*3.2. Investigation of KH2PO4 at Elevated Temperature*

The experiment EXP. 4—KH2PO4 started at an elevated seeding temperature of 56.4 ◦C. After seeding, both optical measurement techniques were able to measure the initial crystal size distribution. After t = 0.5 h the reactor reached a temperature of around 50 ◦C and crystallization occurred in the bypass of the online microscope, which led to a blockage. Therefore, the bypass was closed down and only the shadowgraphic probe was used to evaluate the state of the particulate phase (see Figure 11).

**Figure 10.** ATR-FTIR suspension density in comparison to the optical densities (microscope and probe) based on pixel ratios for Exp. 2—KH2PO4.

**Figure 11.** q0-distributions of Exp. 4—KH2PO4 (**a**) shadowgraphic probe; (**b**) QICPIC; *Solid lines* percentiles shadowgraphic probe; *Dashed lines*—percentiles QICPIC; *yellow and red* −0.15 and 0.85 percentile distribution; *blue*—transient mean sizes of the distribution.

Both principles have a good match with the distributions of the seeds, and similar growth is visible for both techniques at starting conditions. After the bypass was blocked, the shadowgraphic probe recognized a broadening of the mass-based distribution, therefore the larger fractions are no longer visible in the number distribution. This is also caused by nucleation of smaller crystals that are dominant in number compared to the larger grown seeds. This smaller fraction increased rapidly in number and therefore, accounts for about 15% of solid fraction in the mass-based distribution. To sum up, the inline probe can be utilized at conditions where massive nucleation and fast crystal growth leads to blocking of a bypass-based measurement technique, which requires a precise temperature control when withdrawing samples. This shows clearly that the probe opens a new field of applications, where other measurement systems fail.

### *3.3. Crystallization of Thiamin Hydrochloride Monohydrate*

The crystallization of thiamine hydrochloride monohydrate was performed as nucleation from aqueous solution with ethanol as antisolvent. Nucleation was observed by the shadowgraphic probe after approximately t = 0.3 h after adding the antisolvent. These crystals were only a few pixels in width, and a certain time was necessary to overcome the lower detection limit of the probe, therefore an exact time cannot be referred. After t = 0.6 h, a representative amount of crystals was visible within the images. The amount increased significantly up to the time of t = 1 h, when the bypass was put into operation. As the suspension passed the flow cuvette, the online microscope was not capable of setting an autofocus automatically, therefore the focus had to be adjusted manually. An example of the images taken by the optical methods is given in Figure 12.

**Figure 12.** Thiamine hydrochloride (t = 1 h) monohydrate crystals (**a**) online microscope (**b**) shadowgraphic probe. The images are enlarged for a better view of the edges.

The crystals captured with the online microscope appear blurry, without clear edges, and a proper image evaluation is not possible. In addition, the bypass could only be utilized for a few minutes before the suspension flow blocked. In contrast, the shadowgraphic probe was still capable of capturing images with sharp edges and suitable image quality for a crystal size evaluation. For the present case, the telecentric lens in the shadowgrphic probe shows a clear advantage, because no focus has to be adjusted, and the depth of field covers the whole measured volume. After t = 1.22 h, the experiment was ended due to a significant increase in suspension density. At this point even a qualitative evaluation of the captured images was not possible, either with the online microscope nor with the shadowgraphic probe. Figure 13 shows images captured by the shadowgraphic probe of the suspension in different states.

At about t = 1 h the crystals grew as thin needles, as described above. They varied in length and width but were mostly isolated single crystals. Already a few minutes later (t = 1.16 h), the suspension density increased significantly, which may have been supported by natural breakage and secondary nucleation. Hence, the broken crystal pieces increase the total particle number, additionally. Due to the increased number of crystals, single particles have an increased probability of colliding with each other and forming agglomerates, which can be seen at the time mark for t = 1 h in Figure 13c,d.

For this state of the system an image evaluation of the crystals is quite challenging and may not be solved with a conventionally image analysis based on binary object identification, because the thin needles overlap and single crystals cannot be identified [72]. Interestingly, the needles tend to align with the flow direction in the measured volume of the shadowgraphic probe, especially at a higher solid content. Because the gap is comparably small to the vessel, the flow inside the gap is hindered and mostly laminar, even if the flow around it is turbulent. A group of researchers reported an algorithm which was utilized to determine the size of high-aspect-ratio crystals. They found that an irregular alignment hinders clear object detection at a higher solid content [72]. Therefore, the alignment of the needle-like crystals in the shadowgraphic probe could be an opportunity to investigate the crystallization in such difficult systems, with high aspect ratios of the particles.

**Figure 13.** Optical density for the thiamine anti solvent crystallization based on pixel ratios of the shadowgraphic probe images. The images at the bottom were captured with the shadowgraphic probe at different experiment times; (**a**) t = 0.3 h first single crystals, (**b**) t = 1 h recognizable number of crystals, (**c**) t = 1.16 h first agglomerates and increased overlapping of the crystals and (**d**) t = 1.18 h last possible measurement point, afterwards the suspension density was too high to capture further images.

Although a crystal size determination with the presented methods was not possible after t = 1 h, a measurement of the optical suspension density was still possible (see Figure 13).

The optical density shows a rapidly increasing amount of crystals that started at around t = 0.3 h. From that point, the optical density increased with exponential progression. The width and length distributions at different experiment times are shown in Figure 14.

The diagram for t = 0 h was made as a reference, where mostly dust was detected. At t = 0.63 h the first reliable distribution shows that the single needle-like crystals have between 500–1000 μm in length and 20–40 μm width. At t = 0.97 h the crystal sizes are about the same, while their amount has significantly increased. The last diagram at t = 1.18 h depicts only a detection of small particles, which clearly shows that the simple image algorithm that was applied failed to isolate the crystals, and is the limit in PSD evaluation, at least with the methods that were used.

The comparison between the thiamine hydrochloride and KH2PO4 experiments shows clearly that the limiting optical density for image-based measurement systems depends on several properties of the particulate phase, e.g., size, size distribution, and shape. KH2PO4 could be measured up to an optical density of 8% for the shadowgraphic probe and 11% for the online microscope, with a suspension density (FTIR) of 7%. The measurement had already failed for thiamine at a value of 3% to 4% optical density of the probe (see Figure 13, t = 1 h). Hence, a clear suspension, or optical density limit, of application for both utilized techniques cannot be given here. It has to be determined for each substance system individually, and the methods for the image evaluation must be adjusted for the specific case in order to maximize the applicability.

**Figure 14.** Length and width distributions of the needle-like crystals of thiamine hydrochloride monohydrate at different time points of Exp. 5, measured with the shadowgraphic probe. (**a**) measured crystals in the clear solution, (**b**) measurement of the first single needles, (**c**) significant number of crystals, (**d**) failed measurement as result of massive overlapping.
