Apply Controlled Temperature to a Sample Using a Heated AFM Probe

Ztherm™ Local Thermal Analysis (LTA) is an accessory that can be used in combination with the Asylum Research MFP-3D™ Atomic Force Microscope (AFM). Ztherm employs a heated AFM probe to apply controlled temperature to a sample. This technology offers a distinctive understanding, for instance, of the different surface states of sugar particles after grinding, which are of specific importance in chocolate manufacturing.

It is a well-known fact that a fine chocolate has better taste, flavor, melting, and more specifically, a smooth texture and mouth feel without grittiness. Hence, the solid ingredients of chocolate — milk powder, sugar, and cocoa — are ground to a size less than around 30 µm in a liquid phase of lipophilic cocoa butter. This is performed predominantly by using ball or roller mills.1

Apart from chocolate texture, refining is also applicable for the processing behavior during the production process, for instance, relative to the flow characteristics of chocolate masses.2,3 Despite the fact that the main influencing factors are well known,2,4,5 flow characteristics of chocolate masses differ based on the refining technique.3 Flow characteristics are the consequence of the interactions of the particles with one another and also with the surrounding cocoa butter. Grinding the particles leads to not only reduction of particle size but also modification of their surface characteristics. Consequently, the interactions change.

Since the interactions with other particles and with the surrounding phase are determined by surface characteristics such as adhesion, topography, and/or the crystalline or amorphous property of the sucrose, they play a vital role in chocolate manufacturing. To gain insights into the impact of grinding on these characteristics, it is essential to carry out a detailed study of the surfaces of unground and ground sucrose particles. An AFM surface morphology image of a sucrose particle as it appears in unground chocolate mass is illustrated in Figure 1. Sorption experiments have already shown that grinding results in an amorphization of sucrose particles.6,7 However, it is not possible to confirm this amorphization in detail through polarization microscopy, X-ray diffraction, or differential scanning calorimetry (DSC). These methods only provide information related to bulk characteristics of the material, which results in the conclusion that amorphization occurs only at the surfaces of the particles.

Figure 1. Surface morphology of a sucrose particle as it appears in unground chocolate mass. Scan size 5 µm.

Materials and Methods

Grinding of Sucrose Particles

A ball mill containing 10 kg steel balls, with diameters of 1.5 cm, was used to grind 450 g of commercially available crystalline sucrose, with a particle size of ~1 mm, in 550 g molten cocoa butter at a temperature of 45 °C. The grinding process was carried out until a particle size of ≤30 µm and a median diameter of 10–12 µm for 90% of the particles were achieved.

AFM Experiments and Sample Preparation

After separating the sucrose particles from bulk suspension by centrifugation (55 °C, 10 min, 5000 rpm), they were washed with hexane and dried in a dessicator. Subsequently, sucrose particles were glued onto a mica disk. Adhesion force measurements and surface analysis were performed using the MFP-3D AFM with standard silicon cantilevers of a nominal spring constant of 2 N/m (AC 240, Olympus Corp.). Analysis was carried out in a dried nitrogen atmosphere (relative humidity 5%, Humidity Sensing Cell for the MFP-3D AFM).

After carrying out 60 x 60 force-versus-distance-curves per force map of a size of 1 µm² in contact mode, adhesion forces were evaluated. For each tip, the exact cantilever spring constant was established through the thermal noise method.5 Scan velocity was 2.0 µm/s and contact trigger point was 1 V. Two force maps per sucrose particle at different surface areas were inspected, and maps were captured in duplicate. A minimum of 15 sucrose particles were analyzed per sample. Force map data were converted into a histogram of the frequency of adhesion force per map in %. Quantities less than 0.5% of the total number of force curves were not considered.

Oxford Instruments Asylum Research provided the Ztherm LTA. To calibrate the tip, saccharides of different melting temperatures were used. Amorphous sucrose was prepared in accordance with Ref. 8. LTA was carried out using an AN2-200 ThermaLever (Anasys Instruments) with a setpoint of 500 mV, a trigger of 10 nm, and a voltage ramp of 1 V/s. The MFP-3D was used for analyzing images and maps.9

Melting Temperatures of Standard Substances

In order to determine melting temperatures, DSC analysis was carried out using an MDSC 2920 (TA Instruments) and a heating rate of 5 K/min in a temperature range of 50-200 °C in duplicate. After the sample was hermetically sealed in an aluminum crucible (40 µL, Mettler Toledo), it was measured against an empty one as a reference.

Particle Size Analysis

A laser diffraction spectrometer (Malvern Mastersizer 2000, Malvern Instruments) was used to perform particle size analysis of the sucrose particles in suspension. For this purpose, the molten suspension (45 °C) was dispersed into low-viscosity mineral oil using ultrasound.

Results and Discussion

When ground in a lipophilic suspension, the surface state of sucrose is predicted to be partially amorphous. In order to verify this hypothesis, a first experiment was performed to differentiate surface morphology of amorphous particles from crystalline ones. Hence, an entirely amorphous sucrose particle was created and was characterized by AFM. Subsequently, relative humidity was increased to approximately 75% using the Asylum Research MFP-3D Humidity Sensing Cell to induce recrystallization by a saturated solution of sodium chloride in water. Following 12 hours of equilibration, relative humidity was lowered to 50% for accurate measurement conditions, and topography was determined again at the same surface area. This method enables artifact-free AFM imaging with consistent results. The surface morphologies of sucrose before and after humidity treatment are shown in Figure 2, left, and correlated with the tapping mode image after ball-milling (Figure 2, right).

AFM surface morphology images

Figure 2. AFM surface morphology images of amorphous (top left), recrystallized (bottom left) and ball-milled sucrose (right).

The amorphous surface displays cleaved, coarse structures (Figure 2, upper left image). On the other hand, the recrystallized surface appears finer, with step-like structures and almost no cleft could be determined (Figure 2, lower left image). Similar surface structures were found on ball-milled sucrose particles: the image on the right mainly exhibits coarser structures and smooth contours just like the structures found at the surface of amorphous particles. Moreover, it is possible to detect some finer and more step-like structures. These structures can be compared to the recrystallized sucrose surface.

The surface morphology denoted that ball-milled sucrose particles partly consist of both crystalline and amorphous structures. As aforementioned, the amorphous areas are considered to occur only at the external surface layers of the sucrose particles. Hence, the amount of amorphous material is so small that it is lower than the limit of detection for common DSC analysis. Furthermore, DSC cannot locally differentiate between different surface areas and is only appropriate for bulk material characterization. Consequently, another method is used to confirm the results.

As it is possible to distinguish amorphous and crystalline materials by their glass transition or melting temperatures, respectively, the combination of AFM and LTA imaging is a potential tool for detailed characterization of the particle surface characteristics. LTA is a method that has already been effectively used for characterization of mixtures of crystalline and amorphous lactose in the pharmaceutical sector.10,11 This method has now also been adopted in the food sector.

For LTA of sucrose, it is necessary to calibrate the system with chemically similar substances of known melting characteristics. Hence, the melting temperatures of glucose, maltose, and sucrose in addition to the glass transition temperature of amorphous sucrose were determined by DSC. Subsequently, AFM-LTA calibration was carried out. The resistance is the measured quantity of LTA and is a product of the measured electrical current and the applied heating voltage at the softening point of the sample during contact of the tip with the surface of the sample (Ohm’s law). This resistance is now correlated with a softening temperature; Figure 3 shows the resulting calibration function. Correlation between the resistance and the temperature is virtually linear, with a reasonable correlation coefficient of 0.947. It is necessary to repeat the calibration procedure on a daily basis and for each tip.12

Results of LTA calibration using Ztherm LTA

Figure 3. Results of LTA calibration using Ztherm LTA: resistance R measured with AFM tip versus temperature T, where T corresponds to the melting point of low molecular saccharides or glass transition temperature of amorphous sucrose (determined via DSC). Error bars represent the standard deviation of R. The standard deviation of the softening of temperatures was ± 7 °C.

A mean softening temperature of 75 ± 48 °C was detected on a surface area of 25 µm² for ball-milled particles that are supposed to be at least partially amorphous (cf. Figure 2).

The high standard deviation of about 64% again relates to the assumption that only part of the surface is in an amorphous state and the rest is crystalline. Both lower softening temperatures, considering the glass transition of amorphous sucrose (approximately 65°C), and higher softening temperatures, considering the melting temperature of crystalline sucrose (approximately 190 °C), were expected.

Once these particles are washed with acetone,6 softening temperatures of 182 ± 18 °C were found, and the recrystallization process could be verified. The standard deviation in temperature is low when compared to the value before washing with acetone. This also indicates that the surface of ball-milled sucrose is almost fully recrystallized due to acetone.

Another approach was carried out for correlating the different surface morphologies such as those depicted in Figure 2 (right image) with quantitative LTA results. Hence, 36 measurement points were equally distributed across the surface of ball-milled sucrose. The softening temperature of each point was established and correlated with the surface state by a color code: red for amorphous material (60-90 °C), green for the softening temperature of crystalline material (155-190 °C), and orange for a mixed surface state with a softening temperature between 90-155 °C (Figure 4).

These results again prove that the sucrose surface post grinding is partially amorphous (cf. colored dots in Figure 4, right). These results also show that different surface states can exist simultaneously, denoted by green and red dots. Moreover, the transition of one state to the other can, for instance, occur across widths of 2 µm, which is about 2000 sucrose molecules.15 As a result of these images, it can be assumed that some kind of transition zones are present between the crystalline and amorphous states (cf. orange in Figure 4). These are distinguished by a softening temperature between the ones for the crystalline and amorphous states and are indicated by orange dots (approximately 90-155 °C). These results concur with the results obtained from adhesion force measurements.9,16 Shown in Figure 5 are four histograms calculated from adhesion force measurements on two different surface areas, each with ball-milled (red) and unground sucrose (green). Histograms and a representative surface morphology are correlated, where the red-framed image was measured on ball-milled sucrose and the green-framed image one at the surface of the unground particles. There is a consistent arrangement of molecules in the crystalline state of unground sucrose particles. Step-like formations (green framed image) were found at the surface of these highly ordered structures. Corresponding adhesion forces were quantified in the range below 15 nN (green histograms).

Surface morphology of ball-milled sucrose

Figure 4. Surface morphology of ball-milled sucrose (left) and correlation of softening temperatures to surface state by color code (right): 155-190 °C for crystalline in green, 60-90 °C for amorphous in red and 90-155 °C for mixed state in orange, according to [13] and [14]. Imaged in contact mode.

For ball-milled sucrose surfaces, adhesion forces of wider force ranges at higher forces between 10 and 25 reaching up to even 50 nN (dark-red histogram) and also between 15 and 40 nN were detected (light-red histogram). As mentioned before, ball-milled sucrose particles contain both amorphous parts and crystalline areas. The dual amorphous and crystalline character of the ball-milled sucrose surface is thus represented by the higher and broader adhesion force ranges (red histograms in Figure 5).

Figure 5. Surface morphology of unground (green) and ball-milled sucrose particles (red) with corresponding histograms calculated from of adhesion force maps. Scan size 1 x 1 µm. Each row represents the results from one force map, extracted from [14].

Summary and Conclusion

As one of the key components of chocolate, variation in sucrose particles has a significant effect on the production properties of liquid chocolate masses, which in turn has an effect on product quality. Traditional sorption experiments have shown that the grinding process results in amorphous sucrose. However, methods like X-ray diffraction and/or differential scanning calorimetry were unable to detect these structures in detail and confirm the results of sorption experiments. As a result, a comparatively new AFM technique called Ztherm Local Thermal Analysis was used to integrate thermal analysis with adhesion force measurements and morphology characterization.

Adhesion, surface morphology, and thermal behavior of the sucrose surface were characterized. The researchers were able to confirm sorption experiments and verify that grinding causes amorphization of sucrose only by using Ztherm LTA. Additionally, their studies have proven for the first time that particles are not completely amorphous, but that the surface of ball-milled sucrose particles contains crystalline surface areas.

Acknowledgements

This IGF Project of the FEI is/was supported by AiF within the program for promoting the Industrial Collective Research (IGF) of the German Ministry of Economics and Energy (BMWi), on the basis of a resolution of the German Parliament (project AiF 16757 N). The authors are also thankful to Oxford Instruments Asylum Research Wiesbaden, Germany, office for supplying the Ztherm Kit for thermal analysis with AFM. Furthermore, the authors would like to extend their gratitude to August Storck GmbH & Co. KG, Halle, Germany, for providing the cocoa butter.

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