Nathan C. Crawford

Rheology coupled with in situ Raman spectroscopy (rheoRaman) was used to examine the isothermal crystallization of cocoa butter. Two unique isolation protocols were used to form cocoa butter polymorphs form III and form IV. The hyphenation of these two independent analytical techniques, rheology and Raman spectroscopy, enabled a more holistic depiction of the crystallization process and provided unique insights into the formation of cocoa butter polymorphs.

	Cocoa butter is an edible vegetable fat extracted from the cocoa bean. Cocoa butter is commonly used in home and personal care products (such as ointments and lotions) and it is a vital ingredient in chocolate. Cocoa butter forms the continuous phase within chocolate confections and is responsible for the chocolate's texture, snap, gloss, melting behavior, and resistance to fat bloom. These physical characteristics are a direct result of cocoa butter's triacylglycerol (TAG) composition and overall crystalline structure.

	In general, TAG molecules assume a tuning fork configuration and the TAG "forks" assemble to form crystal lattice structures. During crystallization, the TAG molecules slow down as the cocoa butter oil cools and the TAGs come to rest in contact with one another, forming subcrystalline cells (1). After the subcells are formed, they are thermodynamically driven to aggregate into larger and more-stable crystalline structures (2). The self-assembly of subcell structures and their further aggregation is governed by a balance of intra- and intermolecular interactions. Depending on the molecular level packing and orientation of the TAGs, cocoa butter can form different types of crystal lattice structures, commonly referred to as 

	Researchers have identified six unique cocoa butter polymorphs (3)- form I, II, III, IV, V, and VI-each with a distinct molecular signature. Form V is the polymorph most commonly found in commercially available chocolates, and it is believed that all other polymorphs lead to inadequate properties or a shortened shelf life. For example, form IV is often associated with poorly or under-tempered chocolate; whereas form VI is considered to cause fat bloom, the greyish surface coating sometimes observed on old or temperature-abused chocolate. As a result, the chocolate tempering process is critical because it ensures that the cocoa butter has assembled into the desired crystalline polymorph.

	Overall, cocoa butter crystallization is a highly complex, multistage process. Understanding the isothermal crystallization behavior of cocoa butter is vital for improving chocolate manufacturing processes and maintaining product quality. Cocoa butter crystallization has been widely studied using a variety of analytical techniques, including differential scanning calorimetry (DSC), X-ray diffraction (XRD), X-ray scattering, rheology, and Raman spectroscopy (2,4–7). Although cocoa butter crystallization has been thoroughly examined from the molecular, microstructural, and macroscopic levels, a lack of consensus about the existence of some cocoa butter polymorphs, the overall crystallization kinetics (especially in the presence of additives), and the underlying mechanisms of the crystallization process still persist.

	In this study, rheology coupled with in situ Raman spectroscopy was used to examine the isothermal crystallization of cocoa butter. Raman spectroscopy is a highly sensitive, relatively fast, and nondestructive technique that can probe the molecular structure and conformation in both liquid and solid TAG assemblies, as well as intra- and inter-TAG interactions. With simultaneous Raman and rheological measurements, molecular-level interactions and conformational shifts during the isothermal crystallization of cocoa butter were directly correlated with the changes in bulk viscoelastic properties, providing unique insight into the multifaceted crystallization behavior of cocoa butter.

	Commercially available, organic cocoa butter (

	Rheological measurements were performed using a Thermo Scientific HAAKE MARS 60 rheometer equipped with a serrated 35-mm-diameter plate rotor at a gap height of 1 mm. The serrated plate was used to prevent slip at the sample-rotor interface. All measurements were conducted using oscillatory shear, with a frequency of 1 Hz and a constant strain of 0.1%; data were collected every 10 s. Cocoa butter samples were loaded onto the rheometer at 60 °C and allowed to equilibrate for 10 min to erase any crystal structures or shear history from sample loading. After the equilibrium step, samples were exposed to one of the following temperature ramp and isothermal hold procedures to generate cocoa butter polymorph form III or form IV (2):

		Temperature decreased from 60 °C to 14 °C at a rate of 2 °C/min

		Temperature held constant at 14 °C for 60 min

		Temperature decreased from 60 °C to 22 °C at a rate of 10 °C/min

		Temperature held constant at 22 °C for 120 min

	The melt-to-solid phase transition of cocoa butter was probed rheologically using small amplitude oscillatory shear measurements, where the storage modulus G' and loss modulus G" were measured as a function of time. The overall magnitudes of G' and G", as well as the ratio of G"/G' = tan(δ), determine the general viscoelasticity and overall resistance to deformation for a given material. The term "tan(δ)" is also referred to as the loss or damping factor where δ is the phase angle defined as the shift or lag between the input strain and resultant stress sine waves (or vice versa) during an oscillatory shear measurement. Values of tan(δ) less than unity indicate elastically dominant (solid-like) behavior, whereas values greater than unity indicate viscously dominant (liquid-like) behavior. Unlike the individual moduli, tan(δ) can be used to quantify overall brittleness of a material and is commonly used to assess glass transition behavior. In general, as tan(δ) becomes smaller, the more G' deviates from G", and the more brittle (or glass-like) the material becomes.

	Raman spectroscopy measurements were performed using a Thermo Scientific iXR Raman spectrometer. The iXR system used a 532-nm laser operated at 10-mW power at the sample. Data acquisition and processing were carried out using the Thermo Scientific OMNIC for Dispersive Raman software. Sequential Raman spectra (in parallel with the rheological measurements) were collected over a predetermined time window using the time series collection function of the SERIES software within the OMNIC for Dispersive Raman package. Spectra were collected as an average of four 2-s sample exposures.

	The Raman spectrometer (Thermo Scientific iXR Raman spectrometer) and rheometer (Thermo Scientific HAAKE MARS 60) were coupled together using the Thermo Scientific HAAKE rheoRaman module. The iXR Raman spectrometer was free-space coupled to the rheometer with an optical train that used a series of mirrors to direct the incident laser into the rheoRaman module (Figure 1).


	Figure 1: Schematic diagram of the rheoRaman system (MARSXR) showing side and top views of the rheometer sample stage. The iXR Raman spectrometer is free-space coupled to the MARS rheometer using lens tubes and mirrors that direct light into a 20x objective. The objective focuses the incoming laser (green dashed line) and collects the back-scattered Raman light (yellow) coming out of the sample sitting atop the rheometer stage.

	The sample was positioned between a sandblasted glass bottom plate and the serrated 35-mm plate rotor to avoid slippage at the sample–plate interfaces. An electrical heating element within the rheoRaman module provided temperature control from below the sample, while an active electrical hood was used to provide temperature control from above (eliminating the potential for a temperature gradient within the sample). Cooling of the sample was supplied from a temperature-controlled water bath circulator.

) Raman spectra for the liquid-phase cocoa butter and the two investigated solid phase cocoa butter polymorphs (forms III and IV) are shown in Figure 2. Prominent Raman features were observed in both the C–H stretching region (2700–3050 cm

) and the fingerprint region (1000–1800 cm

). More specifically, the lower Raman shift features include the carbonyl (C=O) stretching region (1700–1800 cm

), and the C–C stretching region (1000–1200 cm


	Figure 2: The full Raman spectra for the melt phase and two polymorphs (forms III and IV) of cocoa butter.

	The C-H stretching regions for the melted and solidified cocoa butter specimens are highlighted in Figure 3. Two strong peaks were observed at ~2850 cm

, which are attributed to symmetric and asymmetric CH

 stretching, respectively (2). The symmetric vibrational modes at 2850 cm

 were dominant in the liquid (melt) phase, indicating reduced lateral interactions between CH

 groups and a low degree of intermolecular interactions. Conversely, the asymmetric vibrations at 2882 cm

 were dominant in the solid phase, which suggests increased lateral interactions resulting from closely packed crystalline structures. Thus, the 2850 cm

 bands are strong indicators of amorphous and crystalline content, respectively (8). Subsequently, the 

 peak intensity ratio was used to dynamically track crystal formation during the in situ rheoRaman measurements.


	Figure 3: Raman spectra of the C-H stretching region (2700–3050 cm

) for the melt phase and two polymorphs (forms III and IV) of cocoa butter. 

	Although they are less intense than the C-H stretching region, approximately eight unique spectral features were identified in the fingerprint region (1000–1800 cm

; Figure 4). When comparing the cocoa butter melt state to the crystalline cocoa butter polymorphs, the most significant changes were observed in the C–C stretching region (1000–1200 cm

). Two well-defined features emerged at 1130 cm

 during the solidification process, which originate from the symmetric and asymmetric C-C stretching, respectively (9,10). In the melt phase, all C-C stretching bands were relatively weak and broad because of the disordering effects of methyl gauche conformations. However, as the cocoa butter solidified, the backbone methyl groups were ordered into the trans conformation, signified by the emergence of the peak at 1130 cm

 spectral marker was also used to track the crystalline-phase transition within cocoa butter via in situ rheoRaman measurements.

 Raman spectral range for the melt phase and two polymorphs (forms III and IV) of cocoa butter.

	To form cocoa butter polymorph form III, the temperature was ramped down from 60 °C to 14 °C at a rate of 2 °C/min. During cooling and before reaching the isothermal temperature of 14 °C, both the elastic and viscous moduli (G' and G", respectively) began to increase exponentially at ~20 min and ~20 °C (Figure 5a). From 20 to 25 min, as the temperature was further decreased and then held at 14 °C, G' and G" increased by approximately six and five orders of magnitude, respectively. The rapid increase in the moduli indicates the start of the solidification process, where the cocoa butter specimen transformed from a semisolid material into a firm solid. After the temperature reached the constant value of 14 °C, the elastic modulus began to plateau at 30 min, whereas the viscous modulus reached a maximum at ~25 min and then gradually decreased from ~25 to 55 min. From 55 min and beyond, both G' and G" remained constant. After 55 min, G' was about two orders of magnitude greater than G", suggesting that the cocoa butter was now a rigid, glass-like solid. From a purely rheological standpoint, the cocoa butter appeared to have fully crystallized.


	Figure 5: (a) Rheology: G' and G" (filled and open circles, respectively; plotted on the left y-axis) and the temperature trace (solid line; plotted on the right y-axis) and (b) Raman: the I

 (right y-axis) intensity ratios for cocoa butter during the melt-crystalline transition to polymorph form III from 60 °C to 14 °C. The vertical dashed line at 20 min indicates the increase in both G' and G" and the Raman intensity ratios.

	The rheological measurements for cocoa butter form III were further corroborated using in situ Raman spectroscopy (Figure 5b). Similarly to G' and G", both the 

 peak intensity ratios remained relatively constant during the first ~20 min of the measurement. Then the 

 spectral markers began to abruptly increase at ~20 min, indicating the formation of crystalline structures within the cocoa butter. As the cocoa butter further crystallized, both spectral ratios continued to increase from 20 to 70 min. After ~70 min, the Raman peak intensity ratios began to stabilize as the cocoa butter TAGs arranged into their final crystal structure. Significant scatter in the 

 ratio, especially at the earlier melt state and at the latter part of the crystallization process. This scatter was most likely because of the low intensity of the 1130 cm

 feature in comparison to the strong Raman bands in the C-H stretching region (2850 cm

	Notably, the initial increase in G' and G" directly aligned with the rise of the 1130 and 2882 cm

 spectral ratios. The concurrent increases in both the rheological response and Raman feature intensity suggests that the bulk solidification during the formation of cocoa butter polymorph form III was directly associated with the formation of crystalline structures. Interestingly, as the change in rheological behavior began to subside at 55 min, the Raman spectral features were still increasing and they did not become steady until ~70 min and beyond. It is postulated that although the overall bulk hardening of the cocoa butter had stopped, the crystalline domains were still rearranging at the molecular level and that the cocoa butter had not reached its final crystalline lattice structure until the end of the measurement.

	To form cocoa butter polymorph form IV, the temperature was rapidly decreased from 60 °C to 22 °C at a rate of 10 °C/min. Note that since the cooling step was so brief, only the data from the isothermal step is shown in Figure 6. During the initial portion of the isothermal hold from 0 to 5 min (immediately following the rapid decrease in temperature from 60 °C to 22 °C), both G' and G" increased as the cocoa butter transformed from a melted liquid to a soft semisolid (Figure 6a). This initial increase in modulus is most likely because of a delay between the set temperature and the internal temperature of the loaded sample. After the sample had reached thermal equilibrium and was at the isothermal set point of 22 °C, the moduli were relatively stable from 10 to 25 min. From 25 to 50 min, however, both G' and G" began to gradually increase and then from 50 to 80 min, the moduli rapidly increased, where G' and G" increased by approximately five and four orders of magnitude, respectively. The exponential increase in the moduli indicates a solidification process, where the cocoa butter transformed from a pliable semisolid to a more robust, hardened solid. At 80 min and beyond, growth in the elastic modulus slowed and eventually plateaued, showing no further significant change past 100 min. The viscous modulus, however, reached a slight plateau from 80 to 100 min but continued to decrease from 100 min and beyond.


	Figure 6: (a) Rheology: G' and G" (filled and open circles, respectively; plotted on the left y-axis) and tan(δ) (plotted on the right y-axis) and (b) Raman: the I

 (right y-axis) peak intensity ratios for cocoa butter during isothermal crystallization at 22 °C. The vertical dashed line at 45 min indicates the increase of G' and G", while the dashed line at 65 min indicates the decrease in tan(δ) and increase in the Raman intensity ratios.

	During the increase in G' and G", a rapid decrease in the loss factor tan(δ) was observed from ~65 min and beyond (Figure 6a, right 

-axis). The decrease in the loss factor indicates a deviation in overall magnitude between G' and G". As the cocoa butter hardened, the increase in G' exceeded the increase in G", triggering the decrease in tan(δ). At the end of the 120 min isothermal study, G' was more than a full order of magnitude greater than G" and the loss factor was approaching 0.01, indicating the cocoa butter had transitioned into a brittle glass-like solid.

	The observed rheological behavior was further confirmed using simultaneous Raman spectroscopy (Figure 6b). Initially, both the 

 peak intensity ratios remained unchanged during the first ~65 min of the isothermal study. Then a sharp increase of the 

 ratios began at ~65 min, indicating the formation of crystal structures within the cocoa butter. As the cocoa butter further crystallized, both spectral markers continued to increase from 65 to 100 min. Beyond 100 min, the growth in both Raman features had subsided and the peak intensity ratios began to stabilize.

	Overall, the rate of increase in the 1130 and 2882 cm

 peak intensity ratios were similar to the rate of change for both G' and G" during the formation of cocoa butter polymorph form IV (Figures 6a and 6b). However, there was a noticeable 15–20 min lag between the observed increase in G' and G" and the rise of the Raman intensity ratios. The sharp upturn in G' and G" indicates an increased resistance to deformation (that is, a bulk hardening of the cocoa butter), signaling the start of the solidification process. The Raman spectral markers, on the other hand, are indicators of crystal formation. Thus, the time delay between the rheology and Raman profiles suggests that cocoa butter first hardens into an amorphous solid, followed by a transformation from an amorphous to a crystalline solid. This morphological transformation was signified by the subsequent increase in the Raman band intensities associated with crystal cocoa butter structures (the 1130 and 2882 cm

peaks). The temporal separation of the rheological and Raman spectral profiles indicates a clear distinction between bulk hardening of the cocoa butter and the formation of crystalline domains during the formation of polymorph form IV.

	Interestingly, the increase in the Raman spectral features (

) directly correlated with the observed reduction in tan(δ) (Figures 6a and 6b). The loss factor is an indication of material brittleness and crystalline structures are commonly known to be brittle. Thus, it is reasonable to infer that the formation of crystal domains at the molecular level (as indicated by Raman) coincides with the overall brittleness of the cocoa butter. As a result, the loss factor may be a more revealing indicator of bulk cocoa butter crystallization than G' and G" alone.

	Simultaneous rheology and Raman spectroscopy measurements were used to examine the isothermal crystallization of cocoa butter. This multimodal analytical technique allowed the bulk mechanical properties of cocoa butter (G', G", and tan[δ]) to be directly correlated with conformational changes at the molecular level (

) in real-time. With a slow cooling (2 °C /min) and subsequent isothermal hold at 14 °C, the concurrent increases in both the rheological response and Raman feature intensity suggests that the bulk solidification during the formation of cocoa butter polymorph form III coincided with the formation of crystalline structures. With a rapid cooling (10 °C/min) followed by an isothermal hold at 22 °C, however, there was a noticeable time lag between the rheological response (G' and G") and the Raman spectral profiles during the formation of cocoa butter polymorph form IV. The observed time delay indicates that cocoa butter crystallized by first hardening into an amorphous solid, manifested by a sharp increase in G' and G" while the Raman features remained unchanged. The amorphous solid then underwent a morphological transition to form a crystalline solid, signified by the increase in Raman features associated with crystal cocoa butter structures (1130 and 2882 cm

). Without coupling these two separate analytical techniques, the observed amorphous-solid to crystalline-solid transformation would have been left undetected. Alone, each technique suggests a single stage process, however, only when the two techniques are coupled is the multiphase crystallization process revealed, further exemplifying the unique analytical capability unleashed by hyphenating rheology with in situ Raman spectroscopy.

Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals

 (John Wiley & Sons, Hoboken, New Jersey, 2018).

	(2) S. Bresson, D. Rousseau, S. Ghosh, M. El Marssi, and V. Faivre, 

	(4) K. Dewettinck, I. Foubert, M. Basiura, and B. Goderis, 

	(5) C. Loisel, G. Keller, G. Lecq, C. Bourgaux, and M. Ollivon, 

	(7) K. van Malssen, A. Langevelde, R. Peschar, and H. Schenk, 

	(8) R.G. Snyder, H.L. Strauss, and C.A. Elliger, 

 are with Thermo Fisher Scientific in Madison, Wisconsin. 

 is with Thermo Fisher Scientific in San Jose, California. Direct correspondence to: 

Articles

The hyphenation of rheology and Raman spectroscopy enabled a more holistic depiction of the crystallization process and provided unique 
insights into the formation of cocoa butter polymorph form IV.


Gaining Insight into Cocoa Butter Polymorph Formation Through In Situ Rheology and Raman Spectroscopy

The use of rheology and Raman spectroscopy in a coupled system for obtaining comprehensive insight into materials behavior is presented. Coupling rheology with Raman spectroscopy provides direct correlation of molecular composition and structure to the observed mechanical properties. The approach is extremely useful for studying phase transition behavior, such as the crystallization behavior of polymer melts during processing. It can also provide insight for in situ characterization and monitoring, which can be challenging when working with on-line techniques because only relative flow fields are characterized. Finally, the coupling of the two techniques gives researchers the advantage of maximizing and synchronizing the information gathered from a single measurement, as compared to sequential measurements, as well as enabling the observation of transient information on their materials during fast processing conditions. In this article, we present the experimental details of a rheo-Raman approach and then apply it to polymer melting.

	Rheological measurements are used to study the bulk physical and mechanical properties of complex fluids and soft matter. Conversely, vibrational spectroscopy (like Raman) is a noninvasive diagnostic tool that provides chemical information such as chemical composition, molecular conformation, and bond disruption and formation-to name a few. Most commonly, these two analytical techniques are used ex situ, where rheology is used to measure or induce a physical material change (for example, melting, crystallization, gelation, polymerization, and chemical crosslinking) and chemical analysis is typically performed before and after the observed physical transformation. When applying these two techniques ex situ, however, the underlying chemical changes that are driving the observed physical change are left uncaptured, forcing researchers to speculate on the dynamic physicochemical relationships of their materials. In a recent paper, Kotula and colleagues (1) demonstrated the benefits of coupling a Raman microscope to a rheometer in studies of the recrystallization of high-density polyethylene.

	The in situ combination of rheometry and Raman spectroscopy, termed a 

, allows for real-time, synchronized measurement of both physical and chemical material properties. This in situ analysis also helps preserve sample fidelity; researchers no longer have to wonder if the sample they gathered from their rheometer and loaded into their spectrometer is a “true” representation of their material. In addition, the rheo-Raman approach reduces sample consumption (only one sample is needed for both physical and chemical analysis) and reduces analysis time (complete rheological information and full Raman spectra are collected in unison).

	In this study, we use a combined rheo-Raman approach to investigate the temperature-dependent melting and crystallization of polypropylene as well as the isothermal crystallization process. The melt and crystalline phase transitions of polymeric materials are commonly correlated with variations in viscous and elastic behavior during rheological analysis. In addition, these phase transitions are often associated with molecular conformational changes during spectroscopic investigation. Measurements performed on separate instruments are often challenging to compare because of discrepancies in temperature control, slight deviations in sample composition, and differences in processing history. The in situ rheo-Raman technique, on the other hand, completely eliminates these discrepancies, allowing for a more valid physical and chemical analysis of the melt and crystalline phase transitions.

	Polypropylene (Ineos Olefins and Polymers, USA, R12C-00 random copolymer) pellets were used for this study. The pellets were melted at 190 °C on the rheometer to form a continuous, disk-shaped specimen for testing.

	Rheological measurements were performed using a Thermo Scientific HAAKE MARS III rheometer, equipped with a 20-mm-diameter stainless steel parallel-plate rotor. To extract viscoelastic behavior of the polypropylene, all measurements were conducted in the oscillatory mode. Oscillatory measurements were performed at 1 Hz with a constant strain of 0.1%, and data were collected every 15 s. To soften the polypropylene and help it conform to the measuring geometry, all samples were initially loaded at 190 °C. For the melt-to-crystallization phase transition study, the temperature was decreased from 190 °C to 30 °C, at a rate of 5 °C/min. For the isothermal crystallization studies at 138 °C and 150 °C, the temperature was rapidly decreased from 190 °C to 10 °C above the target temperature. The temperature was then slowly decreased until it reached the test temperature (either 138 °C or 150 °C). The temperature was then held constant for a maximum of 1 h (3600 s) while the isothermal recrystallization process was observed.

	Raman spectroscopy measurements were performed using a Thermo Scientific iXR Raman spectrometer. The Raman spectrometer used a 532-nm, 10-mW laser, a triplet spectrograph providing Raman spectra over the range 3500 to 50 cm

 resolution, and a silicon charge coupled device (CCD) camera cooled to -50 °C. Alignment of the laser, Raman scatter, and aperture selection within the spectrometer were all software controlled. The minimum exposure collection time was 0.1 s. For the data presented here, the exposure collection time was 4 s and two sample exposures were averaged per spectra collection.

	The Raman spectrometer and rheometer were coupled together as shown schematically in Figure 1. The Raman spectrometer was free-space coupled to the rheometer with an optical train of two plane mirrors, which direct the laser light into the Thermo Scientific HAAKE RheoScope module in the base of the rheometer. Within the module, the laser beam was directed into a 20x objective, where the laser light was focused through a 2-mm-thick fused silica window into the sample (perpendicularly to the flow or vorticity plane). Raman scattered light was collected in 180° backscatter geometry using the 20x objective, and traveled back into the spectrometer through the same optical train as the incident laser, and eventually to the spectrograph inside the spectrometer. The Raman excitation laser wavelength can be changed to optimize the laser wavelength to the sample (785- and 455-nm laser sets are also available).

	The sample was positioned between the silica window and the rotor geometry attached to the rheometer measuring head. The objective can be adjusted for interrogation at different penetration depths within the sample, as well as positioned at various radial locations (from the true center to outer edge of the sample). An electrical heating element was positioned below the fused-silica window to provide temperature control during testing. The electric heater allowed for a maximum temperature of 300 °C and a minimum temperature of -5 °C, with a maximum heating-cooling rate of 10 °C/min. An active electrical hood was also used to provide temperature control from above, eliminating the potential for a temperature gradient within the sample. Cooling was provided from a temperature-controlled circulator with 50:50 ethylene glycol–water. All instrumentation was controlled through Thermo Scientific OMNIC and RheoWin software.

	The physicochemical relationships during polymer crystallization are of critical interest for the polymer processing industry. Representative Raman spectra for polypropylene across three different temperatures are shown in Figure 2. In general, the spectrum at 53 °C shows sharp peaks across the examined spectral range. These sharp spectral features suggest a high degree of conformational order, which is indicative of semicrystalline and crystalline structures. As the temperature was increased to 120 °C and 173 °C, these peaks began to broaden and merge together. The broadening of spectral peaks is commonly associated with melt behavior in polymeric materials.

	Specific indicators in the spectra for polypropylene are the intensities of the skeletal deformation of helical chains within the crystal (represented by 808 cm

 peak) and the CH stretching region (characteristic of the 2800–3050 cm

 spectral range). The intensity of the 808 cm

 peak was used as a measure of the crystallinity of polypropylene (2), and the intensity in the CH stretching region was used as a measure of the overall Raman scattering intensity during the crystallization process. For further analysis, the 808 cm

 peak height was normalized by the peak area between 880 and 780 cm

, which contains skeletal chain vibrations of all conformations during the melting-recrystallization process (2). The spectral features in the 2800–3050 cm

 range were integrated into a total peak area. These indicators (normalized helical chain vibration peak height and overall CH stretching peak area) were then tracked throughout the melt- to crystalline-phase transition, allowing for overlay with the in situ rheology data (Figure 3).

	The melt and recrystallization process of polypropylene was probed rheologically using small amplitude oscillatory shear measurements (Figure 3), where the storage modulus (

′′) were measured as a function of temperature. 

′′ are measures of a material’s elastic and viscous behavior, respectively. A liquid-like material will be more viscous than elastic (that is, viscously dominated), and as a result, 

′. Conversely, a solid-like material will display more elastic than viscous behavior (that is, elastically dominated), where 

	Initially, at high temperatures (>150 °C), 

′, signifying the polypropylene specimen was in the melt state (displaying liquid-like behavior). As the temperature was decreased from 150 °C to 100 °C, an abrupt and drastic increase in both 

′′ was observed at ~125 °C. After the temperature had reached 100 °C, 

′ had increased by four orders of magnitude, and 

′′ increased by two orders of magnitude. The temperature range from 150 °C to 100 °C can be viewed as the melt-to-crystalline transition region for this polypropylene specimen. As the temperature was further decreased below 100 °C, 

′′ (more than a full order of magnitude), indicating the polypropylene had transitioned into the semicrystalline or crystalline state (displaying solid-like behavior).

	In addition, the observed plateau regions and increase in 

′′ are in direct agreement with the Raman spectral data (Figure 3). In the melt region (~190–130 °C), the normalized 808 cm

 peak height was near zero and unchanged by the decreasing temperature. However, as the polypropylene melt began to crystallize, the 808 cm

 peak height significantly increased and was in unison with the abrupt increases in both 

′′. Also, as the polypropylene sample started to crystallize, the total peak area of the CH stretching region decreased rapidly before any growth of the 808 cm

 band was observed. The decrease in intensity in the 2800–3050 cm

 spectral region is proposed to be caused by the formation of crystallites in the polypropylene matrix. Crystallites with dimensions on the same order of magnitude as the wavelength of the incident laser light (532 nm) would scatter light and as a result create an overall loss in the Raman signal, as shown in the intensity of the CH stretching bands. The minimum in the CH stretching profile was directly correlated with the observed crossover between 

′′ and with maximum slope of the normalized 808 cm

 peak profile, indicating that the crystallization rate was maximized when the crystallite concentration was greatest. After the observed decrease in intensity and as the crystallization process progressed, the integrated area of the CH stretching region returned to a value close to what was detected in the melt phase.

	To further evaluate the crystallization process of polypropylene, isothermal crystallization studies were performed at 150 °C and 138 °C. The polypropylene samples were heated for a minimum of 5 min at 190 °C (to fully melt the specimen), rapidly cooled at 10 °C/min to 10 °C above the crystallization temperature, and then cooled at a slower rate of 2 °C/min until the set crystallization temperature was reached. The crystallization temperature was then held constant for a maximum of 1 h (3600 s) to observe the isothermal crystallization of polypropylene as a function of time. Simultaneous rheology and Raman spectroscopy data were captured in situ during crystallization at 138 °C and 150 °C (Figures 4 and 5, respectively). For both isothermal temperatures, the polypropylene material was initially viscously dominated 

′′ (displayed liquid-like behavior). However, as time progressed, a crossover in 

′′ was observed; where the moduli increased two to three orders of magnitude. The crossover at 138 °C occurred at 84 s into the isothermal curing process, while it took 2150 s for the crossover to be observed at 150 °C. Also, the observed plateau in 

′′ at later times indicated that the crystallization process had stabilized and was complete.

	Again, the simultaneous rheology and Raman data were in agreement during the isothermal crystallization studies (Figures 4 and 5). The observed increase in the elastic and viscous moduli (

′′, respectively) directly corresponds with the increase in the normalized 808 cm

 peak height. It was similar to the observations in crystallization during the cooling measurement, in which a minimum in the intensity of the overall Raman spectrum was observed as the specimen transitioned from the liquid to the solid state (data not shown).

	A combined rheology and Raman spectroscopy approach was used to analyze polypropylene during the melt-to-crystallization transition process. Combining rheological interrogation with in situ Raman spectral acquisition ensured the sample was exposed to identical experimental conditions (temperature, oscillatory deformation, and so forth) during data collection. This multimodal analytical method allowed the bulk structural properties of polypropylene to be directly correlated with real-time conformational changes at the molecular level. The data indicated that the rate of crystallization reached its maximum when the crystallite concentration was the greatest.

	The observed correlation between the macroscopic and molecular level measurements exemplifies the unique analytical capability unleashed by combining rheology with Raman spectroscopy. Although this work focuses on the melting and crystallization of polypropylene, the underlying principles applied here should be applicable to a wide range of material processes including gelation, polymerization, curing behavior, and other shear-induced phenomena.

		A.P. Kotula, M.W. Meyer, F. De Vito, J. Plog, A.R. Hight Walker, and K.B. Migler, 

 is a Product Manager with Thermo Fisher Scientific in Madison, Wisconsin. 

 is Group Leader Rheology Applications with Thermo Fisher Scientific in Karlsruhe, Germany. 

 is a Rheology Applications Engineer with Thermo Fisher Scientific in Brighton, Colorado. Direct correspondence to: 

The in situ combination of rheometry and Raman spectroscopy allows for real-time, synchronized measurement of both physical and chemical material properties.

Nathan C. Crawford

Articles

Gaining Insight into Cocoa Butter Polymorph Formation Through In Situ Rheology and Raman Spectroscopy

Monitoring Polymer Phase Transitions by Combining Rheology and Raman Spectroscopy