Mohammed Ibrahim

In this article, inline monitoring of active pharmaceutical ingredient (API) crystallinity by Fourier transform near-infrared (FT-NIR) during a hot melt extrusion process is described. The area of the peak at 6100 cm

 was used to track the transitions between different solid dispersions during a hot melt extrusion in real time. The NIR observations are further corroborated by an offline terahertz (THz) Raman imaging analysis. Overall, both high process temperature and high screw speed are conducive to the formation of amorphous acetaminophen (APAP) dispersions, but temperature appears to more directly impact the formation of the most desired amorphous dispersion. FT-NIR is effective in providing real-time information on the solid physical state transformation, but through bulk measurements. Raman microscopy complements FT-NIR by offering direct visualization of the spatial distributions of different solid physical states of the API.

Hot melt extrusion (HME) is gaining increased interest in the pharmaceutical industry because of its high manufacturing efficiency and economic benefits. HME has been utilized to develop versatile dosage forms, especially for poorly soluble active pharmaceutical ingredients (API) (1–3). In an HME process, APIs are compounded with thermoplastic polymers and extruded to form solid dispersion. The polymer matrix acts as a solid solvent for the drug molecules. Amorphous API dispersions in a polymer matrix at the molecular level is highly desirable because the drug is in a dissolved molecular state with no lattice energy to overcome prior to dissolution. Furthermore, amorphous API dispersions can provide a physical barrier to recrystallization or aggregation of drug particles and reduce the risk of physical change post-manufacture.

To ensure the consistency and the product quality, it is imperative to have a thorough understanding of the critical process parameters, such as screw design, rotational speed, and zone temperature, in real time as well as their impact on the quality of the final products. To that end, Fourier transform near-infrared (FT-NIR) spectroscopy in conjunction with multivariate analysis (MVA) techniques have long been used as an in-process analytical tool to provide molecular level chemical information through direct measurements of the material being processed (4–6). Recently, Repka and others reported the successful implementation of FT-NIR for inline API concentration and process stability monitoring during HME processes (7,8).

Another integral part of the overall HME process design and optimization is characterizing the solid physical state of extrudates (amorphous, crystalline, or partially amorphous), which provides critical information on product stability during shelf life, crystallization propensity, drug dissolution, and drug bioavailability, as well as process efficiency (2). Previously, we reported a method employing terahertz (THz) Raman imaging to characterize the solid state of the acetaminophen (APAP) within a solid dispersion prepared by HME (9). The low-frequency Raman shift region of ≤ 6 THz or ≤ 200 cm

, is sensitive to the variations in crystal structures, such as lattice form and orientation. Therefore, THz Raman spectroscopy readily identifies different polymorphs of APIs and monitors API crystallization and dissolution processes (10–12).

In this article, we describe the use of inline FT-NIR to characterize the solid physical states transformation of acetaminophen (APAP) in hydroxypropyl methylcellulose (HPMC) matrix prepared by HME. By tracking the changes in the peak area associated with crystalline APAP during HME, the underlying transitions between different solid dispersions under different process conditions are rationalized. The FT-NIR data is further corroborated by an offline THz Raman imaging analysis.

Acetaminophen (APAP) (Spectrum Chemicals), a crystalline biopharmaceutics classification system (BCS) I drug with a melting point of 169°–170 °C, was chosen as the model API. Hydroxypropyl methylcellulose (HPMC) (Ashland) E5 grade, a common hydrophilic matrix material for amorphous solid dispersions, was used as the polymer matrix.

An 11-mm twin-screw extruder, Thermo Scientific Process 11 was used for the HME processes. Figure 1 is a schematic showing the screw profile design as well as the process configuration. APAP and HPMC were tumbling mixed for 30 min before fed into the extruder at the Zone 1 position. The Zones 2 through 8 were mixing, conveying and discharge zones. The Zone 9 is a die that reduces the barrel configuration to a single circular orifice with 5 mm in diameter.

FIGURE 1: Schematic of a Process 11 twin screw extruder configured in this study.

The blending ratio of APAP and HPMC was 30:70 (w/w). Four different HME processes were performed with two different screw speeds at two different temperatures, as shown in Table I. For each HME process, after the extrusion reached the steady state, the extrudates from the die (zone 9) was collected and pressed into thin films using a hydraulic press with heating panels at 40 °C to be analyzed by Raman imaging.

A Thermo Scientific Antaris II MDS FT-NIR spectrometer equipped with a diffuse reflectance probe was used for spectral acquisition. The probe and a metal reflector were screwed opposite into the sensor ports of the die (zone 9) with a gap of 4 mm. NIR spectra were collected every 20 s with the spectral range of 4000–10,000 cm

 spectral resolution, and 16 scans were co-added (8 s scan time). FT-NIR data acquisition was performed using the Thermo Scientific Result v3 software package. Data processing and modeling was performed using the Thermo Scientific TQ Analyst software.

To track the polymorphous change of APAP during the extrusion processes, the spectral peak at 6100 cm

 was chosen as the indicator for crystalline APAP. All spectral data was first processed with 11-data point and third-order Savitzky–Golay smoothing. The peak area was then integrated between 6055.39–6124.81 cm

Raman spectroscopy and Raman imaging were performed using a Thermo Scientific DXR3xi Raman imaging microscope, using a 532 nm laser operated at 8 mW power at the sample. Data acquisition and processing were carried out using the Thermo Scientific OMNICxi software.

Raman images were obtained by the Correlation Profiling option of the OMNICxi software, using the THz region (≤ 200 cm

) of the crystalline APAP Raman spectrum as a reference. The correlation profile shows the correlation between the spectrum at each pixel (sampling point) in the Raman image and the specified reference spectrum. Each color in the rainbow-colored intensity scale represents a degree of correlation with the reference spectrum. The higher the intensity value, the greater similarity to the reference spectrum. A value of 1.0 indicates that the sample spectrum and reference spectrum are identical and is displayed as red. For the purpose of direct comparison, a correlation range of 0.75–1 were applied to all the Raman images.

As shown in Figure 2, all FT-NIR spectra have strong features in the first overtone C–H stretching region around 6000 cm

. The amorphous and crystalline APAP, however, are noticeably different (Figure 2). The crystalline APAP spectrum has a sharp peak around 6100 cm

, whereas the amorphous APAP has a broad feature around 6600 cm

FIGURE 2: (top) FT-NIR spectra acquired during the extrusion processes. (bottom) FT-NIR spectra of amorphous and crystalline APAP.

Spectral peak wavelengths are predominately determined by molecular structures, interactions among molecules, as well as their environment, and peak intensities are often proportional to component concentrations. Therefore, the area of crystalline APAP peak at 6100 cm

 is an indicator for the concentrations of crystalline APAP in the samples. When the extrusion process was operated at 130 °C and 50 rpm (PRC 1), the spectral peak at 6100 cm

 is apparent. As the extrusion condition moved to a higher screw speed (PRC 2), a higher extrusion temperature (PRC 3), or a combination of both (PRC 4), the crystalline peak at 6100 cm

 decreased while the amorphous peak at 6600 cm

 increased concurrently, indicating a solid physical state transformation from crystalline to amorphous. A high temperature promotes amorphization as well as diffusion of the API molecules through the polymer chains for adequate mixing. On the other hand, increasing the screw speed increases the dissolution of the drug into a molten carrier and dispersion by imparting the energy generated by the friction between screws and extruded material.

Figure 3a shows the calculated peak areas of crystalline APAP under different HME process conditions. Under PRC 1 (130 °C, 50 rpm), the extrudates exhibit the highest crystallinity. When the screw speed was increased to 200 rpm while maintaining the same temperature at 130 °C (PRC 2), the crystallinity decreased. When the process conditions were changed back to PRC 1, the crystallinity also reverted to its original value, suggesting the reversible nature of the transition from PRC 1 to PRC 2 and back to PRC 1. Next, the temperature was increased to 170 °C while maintaining the screw speed at 50 rpm (PRC 3). The crystallinity decreased again, but to a level below the value observed in PRC 2, as indicated by the gap bracketed by the two green arrows in Figure 3a. Clearly, PRC 3 resulted in an overall more amorphous solid dispersion than PRC 2. However, increasing the screw speed to 200 rpm (PRC 4) did not further decrease the crystallinity. Finally, the process was changed back to PRC 1. Interestingly, the crystallinity remained at the low value and did not change to the value previously observed in PRC 1.

FIGURE 3: (a) The top shows screw speed vs. time; the middle of the figure shows process temperature vs. time; and the bottom of the figure shows the area of the peak at 6100 cm

 vs. time. (b) Illustration of the three different types of solid dispersion.

A two-component, API-polymer carrier solid dispersion can form multiple structures depending on their composition and process- ing history (13), as illustrated in Figure 3b. The drug can be molecularly dispersed within the polymer matrix, forming a thermodynamically stable, homogeneous solid solution (SS), the most desirable structure of solid dispersions. Another form is a dispersion of crystalline drug particles in a polymer matrix, referred to as a crystalline solid dispersion (CSD). Alternatively, an intermediate, meta-stable structure where amorphous drug aggregates are dispersed in a polymer matrix (ASD) can be formed. The initial process changes from PRC 1 to PRC 2 and back to PRC 1 likely induced a transition of CSD to ASD and back to CSD, at least in portions of the sample, causing the rise and fall of the overall crystallinity value. Because ASD is a meta-stable state, it can be converted to the equilibrium CSD provided that the thermodynamic driving force is sufficient to overcome the Gibbs free energy barrier. The later transitions of PRC 1 to PRC 3 by raising the temperature to 170 °C resulted in the formation of SS, indicated by the lowest crystallinity value. Once the solid dispersion reached the SS, the polymer matrix provided both a physical barrier to aggregation and stabilization through the drug–polymer interaction. As the result, the API stays in that solid solution. Further transitions from PRC 3 to PRC 4 then back to PRC 1 did not change the crystallinity.

The observations in the FT-NIR experiments were further corroborated by THz Raman imaging analysis. At 130 °C and 50 rpm (PRC 1, Figure 4a), agglomeration of crystalline APAP (CSD, red regions), partially amorphous dispersion (ASD, green regions), and amorphous dispersion (SS, blue regions) coexist. Note that the processing temperature was well below the temperature of APAP (169 °C), and the energy imparted to APAP by screw rotation was not enough to accomplish complete amorphization. At this low processing temperature, even with a higher screw speed of 200 rpm (PRC 2, Figure 4b), the sample still contains noticeable amount of partially amorphous dispersion (ASD). Figures 4a and 4b are in sharp contrast to those of the extrudates processed at a higher temperature of 170 °C (PRC 3 and PRC 4; Figures 4c and 4d). By raising the temperature, near-complete amorphous APAP dispersion (SS) were formed with both screw speeds. The comparison between the four processes clearly illustrate that screw speed is a critical process variable that dictates the energy supply to the extruded material, the degree of mixing, and, to a lesser extent, the residence time. Increasing screw speed increases dissolution of drug into a molten carrier and dispersion. However, the higher temperature is more conducive to the formation of SS.

FIGURE 4: Raman images of extrudates from different HME processes. Images (a–c) were collected using a 50× objective and a 5 μm pixel size. Image (d) was collected using a 100× objective and a 0.5 μm pixel size.

In this study, inline monitoring of API crystallinity by FT-NIR during an HME process was demonstrated. The crystallinity of APAP, measured by the peak area at 6100 cm

, shows three distinct values, which are speculated to be associated with three different forms of solid dispersion: crystalline solid dispersion, amorphous solid dispersion, and solid solution. Overall, high process temperature and high screw speed are conducive to the formation of amorphous APAP dispersions, but temperature appears to have a more direct impact on the formation of the desired amorphous APAP dispersion. These observations are further corroborated by an offline THz Raman imaging analysis. The extrudates with the highest crystallinity value (least amorphous) show the coexistence of appreciable amount of CSD with ASD and SS, whereas the extrudates with the lowest crystallinity value (most amorphous) contain predominantly SS. FT-NIR is effective in providing real-time information on the solid physical state transformation, but through bulk measurements. Raman microscopy complements FT-NIR by offering direct visualization of the distribution of different solid physical states of the API.

(1) M.A. Repka, S. Bandari, V.R. Kallakunta, A.Q. Vo, H. McFall, M.B. Pimparade, and A.M. Bhagurkar, 

(2) S.D. Schaber, D.I. Gerogiorgis, R. Ramachandran, J.M.B. Evans, P.I. Barton, and B.L. Trout, 

(3) R. Censi, M.R. Gigliobianco, Cr. Casadidio, and P. Di Martino, 

(4) N. Furuyama, S. Hasegawa, T. Hamaura, S. Yada, H. Nakagami, E. Yonemochi, and K. Terada, 

(6) A. Paudel, D. Raijada, and J. Rantanen, 

(7) A.Q. Vo, H. He, J. Zhang, S. Martin, R. Chen, M.A. Repka, 

(8) A.Q. Vo, G. Kutz, H. He, S. Narala, S. Bandari, and M.A. Repka, 

(9) M. Ibrahim, J. Zhang, M.A. Repka, and R. Chen, 

(11) A. Paudel, D. Raijada, and J. Rantanen, 

(12) F. Tres, K. Treacher, J. Booth, L.P. Hughes, S.A.C. Wren, J.W. Aylott, and J. C. Burley, 

 are with Thermo Fisher Scientific. Direct correspondence to: 

Articles

Inline FT-NIR and offline terahertz Raman imaging analysis are used to characterize active pharmaceutical ingredient (API) crystallinity and to monitor different solid physical states of the API, to control process parameters of hot melt extrusion.

Solid Physical State Transformation in Hot Melt Extrusion Revealed by Inline Near-Infrared (NIR) Spectroscopy and Offline Terahertz (THz) Raman Imaging

In this study, several strains of yeast cells, both wild-type (WT) and engineered, were analyzed by Raman imaging microscopy. Two biologically active compounds produced within the engineered yeast cells were readily identified by their characteristic Raman spectral peaks. The locales and the relative abundance of those biologically active compounds were visualized from the Raman microscopic images of the individual cells. Raman imaging microscopy further reveals the differences in efficiencies with which the biologically active compounds were produced by different yeast cell strains.

In recent years, bioengineering of yeast cells has made them a “biological cell factory” for producing a range of chemicals, including biologically active compounds through fermentation processes. This “biological cell factory” can efficiently convert starch, sucrose, or lignocellulose-based raw materials into precursor metabolites, which can further be converted into the products of interest (1). Bulk chemical analyses, such as gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR), can be used for screening fermentation cultures, assessing product yield, and delivering process optimization. These methods, however, are destructive, time-consuming, and insufficient, because they fail to provide specificity at the cellular level (2). Fluorescence microscopy-based imaging is capable of visualizing large or small molecules at the cellular level, but the success of this approach largely hinges on the photostability and brightness of fluorescent labels, their possible perturbations to the biological system, and the methods by which the labels are attached (3). Confocal Raman microscopy-based imaging has emerged as a great alternative (3,4). Raman spectroscopy provides chemical annotations to the constituents within the cell, and the high spatial resolution intrinsic to confocal microscopy provides cellular level information. In contrast to the fluorescence microscopy-based imaging approach, Raman imaging is nondestructive and requires minimal sample preparation.

In this study, wild-type (WT) and several strains of engineered yeast cells were analyzed by Raman imaging. Two biologically active compounds produced within the engineered yeast strains were readily identified. Furthermore, it was revealed that different strains produced the two compounds with different efficiencies. Raman images of the individual cells show the locales as well as the relative abundance of those compounds. The information extracted provides valuable information that helps optimize the fermentation process.

A 2 μL sample of the WT and each engineered yeast cell strain sample was transferred to an aluminum-coated, 12-positions slide, and then it was air dried prior to the Raman imaging experiments. The two biologically active reference compounds are denoted as “compound 1” and “compound 2”, respectively. Compound 1 is a terpene and compound 2 is a sterol. For the two reference compounds in solid powder form, a small amount of each was transferred to a glass slide, and the Raman spectra were collected.

The Raman spectra and the Raman area images (for an area containing one or two cells within each sample) were acquired using a Thermo Scientific DXR3xi Raman Imaging Microscope. The key experimental parameters for Raman imaging are listed below:

Raman images were obtained by the correlation profiling option of the OM-NICxi software, using a representative Raman spectrum from within a cell as the reference. The correlation profile shows the correlation between the spectrum at each pixel (sampling point) in the Raman image and the specified reference spectrum. Each color in the rainbow-colored intensity scale represents a degree of correlation with the reference spectrum. The higher the intensity value, the greater similarity to the reference spectrum.

Figure 1 shows the representative Raman spectra of the two biologically active compounds, compound 1 (pink trace, top) and compound 2 (red trace, bottom). Both compounds have prominent features in the C-H stretching region (~2900 cm

). The two strong peaks attributed to the C=C stretch- ing vibrations are located at 1667 cm

, respectively, for compound 1 and compound 2. The downshift of the C=C stretching peak in compound 2 (1605 cm

) is because of a ring structure. There are no strong interfering Raman peaks from the cellular matrices in this frequency range. Hence, these peaks are chosen as the Raman markers to distinguish the two biologically active compounds in the yeast cell samples by Raman imaging.

FIGURE 1: Raman spectra of the pure biologically active compounds, (a) compound 1, and (b) compound 2. These two compounds can be clearly distinguished by the presence of strong C=C peaks at 1667 cm

Figure 2 shows the Raman image of a WT yeast cell (Figure 2a), the representative Raman spectra from the different locations of the imaged region (Figure 2b), and the representative Raman spectra of compound 1 and compound 2. As can be seen in Figure 2a, the cells (in red) can be spectroscopically differentiated from the surrounding media (in blue), with a clearly delineated contour in green color. As expected, the cells are devoid of either compound 1 or compound 2 (Figure 2c). Raman image of the strain B yeast cells is shown in Figure 3. Both compound 1 and compound 2 are produced in the cell, as evidenced by Figures 3b and 3c. Interestingly, for the same strain B, there are cell-to-cell variations in the level of produced compounds. Based on the relative peak intensities, the cell in the upper- left corner appears to produce more compound 1 than compound 2, whereas the cell in the center produces similar amounts of compound 1 and compound 2.

FIGURE 2: Raman image of a wild-type (WT) yeast cell. (a) Raman image of a selected region for the WT yeast cells. The Raman image was generated by correlation profiling. (b) Raman spectra from the spots S-1 and S-2 on the Raman image. (c) Raman spectra of S-1 and S-2, as well as compounds 1 and 2 displayed in the 1540–1750 cm

 region. The Raman spectra for the cells were normalized with respect to the C-H stretching peaks near 3000 cm

FIGURE 3: Raman image of the strain B yeast cells. (a) Raman image of a selected region for the strain B yeast cells. The Raman image was generated by correlation profiling. (b) Raman spectra from the spots S-1, S-2 and S-3 on the Raman image. (c) Raman spectra of S-1, S-2 and S-3, as well as compounds 1 and 2 displayed in the 1540–1750 cm

 region. The Raman spectra for the cells are normalized with respect to the C-H stretching peaks near 3000 cm

Figure 4 summarizes the Raman imaging analyses of the three different strains of yeast cells—strain A, strain C, and strain D—in terms of their yield in producing compound 1 and compound 2. At a quick glance, strain A appears to be a low producer of compound 1 and does not produce compound 2. Strains C and D, on the other hand, appear to produce both compound 1 and compound 2, but strain D has a higher yield for compound 1 than for compound 2.

FIGURE 4: Raman images of three different strains of yeast cells. (Top row) Raman images from the selected regions for the three different strains: strain A, strain C, and strain D. The Raman images were generated using correlation profiling. (Bottom row) Raman spectra (1540–1750 cm

 region) from the spots on the top corresponding Raman images. The Raman spectra are normalized with respect to the C-H stretching peaks near 3000 cm

Figure 5 summarizes the yield with which compound 1 and compound 2 are produced by the four different engineered strains of yeast cells (strains A–D). Strain A (blue) appears to be a low producer of compound 1 and does not produce compound 2. Strains B, C, and D all produce both compound 1 and compound 2. Strain B displays nonuniform levels of produced compounds from cell-to-cell, some cells (pink) producing higher level of compound 1 than the others (cyan). Strain C (green) seems to produce higher level of compound 2, and strain D (red) has the highest yield for compound 1. Caution should be exercised when assessing the product yields from the Raman peak intensity. A more rigorous, quantitative analysis requires the respective Raman cross sections for the compounds 1 and 2 be properly accounted for.

FIGURE 5: Raman spectra of the strains A–D of the yeast cells. (a) Raman spectra (1540–1750 cm

 region) from the cells of the strains A–D, shown in the offset mode, and (b) in the overlayed mode. The Raman spectra are normalized with respect to the C-H stretching peaks near 3000 cm

In this study, Raman imaging was successfully applied to analyze different strains of yeast cells, both WT and engineered. The target bioactive compounds produced by the engineered yeast cells can be readily identified and differentiated by their respective Raman spectra, allowing for assessment of the product yields of different yeast strains by Raman imaging. Raman images of the individual cells from the four engineered yeast cell strains indicate that these strains produce compounds 1 and 2 with different efficiencies. Raman imaging can also be used to estimate the relative yield of the bioactive compounds. A quantitative analysis of product yield, however, requires a more systematic study on the Raman cross-sections of those compounds. The information obtained from the Raman imaging will be beneficial for a deeper understanding of the fermentation process and for optimization of the process for higher product yield.

The authors would like to acknowledge Dr. Michael Leavell and his colleagues at Amyris for providing the samples and many insightful discussions.

(1) Y. Chen, L. Daviet, M. Schalk, V. Siewers, and J. Nielsen, 

(2) R. Miyaoka, M. Hosokawa, M. Ando, T. Mori, H.O. Hamaguchi, and H. Takeyama, 

(4) L. Mikoliunaite, R.D. Rodriguez, E. Sheremet, V. Kolchuzhin, J. Mehner, A. Ramanavicius, and D.R.T. Zahn, 

 are with Thermo Fisher Scientific in Madison, Wisconsin. 

 was formerly a scientist with Amyris in Emeryville, California, when this work was being conducted. Direct correspondence to: 

Using Raman imaging, wild-type and engineered yeast cells were compared for their ability to produce bioactive compounds. Raman imaging microscopy is able to visualize locales, relative abundance, and production efficiencies of biologically active compounds for the individual yeast cells.

Tracking Bioactive Compounds Produced by Genetically Engineered Yeast Cells Using Raman Imaging

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, 

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

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.


Mohammed Ibrahim

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