The nucleation effect of N,N′-bis(benzoyl) alkyl diacid dihydrazides on crystallization of biodegradable poly(l-lactic acid)

Five N,N??-bis(benzoyl) alkyl diacid dihydrazides were synthesized from benzoyl hydrazine and alkyl diacyl dichloride which were derived from alkyl diacid via acylation. PLLA/N,N??-bis(benzoyl) alkyl diacid dihydrazide samples were prepared by melt blending and hot-press forming process. The nucleation effect of N,N??-bis(benzoyl) alkyl diacid dihydrazide on crystallization of biodegradable poly(l-lactic acid) (PLLA) was investigated using differential scanning calorimetry (DSC) and vicat softening analysis. The results showed that five N,N??-bis(benzoyl) alkyl diacid dihydrazides acted as powerful nucleating agent for PLLA; with incorporation of N,N??-bis(benzoyl) alkyl diacid dihydrazide, the crystallization peak became sharper and shifted to higher temperature as the degree of supercooling decreased at a cooling rate of 1?°C/min from melt. The nucleation activities of five N,N??-bis(benzoyl) alkyl diacid dihydrazides were quantitatively determined. It is shown that N,N??-bis(benzoyl) suberic acid dihydrazide has higher nucleating activity than the other N,N??-bis(benzoyl) alkyl diacid dihydrazides. In the presence of N,N??-bis(benzoyl) alkyl diacid dihydrazide, the melting behavior of PLLA is affected significantly. In addition, the thermal stability of PLLA/0.8?% N,N??-bis(benzoyl) alkyl diacid dihydrazide is tested and reported. Compared to the neat PLLA, the onset degradation temperature of PLLA/0.8?% N,N??-bis(benzoyl) alkyl diacid dihydrazide samples has been decreased significantly.     

Crystallization behavior of biodegradable poly(L‐lactic acid) filled with a powerful nucleating agent: N,N′‐bis(benzoyl) suberic acid dihydrazide

N,N′‐Bis(benzoyl) suberic acid dihydrazide (NA) as nucleating agent for poly(L ‐lactic acid) (PLLA) was synthesized from benzoyl hydrazine and suberoyl chloride, which was deprived from suberic acid via acylation. PLLA/NA samples were prepared by melt blending and a hot‐press forming process. The nonisothermal and isothermal crystallization, spherulite morphology, and melting behavior of PLLA/NA with different contents of NA were investigated with differential scanning calorimetry, depolarized‐light intensity measurement, scanning electron microscopy, polarized optical microscopy, and wide‐angle X‐ray diffraction. With the incorporation of NA, the crystallization peak became sharper and shifted to a higher temperature as the degree of supercooling decreased at a cooling rate of 1°C/min from the melt. Nonisothermal crystallization indicated that the presence of NA accelerated the overall PLLA crystallization. In isothermal crystallization from the melt, the presence of NA affected the isothermal crystalline behaviors of PLLA remarkably. The addition of NA led to a shorter crystallization time and a faster overall crystallization rate; this meant that there was a heterogeneous nucleation effect of NA on the crystallization of PLLA. With the addition of 0.8% NA, the crystallization half‐time of PLLA/NA decreased from 26.5 to 1.4 min at 115°C. The Avrami theory was used to describe the kinetics of isothermal crystallization of the PLLA/NA samples. Also, with the presence of NA, the spherulite number of PLLA increased, and the spherulite size decreased significantly. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Crystallization behavior and crystallite morphology control of poly(L‐lactic acid) through N,N′‐bis(benzoyl)sebacic acid dihydrazide

Adding a nucleating agent is one of the best ways to accelerate the crystallization rate of poly(L ‐lactic acid) (PLLA) so as to obtain a high degree of crystallinity during the process, which will improve the heat distortion temperature of final products. In the work reported, N, N′‐bis(benzoyl)sebacic acid dihydrazide (BSAD) was synthesized and used as a nucleating agent for PLLA. Isothermal and non‐isothermal crystallization behaviors were investigated using differential scanning calorimetry (DSC). The addition of BSAD successfully enhances the crystallization rate of PLLA. A unique phase separation behavior of PLLA/BSAD blends is found from DSC as well as from polarized optical microscopy, which explains the difference of optimal BSAD concentration between isothermal and non‐isothermal crystallization. This is the first recording of a phase separation peak in PLLA/nucleating agent blends using DSC. In thermogravimetric analysis, the enhanced thermal stability indicates that there are strong hydrogen bonds between BSAD and PLLA matrix. BSAD can dissolve in PLLA melt below its melting point through intermolecular hydrogen bonding with PLLA and self‐assemble upon cooling, leading to the surface being capable of nucleating PLLA. Different phase separation temperatures can be used to control the morphology of BSAD, which finally determines the crystallite morphology of PLLA. © 2012 Society of Chemical Industry     

Rapid crystallization of poly(l-lactic acid) induced by a nanoscaled zinc citrate complex as nucleating agent

A nanoscaled zinc citrate complex (ZnCC) was synthesized by the reaction of zinc acetate and citric acid using solution method. As a new eco-friendly nucleating agent, ZnCC was introduced into poly(l-lactic acid) (PLLA) via melt blending. The nonisothermal and isothermal crystallization, melting behavior, crystalline morphology and mechanical properties of the PLLA/ZnCC blends were investigated. It is found that ZnCC exhibits much more prominent nucleation activity on the crystallization of PLLA than conventional nucleating agent talc and commercial zinc citrate (ZnCit). By loading 0.05 wt% ZnCC, PLLA can complete crystallization upon cooling at 10 °C/min, and the crystallization peak shifts to a higher temperature with increasing ZnCC content. In the case of isothermal crystallization from the melt, the addition of ZnCC leads to a shorter crystallization time and a faster overall crystallization rate. Besides, the nucleation density of PLLA increases and the spherulite size decreases significantly in the presence of ZnCC. Epitaxy is the possible mechanism to elucidate the nucleation phenomenon of PLLA/ZnCC system. The tensile results show that ZnCC has a plasticization effect on the amorphous PLLA. Through a short-time annealing procedure, the mechanical properties such as tensile modulus and storage modulus of PLLA are improved by the addition of ZnCC.     

Synergistic effects of N,N′‐bis (benzoyl) sebacic acid dihydrazide and talc on the physical and mechanical behaviors of poly(l‐latic acid)

The important practical problem of poor heat stability of poly(l ‐lactic acid) (PLLA) is addressed by the addition of N, N′‐bis (benzoyl) sebacic acid dihydrazide (BSAD) and talc as a nucleating agent system. The idea of incorporating talc into the PLLA/BSAD composites is that talc can provide supplementary nucleation effect with very small amount of BSAD (0.2 wt %) and therefore can improve the heat deflection resistance of PLLA materials. Effects of BSAD/talc on morphology, crystallization behavior, heat resistance, and mechanical properties of PLLA/BSAD/talc were investigated after annealing processes. The results indicated that the BSAD/talc system increased the crystallinity from 6.0% of pure PLLA to a maximum 42.9% by the synergistic effects of BSAD and talc increasing the growth of spherulites and nucleation density, respectively. After annealing at different temperatures, the heat deflection temperature (HDT) of PLLA was improved dramatically due to synergistic effects of BSAD/talc between restricted chain movement and acceleration of crystallization. At high temperature (above T_g), the thermo‐mechanical properties of PLLA is mainly determined by the crystallinity and the reinforcement effect of talc acted as a filler. Moreover, effects of BSAD/talc on mechanical properties were discussed. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41454.     

异烟肼衍生物对聚L-乳酸性能的影响

以异烟肼和均苯三酸为原料合成了新型均苯三酸三异烟肼,考察了其对聚L-乳酸(PLLA)结晶性能和热稳定性的影响。结果表明:均苯三酸三异烟肼起到了异相成核的作用,能有效提升PLLA的结晶速率,其中添加质量分数2.0%的均苯三酸三异烟肼可使PLLA有最大非等温结晶焓,但过量的均苯三酸三异烟肼却不利于PLLA的结晶。均苯三酸三异烟肼的加入不会改变PLLA的热分解行为,但随着均苯三酸三异烟肼含量的增加,其起始分解温度下降。     

Poly(l‐lactic acid) with the organic nucleating agent N,N,N′‐tris(1H‐benzotriazole) trimesinic acid acethydrazide: Crystallization and melting behavior

N,N,N′‐Tris(1H‐benzotriazole) trimesinic acid acethydrazide (BD) was synthesized from 1H‐benzotriazole acetohydrazide and trischloride to serve as an organic nucleating agent for the crystallization of poly(l ‐lactic acid) (PLLA). First, the thermogravimetric analysis of BD exhibited a high thermal decomposition temperature; this indicated that BD maybe used as a heterogeneous nucleating agent of PLLA. Then, the effect of BD on the crystallization and melting behavior of PLLA was investigated through differential scanning calorimetry, depolarized light intensity measurements, and wide‐angle X‐ray diffraction. The appearance of a nonisothermal crystallization peak and increases in the glass‐transition temperature and the intensity of the diffraction peak suggested that the presence of BD accelerated the overall PLLA crystallization. Upon cooling at a rate of 1°C/min, the addition of just 0.5 wt % BD to PLLA increased the onset crystallization temperature from 101.4 to 111.3°C, and the nonisothermal crystallization enthalpy increased from 0.1 to 38.6 J/g. The isothermal crystallization behavior showed that the crystallization half‐time of PLLA with 0.5 wt % BD (PLLA/0.5% BD) decreased from 49.9 to 1.1 min at 105°C. However, the equilibrium melting point of PLLA/0.5% BD was lower than that of the pristine PLLA; this resulted from the increasing nucleating density of PLLA. The melting behavior of PLLA/0.5% BD further confirmed that BD improved the crystallization of PLLA, and the double‐melting peaks of PLLA/0.5% BD were assigned to melting–recrystallization. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42402.     

Low-molecular weight aliphatic amides as nucleating agents for poly (L-lactic acid): Conformation variation induced crystallization enhancement

Two kinds of low molecular weight aliphatic amides, N, N′-ethylenebis (12-hydroxystearamide) (EBH) and N, N′-ethylenebisstearamide (EBSA), have been selected in present study to mediate the crystallization behavior of poly (L-lactic acid) (PLLA). The results showed that the crystallization rate of PLLA was significantly improved with the addition of EBH and EBSA, and EBH presented a stronger nucleating efficiency. The correlation between the variation of chain conformation during the early stages of isothermal crystallization and the enhancement of crystallization rate for pure PLLA and its mixtures was investigated by time-resolved FTIR. The formation of interchain conformational-ordered structure and intrachain 10₃ helix structure for amide-doped PLLA preceded that for pure PLLA, suggesting a stimulatory nucleating effect of EBH and EBSA. In the case of PLLA/EBH, the interchain interactions of –(COC + CH₃) and –CH₃ groups were faster than the –(CH₃+CC) intrachain interactions, while the interchain interactions and the intrachain 10₃ helix formation were nearly synchronous for PLLA/EBSA. The hydrogen bond interaction between hydroxyl groups in EBH and the carbonyl groups in PLLA was proposed to be an important factor influencing the conformation variation during isothermal crystallization of PLLA.     

Preparation and characteristics of a novel nano‐sized calcium carbonate (nano‐CaCo3)‐supported nucleating agent of poly(L‐lactide)

Nano‐sized calcium carbonate (nano‐CaCO₃)‐supported nucleating agent for poly(L ‐lactide) (PLLA) was prepared by supporting calcium phenylphosphonate (PPCa) on nano‐CaCO₃ surface. The thermal properties of phenylphosphonic acid (PPOA) and nano‐CaCO₃‐supported nucleating agent and its dispersion in PLLA matrix were investigated by differential scanning calorimetry and field emission scanning electron microscopy. The results indicated that the formation of nucleating agent supported on nano‐CaCO₃ was attributed to the chemical reaction between nano‐CaCO₃ and PPOA. The nano‐CaCO₃‐supported nucleating agents were dispersed evenly in the PLLA matrix even with 5 wt% loading. The supported nucleating agent was added to PLLA to examine its nucleating ability for PLLA. The results of the investigation showed that the nano‐CaCO₃‐supported nucleating agent exhibited higher nucleation ability compared to PPCa nucleating agent. POLYM. ENG. SCI., 2012. © 2012 Society of Plastics Engineers     

Melt preparation and nucleation efficiency of polylactide stereocomplex crystallites

A melt blending procedure was developed for the preparation of poly(l-lactide) (PLLA)/poly(d-lactide) (PDLA) stereocomplex crystallites dispersed in a PLLA matrix. All PLLA/PDLA blends were prepared in a batch melt mixer with ≥95% PLLA. Three PDLA homopolymers with a range of molecular weights were used as the minority (≤5%) component. The presence of the stereocomplex in the PLLA matrix was verified by differential scanning calorimetry (DSC) and optical microscopy. The effectiveness of the in situ formed stereocomplex crystallites for nucleating PLLA crystallization was evaluated using self-nucleation and non-isothermal DSC methods. With only 3 wt% of the 14 kg mol⁻¹ PDLA, nucleation efficiencies near 100% could be obtained. In addition, fast crystallization kinetics were observed in isothermal crystallization experiments at 140 °C. The stereocomplex crystallites were much more effective at enhancing the crystallization rate of PLLA compared to talc, a common nucleating agent.     

Nucleation in polymer crystallization: A physical or a chemical mechanism?

Primary nucleation is in general heterogeneous in polymer crystallization. In unseeded polymers, the crystallization originates from a very small concentration (<0.1 percent) of unknown submicroscopic heterogeneous nuclei. Only in three cases have the nucleation mechanisms been identified with precision. The first example is self-nucleation, which relates to the nucleation of a polymer melt by fragments of its own crystals previously present in the melt. Another example of heterogeneous nucleation is represented by epitaxial nucleation of polymers on inorganic and organic substrates recently established by Lotz and Wittmann. More recently, it was discovered that finely divided organic salts added to reactive polymers do not behave as inert heterogeneous substrates but rather dissolve and cut molten macromolecules producing ionic chain ends which precipitate into the melt and form organized aggregates which are the true nucleating species. It is clearly shown that mechanisms of physical and chemical origin are involved in heterogeneous primary nucleation of polymer crystallization.     

Enhancement of drawdown force in polypropylene containing nucleating agent

The effect of the nucleation efficiency of three commercial nucleating agents, such as 1,3:2,4-bis(3,4-dimethylbenzylidene)sorbitol (DMDBS), sodium 2,2′-methylene-bis-(4,6-di-tert-butylphenyl)phosphate (SMBP), and magnesium silicate (talc), on the melt-stretching performance of isotactic polypropylene (PP) is studied. It is found that the addition of 0.5 wt % of a nucleating agent enhances the crystallization temperature and that effect is pronounced for DMDBS and SMBP. Furthermore, DMDBS is more efficient than the other tested nucleating agents in enhancing the drawdown force, defined as a force required for stretching a molten strand. Nanofibers of DMDBS, which show significant alignment in the flow direction, are responsible for the rapid crystallization of PP in the flow field leading to an increase of drawdown force. The stretched strand shows a highly oriented structure in which the α-form crystals orient to the flow direction. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47295.     

The electrical properties and crystallization of stereocomplex poly(lactic acid) filled with carbon nanotubes

Multi-walled carbon nanotubes (MWCNTs) filled poly(l-lactic acid) (PLLA) and PLLA/poly(d-lactic acid) (PDLA) composites were prepared through a directly melt mixing process. A special crystalline structure of stereocomplex was formed by PLLA and PDLA, which was easily found when mixing two polymers with identical chemical composition but different steric structures. The electrical conductivities were greatly improved by the formation of stereocomplex compared to that of PLLA/MWCNT composites at same MWCNT content. The percolation threshold of the PLLA/PDLA/MWCNT composite at a PLLA/PDLA weight ratio of 50/50 was 0.35 wt%, while being 1.43 wt% of PLLA/MWCNT composites. The X-ray diffraction, non-isothermal and isothermal crystallization results showed that the formation of stereocomplex greatly increased the crystallinity of the composites, meanwhile MWCNTs acted as heterogeneous nucleating agent, which significantly accelerated the nucleation and spherulite growth. Therefore, the PLLA/PDLA/MWCNT composites have a very low percolation threshold due to the volume exclusion effect.     

Polypropylene-organoclay nanocomposites containing nucleating agents

Polypropylene/organoclay nanocomposites containing nucleating agents, viz., aluminum hydroxybis[2,2-methylenebis(4,6-di-tert-butylphenyl) phosphate (NA21) and 1,3:2,4-bis(3,4-dimethylbenzylidene)sorbitol (Millad 3988), were prepared by direct melt intercalation in a twin-screw extruder. Nucleating agents were added to polypropylene during compounding and their effect on the properties of the nanocomposites was studied. X-ray diffraction (XRD) and transmission electron microscopy (TEM) exhibited clay layers to be intercalated and partially exfoliated. The expansion of inter-gallery distance of the clay layers was governed by the interaction between polypropylene, compatibilizer, and different nucleating agents. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicated higher thermal stability and crystallization temperature for nanocomposites compared to virgin polymer. Even a small addition of the nanoscale filler with 0.2% nucleating agents was found to promote concurrently several PP material properties, including improved tensile characteristics, higher Young’s modulus, increased thermal stability and rate of crystallization.     

The effect of inorganic and organic nucleating agents on the electrical breakdown strength of polyethylene

Altering the morphology of polyethylene affects physical and electrical properties with reduced spherulite size correlating with higher electrical breakdown strength. Nucleating agents in polyethylene influence the final crystal morphology by increasing the number of spherulites and reducing spherulite size. Few studies are available that relate the nucleating activity to improved electrical breakdown strength. Although nanosilica is known to improve electrical breakdown strength of polyethylene in addition to serving as a nucleating agent, previous studies have not fully addressed the relationship between the improved breakdown strength and nucleating activity. In this article, direct current electrical breakdown strength and nucleation effects on morphology are assessed on a single set of controlled polyethylene compositions containing two types of surface treated nanosilica particles. The results are compared to composites with two types of organic nucleating agents 1,3:2,4‐bis(3,4‐dimethylbenzylidene) sorbitol or calcium 1,2‐cyclohexanedicarboxylate (CDA). CDA was the most effective organic nucleating agent and the hexamethyldisilizane treated nanosilica was the most effective inorganic nucleating agent in reducing spherulite sizes in low density polyethylene (LDPE). Reduced spherulite sizes in nucleated samples correlated with increased breakdown strength and lower conduction current compared to the neat LDPE. The LDPE sample with CDA also had the highest increase in crystallization temperature indicating stronger nucleating agent performance than the nanosilica and 1,3:2,4‐bis(3,4‐dimethylbenzylidene) sorbitol composite samples. The addition of these inorganic and organic nucleating agents all resulted in improvements in electrical breakdown strength. The results show that nucleation deserves more attention as a potential cause for improved breakdown strength observed with silica and organic nucleating agents. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46325.     

Poly(L‐lactic acid)/silicon dioxide nanocomposite prepared via the in situ melt polycondensation of L‐lactic acid in the presence of acidic silica sol: Isothermal crystallization and melting behaviors

In a previous article, we reported the preparation and characterization of a nanocomposite of poly(L ‐lactic acid) (PLLA) and silica via the in situ melt polymerization of L ‐lactic acid in the presence of acidic silica sol. In this study, the isothermal crystallization and melting behaviors of a PLLA/silicon dioxide (SiO₂) nanocomposite with 5 wt % well‐dispersed SiO₂ nanoparticles (PLLASN5) and pure PLLA were comparatively studied with differential scanning calorimetry and polarized optical microscopy. The SiO₂ nanoparticles acted as nucleation agents in the PLLA matrix and enhanced its nucleation rate and overall crystallization rate, especially at high crystallization temperatures. However, no deleterious effect on the crystal morphology or crystallinity was observed. The crystals that formed at a low temperature were imperfect; therefore, double melting peaks occurred during the second heating scan because of melt recrystallization. With the crystallization temperature increasing, the crystals became increasingly perfect; as a result, the low melting peak increased and shifted to a higher temperature. The existence of SiO₂ nanoparticles had no effect on the equilibrium temperature of the PLLA matrix. Pure PLLA and PLLASN5 have the same equilibrium temperature of 171.5°C. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Optimizing crystallinity of engineered poly(lactic acid)/poly(butylene succinate) blends: The role of single and multiple nucleating agents

This work deals with the design and experimental development of formulations based on bioplastic materials optimized for the production processes of high-performance packaging products, suitable for food contact and characterized by high thermo-mechanical stability. For this purpose, mixtures of poly (L-lactic acid) (PLLA)/poly (butylene succinate) (PBS) (PLA/PBS) containing micro-lamellar talc as reinforcing agent and inorganic nucleating agent have been obtained on a pre-industrial scale. Through the addition in formulation of two organic nucleating agents, ethylene (bis)stereamide (EBS) and poly (D-lactic acid) (PDLA), evaluated separately and in combination with each other, four different PLA/PBS formulations were produced by means of a co-rotating twin screw extruder. The effects on the crystallinity of the materials obtained first in the form of pre-dried pellets, then in the form of films by casting extrusion and, finally, as thermoformed items were compared, according to the variation of the chosen nucleation system. The effect on the crystallinity of the materials was assessed by differential scanning calorimetry (DSC) and by Fourier transform infrared analysis in attenuated total reflection mode (ATR-FTIR). This approach aims to provide data for the further investigation on the processability and the thermo-mechanical properties of end-goods achievable by converting processes (melt processing) of the bioplastic compounds.     

一种成核剂对聚乳酸结晶行为的影响

通过示差扫描量热法研究了一种新型成核剂对聚乳酸结晶行为的影响,并通过Avrami方程对其结晶动力学进行分析。结果显示,当聚乳酸中加入0.3%质量分数的成核剂,即可使其半结晶时间由61.4 min缩短至9.2 min。通过偏光显微镜对聚乳酸及含成核剂的聚乳酸的晶体形貌观察,发现成核剂可以显著的提高聚乳酸的结晶速率,相同时间内,球晶数量显著增多,晶粒尺寸明显减小。     

Crystallization behavior and nucleation analysis of poly(l‐lactic acid) with a multiamide nucleating agent

To accelerate the crystallization of poly(L ‐lactic acid) (PLLA) and enhance its crystallization ability, a multiamide nucleator (TMC) was introduced into the PLLA matrix. The thermal characteristics, isothermal and nonisothermal crystallization behavior of pure PLLA and TMC‐nucleated PLLA were investigated by differential scanning calorimetry. The determination of thermal characteristics shows that the addition of TMC can significantly decrease the onset temperature of cold crystallization and meanwhile elevate the total crystallinity of PLLA. For the isothermal crystallization process, it is found that the overall crystallization rate is much faster in TMC‐nucleated PLLA than in pure PLLA and increases as the TMC content is increased, however, the crystal growth form and crystalline structure are not influenced much despite the presence of TMC. In the case of nonisothermal crystallization, the nucleation efficiency and nucleation activity were estimated and the results indicate that excellent nucleation‐promoting effect could be achieved when the weight percentage of TMC is chosen between 0.25% and 0.5%. Polarized optical microscopy observation reveals that the nuclei number of PLLA increases and the spherulite size reduces greatly with the addition of TMC. POLYM. ENG. SCI., 2012. © 2011 Society of Plastics Engineers     

Effect of nucleation and plasticization on the stereocomplex formation between enantiomeric poly(lactic acid)s

The effect of nucleation and plasticization on the stereocomplex formation between poly(l-lactic acid) (PLLA) and poly(d-lactic acid) (PDLA) was investigated in blends where PDLA is added as a minor phase in a major phase of PLLA. The use of small amounts of PDLA is aimed at creating a high melting point stereocomplex phase that in turn can serve as nucleating agent for the major phase of PLLA. Blends containing 5% PDLA with talc or organic phosphonate as nucleants and polyethylene glycol as plasticizer were prepared via melt-blending. Their crystallization behavior was investigated through Differential Scanning Calorimetry (DSC) using various thermal histories. Two peculiar stereocomplex melting endotherms were found. The peak temperature and enthalpy of these two endotherms were correlated to prior isothermal crystallization temperature. The different endotherms were also associated with two different crystalline morphologies observed by optical microscopy and referred to as Network and Spherulitic morphologies. The influence of plasticization and of heterogeneous nucleation on these morphologies was investigated through optical microscopy and calorimetric observations.