cathode. The material of the cathode is normally tungsten or copper. The target cathode emits X-ray beams which fall onto the sample. Fig. 16.16 illustrates the basic anatomy of an X-ray source. The beam size has significance in the radiograph and resolution; the finer the electron beam, the smaller the spot size of the X-rays and we obtain a better-resolution radiograph. The emitted X-rays have the specific shape of the projection. In general, the shape is likely to be a cone where the origination point is the spot on the target and the beam diverges in a conical shape. The second phase of the process is the absorption of X-ray beams in the sample. The exceptions are classified as partial absorption and differential absorption. Partial absorption corresponds to the photons observed in the specimen during the process. The differential absorption varies depending on the contrast. If there is no differential absorption the result comes out as a homogeneous gray level. The unabsorbed X-rays are recorded by the detector. In the third phase, rotation of the sample is very important. In this phase, the incident X-rays judge the total specimen in various dimensions and angles. This provides a better microstructure assessment and improved cross-sectional radiographs.

508 Chapter 16 The testing is mostly done in the case of porous gel samples. For porous gels, preparation freeze-drying has been chosen for uniform pore size. Hydroxyethyl methacrylate (HEMA)based aero gel has been characterized by micro-CT to assess the pore size and microstructure [115]. The electron microscopic images of the porous morphology of aerogels were initially shown. The main disadvantage of scanning electron microscopic images is their restriction to quantitative estimation of porosity and void volume present inside the matrix. Thus micro-CT is the ideal choice in such cases where quantitative requirement of porosity and pore volume is mandatory. Micro-CT also showed the interconnectivity of pores. As the porosity is a fundamental quality to evaluate aerogels and other porous type materials, micro-CT could correlate the other physical features. Thus, in an indirect fashion, micro-CT could explain the strength, compressibility, swelling behavior, kinetics, small molecular imbibition, and other diffusion-based data reduction. The homogeneous pore size in the horizontal as well vertical direction of a specimen also has been performed in another work [22].

16.9 Electrical characterizations General polymers are insulating in nature. However, for the scenario of hydrogel research the situation is different to an extent. Polyelectrolyte hydrogels are slightly different in their external stimuli responsiveness. In order to impose electro-conductivity in hydrogels, generally two strategies are adopted; one is using conducting polymer as an electron carrier and another is using conducting inclusions as an electron transporter. The second method is much better because of the ease of synthesis, fabrication, and simple purification methods. Such hydrogels are capable of controlled release of small molecules, namely, drugs, enzymes, and other endogenous chemicals. That is why these types of devices are called “intelligent” drug-delivery systems. Precise control of drugs in the human body is very important as an excess/inadequate proportion of drug could be detrimental for better physiological fitness. Now the question is why electro-stimulation is becoming so important in technology research? Electro-stimulation has a precise magnitude of current which can influence controlled release of small molecules even in in vivo systems—sensationally called iontophoresis and electroporation. This device is installed as a transdermal and dermal delivery unit [116]. This is not only limited to the area of laboratory research, it is also marketed with the trade name of Iontocaine, which is a medical unit for pulsatile release of lignocaine by iontophoresis. When a patient needs to be administered drugs, this small iontophoretic device can be attached to his/her arm as a dermal patch with the prescribed format of medications. Electrodes are normally attached to the conducting patch and the electrode terminals are connected to form a complete circuit for electro-stimulation. This drug-delivery technique reduces the requirement for surgical or invasive treatments. In an electro-responsive system, the hydrogels behave differently. Most often it is seen as a volume change, that is, swelling. Moreover, there are several observed physical changes to

Characterization tools and techniques of hydrogels 509 hydrogels, including deswelling, bending, and sometimes shape recovery behaviors. This behavior of hydrogels can result in them being used as actuators. Hydrogel bending is monitored in the case of mechanical devices, for example, valves, artificial limbs, soft robotics, molecular machines, and biomedical implanted devices [117]. Another method of interest is electro-conducting hydrogels with conductive inclusion or fillers. This area is based on a special theory of an electronic transportation phenomenon inside the hydrogel matrix [118]. These foreign inclusions liable for electronic transportation are called conductive fillers [119121]. The conductive fillers are mostly in nano dimensions. Nanofillers with electronic conduction are supported by the theory of electrical percolation [122124]. Electrical percolation is a special physical hypothetical phenomenon where a minimum number of conducting 3D networks, like pathways, are formed in the fabricated nanofiller-based hydrogels. Nanofiller-based hydrogels are called nanocomposite hydrogels. Percolation threshold is an effective term to designate a conducting hydrogel which reveals the quantitative estimation of required filler to gain an electric flow inside a hydrogel matrix. An IPN-based conducting gel coating was also reported which implied foreign inclusion enhanced electrical conductivity [125127]. For electrical responsiveness, the hydrogels were tested under diffident setups. The designs are depicted in Fig. 16.17. The device under test (DUT) is normally placed either in a phosphate buffer system or saline media. The environment of the electrodes applied should be in the conducting region in such a fashion that there will not be any interruption to the current flowing to the conducting patches. The electrodes applied are normally platinum or carbon. Sometimes noncontacting mode electrodes are also applied where the environment of the DUT has been kept in conducting media, such as electrovalent salts. For polyelectrolyte gel special when external electric field has been applied gel shows volume change. Polyelectrolyte gels are categorized as ionic as well as cationic gels; where ionic gels generally collapse or shrink at the anode end and the cationic gels shrink at the cathode end. This volume change of polyelectrolyte hydrogels is generally diffusion

Figure 16.17 Experimental setups of various electro-responsive drug-delivery systems.

510 Chapter 16 controlled. This negative volume change is described as hydrogel swelling. Such anisotropy in this hydrogel phenomenon can be monitored visually. Fluctuations in the gel dimensions are seen even in very low-magnitude electric currents. The volume change becomes significant when the externally applied voltage is high. However, the swelling is not always linear or proportional to the externally applied field. At a higher voltage when the gels start to the swell up to a certain degree, the resistivity of the intrinsic charges increases because of the decrease in free water present in the hydrogel matrix. Gong et al. proposed that the maximum amount of swelling is dependent on the charge transport throughout the hydrogel matrix, and not solely dependent on the externally applied voltage [128]. As per their hypothesis to evaluate the electrical property of hydrogel it is more appropriate to calculate the charge rather than the externally applied voltage. When the external electric field is off, the external environmental fluid molecules imbibes into the gel matrix. Hence this can be called a switch “onoff” phenomenon, which is dependent on the external electric field. The cyclic “onoff” experiment also has significance in calculating the gel strength and the longevity of the hydrogel specimens in service life. The electro-responsive character of polyelectrolyte hydrogels is dependent on many factors such as the experimental setup, composition of hydrogel, charge density, degree of cross-linking, pendant chains, time of swelling equilibrium, and the extent of hydrophilicity present in the matrix. Beside these, several divisions are also reported. Tanaka et al. reported a negative volume change to PAAm gel in a wateracetone mixture [129]. It was proposed that this volume change can be understood by means of a phase transition of the gel matrix. The phase transition is an accumulative effect of several competitive forces. The significant competitive forces are positive osmotic pressure of the counter ions, negative pressure due to interpolymer affinity, and rubber-like elasticity of the polymer spatial network. For electro-induced characterization the deswelling and swelling take place simultaneously when the gel deswells at one electrode at the same time the gel as swelling at the other electrode in the device. Such a volume change obeys three basic principles: first is the stress gradient generated in the hydrogel after application of an external electric field; second is localized pH variation in close proximity to the electrodes; and last is electro-osmosis of water molecules associated with the electrophoresis phenomenon. Another electro-induced responsive system has been reported by Kwon et al. [130]. They named the system electro-induced gel erosion. This theory proposed the erosion rather than any kind of deswelling after application of an electric field. When water-soluble monomers and polymers are mixed and interact chemically or by means of any kind of physical interactions, water-insoluble 3D polymeric networks have been formed gradually. Surface erosion from hydrogels occurs when one end of the hydrogel specimen is attached to an electrode and the other end is in a noncontact position keeping the distance of 1 cm apart in between them. When an electric field is applied, the free end starts to erode and produces lots of ions in the environment. Erosion from the gel surface results in disruption of

Characterization tools and techniques of hydrogels 511 physical and ionic cross-linking between the polymeric segments followed by leaching of drug molecules from the gel matrix. Because of this erosion, mass loss of the gel sample is observed and follows the zero-order release kinetics. This process continues until 80% of the initial mass has been lost from the matrix. This also ruptures the dimensional stability and shape of the cell matrix. However, there are several disadvantages to electro-responsive gels as drug carriers. These limitations include typically delayed release and response times, gel fatigue, and a nonlinear relationship between cumulative release (% release) and applied current. However, the slow release can be overcome by minimizing the amount of gel. Hydrogel microspheres or injectable hydrogels can be tuned to achieve the best electrostimulation results. Though electro-responsive drug-delivery devices are immense area of interest in the biomedical field, this field has not been fully explored in the pharmaceutical market.

16.10 Conclusion Hydrogels are classified as soft materials by materials scientists. In this chapter, the extensive testing of soft polymeric hydrogels has been illustrated. Porous hydrogels have some drawbacks due to improper stress distribution inside the matrix. Thus, characterization-based discussions have the utmost significance in the area of hydrogels. The wide areas of hydrogel applications need initial quality control before market promotion. The required physicomechanical features can be obtained after proper characterizations of hydrogels. Porous hydrogels are extensively used in superabsorbent-like applications. Toughness hydrogels have high importance in biomedical fields and some specific bio-glue applications. Moreover, very recently, conducting hydrogels also have gained immense interest in electric pulsatile drug delivery, thus electrical characterizations of hydrogel also had been discussed in this chapter. To summarize, the vital experimental tools and their related characterizations are the choice of materials scientists before the recommendations of any materials.

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