Investigation of pH-dependent swelling behavior and kinetic parameters of novel poly(acrylamide-co-acrylic acid) hydrogels with spirulina

2.1 Materials

Acrylamide (AAm), acrylic acid (AAc), N,N-methylene bisacrylamide (BAAm), andammonium persulfate (APS) and sodium metabisulfite (SMBS) as a redox initiator pair were purchased from Merck (Darmstad, Germany) and used without any further purification. Spirulina was supplied by Egert Natural Products Ltd. Co. (Izmir, Turkey).

2.2 Preparation of the hydrogels

The poly(acrylamide-co-acrylic acid)-spirulina hydrogels were prepared using the following procedure via a free radical polymerization reaction. Firstly, a calculated amount of spirulina (0.0030 g) was dispersed in approximately 5 ml of deionized water. Then nonionic comonomer (AAm, 0.587 g) and the crosslinker (BAAm, 1 mol% with respect to the monomer) were added to the mixture. After a few minutes, the ionic comonomer (AAc) was pipetted into the solution at various specific volumes, which corresponded to mole ratios of 1–5 mol% of the total amount of the monomer. Finally, the initiator pair, APS (0.25 mol% based on the monomer) and SMBS (0.1 mol% based on the monomer), was put into the mixture. After mixing, the total volume was brought to 10 ml. The resultant mixture was poured into air-tight glass tubes and allowed to form hydrogels at 35°C for 24 h. The hydrogels obtained at the end of this procedure are referred here as AAm-XAAc and AAm-XAAc-Ysp, where X and Y represent the AAc mole percent and spirulina weight percent ratios to the monomer, respectively. The composition of the hydrogels is given in Table 1.

2.3 Swelling studies

Swelling ratios of the hydrogels were calculated by weighing water-swollen hydrogels at regular time intervals. All the measurements were conducted at 25°C. The percent swelling was calculated from the following equation:

where S_w%, m_s and m_d are the percent swelling, weight of swollen hydrogels and weight of dry hydrogels, respectively. The swelling measurements of all hydrogels were repeated at different pH values, which varied between 3 and 10, using buffer solutions. The swelling ratios as a function of pH were plotted against the pH values.

2.4 Mechanical tests

The compression strengths of the cylindrical hydrogels were measured by performing a uniaxial compression experiment using a Zwick/Roell Z1.0 universal testing machine (Zwick GmbH & Co., KG, Ulm, Germany) equipped with a 50-N load cell. The data were analyzed using the Zwick/Roell testXpert II software. All the mechanical measurements were carried out with equilibrium water-swollen hydrogels, which were cut into cylindrical samples of about 1 cm in thickness and varied between 1 and 1.5 cm in diameter. The samples were compressed at a rate of 3 mm/min, and the resultant deformation was monitored. The measurements were conducted until the samples fractured. The compression force vs. the deformation percentage was obtained from the original output of the instrument.

2.5 Morphological analysis

The morphological structures of the hydrogels in freeze-dried form were characterized by using an ESEM-FEG/EDAX Philips XL-30 instrument (Philips, Eindhoven, The Netherlands).

3.1 Swelling behavior of the hydrogels

The swelling behavior of the AAm-AAc and AAm-AAc-sp hydrogels at pH 5 is shown in Figure 1A and B.

Figure 1

Swelling curves of AAm-AAc and AAm-AAc-0.5sp (A); AAm-2AAc-sp hydrogels (B) in a buffer solution of pH 5.

It was found that, although they had the same AAc content, the hydrogels with spirulina were much more swollen than those without the microalgae. It is clearly seen that the biggest difference in swelling degrees between the hydrogels with the same AAc content was obtained by AAm-2AAc and AAm-2AAc-0.5sp. The swelling degree of the AAm-2AAc-0.5sp hydrogel was found to be four times higher than that of the AAm-2AAc hydrogel. These increments in the swelling capacity can be ascribed to the formation of additional hydrogen bonds between water and the functional groups on the surface of spirulina cell walls such as amide, hydroxyl and carboxyl groups. In contrast, the swelling values of the AAm-AAc hydrogels increased with the increase in AAc content, which was predictable owing to the increase in the ionizable groups in the hydrogel network at pH 5 (23). However, in terms of the AAm-AAc-sp hydrogels, the swelling degrees were found to increase with the increase in AAc content of up to 2 mol% and then started to decrease. This may be attributed to the suggestion that there was a large quantity of acrylic acid as well as of protein and polysaccharide molecules in the structure of spirulina, resulting in the increase in the solubility of the microalgae in AAc (27). This condition was also observed during the preparation of the hydrogels. When the microalgae were dispersed in the monomer solution, the color of the solution became green and turned to a pale yellow in the hydrogel after the polymerization reaction. On the contrary, in our previous study, the solution in which spirulina was dispersed in the acrylamide monomer was colorless (26). Thus, the increase in swelling can be ascribed to the increase in AAc ratio, which resulted in high amounts of the dissolved part of spirulina in the hydrogels. This can cause the functional groups of spirulina (carboxyl, sulfate, phosphate, amine, etc.) to become more effective for swelling owing to their charges. In contrast, after the addition of 2 mol% of AAc, the gel had a lower swelling degree and the volume caused the charged functional groups of spirulina to become closer to each other. Thereby, the electrostatic interactions between ions with opposite signs increased and began to affect the swelling behavior of the gels adversely. The reason for the low swelling can be explained by the fact that, while the ions with opposite signs were attracting each other, they became ineffective for swelling because of the loss in their charges. Also, it should be noted that almost all the functional groups in the hydrogels at pH 5 were of ionic forms.

In the second part of the swelling experiments, the effect of spirulina amount on the swelling behavior was investigated. For this purpose, the AAm-2AAc-sp hydrogels containing different amounts of spirulina were synthesized and the swelling degrees were measured at pH 5. The swelling curves of AAm-2AAc-0.5sp, AAm-2AAc-0.75sp, AAm, AAm-2AAc-1sp and AAm-2AAc-1.5sp can be seen in Figure 1B. It is worth noting that the insertion of spirulina biomass into the hydrogels at 1% loading resulted in a rise in equilibrium swelling values of up to 33,000%. This result may be attributed to the good distribution of spirulina and its aforementioned functional groups as additional junctions for hydrogen bonding. This extreme swelling, in contrast, decreased to a lower value at 1.5% loading of spirulina. This may be ascribed to both the increase in the hydrophobic group in spirulina and the disruption of the good distribution of spirulina in the hydrogel network; however, it is obvious that these hydrogels have a higher value compared to the hydrogels without spirulina. In contrast, the decrease in the swelling degree of this hydrogel can also be explained by the possible interaction between the opposite charged functional groups of AAc and spirulina, which resulted in the loss of the charged ions that were responsible for the swelling.

3.2 Swelling kinetic characteristics

The swelling mechanism of the hydrogels is described by a second-order kinetics that has been proposed by Schott (28) with the following equation:

where S is the swelling percent at time t, B is the inverse of equilibrium swelling percent (B=1/S_eq) and A is a constant that gives the swelling constant: k_s (A=1/S_eq²×k_s). When Equation 2 is arranged, it takes the form below:

The t/S values in Equation 3 were obtained by using the experimental swelling ratios (S_w%) of the AAm-AAc and AAm-AAc-sp hydrogels at a related time, which were plotted against time t (Figure 2A and B).

Figure 2

Swelling kinetic relations of the AAm-AAc and AAm-AAc-0.5sp hydrogels at different ratios of AAc (A); AAm-2AAc-sp hydrogels with various amounts of spirulina (B).

It can be seen clearly from the straight lines in Figure 2A and B that all the swelling values showed a good correlation with Equation 3. The kinetic parameters that define the swelling mechanism of the hydrogels are the swelling rate constant (k_s); the initial swelling rate (k_is), which is the product of k_s×S_eq²; and the theoretical equilibrium swelling (S_eq). These parameters were calculated from the slope and intercept of the lines in Figure 2 and given in Table 2.

The data in Table 2 show that the swelling rate constant k_s decreased with the AAc content. This means that the swelling process proceeded faster at high amounts of AAc via an increasing number of free ions. In contrast, the AAm-AAc-0.5sp hydrogels had lower k_s values than the AAm-AAc hydrogels and k_s decreased with the increase in AAc content, up to 3% AAc, namely, AAm-3AAc-0.5sp. This observation can be attributed to the number of effective ions for swelling, which hastened the swelling process. However, the further increase in AAc content in the AAm-AAc-0.5sp hydrogels resulted in the decrease in k_s values due to the ineffectivity of some ions in the polymer network. Moreover, the kinetic parameters (k_s) of the AAm-2AAc-Sp hydrogels with 0.5–1.5% spirulina increased, up to 1% spirulina, but they decreased for the AAm-2AAc-1.5sp hydrogels because of the number of effective ions. In addition, the theoretical swelling degrees (S_eq) in Table 2 were found to be close to the S_w% values (experimental swelling percent), as expected.

Equation 3 may not explain properly the kinetics of the initial swelling stage, which is essentially important for the various applications of hydrogels in biomedical, pharmaceutical, environmental and agricultural engineering fields, etc. (29). For the initial phase of the swelling (60% of swelling curves), the swelling kinetic behavior was defined as zero-order kinetic, which is expressed in following equation:

where F, m_s and m_d represent the power of swelling, the weight of swollen hydrogels and weight of dry hydrogels, respectively; whereas n, t and k represent the diffusion exponent, diffusion time and swelling constant related to the polymer structure, respectively (30). However, in order to calculate the diffusion exponent values, n, Equation 4 was converted to the following logarithmic form:

The graph drawn between lnF and lnt yielded straight lines, and the slope of these lines gave n, the diffusion exponent, which determines the diffusion type. Generally, there are three types of diffusion, basically, according to the relative rates of diffusion (R_diff) and polymer relaxation (R_relax). n=0.5 indicates a Fickian diffusion mechanism (Case I) in which the rate of diffusion is much smaller than the rate of relaxation (R_diff«R_relax, system controlled by diffusion); n=1.0 indicates Case II, where the diffusion process is much faster than the relaxation process (R_diff»R_relax, system controlled by relaxation); and 0.5<n<1.0 indicates non-Fickian (anomalous) diffusion mechanism, which describes those cases where the diffusion and relaxation rates are comparable (R_diff≈R_relax) (21, 31). In some cases, values of n>1 had been observed, which were evaluated as super Case II kinetics (32). When the water penetration rate is way below the polymer chain relaxation rate, it is possible to report the n values below 0.5. This condition, which is classified also as Fickian diffusion, is known as “less Fickian” behavior (33, 34). However, some studies claimed that, for cylindrical shapes, n=0.45–0.50 and it corresponds to Fickian diffusion, whereas 0.50<n<1 indicates that the diffusion is non-Fickian.

The results obtained in Figure 3A showed that the diffusion exponent, n, for cylindrical AAm-AAc hydrogels ranged between 0.402 and 0.485, which correspond to less-Fickian and Fickian type of diffusion, whereas that for AAm-AAc-Sp hydrogels varied between 0.393 and 0.546, which indicate less-Fickian and non-Fickian (anomalous) diffusion, respectively.

Figure 3

Plots of lnF against lnt for AAm-AAc and AAm-AAc-0.5sp (A); AAm-2AAc-sp hydrogels with various amounts of spirulina (B) in a buffer solution of pH 5.

For the ionic hydrogels, the increase in the degree of ionization of the functional groups resulted in electrostatic repulsion between the ions, resulting in chain expansion and macromolecular chain relaxation. Hence, the swelling mechanism becomes more relaxation controlled when the ionization of hydrogels increases (21, 35). Herein, the same behavior for the hydrogels was also observed. The diffusion exponent (n) values increased gradually with the AAc content, which resulted in the increase in the number of ionized functional groups in the AAm-AAc hydrogels. In the case of AAm-AAc-0.5sp hydrogels, these values increased by the number of effective ions in the polymer network, as shown in Table 3. In contrast, the hydrogels with different spirulina content exhibited a similar behavior to the AAm-AAc-0.5sp hydrogels, as shown in Figure 3B. It can be concluded that increasing the number of ionized functional groups for all hydrogels caused the swelling mechanism to be become more relaxation controlled and increased the diffusion exponent n values. Table 3 presents the n diffusion exponent of all hydrogels. Hence, it may be said that the n diffusion exponent is affected strongly by the hydrogel composition, which determines the number of ionized functional groups.

However, the diffusion exponent is not sufficient by itself to define the diffusion mechanism of hydrogels; the diffusion coefficient (D) value must also be calculated. Various methods for its calculation have been proposed (36–38). Among them, which is the most known and used method, is the “short time approximation method”. In this method, only the first 60% of swelling is used (28). The diffusion coefficients of the cylindrical AAm-AAc and AAm-AAc-sp hydrogels were calculated from the following relation:

where l is the radius or thickness of the cylindrical hydrogel sample. When F values are plotted against t^1/2, straight lines occur and the D diffusion coefficient can be calculated from the slope of these straight lines, which can be seen in Figure 4A and B for the prepared hydrogels in the entire study.

Figure 4

Plots of F against t^1/2 for AAm-AAc and AAm-AAc-0.5sp (A); AAm-2AAc-sp hydrogels with various amounts of spirulina (B) in a buffer solution of pH 5.

The calculated D values are given in Table 4. It was found that the AAm-2AAc-1sp hydrogel had the maximum D values, whereas the AAm-1AAc hydrogel had the minimum D values, which corresponded to 4.319 and 0.107, respectively. It was an expected result since the AAm-2AAc-1sp hydrogel with the maximum diffusion coefficient was the most swollen hydrogel with the highest number of effective ions compared to the other hydrogels. Thus, the AAm-2AAc-1sp hydrogel showed the fastest swelling. On the contrary, the AAm-1AAc hydrogel had less effective ions than the other hydrogels, which resulted in slow water absorption rate and the lowest diffusion coefficient. It may be concluded from the D values in Table 4 that the swelling results in Figure 1 prove that the D value was maximum for AAm-2AAc-1sp and minimum for AAm-1AAc. As a result of this, the number of effective ions increased the rate of diffusion and diffusion coefficient (D) proportionally.

3.3 Effect of pH on swelling behavior and swelling kinetics

In other parts of the swelling experiments, the effect of pH on kinetic constants was investigated. For this, swelling measurements were repeated at six different pH values varied between 3 and 10. Figure 5A and B shows the swelling percentages of the hydrogels related to the change in pH.

Figure 5

Effect of pH of the buffer solution on the swelling of AAm-AAc and AAm-AAc-0.5sp hydrogels (A); AAm-2AAc-sp hydrogels with various amounts of spirulina (B).

It is well known that AAc is a weak acid and does not ionize at low pH. The pK_a value of AAc was reported to be 4.66, which means that AAc molecules ionize at pH values above 4.66 (39). Hence, at pH value of >4.66, AAc units in the hydrogels became ionized and the number of ions increased by increasing the pH. It is clearly seen from Figure 5A that the swelling degrees of AAm-AAc hydrogels at pH >4 are higher than those at pH ≤4. However, the AAm-AAc-sp hydrogels exhibited a different pH-dependent swelling behavior, as seen in Figure 5A. The hydrogels had relatively low swelling values up to pH 5, whereas they reached the maximum degree at pH 5. But further increase in pH caused the swelling values of the hydrogels to decrease. This result shows that the AAm-AAc-sp hydrogels have both positive and negative ions unlike the AAm-AAc gels and so they have an amphoteric characteristic. This fact can be explained by the negatively charged carboxyl and positively charged amine groups coming from the AAc units and amino acids of the protein molecules in spirulina. In contrast, both the positive and negative ions in the AAm-AAc-sp hydrogels are weak and ionize at certain pH values. In the meantime, it must be stated here that spirulina has a complicated structure and it does not have a specific pK_a or pK_b value. It is formed from various molecules such as proteins, polysaccharides, lipids and vitamins, etc., which have several functional groups like carboxyl, hydroxyl, amine and phosphate. Although the pK_a or pK_b values for spirulina cannot be determined easily, it is still possible to explain the pH-dependent swelling behavior of the AAm-AAc-sp hydrogels.

It was found that the most swollen hydrogel was AAm-2AAc-0.5sp over the entire range of pH (Figure 5B). This might be due to the higher effective ion number for this hydrogel composition at all pH values. Moreover, the swelling degrees of the AAm-AAc-sp hydrogels in the basic condition were slightly higher than those in acidic solution. For example, while the swelling degrees of the AAm-1AAc-0.5sp, AAm-2AAc-0.5sp, AAm-3AAc-0.5sp and AAm-5AAc-0.5sp hydrogels at pH 4 were 3367, 3489, 2901 and 477, respectively, these values were found to be a little higher at pH 10, with values of 4333, 5880, 4128 and 3984, respectively. This finding shows a proportional increase in AAc content. This may due to the higher number of negative ions that correspond to the carboxyl groups than the number of positive ions that originated from the amine groups. Considering the composition of the hydrogels, the carboxyl groups originated from both AAc and spirulina, while the amine groups originated from spirulina only. Based on the swelling results, it was found that the swelling degrees were influenced strongly by pH changes (Figure 5B).

In contrast, it was found that not only was pH affected by the swelling degrees but also had influenced the diffusion parameters of the hydrogels. The diffusion exponent (n) and the diffusion coefficient (D) values calculated at different pH values are listed in Tables 3 and 4. As can be seen in the table, the increase in pH increased both the n and the D values of the AAm-AAc hydrogels, especially at pH >4, whereas those of the AAm-AAc-sp gels increased up to pH 5. However, further increase caused both the diffusion constants to decrease. The data in Table 3 show that the diffusion rate, and thus the diffusion exponent and coefficients, is strongly coherent with the number of effective ions that do not pull ions with opposite signs in the polymer network and varies with the pH values of the swollen media.

3.4 Reversible response of the AAm-AAc-sp hydrogel to pH

To evaluate the pH reversibility of the AAm-AAc-sp hydrogels, the swelling-deswelling behavior was investigated. In Figure 5A, it can be seen that the AAm-2AAc-0.5sp hydrogel has significantly different swelling values at pH 3 and pH 5, which correspond to the minimum and maximum swelling values, respectively. For this reason, the AAm-2AAc-0.5sp hydrogel was chosen to examine the pH-responsive behavior of the prepared gels. The swelling measurements were performed by alternately changing, at regular time intervals, the solutions whose pH values were 3 and 5. The swelling degrees of the AAm-2AAc-0.5sp hydrogel at pH 3 and pH 5 were close to each other up to 1500 min. After this time, the gel continued to swell at pH 5, while it showed an almost constant value at pH 3. It might be explained by the ongoing ionization of the functional groups in spirulina. Reversible swelling-deswelling cycles could be performed at long time intervals due to the slow diffusion, as explained previously. It was observed that the hydrogel exhibited pH-reversible swelling behavior (on-off switching effect) (Figure 6). Based on these results, AAm-2AAc-0.5sp hydrogels can be used in drug delivery process when slow release is required in neutral media.

Figure 6

pH-Reversible swelling behavior of the AAm-2AAc-0.5sp hydrogel.

3.5 Mechanical properties

The mechanical properties of the samples were investigated by performing a uniaxial compression test. The compression force-deformation data of the hydrogels are shown in Table 1. For the AAm-AAc hydrogels, the maximum compression strength was achieved by AAm-1AAc, while the minimum compression strength belonged to AAm-5AAc. Accordingly, the compression strength values of these hydrogels decreased with increasing AAc amount. These results are in good agreement with their swelling degrees (Figure 1A). The compression strength of the swollen AAm-AAc-0.5sp hydrogels is also given in Table 4. It is worth noting from the table that spirulina-loaded hydrogels have lower compression strength values than the hydrogels without spirulina for the same amount of AAc. This decrease in the mechanical performance can be ascribed to the physical and reversible noncovalent interactions between the main matrix and spirulina, which leads to more viscous characteristics. In the 0.5% spirulina-loaded hydrogels, the compression strength increased as AAc content increased up to 2%, and the AAm-2AAc-0.5sp hydrogel was found to have the highest compression strength value among all the 0.5% spirulina-loaded hydrogels. In terms of 3% and 5% AAc, the compression strength decreased as the AAc content increased. This increment/decrement tendency in compression strength was also observed for the maximum swelling values of the same hydrogels in the same manner. So, this cannot be explained by the above mentioned inverse relationship between swelling and compression strength. This situation can be explained in such a way that AAc content affects the dissolution part amount of spirulina in the hydrogel, directly. Therefore, the increase in AAc content in the hydrogel causes much more part of spirulina dissolved and thereby a high number of effective ions which originated from spirulina were formed. It can be said that, when 2% AAc is used as the optimized ratio, a relatively higher amount of spirulina dissolves in the system, which results in a more homogeneous hydrogel structure leading to high mechanical strength. This was also in good agreement with the scanning electron microscopy (SEM) image of the hydrogel showing much smaller pores with a homogeneous pore size and distribution. But the higher amount of AAc content resulted in the decrease in compression strength values. This decline can be due to the excessive dissolved part of spirulina at higher AAc content, which can lead to ion-dipole or dipole-dipole interactions between spirulina and the polymer chains, leading to additional physical reversible crosslinks. These non-covalent interactions between polymer chains and spirulina can cause a more viscous characteristic, leading to a more heterogeneous crosslinked system and, thereby, a decrease in compression strength. For AAm-2AAc-sp hydrogels containing different amounts of spirulina, higher and lower compression strengths were achieved for the AAm-2AAc-0.5sp and AAm-2AAc-1sp hydrogels, respectively. It can be seen in Table 1 that, as spirulina content increased, the compression strength values decreased up to 1% spirulina. The decrease in compression strength values observed in the AAm-2AAc-sp hydrogels as compared with the AAm-2AAc hydrogels can be ascribed to the plasticizing effect of too much water in their equilibrium swollen state. This is highly consistent with the lowest compression strength of the AAm-2AAc-1sp hydrogel with the highest value. The low mechanical performance of the hydrogels with increasing spirulina may also be attributed to the above mentioned reason, which results in more viscous characteristics of the hydrogels with the help of the physical reversible crosslinks. The tendency for an increase in compression strength for 1.5% spirulina loading, in contrast, can be caused by the presence of possibly too many physical crosslinks in the system, which can help in sharing the applied compressive forces between the chemical crosslinks. It can be concluded from the compression test results that the optimum composition for the hydrogel with maximum swelling at pH 5 and moderately high mechanical strength is the AAm-2AAc-0.5.

3.6 Morphological structures

SEM images of the swollen hydrogels in Figure 7 represent the morphologies of the AAm-AAc hydrogels, whose AAc content varied between 1% and 5% mol, whereas the microstructures of the AAm-AAc-0.5sp hydrogels are shown in Figure 8. The AAm-AAc hydrogels had smaller pores, approximately 50 μm in size, compared to the AAm-AAc-0.5sp hydrogels, with 500 μm in pore size.

Figure 7

SEM images of the AAm-1AAc-0.5sp (A), AAm-2AAc-0.5sp (B), AAm-3AAc-0.5sp (C) and AAm-5AAc-0.5sp (D) hydrogels. The scale bars are 500 μm.

Figure 8

SEM images of the AAm-1AAc (A), AAm-2AAc (B), AAm-3AAc (C) and AAm-5AAc (D) hydrogels. The scale bars are 500 μm.

For the AAm-AAc hydrogels containing no spirulina, it was observed that their pore sizes became smaller with increasing AAc content. The AAm-1AAc hydrogels had many open small pores, approximately 100 μm in size, and they were distributed homogeneously. But, interestingly, the increase in AAc content caused the distribution of pores to be more heterogeneous. For instance, open pores with sizes varying between 50 and 250 μm were observed in the AAm-2AAc hydrogels. Similarly, in the AAm-3AAc and AAm-5AAc hydrogels, small pores that were also heterogeneously distributed were observed. However, most of them appeared to be close to each other. It is well known that big open pores provide high swelling values to hydrogels. But this observation is different in poly(acrylamide-co-acrylic acid) gels. AAm-AAc hydrogels are composed of AAc and AAm units whose functional groups are carboxylic acid and amide, respectively. These carboxylic acid and amide groups form hydrogen bonds that form extra crosslinks in the polymer network (36). Thus, as AAc content increased, the pores of the hydrogels became smaller, as seen in the SEM images in Figure 8. In contrast, the swelling degrees gradually increased from 1% to 5%AAc, owing to the number of carboxyl ions, which were responsible for the high swelling degree. However, carboxyl ions in the polymer network also repelled each other, resulting in bigger pores. Hence, this repulsion was weakened by the hydrogen bonds; in other words, H-bonds prevented the repulsion of carboxyl ions to some extent.

For AAm-AAc-0.5sp, the pores became larger and had an extended shape, with increasing AAc content of up to 3%. After that ratio of AAc, the pore sizes became smaller (Figure 7). Moreover, the pore distribution was heterogeneous for all samples with spirulina and heterogeneity increased with increasing AAc content. For instance, while AAm-2AAc-0.5sp hydrogels had relatively closed pores with sizes of approx. 500 μm, this value was between 100 and 200 μm for AAm-5AAc-0.5sp with relatively open pores.

The effect of spirulina amount on the morphological structure of poly(acrylamide-co-acrylic acid) hydrogels can be seen clearly in Figure 9. As spirulina content was increased, the pores distributed more heterogeneously and appeared to be relatively close to each other. Since spirulina also has AAc content naturally, it helps to dissolve more spirulina into the gel. Although the pore size became smaller with higher amount of spirulina, the increase in the number of functional groups let the gels swell more.

Figure 9

SEM images of the AAm-2AAc-0.5sp (A), AAm-2AAc-0.75sp (B), AAm-2AAc-1sp (C) and AAm-2AAc-1.5sp (D) hydrogels. The scale bars are 500 μm.