Technical Note | ElastoSens™ Bio
How Sample Volume Affects Viscoelastic Properties of Thermosensitive Polymers
by Maya Salame and Dimitria Camasão
Application Scientists, Rheolution Inc.
Summary
- Material-dependent response: Sensitivity to volume and temperature varies by material, influencing gelation and mechanical properties.
- Controlling temperature kinetics is important: The rate at which a sample reaches its target temperature can influence the timing of gelation.
- Consistency in experimental setup matters: Replicating conditions such as sample volume or starting temperature is essential for generating reproducible data.
Introduction
Thermosensitive polymers are materials that often exhibit reversible changes in their physical state in response to temperature, allowing control over their mechanical and structural properties. These transitions typically involve a shift from a liquid solution to a gel, driven by molecular interactions such as hydrogen bonding, hydrophobic effects, and polymer–solvent affinities.
Hydrogels derived from thermosensitive polymers are widely used in biomedical, pharmaceutical, and soft material applications due to their high water content and tunable viscoelasticity. Common examples include Pluronic® F-127, a synthetic triblock copolymer that gels upon heating; agar, a naturally derived polysaccharide that gels upon cooling; and gelatin, a natural biopolymer that can undergo reversible gelation with temperature.
The shear storage modulus (G′) is a key parameter for characterizing the mechanical behavior of hydrogels, representing the elastic component of their response. Although G′ is theoretically regarded as an intrinsic material property—independent of sample volume—experimental measurements can vary due to differences in gelation kinetics. These variations can arise from thermal gradients, which can temporarily affect how the network forms during gelation. In this study, three thermosensitive hydrogels—Pluronic® F-127, agar, and gelatin—were evaluated to determine their susceptibility to volume-dependent effects.
Protocol
Three thermosensitive hydrogels were evaluated: Pluronic® F-127 (Poloxamer 407, Sigma-Aldrich, USA), agar (Sigma-Aldrich, USA), and gelatin type B from bovine skin (Sigma-Aldrich, USA). Pluronic F-127 was prepared at 20% w/v by dissolving the polymer in deionized water at 4 °C overnight, followed by gentle stirring for 5 minutes prior to testing to ensure homogeneity. The solution was stored at 4 °C until use. Agar was prepared at 1% w/v by dispersing the powder in deionized water and heating the solution to boiling (95–100 °C) with continuous stirring until fully dissolved. The solution was then maintained at 60 °C in a water bath to keep it in the liquid state prior to testing. Gelatin was prepared at 15% w/v by dispersing the powder in deionized water and incubating the mixture at 60 °C for approximately 2 hours, with intermittent stirring, until fully dissolved. The solution was kept at 60 °C until testing.
All measurements were conducted using the ElastoSens™ Bio system. The effect of sample volume on gelation behavior was assessed by comparing 3 mL and 6 mL samples under multiple temperature protocols. Each condition was tested in triplicate. For Pluronic F-127, which gels upon heating, samples were tested under two conditions: (1) at a constant chamber temperature of 30 °C, and (2) under a pre-heated protocol in which the chamber was initially set to 50 °C and then lowered to 30 °C at the start of the test sequence. For agar, which gels upon cooling, samples were first liquefied at 60 °C and then tested under two cooling conditions: (1) at a constant chamber temperature of 15 °C, and (2) under a pre-cooling protocol where the chamber was initially set to 10 °C, followed by an increase to 15 °C once the sample was loaded. Gelatin samples were prepared under the same cooling conditions as agar; however, because gelatin takes longer to reach its end stiffness in these conditions, they were incubated for 3 hours (following pre-cooling protocol) before being tested for 2 minutes. Gelation kinetics measurements for gelatin were performed in a separate experiment.
Table 1: Additional protocol details for thermosensitive hydrogel testing using the ElastoSens™ Bio system.
*Gelatin samples were incubated for 180 min at the testing temperature, then tested for 2 min.
An additional temperature profiling test was conducted under the same conditions used for G′ measurements. A digital thermometer probe was inserted into the center of the sample to directly monitor internal temperature changes during both heating and cooling protocols. The test was performed using both 3 mL and 6 mL volumes for each sample, under constant and pre-heated/pre-cooled chamber conditions, mirroring the mechanical testing sequence.
Results and Discussion
1. Pluronic® F-127 (20% w/v): Heating-Induced Gelation
Figure 1 presents the shear storage modulus (G′) of Pluronic® F-127 over time under the two heating protocols: lower heating speed (left) and higher heating speed (right). As expected, under identical temperature conditions, gelation occurred more quickly in the 3 mL samples than in the 6 mL samples (second row). This is consistent with their respective temperature profiles during the test, where the 3 mL samples reached equilibrium temperature more rapidly (first row). When comparing lower and higher heating speeds, the final G′ values were similar across all conditions (third row). Similarly, no significant differences were observed in the final shear loss modulus (G″) (bottom row), except for 3 mL at lower heating speed and 6 mL at higher cooling speed (p<0.05).
Overall, these results demonstrate that while the gelation kinetics of Pluronic® F-127 are influenced by the sample’s volume (i.e., temperature profile), its final mechanical properties remain unaffected. The time required to reach the final G’ depends on gelation kinetics; if a mechanical test is performed before gelation is complete, the measured modulus may differ.
Figure 1. Effect of sample volume and heating speed on Pluronic® F-127 gelation kinetics.
Temperature profiles and shear storage modulus (G′) evolution over 45 minutes for 20% w/v Pluronic® F-127 hydrogels measured with the ElastoSens™ Bio. G′ time-course curves show the mean (solid line, n = 3) with ± standard deviation (dotted lines). Samples of 3 mL and 6 mL were tested under two conditions in triplicate: lower heating speed (left) and higher heating speed (right). Bar graphs summarize the final G′ and shear loss modulus (G″) values; significance is indicated as p < 0.05 (*) or not significant (ns).
2. Agar (1% w/v): Cooling-Induced Gelation
Figure 2 presents the shear storage modulus (G′) of 1% agar under the two cooling protocols: lower cooling speed (left) and higher cooling speed (right). Under identical temperature conditions, cooling proceeded more quickly in 3 mL samples than in 6 mL samples (first row), consistent with their different thermal profiles in which the smaller volume reached equilibrium faster. We can observe a difference in kinetics for each condition, however, once gelation was complete, the final G′ values were similar across all conditions (second row), even when different heating speeds were used. Similarly, no significant differences were observed in the final shear loss modulus (G″) (bottom row).
Similar to before, these findings indicate that while sample volume influences the thermal profile of 1% agar and its gelation kinetics, it does not alter the final mechanical properties.
Figure 2. Effect of sample volume and cooling speed on 1% agar.
Temperature profile and shear storage modulus (G’) evolution over 45 minutes for 1% agar measured with the ElastoSens™ Bio. G′ time-course curves show the mean (solid line, n = 3) with ± standard deviation (dotted lines). Samples of 3 mL and 6 mL were tested under two conditions in triplicate: lower cooling speed (left) and higher cooling speed (right). Bar graphs show final G′ and shear loss modulus (G″) values (mean ± SD), with no significant differences observed between volumes.
3. Gelatin (15% w/v): Cooling-Induced Gelation with Longer Time Frame
Figure 3 presents the temperature profiles (top row) for 15% gelatin during cooling (middle row) under lower cooling speed (left) and higher cooling speed (right) protocols. In both cases, 3 mL samples cooled quicker than 6 mL samples under identical conditions. This difference in cooling profiles was accompanied by corresponding changes in gelation kinetics.
Under the lower cooling speed protocol, both volumes had comparable gelation behavior, with similar G′ evolution over time. In contrast, under the higher cooling speed protocol, a clearer difference between volumes was observed. While the 3 mL samples reached a final G′ comparable to that obtained at lower cooling speed, the 6 mL samples plateaued at a lower final G′, resulting in a significant difference between volumes (** p < 0.001). Additionally, comparison of the 6 mL samples across cooling speeds revealed a significant decrease in final G′ at higher cooling speed ( p < 0.01), highlighting the combined influence of volume and cooling rate.
The final shear loss modulus (G″) values (bottom row) were generally similar across conditions, with most comparisons remaining not significant. The only notable difference was again observed for the 6 mL samples (** p < 0.01).
Overall, these results demonstrate that gelatin is more sensitive to sample volume than agar or Pluronic® F-127. As with the other materials, its gelation kinetics are influenced by sample volume; however, unlike the others, the final G′ values can also differ when evaluated over a similar timeframe, with larger volumes exhibiting a reduced final modulus.
Figure 3. Effect of sample volume and cooling speed on 15% gelatin.
Temperature profiles and shear storage modulus (G′) over 120 min measured with the ElastoSens™ Bio. G′ time-course curves show the mean (solid line, n = 3) with ± standard deviation (dotted lines). Samples (3 mL and 6 mL) were tested in triplicate under two cooling conditions: lower cooling rate (left) and higher cooling rate (right). Bar graphs summarize the final G′ and shear loss modulus (G″) values (mean ± SD). Statistical significance is indicated as ns (not significant), **p < 0.01, and ***p < 0.001.
Conclusion
This study suggests that the effects of sample volume and heating or cooling speed depend on the material. Pluronic® F-127 and agar reached similar final shear storage modulus (G′) across volumes, with heating and cooling speed mainly affecting gelatin kinetics. In contrast, larger volumes in gelatin showed greater variability and lower final G′ at higher cooling speed, likely due to its slower network formation.
These results highlight the need to control variables such as sample volume, formulation, and loading temperature to ensure reproducibility—particularly for temperature-sensitive systems. Timing is also an important factor, as measurements taken before gelation is complete can yield different results compared to those taken at the final stiffness. The ElastoSens™ Bio enables real-time, non-destructive measurement of mechanical properties, providing valuable insight into how various factors influence material performance.
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