Materials Science and Engineering C 44 (2014) 371–379
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An investigation into environment dependent nanomechanical properties of shallow water shrimp (Pandalus platyceros) exoskeleton Devendra Verma, Vikas Tomar ⁎ School of Aeronautics and Astronautics, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 4 January 2014 Received in revised form 26 July 2014 Accepted 8 August 2014 Available online 15 August 2014 Keywords: Shrimp exoskeleton Nanomechanics Environment dependence Biomimetic Liquid cell
a b s t r a c t The present investigation focuses on understanding the influence of change from wet to dry environment on nanomechanical properties of shallow water shrimp exoskeleton. Scanning Electron Microscopy (SEM) based measurements suggest that the shrimp exoskeleton has Bouligand structure, a key characteristic of the crustaceans. As expected, wet samples are found to be softer than dry samples. Reduced modulus values of dry samples are found to be 24.90 ± 1.14 GPa as compared to the corresponding values of 3.79 ± 0.69 GPa in the case of wet samples. Hardness values are found to be 0.86 ± 0.06 GPa in the case of dry samples as compared to the corresponding values of 0.17 ± 0.02 GPa in the case of wet samples. In order to simulate the influence of underwater pressure on the exoskeleton strength, constant load creep experiments as a function of wet and dry environments are performed. The switch in deformation mechanism as a function of environment is explained based on the role played by water molecules in assisting interface slip and increased ductility of matrix material in wet environment in comparison to the dry environment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies of natural materials have often provided insights into the design and development of new class of materials [1,2]. In continuation to the exploration of properties of natural materials, a new area that focuses on the study of crustacean family (lobsters, crabs, shrimps etc.) has emerged. Several studies have been performed on the natural crustacean (lobsters, crabs) shells in order to understand correlations between structure and properties of such materials [3–15]. Exoskeleton of crustaceans has a well-defined hierarchical structure [6–11]. It consists of chitin based fibrils coated in proteins at nanometer level. Such fibrils bind together to form fibers. These fibers are then woven together to form chitin–protein layers. Such layers are stacked in a twisted plywood structure known as the Bouligand pattern [6–11]. Studies by Seki et al. [12,13] have concentrated on a similar kind of exoskeleton found in toucan beak. It was found to be a sandwich structure with the keratin exterior and a closed cell fibrous network made up of calcium rich proteins. Chen et al. [16–19] have also thoroughly investigated beetle forewing to be able to mimic its design for better sandwich panel structures. The crustacean family consists of several different members including shrimps, lobsters, and crabs [14]. Most of the studies discussed so far have been performed on exoskeletons of crabs and lobsters [3–11]. An extensive examination of the literature regarding exoskeleton property measurements of such species will reveal that there have been limited studies focusing on nanomechanical properties of ⁎ Corresponding author. Tel.: +1 765 494 3423. E-mail address: [email protected] (V. Tomar).
http://dx.doi.org/10.1016/j.msec.2014.08.033 0928-4931/© 2014 Elsevier B.V. All rights reserved.
shrimp exoskeleton as a function of change in environment from wet to dry. For example ref. [20] is the first paper that presented such nanomechanical data for mantis shrimp in dry conditions. Focus of the present work is on measuring the reduced modulus and hardness of shallow water shrimp (Pandalus platyceros) exoskeleton. In further sections, the use of the term nanomechanical properties should be considered as reduced modulus and hardness measurements performed using nanoindentation experiments. Chen et al. [3] have shown that the presence of water results in increased toughness of crab shells. However, how such environment affects shrimp exoskeleton properties is not clear. The present work focuses on such measurements. The design and development of new materials rest on the choice of experimental techniques applied to obtain data on which predictive models are based. Specifically, in the case of natural materials, there are certain uncertainties associated with mechanical property determination as most such experiments are conducted under ambient conditions. On the other hand, it is well established that hydrated state of natural materials exhibits a large variation in its properties as compared to the dry state [3,21–28]. Various studies have focused on finding chemical composition of shrimp exoskeleton [29–35]. Shahidi and Synowiecki [32] compared chitin, protein, and carotenoid contents of shrimp and crab exoskeletons. Protein content in the shrimp exoskeleton was found to be almost double of that found in the crab exoskeleton. Studies suggest that moisture content in shrimp exoskeleton ranges from 10% to 15% by weight. Earlier, different molecular simulations have shown that the protein mechanical properties (i.e. modulus and hardness) significantly change when exposed to water [36,37]. It has also been reported that moisture content plays a major role in affecting
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the mechanical properties [21–24] of bone. Based on such observations, it is expected that the natural water content in shrimp exoskeleton would lead to a significant change in mechanical properties as a function of environment change from dry to wet. Studies reported in the case of crab and lobster exoskeletons have so far been performed under normal atmospheric conditions. Not many studies compare nanomechanical properties at high pressures. However, such creatures exist under conditions of high constant pressure. It is, therefore, important to understand how constant load applied for long time duration (creep) in wet and dry environments would affect material properties as well as what would be the underlying deformation mechanism under such boundary conditions. The present work, therefore, also focuses on performing creep measurements in dry and wet conditions.
The shallow water shrimp exoskeleton has μm scale thickness as well as significant heterogeneity. In such material, traditionally performed uniaxial tests will only provide overall mechanical strength information while nanoindentation has the capability to give site specific data with minimal sample preparation. This makes nanoindentation a standard experimental technique to find modulus and hardness of such materials at nano- and microscale. Fig. 1 shows a standard indentation curve and parts of curve used for material property calculations. The experimental procedure involves indenting the surface of material being tested by increasing the force in small steps until the predetermined peak load (Pmax) or peak depth (hmax) is achieved. The Berkovich indenter is used in the present work. During the unloading process only elastic recovery happens. The unloading part is used for predicting the material properties using a framework based on contact mechanics [38,39]. During experiments maximum indentation load Pmax and maximum area of indentation A are measured. Area A is also referred to as the maximum projected area of elastic contact. The hardness, H is given as P max : A
ð1Þ
The contact area function for ideal Berkovich indenter used in the present work as a function of contact depth hc (described later) is given as pffiffiffi 2 2 2 A ¼ 3 3hc tan 65:3 ≈ 24:5hc :
S¼
pffiffiffi dP ¼ 1:17Er A: dh
ð3Þ
Here, S is the stiffness measured experimentally from the slope of the unloading curve. Based on known S and A values, Er can be calculated. The contact depth, hc, is such that hc ¼ hmax −0:75
P max : S
ð4Þ
According to Oliver and Pharr [38], the relationship between load P and depth h during an indentation experiment can be described as
2. Method
H¼
Reduced modulus, Er, is related to slope, S, of the upper part of unloading curve given as
ð2Þ
Fig. 1. Indentation curve showing parts used for stiffness, creep and thermal drift calculations.
1:5 P ¼ a h−h f :
ð5Þ
Here, a is an empirical fitting variable [38] determined individually for each indentation experiment based on fitting of the load–depth curve to Eq. (5). The depth hf is the indentation depth after unloading is complete. The contact stiffness S as a function of depth h can be calculated as, S¼
1=2 dP ¼ 1:5a h−h f : dh h¼hmax
ð6Þ
Reduced modulus, Er, is used to represent material strength in this work. 2.1. Aqueous environment experiment setup Experiments were performed in a multi-module mechanical tester (NanoTest, Micro Materials Ltd.), [40,41]. The liquid cell set up, Fig. 2, was used to perform the indentation experiments in wet environment. The same set up was used for dry samples without the use of liquid. The instrument consists of a vertical pendulum pivoted on a frictionless spring. An indenter is attached to the pendulum that indents samples horizontally. The force on pendulum is applied through the magnetic coils which allows for a very high sensitivity during experiments. The depth of indentation during experiments is measured by capacitor plates located behind the indenter. Before the experiments, calibrations are performed to get accurate measurements of load and depth. Depth calibration, load calibration, and frame compliance experiments are performed before conducting actual experiments. Tip radius of the Berkovich indenter is 20 nm. During indents, samples were mounted on stage firmly to avoid any movement during experiments. When using liquid cell, indenter is mounted on an extension to the pendulum which allows indenter to be immersed in liquid while performing indents. In this way a direct comparison of the properties measured during indentation on dry samples and on liquid samples can be performed.
Fig. 2. Photograph of instrument set-up for liquid cell indentation tests.
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This setup enables the measurement of properties at high level of accuracy in the original hydrated state. Experiments were performed on dry and wet samples at different loads. Indentation locations were chosen randomly and a series of indents at 10 points in each sample were performed. Experiments were performed on 10 different samples to capture possible variations arising due to sample heterogeneity. Indents were performed at peak loads of 5, 10, 15, 20 and 25 mN. A total of 50 indentations were performed for obtaining each data point. DI (de-ionized) water was used as liquid. Indentation depth was in the range of 900 nm to 1100 nm for dry samples and from 2500 nm to 2800 nm for wet samples. All creep measurements were performed at 10 mN peak load. The creep strain rate of soft materials is higher than that of hard materials. During indentation creep experiment, Fig. 1, a nose section will appear at the highest load leading to wrong calculations of modulus, [41–44]. To avoid it, a hold time is set at a maximum load that corrects the nose and gives correct elastic modulus measurements. A dwell period of 100 s was applied during indentation experiments to collect creep data. Indentation data for reduced modulus and hardness measurements was also corrected. Creep response is related to microstructural processes and conditions under which creep tests are performed. It is affected by temperature, pressure, and stress level during experiments [46]. Activation volume, V, is one other parameter that is helpful in identifying a proper creep mechanism. V is given as
V ¼ kB T
∂ ln ε˙ ∂σ ε;T
ð7Þ
here, kB is the Boltzmann's constant, T is the temperature in Kelvin, ε˙ is the creep strain rate, σ is the stress, and ε is the creep strain.
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2.4. Indentation size effect The indentation size effect according to the Meyer's law [47–49] is a change in the measured properties as a function of indentation depth. Modulus and hardness values are plotted with different indentation depths in Fig. 4(a) and (b), respectively. As shown, the measured values are similar at different depths. The indenter tip radius is 20 nm and indentation depths in current experiments were up to 1 μm. It leads to an area of indentation during experiments of order of microns. 2.5. Substrate effect The substrate could have a major effect during measurements on thin samples. The general rule is that indentation depth should be less than 10% of the total thickness of sample [50–52]. The thickness of current samples is 110 ± 10 μm and indentation depth at the peak load of 10 mN was 1 μm for dry samples which satisfies the 10% rule. 2.6. Pile-up effect Pile-up during indentation can have an adverse effect on the values of modulus and hardness measurement as investigated by A. Bolshakov et al. [53,54]. SEM images were obtained after performing indentations at different loads. Fig. 5(a) shows the image of indent performed at 10 mN load. Fig. 5(b) shows the indent mark for 200 mN load. A pileup is observed at very high loads of 200 mN as shown in Fig. 5(b). Indentations were performed at 10 mN load and no pile-up was observed as shown in Fig. 5(a). 3. Results and discussion 3.1. Structure of exoskeleton
2.2. Sample preparation Whole fresh unfrozen shallow water shrimps were obtained in order to prepare the samples. Shrimps were immediately stored in frozen condition after procurement. Fig. 3 shows the sample preparation steps. To prepare samples, shrimps were taken out and the carapace was removed carefully without mechanically straining or producing any fracture in it. Carapace is the exoskeleton of shrimp that protects cephalothoracic region of shrimp. Cephalothoracic is the front part of shrimp that contains vital parts of shrimp including mouth, eyes, and antennas. Carapace will be referred to as shrimp exoskeleton further in this paper. Exoskeleton was then dried in open for 3 days. 2.3. Factors affecting indentation measurements Different factors that could adversely affect the nanoindentation experiments are briefly described next.
SEM images were obtained to get a detailed understanding of the composition of examined shrimp exoskeleton. Fig. 6 shows a SEM image of the full cross section of one of the examined shrimp exoskeletons. As shown, there is a clear non-uniformity in the stacking density and thickness of the layers across cross section. Each visible layer is a layer of woven α-chitin–protein fiber network, the characteristic feature of crustacean exoskeleton [7–9]. It consists of different kinds of proteins and minerals. A closer look at Fig. 6 shows that the thicknesses of layers have a gradient. Starting from the bottom, these layers increase in thickness slowly and then have a stacking of layers of similar thickness till half of the total thickness. At this point there is an interface where thickness of the layers increases. Such distribution of layers suggests a possible contribution of the stacking sequence in the overall elastic properties of the shrimp exoskeleton. Fig. 7(a, b, c, d)shows the magnified view of the regions shown in Fig. 6 with different layer thicknesses across exoskeleton cross section. Fig. 7(a) is a magnified image of the top surface. In the top
Fig. 3. Sample preparation (a) whole shrimp (b) SEM image of sample surface.
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Fig. 4. Variation of (a) reduced modulus and (b) hardness with indentation depth.
most regions, layers are made up of the same kind of material and have minimal thickness gradient in design. The top region has low porosity and high level of mineral contents. The maximum thickness of layers is up to 2 μm and minimum thickness at the top layer drop downs to 500 nm. Fig. 7(b) shows a magnified region in the top half of cross section with layers of uniform thickness of 5 μm. Fig. 7(c) shows a SEM image of the mid to bottom region. The thickness of layers is 1 μm in this region. The minimum thickness is found in the bottom most region with value of the order of 50 nm to 100 nm as observed in Fig. 7(d). The regions in Figs. 7(b–c) are made up of thicker layers as compared to those shown in Fig. 7(d). There is a clear difference in the density of layers per unit thickness. The bottom layers are more closely packed. Fig. 8(a) shows the interface at the mid region of Fig. 6 with the thickness of layers gradually decreasing to almost one fifth as compared to the top half region. Fig. 8(b) shows the twisted layers of cross sectional structure showing Bouligand structure. These layers are formed by the helicoidal stacks of α-chitin–protein planes. The thickness of each layer has 180° rotated layers of α-chitin–protein planes. Fig. 9 shows the EDX analysis of analyzed samples. Fig. 9(a) shows the collected spectrum from the sample. It shows the high content of oxygen and carbon which are the main constituents of proteins [13]. It also shows a higher quantity of calcium which means that there is a high content of calcium based minerals present in shrimp exoskeleton. These observations can be further verified from Table 1 giving the quantitative elemental composition. This is in agreement with the chemical composition obtained by several other researchers [29–35]. The elemental map across cross section shows the distribution of C, O and Ca across the cross section in Fig. 9. Ca is uniformly distributed across the
region but the distribution of C and O elements changes across layers showing protein rich regions. On comparing the structure of shrimp exoskeleton with that of crabs and lobster as obtained by Raabe et al. [8,9] the thickness of the layers in shrimp exoskeleton is found to be smaller. It suggests that mechanical properties (i.e. modulus and hardness) of shrimp would be different due to the difference at the micro-scale level in design and due to the difference in stacking density. Mechanical properties (i.e. modulus and hardness) of regions with thinner layers and higher packing density were higher as compared to regions with thicker layers [9]. It also suggests that mechanical properties (i.e. modulus and hardness) in this case would be even higher (or stronger) than the properties reported by Raabe et al. [8,9] for lobster. 3.2. Mechanical properties of the exoskeleton of dry and wet samples Fig. 10(a) shows a comparison of reduced modulus of the examined shrimp exoskeleton in dry and wet environments. Hardness values are plotted in Fig. 10(b). As determined from the chemical composition of shrimps, moisture content amounts are found to be 10%–15% of the weight of shrimp exoskeleton. Fig. 10(a) and (b) shows that there is a significant difference in the properties of wet and dry samples. This difference is directly attributable to the presence of water. As expected, the presence of water decreases the reduced modulus and hardness of the material. Shrimps contain 60% by weight of proteins in their shells. It has been shown earlier in literature that the presence of water helps preferential bonding in proteins that leads to interlayer sliding. Such sliding enables increased toughness and reduced hardness [36,37]. Protein molecules bind water in their molecules and have higher volume in
Fig. 5. SEM image of indentation mark on shrimp exoskeleton at (a) 10 mN and (b) 200 mN.
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Fig. 6. SEM image of full cross section of one of the examined shrimp exoskeleton with legends showing the zoomed in images shown in next figures.
wet samples [55,56]. Various biomedical material experiments have discussed such softening [57–59]. The presence of water also reduces mechanical properties (i.e. modulus and hardness) by adversely
affecting the mineralization of calcium minerals present in shrimp exoskeleton. In dry samples, water molecules are lost and proteins bind together more closely. Water molecules also decrease the density of
Fig. 7. Magnified view of the regions with different layer thicknesses across cross section from (a) top to (b, c) middle regions to (d) bottom.
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Fig. 8. (a) SEM image of the interface of layers of different thicknesses through the cross section of shrimp exoskeleton and (b) twisted layers of examined exoskeleton showing the Bouligand structure.
protein network by increasing their volume through absorption induced swelling [60]. Materials with increased porosity have lower mechanical properties (i.e. modulus and hardness). In addition to the above factors, chitin molecules have been found to be highly hydrophobic and insoluble in water [61] that makes the wet samples to have lower mechanical properties (i.e. modulus and hardness) as compared to the dry samples. The significant change in mechanical properties of
dry and wet samples can be conclusively attributed to the above discussed effects of water on the constituents of shrimp shell. Fig. 11 shows dwell data for the creep tests plotted for dry and wet samples. As discussed earlier, creep dwell tests are performed to develop an understanding of the effect of constant pressure environment on material deformation behavior. Table 2 lists the creep strain rate of dry and wet samples. Creep rate is higher for wet samples. It is expected
Fig. 9. EDX analysis of cross section of shrimp (a) spectrum and elemental distribution map for carbon(C), oxygen(O) and calcium(Ca). Scale bars = 10 μm.
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Table 1 Quantitative data of EDX analysis on the cross section of examined shrimp exoskeleton. Element
C
O
Ca
Atomic %
45.55
39.78
12.72
that soft material will creep at higher creep rate at the same load. A higher elastic constant leads to better creep resistance [46].
3.3. Constant stress deformation (creep) mechanism Shrimp exoskeleton is made up of polymeric protein fibers woven together and the spaces between these fibers are filled with minerals. It is expected that creep mechanisms experienced by shrimp exoskeleton can be closely represented by polymeric creep mechanisms. The deformation of shrimp exoskeleton exhibits viscoelastic response due to its polymer like design. During deformation, the flow of stress will be carried through fibers and filler material. The extent of deformation in material will depend on constraints provided by filler material to the stretching of polymer chains and also on the load transfer capacity between polymer chains and distributed bio-minerals [62–64]. This additional constraint in the material is dependent on the polymer type, length of polymer chains, distribution and type of filler material. Polymers with added constraints due to an additional filler material show decreased creep strain rate behavior [62]. In addition, viscoelastic deformations in composites depend on a variety of factors including laminate layup, stress level, frequency of loading, humidity, temperature and pressure thermal history [65]. A long term prediction for the strength and stiffness of composites is required for a better aid in the material design. Short term creep tests are fit for obtaining appropriate viscoelastic constitutive models to predict long term creep behavior. The duration of the short time tests depends on the desired maximum time prediction and accuracy required at maximum prediction time. Viscoelastic damage in composites relates to fracture mechanisms as well as damage accumulation within individual layers during creep. The reinforcing fibers do not alter the creep mechanism in matrix material. However, such fibers do alter the creep behavior by increasing resistance to the cooperative chain segmental mobility of polymer chain segments. Viscoplastic response of polymer composites arises mainly from the matrix viscoplasticity. In the present experiment, creep experiments were performed for a duration of 100 s. The creep strain rate increases in wet samples. It could be attributed to the relaxation of polymer chains and
Fig. 11. Plot of dwell data for wet and dry samples.
decrease in the matrix/fiber interface strength with high moisture content. 3.3.1. Activation volume Arrhenius type flow function at low temperatures is used to express creep strain rate [46]. It is given as ΔGðσ Þ ε˙ ¼ ε˙ o exp − kB T
ð8Þ
here, ε˙ is the strain rate. ε˙o is the reference strain rate that depends on material type, material microstructure and stress level. ΔG is the change in Gibbs free energy of the system to bring deformations to its saddle point from the equilibrium position, kB is the Boltzmann's constant, and T is the temperature in Kelvin. Thermal activation volume is defined as the volume involved in the deformation of material by locally acting stress. The expression for thermal activation volume used in this work is given by Schoeck [66]. The polymers during deformations are initially in equilibrium and have to move from saddle point configuration to be able to move larger distances during creep deformation. Creep mechanism dominating the deformation is dependent on the excess free volume available for deformation. This excess free volume is in turn related to thermal activation volume. Thermal activation volume measurements during creep experiments help in determining creep mechanisms [66].
Fig. 10. Comparison of (a) reduced modulus and (b) hardness of wet and dry samples.
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Table 2 Creep strain rate and thermal activation volume for dry and wet samples of shrimp exoskeleton. Creep rate (nm/s)
Dry Wet
Thermal activation volume (nm3)
Average
Error
Average
Error
0.210 1.602
0.021 0.137
0.060 0.100
0.004 0.005
Table 2 lists the activation volumes for wet and dry samples. Activation volume in the case of the wet sample is higher as compared to the dry samples. This is expected as the extent of deformation in wet samples is higher than that in dry samples. The order of activation volume is in the range of 10−2 nm3 in dry samples and in the range of 10−1 nm3 in wet samples. Observed variations could be attributed to the complex structure of exoskeleton. Values could also depend on the site of indentation. Creep is more likely to be associated with the vacancies present and loose packing of the chitin based fiber layers. The deformation mechanisms are generally polymer chain relaxations and interlaminar slip. Higher creep rate in wet environments can be related to lower mechanical properties (i.e. modulus and hardness) of wet samples. A discussion about differences in mechanical properties (i.e. modulus and hardness) is given in the previous section. Based on the previous studies, [46], most probable creep mechanism in the current work is the collapsing of interstitial vacancies of α-chitin layers at the nanoscale, interface slip, and increased ductility of matrix material. With the increase in moisture content, polymer chains are expected to be in more relaxed state. Matrix material which is ceramic and mineral based particles will also experience reduction in stiffness. It will allow fibers to move more than usual and is responsible for an increased creep strain rate. 4. Conclusions The present work is focused on analyzing environment dependent properties of shrimp exoskeleton. Nanomechanical properties (i.e. modulus and hardness) were measured using indentation experiments. The difference in the mechanical properties of dry and wet samples is correlated with the microstructure and composition of samples. The presence of water helps preferential bonding in proteins that leads to interlayer sliding. Such sliding enables increased toughness and reduced hardness. Protein molecules bind water in their molecules and have higher volume in wet samples. The presence of water also reduces the mechanical properties by adversely affecting the mineralization of calcium minerals present in shrimp shell. In dry samples, water molecules are lost and the proteins bind together more closely. Water molecules also decrease the density of protein network by increasing the volume through absorption induced swelling. Materials with increased porosity have lower mechanical strength. In addition to these factors, chitin molecules have been found to be highly hydrophobic and insoluble in water that makes wet samples to have lower mechanical strength as compared to dry samples. The analyses also indicate that wet environment under constant pressure induces higher activation volume for deformations in the exoskeleton. This leads to reduced strength but increased toughness of exoskeleton. The constant high pressure deformation occurs through the relaxation of polymer chains of α-chitin layers at nanoscale and decreased matrix viscoplasticity. Overall, while dry samples show strength, wet samples show a better combination of strength with toughness. Acknowledgments Authors would like to thank Dr. Ming Gan for help with the nanoindentation. Also, authors would like to acknowledge excellent technical
assistance of Dr. Christopher J. Gilpin, Chia-Ping Huang and Laurie Mueller with Scanning Electron Microscopy at Purdue University. Support from NSF grant CMMI-1131112 (Program Manager: Dr. Dennis Carter) is gratefully acknowledged.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
An investigation into environment dependent nanomechanical properties of shallow water shrimp (Pandalus platyceros) exoskeleton Devendra Verma, Vikas Tomar ⁎ School of Aeronautics and Astronautics, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 4 January 2014 Received in revised form 26 July 2014 Accepted 8 August 2014 Available online 15 August 2014 Keywords: Shrimp exoskeleton Nanomechanics Environment dependence Biomimetic Liquid cell
a b s t r a c t The present investigation focuses on understanding the influence of change from wet to dry environment on nanomechanical properties of shallow water shrimp exoskeleton. Scanning Electron Microscopy (SEM) based measurements suggest that the shrimp exoskeleton has Bouligand structure, a key characteristic of the crustaceans. As expected, wet samples are found to be softer than dry samples. Reduced modulus values of dry samples are found to be 24.90 ± 1.14 GPa as compared to the corresponding values of 3.79 ± 0.69 GPa in the case of wet samples. Hardness values are found to be 0.86 ± 0.06 GPa in the case of dry samples as compared to the corresponding values of 0.17 ± 0.02 GPa in the case of wet samples. In order to simulate the influence of underwater pressure on the exoskeleton strength, constant load creep experiments as a function of wet and dry environments are performed. The switch in deformation mechanism as a function of environment is explained based on the role played by water molecules in assisting interface slip and increased ductility of matrix material in wet environment in comparison to the dry environment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies of natural materials have often provided insights into the design and development of new class of materials [1,2]. In continuation to the exploration of properties of natural materials, a new area that focuses on the study of crustacean family (lobsters, crabs, shrimps etc.) has emerged. Several studies have been performed on the natural crustacean (lobsters, crabs) shells in order to understand correlations between structure and properties of such materials [3–15]. Exoskeleton of crustaceans has a well-defined hierarchical structure [6–11]. It consists of chitin based fibrils coated in proteins at nanometer level. Such fibrils bind together to form fibers. These fibers are then woven together to form chitin–protein layers. Such layers are stacked in a twisted plywood structure known as the Bouligand pattern [6–11]. Studies by Seki et al. [12,13] have concentrated on a similar kind of exoskeleton found in toucan beak. It was found to be a sandwich structure with the keratin exterior and a closed cell fibrous network made up of calcium rich proteins. Chen et al. [16–19] have also thoroughly investigated beetle forewing to be able to mimic its design for better sandwich panel structures. The crustacean family consists of several different members including shrimps, lobsters, and crabs [14]. Most of the studies discussed so far have been performed on exoskeletons of crabs and lobsters [3–11]. An extensive examination of the literature regarding exoskeleton property measurements of such species will reveal that there have been limited studies focusing on nanomechanical properties of ⁎ Corresponding author. Tel.: +1 765 494 3423. E-mail address: [email protected] (V. Tomar).
http://dx.doi.org/10.1016/j.msec.2014.08.033 0928-4931/© 2014 Elsevier B.V. All rights reserved.
shrimp exoskeleton as a function of change in environment from wet to dry. For example ref. [20] is the first paper that presented such nanomechanical data for mantis shrimp in dry conditions. Focus of the present work is on measuring the reduced modulus and hardness of shallow water shrimp (Pandalus platyceros) exoskeleton. In further sections, the use of the term nanomechanical properties should be considered as reduced modulus and hardness measurements performed using nanoindentation experiments. Chen et al. [3] have shown that the presence of water results in increased toughness of crab shells. However, how such environment affects shrimp exoskeleton properties is not clear. The present work focuses on such measurements. The design and development of new materials rest on the choice of experimental techniques applied to obtain data on which predictive models are based. Specifically, in the case of natural materials, there are certain uncertainties associated with mechanical property determination as most such experiments are conducted under ambient conditions. On the other hand, it is well established that hydrated state of natural materials exhibits a large variation in its properties as compared to the dry state [3,21–28]. Various studies have focused on finding chemical composition of shrimp exoskeleton [29–35]. Shahidi and Synowiecki [32] compared chitin, protein, and carotenoid contents of shrimp and crab exoskeletons. Protein content in the shrimp exoskeleton was found to be almost double of that found in the crab exoskeleton. Studies suggest that moisture content in shrimp exoskeleton ranges from 10% to 15% by weight. Earlier, different molecular simulations have shown that the protein mechanical properties (i.e. modulus and hardness) significantly change when exposed to water [36,37]. It has also been reported that moisture content plays a major role in affecting
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the mechanical properties [21–24] of bone. Based on such observations, it is expected that the natural water content in shrimp exoskeleton would lead to a significant change in mechanical properties as a function of environment change from dry to wet. Studies reported in the case of crab and lobster exoskeletons have so far been performed under normal atmospheric conditions. Not many studies compare nanomechanical properties at high pressures. However, such creatures exist under conditions of high constant pressure. It is, therefore, important to understand how constant load applied for long time duration (creep) in wet and dry environments would affect material properties as well as what would be the underlying deformation mechanism under such boundary conditions. The present work, therefore, also focuses on performing creep measurements in dry and wet conditions.
The shallow water shrimp exoskeleton has μm scale thickness as well as significant heterogeneity. In such material, traditionally performed uniaxial tests will only provide overall mechanical strength information while nanoindentation has the capability to give site specific data with minimal sample preparation. This makes nanoindentation a standard experimental technique to find modulus and hardness of such materials at nano- and microscale. Fig. 1 shows a standard indentation curve and parts of curve used for material property calculations. The experimental procedure involves indenting the surface of material being tested by increasing the force in small steps until the predetermined peak load (Pmax) or peak depth (hmax) is achieved. The Berkovich indenter is used in the present work. During the unloading process only elastic recovery happens. The unloading part is used for predicting the material properties using a framework based on contact mechanics [38,39]. During experiments maximum indentation load Pmax and maximum area of indentation A are measured. Area A is also referred to as the maximum projected area of elastic contact. The hardness, H is given as P max : A
ð1Þ
The contact area function for ideal Berkovich indenter used in the present work as a function of contact depth hc (described later) is given as pffiffiffi 2 2 2 A ¼ 3 3hc tan 65:3 ≈ 24:5hc :
S¼
pffiffiffi dP ¼ 1:17Er A: dh
ð3Þ
Here, S is the stiffness measured experimentally from the slope of the unloading curve. Based on known S and A values, Er can be calculated. The contact depth, hc, is such that hc ¼ hmax −0:75
P max : S
ð4Þ
According to Oliver and Pharr [38], the relationship between load P and depth h during an indentation experiment can be described as
2. Method
H¼
Reduced modulus, Er, is related to slope, S, of the upper part of unloading curve given as
ð2Þ
Fig. 1. Indentation curve showing parts used for stiffness, creep and thermal drift calculations.
1:5 P ¼ a h−h f :
ð5Þ
Here, a is an empirical fitting variable [38] determined individually for each indentation experiment based on fitting of the load–depth curve to Eq. (5). The depth hf is the indentation depth after unloading is complete. The contact stiffness S as a function of depth h can be calculated as, S¼
1=2 dP ¼ 1:5a h−h f : dh h¼hmax
ð6Þ
Reduced modulus, Er, is used to represent material strength in this work. 2.1. Aqueous environment experiment setup Experiments were performed in a multi-module mechanical tester (NanoTest, Micro Materials Ltd.), [40,41]. The liquid cell set up, Fig. 2, was used to perform the indentation experiments in wet environment. The same set up was used for dry samples without the use of liquid. The instrument consists of a vertical pendulum pivoted on a frictionless spring. An indenter is attached to the pendulum that indents samples horizontally. The force on pendulum is applied through the magnetic coils which allows for a very high sensitivity during experiments. The depth of indentation during experiments is measured by capacitor plates located behind the indenter. Before the experiments, calibrations are performed to get accurate measurements of load and depth. Depth calibration, load calibration, and frame compliance experiments are performed before conducting actual experiments. Tip radius of the Berkovich indenter is 20 nm. During indents, samples were mounted on stage firmly to avoid any movement during experiments. When using liquid cell, indenter is mounted on an extension to the pendulum which allows indenter to be immersed in liquid while performing indents. In this way a direct comparison of the properties measured during indentation on dry samples and on liquid samples can be performed.
Fig. 2. Photograph of instrument set-up for liquid cell indentation tests.
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This setup enables the measurement of properties at high level of accuracy in the original hydrated state. Experiments were performed on dry and wet samples at different loads. Indentation locations were chosen randomly and a series of indents at 10 points in each sample were performed. Experiments were performed on 10 different samples to capture possible variations arising due to sample heterogeneity. Indents were performed at peak loads of 5, 10, 15, 20 and 25 mN. A total of 50 indentations were performed for obtaining each data point. DI (de-ionized) water was used as liquid. Indentation depth was in the range of 900 nm to 1100 nm for dry samples and from 2500 nm to 2800 nm for wet samples. All creep measurements were performed at 10 mN peak load. The creep strain rate of soft materials is higher than that of hard materials. During indentation creep experiment, Fig. 1, a nose section will appear at the highest load leading to wrong calculations of modulus, [41–44]. To avoid it, a hold time is set at a maximum load that corrects the nose and gives correct elastic modulus measurements. A dwell period of 100 s was applied during indentation experiments to collect creep data. Indentation data for reduced modulus and hardness measurements was also corrected. Creep response is related to microstructural processes and conditions under which creep tests are performed. It is affected by temperature, pressure, and stress level during experiments [46]. Activation volume, V, is one other parameter that is helpful in identifying a proper creep mechanism. V is given as
V ¼ kB T
∂ ln ε˙ ∂σ ε;T
ð7Þ
here, kB is the Boltzmann's constant, T is the temperature in Kelvin, ε˙ is the creep strain rate, σ is the stress, and ε is the creep strain.
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2.4. Indentation size effect The indentation size effect according to the Meyer's law [47–49] is a change in the measured properties as a function of indentation depth. Modulus and hardness values are plotted with different indentation depths in Fig. 4(a) and (b), respectively. As shown, the measured values are similar at different depths. The indenter tip radius is 20 nm and indentation depths in current experiments were up to 1 μm. It leads to an area of indentation during experiments of order of microns. 2.5. Substrate effect The substrate could have a major effect during measurements on thin samples. The general rule is that indentation depth should be less than 10% of the total thickness of sample [50–52]. The thickness of current samples is 110 ± 10 μm and indentation depth at the peak load of 10 mN was 1 μm for dry samples which satisfies the 10% rule. 2.6. Pile-up effect Pile-up during indentation can have an adverse effect on the values of modulus and hardness measurement as investigated by A. Bolshakov et al. [53,54]. SEM images were obtained after performing indentations at different loads. Fig. 5(a) shows the image of indent performed at 10 mN load. Fig. 5(b) shows the indent mark for 200 mN load. A pileup is observed at very high loads of 200 mN as shown in Fig. 5(b). Indentations were performed at 10 mN load and no pile-up was observed as shown in Fig. 5(a). 3. Results and discussion 3.1. Structure of exoskeleton
2.2. Sample preparation Whole fresh unfrozen shallow water shrimps were obtained in order to prepare the samples. Shrimps were immediately stored in frozen condition after procurement. Fig. 3 shows the sample preparation steps. To prepare samples, shrimps were taken out and the carapace was removed carefully without mechanically straining or producing any fracture in it. Carapace is the exoskeleton of shrimp that protects cephalothoracic region of shrimp. Cephalothoracic is the front part of shrimp that contains vital parts of shrimp including mouth, eyes, and antennas. Carapace will be referred to as shrimp exoskeleton further in this paper. Exoskeleton was then dried in open for 3 days. 2.3. Factors affecting indentation measurements Different factors that could adversely affect the nanoindentation experiments are briefly described next.
SEM images were obtained to get a detailed understanding of the composition of examined shrimp exoskeleton. Fig. 6 shows a SEM image of the full cross section of one of the examined shrimp exoskeletons. As shown, there is a clear non-uniformity in the stacking density and thickness of the layers across cross section. Each visible layer is a layer of woven α-chitin–protein fiber network, the characteristic feature of crustacean exoskeleton [7–9]. It consists of different kinds of proteins and minerals. A closer look at Fig. 6 shows that the thicknesses of layers have a gradient. Starting from the bottom, these layers increase in thickness slowly and then have a stacking of layers of similar thickness till half of the total thickness. At this point there is an interface where thickness of the layers increases. Such distribution of layers suggests a possible contribution of the stacking sequence in the overall elastic properties of the shrimp exoskeleton. Fig. 7(a, b, c, d)shows the magnified view of the regions shown in Fig. 6 with different layer thicknesses across exoskeleton cross section. Fig. 7(a) is a magnified image of the top surface. In the top
Fig. 3. Sample preparation (a) whole shrimp (b) SEM image of sample surface.
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Fig. 4. Variation of (a) reduced modulus and (b) hardness with indentation depth.
most regions, layers are made up of the same kind of material and have minimal thickness gradient in design. The top region has low porosity and high level of mineral contents. The maximum thickness of layers is up to 2 μm and minimum thickness at the top layer drop downs to 500 nm. Fig. 7(b) shows a magnified region in the top half of cross section with layers of uniform thickness of 5 μm. Fig. 7(c) shows a SEM image of the mid to bottom region. The thickness of layers is 1 μm in this region. The minimum thickness is found in the bottom most region with value of the order of 50 nm to 100 nm as observed in Fig. 7(d). The regions in Figs. 7(b–c) are made up of thicker layers as compared to those shown in Fig. 7(d). There is a clear difference in the density of layers per unit thickness. The bottom layers are more closely packed. Fig. 8(a) shows the interface at the mid region of Fig. 6 with the thickness of layers gradually decreasing to almost one fifth as compared to the top half region. Fig. 8(b) shows the twisted layers of cross sectional structure showing Bouligand structure. These layers are formed by the helicoidal stacks of α-chitin–protein planes. The thickness of each layer has 180° rotated layers of α-chitin–protein planes. Fig. 9 shows the EDX analysis of analyzed samples. Fig. 9(a) shows the collected spectrum from the sample. It shows the high content of oxygen and carbon which are the main constituents of proteins [13]. It also shows a higher quantity of calcium which means that there is a high content of calcium based minerals present in shrimp exoskeleton. These observations can be further verified from Table 1 giving the quantitative elemental composition. This is in agreement with the chemical composition obtained by several other researchers [29–35]. The elemental map across cross section shows the distribution of C, O and Ca across the cross section in Fig. 9. Ca is uniformly distributed across the
region but the distribution of C and O elements changes across layers showing protein rich regions. On comparing the structure of shrimp exoskeleton with that of crabs and lobster as obtained by Raabe et al. [8,9] the thickness of the layers in shrimp exoskeleton is found to be smaller. It suggests that mechanical properties (i.e. modulus and hardness) of shrimp would be different due to the difference at the micro-scale level in design and due to the difference in stacking density. Mechanical properties (i.e. modulus and hardness) of regions with thinner layers and higher packing density were higher as compared to regions with thicker layers [9]. It also suggests that mechanical properties (i.e. modulus and hardness) in this case would be even higher (or stronger) than the properties reported by Raabe et al. [8,9] for lobster. 3.2. Mechanical properties of the exoskeleton of dry and wet samples Fig. 10(a) shows a comparison of reduced modulus of the examined shrimp exoskeleton in dry and wet environments. Hardness values are plotted in Fig. 10(b). As determined from the chemical composition of shrimps, moisture content amounts are found to be 10%–15% of the weight of shrimp exoskeleton. Fig. 10(a) and (b) shows that there is a significant difference in the properties of wet and dry samples. This difference is directly attributable to the presence of water. As expected, the presence of water decreases the reduced modulus and hardness of the material. Shrimps contain 60% by weight of proteins in their shells. It has been shown earlier in literature that the presence of water helps preferential bonding in proteins that leads to interlayer sliding. Such sliding enables increased toughness and reduced hardness [36,37]. Protein molecules bind water in their molecules and have higher volume in
Fig. 5. SEM image of indentation mark on shrimp exoskeleton at (a) 10 mN and (b) 200 mN.
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375
Fig. 6. SEM image of full cross section of one of the examined shrimp exoskeleton with legends showing the zoomed in images shown in next figures.
wet samples [55,56]. Various biomedical material experiments have discussed such softening [57–59]. The presence of water also reduces mechanical properties (i.e. modulus and hardness) by adversely
affecting the mineralization of calcium minerals present in shrimp exoskeleton. In dry samples, water molecules are lost and proteins bind together more closely. Water molecules also decrease the density of
Fig. 7. Magnified view of the regions with different layer thicknesses across cross section from (a) top to (b, c) middle regions to (d) bottom.
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Fig. 8. (a) SEM image of the interface of layers of different thicknesses through the cross section of shrimp exoskeleton and (b) twisted layers of examined exoskeleton showing the Bouligand structure.
protein network by increasing their volume through absorption induced swelling [60]. Materials with increased porosity have lower mechanical properties (i.e. modulus and hardness). In addition to the above factors, chitin molecules have been found to be highly hydrophobic and insoluble in water [61] that makes the wet samples to have lower mechanical properties (i.e. modulus and hardness) as compared to the dry samples. The significant change in mechanical properties of
dry and wet samples can be conclusively attributed to the above discussed effects of water on the constituents of shrimp shell. Fig. 11 shows dwell data for the creep tests plotted for dry and wet samples. As discussed earlier, creep dwell tests are performed to develop an understanding of the effect of constant pressure environment on material deformation behavior. Table 2 lists the creep strain rate of dry and wet samples. Creep rate is higher for wet samples. It is expected
Fig. 9. EDX analysis of cross section of shrimp (a) spectrum and elemental distribution map for carbon(C), oxygen(O) and calcium(Ca). Scale bars = 10 μm.
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Table 1 Quantitative data of EDX analysis on the cross section of examined shrimp exoskeleton. Element
C
O
Ca
Atomic %
45.55
39.78
12.72
that soft material will creep at higher creep rate at the same load. A higher elastic constant leads to better creep resistance [46].
3.3. Constant stress deformation (creep) mechanism Shrimp exoskeleton is made up of polymeric protein fibers woven together and the spaces between these fibers are filled with minerals. It is expected that creep mechanisms experienced by shrimp exoskeleton can be closely represented by polymeric creep mechanisms. The deformation of shrimp exoskeleton exhibits viscoelastic response due to its polymer like design. During deformation, the flow of stress will be carried through fibers and filler material. The extent of deformation in material will depend on constraints provided by filler material to the stretching of polymer chains and also on the load transfer capacity between polymer chains and distributed bio-minerals [62–64]. This additional constraint in the material is dependent on the polymer type, length of polymer chains, distribution and type of filler material. Polymers with added constraints due to an additional filler material show decreased creep strain rate behavior [62]. In addition, viscoelastic deformations in composites depend on a variety of factors including laminate layup, stress level, frequency of loading, humidity, temperature and pressure thermal history [65]. A long term prediction for the strength and stiffness of composites is required for a better aid in the material design. Short term creep tests are fit for obtaining appropriate viscoelastic constitutive models to predict long term creep behavior. The duration of the short time tests depends on the desired maximum time prediction and accuracy required at maximum prediction time. Viscoelastic damage in composites relates to fracture mechanisms as well as damage accumulation within individual layers during creep. The reinforcing fibers do not alter the creep mechanism in matrix material. However, such fibers do alter the creep behavior by increasing resistance to the cooperative chain segmental mobility of polymer chain segments. Viscoplastic response of polymer composites arises mainly from the matrix viscoplasticity. In the present experiment, creep experiments were performed for a duration of 100 s. The creep strain rate increases in wet samples. It could be attributed to the relaxation of polymer chains and
Fig. 11. Plot of dwell data for wet and dry samples.
decrease in the matrix/fiber interface strength with high moisture content. 3.3.1. Activation volume Arrhenius type flow function at low temperatures is used to express creep strain rate [46]. It is given as ΔGðσ Þ ε˙ ¼ ε˙ o exp − kB T
ð8Þ
here, ε˙ is the strain rate. ε˙o is the reference strain rate that depends on material type, material microstructure and stress level. ΔG is the change in Gibbs free energy of the system to bring deformations to its saddle point from the equilibrium position, kB is the Boltzmann's constant, and T is the temperature in Kelvin. Thermal activation volume is defined as the volume involved in the deformation of material by locally acting stress. The expression for thermal activation volume used in this work is given by Schoeck [66]. The polymers during deformations are initially in equilibrium and have to move from saddle point configuration to be able to move larger distances during creep deformation. Creep mechanism dominating the deformation is dependent on the excess free volume available for deformation. This excess free volume is in turn related to thermal activation volume. Thermal activation volume measurements during creep experiments help in determining creep mechanisms [66].
Fig. 10. Comparison of (a) reduced modulus and (b) hardness of wet and dry samples.
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Table 2 Creep strain rate and thermal activation volume for dry and wet samples of shrimp exoskeleton. Creep rate (nm/s)
Dry Wet
Thermal activation volume (nm3)
Average
Error
Average
Error
0.210 1.602
0.021 0.137
0.060 0.100
0.004 0.005
Table 2 lists the activation volumes for wet and dry samples. Activation volume in the case of the wet sample is higher as compared to the dry samples. This is expected as the extent of deformation in wet samples is higher than that in dry samples. The order of activation volume is in the range of 10−2 nm3 in dry samples and in the range of 10−1 nm3 in wet samples. Observed variations could be attributed to the complex structure of exoskeleton. Values could also depend on the site of indentation. Creep is more likely to be associated with the vacancies present and loose packing of the chitin based fiber layers. The deformation mechanisms are generally polymer chain relaxations and interlaminar slip. Higher creep rate in wet environments can be related to lower mechanical properties (i.e. modulus and hardness) of wet samples. A discussion about differences in mechanical properties (i.e. modulus and hardness) is given in the previous section. Based on the previous studies, [46], most probable creep mechanism in the current work is the collapsing of interstitial vacancies of α-chitin layers at the nanoscale, interface slip, and increased ductility of matrix material. With the increase in moisture content, polymer chains are expected to be in more relaxed state. Matrix material which is ceramic and mineral based particles will also experience reduction in stiffness. It will allow fibers to move more than usual and is responsible for an increased creep strain rate. 4. Conclusions The present work is focused on analyzing environment dependent properties of shrimp exoskeleton. Nanomechanical properties (i.e. modulus and hardness) were measured using indentation experiments. The difference in the mechanical properties of dry and wet samples is correlated with the microstructure and composition of samples. The presence of water helps preferential bonding in proteins that leads to interlayer sliding. Such sliding enables increased toughness and reduced hardness. Protein molecules bind water in their molecules and have higher volume in wet samples. The presence of water also reduces the mechanical properties by adversely affecting the mineralization of calcium minerals present in shrimp shell. In dry samples, water molecules are lost and the proteins bind together more closely. Water molecules also decrease the density of protein network by increasing the volume through absorption induced swelling. Materials with increased porosity have lower mechanical strength. In addition to these factors, chitin molecules have been found to be highly hydrophobic and insoluble in water that makes wet samples to have lower mechanical strength as compared to dry samples. The analyses also indicate that wet environment under constant pressure induces higher activation volume for deformations in the exoskeleton. This leads to reduced strength but increased toughness of exoskeleton. The constant high pressure deformation occurs through the relaxation of polymer chains of α-chitin layers at nanoscale and decreased matrix viscoplasticity. Overall, while dry samples show strength, wet samples show a better combination of strength with toughness. Acknowledgments Authors would like to thank Dr. Ming Gan for help with the nanoindentation. Also, authors would like to acknowledge excellent technical
assistance of Dr. Christopher J. Gilpin, Chia-Ping Huang and Laurie Mueller with Scanning Electron Microscopy at Purdue University. Support from NSF grant CMMI-1131112 (Program Manager: Dr. Dennis Carter) is gratefully acknowledged.
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