Centrifugal Step‐Emulsification Platform for High‐Throughput Emulsion Screening

ABSTRACT

A centrifugal emulsification platform enabling the parallel generation of emulsions directly in standard 96‐well plates is introduced. Upon centrifugation, the dispersed phase is driven through capillaries into the continuous phase, where step emulsification yields droplets by clean pinch‐off. The system achieves high reproducibility, with success rates above 90%. The platform's utility is demonstrated by screening silicone‐oil/water emulsions across siloxane surfactants and NaCl gradients, and by mapping fluorinated emulsion stability as a function of salinity and the mixing ratio of two fluorinated surfactants of different chain lengths. In contrast to lab‐on‐a‐disc systems or single‐tube centrifugal devices, this approach combines minimal hardware requirements with high throughput and seamless compatibility with automated liquid‐handling and imaging workflows. Its simplicity and scalability make it suited for closed‐loop optimization, accelerating the discovery of sustainable non‐fluorinated droplet systems and enabling systematic studies of emulsion stability across diverse formulation spaces.

A high‐throughput centrifugal emulsification platform generates reproducible droplets directly in standard 96‐well plates via step emulsification. Compatible with automation, it enables parallel screening and stability mapping of diverse emulsion formulations, accelerating closed‐loop optimization and sustainable droplet‐system discovery.

1. Introduction

Emulsions, dispersions of one liquid phase within another immiscible liquid phase, are essential in a wide range of applications in food science, cosmetics, pharmaceuticals, and biotechnology [1, 2, 3, 4]. Their performance in these applications depends critically on droplet size distribution, stability against coalescence or phase separation, and compatibility with functional additives [5, 6]. Achieving optimal emulsion properties requires careful selection of the continuous phase, dispersed phase, and surfactants, as well as precise control of processing conditions [3, 7].

In biphasic microfluidics and droplet‐based screening systems, these requirements are often met through the use of perfluorinated oils and surfactants, valued for their exceptional droplet stability, chemical inertness, and compatibility with biological materials [8, 9, 10, 11]. However, such compounds impose a severe environmental burden due to their persistence, bioaccumulation, and potential toxicity [12]. Regulatory pressure to restrict and phase out per‐ and polyfluoroalkyl substances (PFAS) is intensifying worldwide, creating an urgent need for non‐fluorinated alternatives [13, 14, 15, 16]. Identifying suitable replacements remains highly challenging, as few oil–surfactant combinations achieve the same stability, biocompatibility, and ease of formulation provided by fluorinated systems in droplet microfluidics.

Replacing fluorinated systems requires the rapid evaluation of large formulation spaces encompassing multiple oil–surfactant combinations and environmental conditions. Conventional methods for testing emulsion stability are poorly suited to this task. Standard bottle tests consume tens to hundreds of milliliters of sample and require days to reveal changes in emulsion stability [17]. More advanced techniques, such as multiple light scattering (e.g., Turbiscan) or analytical centrifugation (e.g., LUMiSizer), provide quantitative readouts but rely on specialized instrumentation and process only a limited number of samples per run [6, 18]. Dynamic light scattering, microscopy, and rheological measurements offer complementary insights, yet all are inherently sequential, demanding specialized equipment and significant labor per sample. Consequently, the analysis phase becomes a major bottleneck, necessitating scalable, parallelized methods that can rapidly evaluate emulsion performance under varied conditions and accelerate the discovery of sustainable, application‐specific formulations.

Centrifugal emulsification represents a promising approach to address this challenge. In lab‐on‐a‐disc devices, rotation drives step‐ or cross‐flow emulsification within microfabricated channels, yielding highly monodisperse droplets and enabling integration of unit operations such as mixing, valving, and metering [19, 20, 21]. Despite these advantages, reliance on custom‐fabricated cartridges limits accessibility and complicates integration with standardized liquid‐handling workflows. In contrast, tube‐based centrifugal methods emphasize simplicity and low cost. Buoyancy‐driven emulsification modules assembled from tubes and needles have enabled reproducible droplet formation for digital PCR and related assays, while centrifugal homogenizers generate milliliter‐scale emulsions by forcing multiphase mixtures through narrow orifices using readily available components [22, 23]. These designs eliminate the need for pumps or specialized equipment, but typically operate on single tubes and lack compatibility with multiwell formats. This gap motivated us to develop alternative centrifugal designs that preserve simplicity while interfacing directly with standardized plate formats.

Here, we present a novel centrifugal emulsification platform compatible with standard 96‐well plates, enabling parallel droplet generation across all wells. Two capillary orientations (vertical and 30 tilt) accommodate liquid phases of differing densities, allowing droplet settlement either above or below the continuous phase while maintaining compatibility with automated liquid‐handling systems. We demonstrate the platform's utility by screening emulsions prepared with different siloxane‐based surfactants, varying both their concentrations and the NaCl content of the dispersed phase. In addition, we demonstrate its versatility by mapping fluorinated emulsion stability through systematic variation of salinity and the mixing ratio of two fluorinated surfactants differing in chain length. The platform's straightforward, alignment‐free design supports seamless automation and paves the way for closed‐loop workflows in which image‐based emulsion analysis directly drives formulation optimization.

2. Results and Discussion

2.1. Platform Design and Workflow Integration

During centrifugation, droplet formation occurs as the aqueous phase is forced through capillaries into the preloaded oil phase (Figure 1a). Rotation generates a radial pressure gradient that drives flow through each needle. Pinch‐off occurs directly at the needle tip and is governed by interfacial tension, buoyancy, and inertial forcing. Depending on the relative densities of the two phases, droplets either migrate downward toward the bottom of the well or upward toward the oil–air interface.

FIGURE 1.

Centrifugal emulsification platform and holder designs. (a) Schematic of droplet formation for density‐matched configurations. When ${\rho }_{aq}>{\rho }_{oil}$, droplets detach and sink to the bottom of the well. When ${\rho }_{aq}<{\rho }_{oil}$, droplets detach from an inclined needle and rise toward the oil–air interface due to buoyancy. (b) Three‐dimensional CAD renderings of the holder systems: the vertical configuration (top) with needles oriented perpendicular to the plate, and the tilted configuration (bottom) with needles inclined at 30 relative to vertical. (c) Schematic of the centrifugal workflow. The holder, positioned on a standard 96‐well plate, is loaded into a centrifuge; rotation generates radial forcing that drives the aqueous phase through the capillaries into the oil, producing droplets in each well. (d) Representative bright‐field images of emulsions generated using the two configurations. The vertical configuration (left) produced water‐in–1 cSt silicone‐oil emulsions stabilized with 1% siloxane surfactant. The tilted configuration (right) produced water‐in–fluorinated‐oil emulsions stabilized with 1% fluorosurfactant.

These two behaviors are addressed by distinct holder designs (Figure 1b). In the vertical configuration, used when the aqueous phase is denser than the oil, the needles are oriented perpendicular to the plate so that detached droplets descend and accumulate at the bottom of the well. In the tilted configuration, used for denser oils, the needles are fixed at a 30 inclination to allow droplets to detach cleanly and rise by buoyancy. The chosen tilt angle provides sufficient immersion depth of the needles while maintaining enough tilt for droplets to pinch off cleanly and move into the bulk, rather than wetting and following the capillary surface. Detailed CAD designs for both holders are provided in Supplementary Note S1.

These principles are implemented in a simple, alignment‐free platform (Figure 1c). A standard 96‐well plate functions both as a reservoir for the preloaded oil phase and as the site of emulsion formation. The removable holder carries 96 stainless‐steel needles, each manually inserted into a dedicated bore to ensure one‐to‐one needle–well correspondence. No seals, clamps, or mechanical alignment features are required; the holder rests directly on the plate, and centrifugal forces maintain structural integrity during spinning. Prior to centrifugation, the aqueous phase is introduced into the holder containing the needles and the oil phase into the wells of the plate, using an automated liquid handling system (Supplementary Note S2). Because each needle addresses a single well, emulsions are generated simultaneously across the entire plate, with droplet size primarily dictated by needle inner diameter and local flow resistance.

Once emulsions are generated, the plate is transferred to a plate reader for automated imaging of individual wells. The instrument allows z‐axis positioning, enabling visualization of emulsions at different depths within each well. Representative images are shown in Figure 1d: emulsions formed in oil phases less dense than water were imaged at the bottom of the well (left panel), whereas emulsions formed in denser oil phases were imaged at the oil‐emulsion interface (right panel). The emulsion in the left panel was produced with the vertical holder configuration using 1% siloxane‐based surfactant in 1 cSt silicone oil with water as the dispersed phase. In turn, the emulsion in the right panel was obtained with the tilted holder configuration using 1% fluorinated surfactant in fluorinated oil with water as the dispersed phase.

2.2. Platform Validation

The performance of the platform was validated through five independent emulsification cycles for each holder configuration (vertical and tilted), yielding a total of 480 emulsions per design. In each run, all 96 wells of a plate were filled with 150 $\mu \mathrm{L}$ of oil, while 50 $\mu \mathrm{L}$ of the aqueous phase was dispensed through the needles during centrifugation. The vertical configuration was tested with 1 cSt silicone oil containing 1% siloxane‐based surfactant (ABE‐3642), whereas the tilted configuration employed a fluorinated oil (TMC 7500) supplemented with 1% fluorosurfactant. In both cases, water served as the dispersed phase. Centrifugation speed and duration were optimized to 600 rpm for 15 min in the vertical configuration and 800 rpm for 30 min in the tilted configuration, as excessive forcing can induce a transition from droplet pinch‐off to jetting, a known hydrodynamic instability that produces irregular and polydisperse structures. To further rationalize this operating window, we added a dimensionless scaling analysis based on the capillary number (Supplementary Note S3). The analysis relates centrifugation speed, needle inner diameter, immersion depth, and density‐dependent counter‐pressure to the transition between capillary‐controlled pinch‐off and jetting. For both holder geometries, the validated operating conditions remained below the approximate jetting guideline of Ca $\approx {10}^{-2}$ [24], while maintaining practical emulsification times. The higher speed and longer duration required for the tilted configuration are consistent with the reduced effective pressure head caused by the inclined needle geometry and the stronger counter‐pressure from the denser fluorinated oil. Average droplet diameters, extracted from the representative images in Figure 1d, were 0.34 $\pm$ 0.01 mm for the silicone‐oil system and 0.29 $\pm$ 0.01 mm for the fluorinated‐oil system.

To quantify platform performance, the emulsions generated in each well were classified into three categories: good (blue), poor (yellow), or no emulsion (red), with representative images and their classification shown in Figure 2a. Across all trials, the platform demonstrated high reliability, with success rates (defined as the proportion of wells yielding good emulsions) ranging from 90% to 95% for the vertical holder configuration and from 91% to 95% for the tilted holder configuration. Figure 2b presents 96‐well plates with wells color‐coded according to the introduced classification, illustrating the emulsification results for single runs with both holder configurations. The remaining emulsion maps (four for each configuration) and the corresponding raw emulsion images are provided in Supplementary Notes S4 and S5.

FIGURE 2.

Classification of emulsions and platform validation. (a) Representative bright‐field images of emulsions classified as good (blue), poor (yellow), or no emulsion (red). Labels (e.g., A1, C11, B2) correspond to specific well positions in the 96‐well plates I and II shown in panel (b). (b) Plate emulsification maps from single runs obtained for the two holder configurations. Each well is color‐coded according to the classification scheme in (a). The remaining eight runs (four for each configuration), together with the corresponding raw emulsion images, are provided in Supplementary Notes S4 and S5.

Failures were limited to a few wells per plate and were most often attributed to physical defects such as needle clogging or deformation during manual insertion, rather than to shortcomings in the holder design. Between runs, needles were cleaned using a standardized protocol involving sonication in water, followed by sonication in 90% ethanol, drying, and oxygen plasma treatment. The impact of alternative cleaning protocols on the quality of generated emulsions is detailed in Supplementary Note S6.

2.3. Platform Application in Studying Emulsion Stability

2.3.1. Application of the Vertical Configuration to Siloxane‐Based Emulsion Stability Studies

To demonstrate the utility of the vertical holder configuration, we investigated the stability of water‐in‐oil emulsions formed in 1 cSt silicone oil with eight siloxane‐based surfactants: ABE‐3642, CMS‐221, CMS‐222, CMS‐626, DBE‐224, DBE‐311, DBE‐621, and ABP‐263. Chemical structures of the surfactants are provided in Figure S22. Each surfactant was tested at a concentration of 1%, with twelve replicate emulsions per condition filling all 96 wells of a plate. Replicates ensured robustness against potential malfunctions such as needle clogging.

Figure 3a shows representative raw images of each emulsion type; the complete dataset, including 88 additional images and the corresponding color‐coded plate map, is provided in Supplementary Note S7. Surfactants ABE‐3642, CMS‐221, DBE‐224, and ABP‐263 consistently yielded stable emulsions, whereas CMS‐222 produced predominantly poor emulsions. In contrast, CMS‐626, DBE‐311, and DBE‐621 failed to generate emulsions under the tested conditions. These trends are consistent with differences in amphiphilic balance and interfacial adsorption: the four successful surfactants possess siloxane backbones and moderate polyether content that support rapid adsorption and stable interfacial film formation in silicone oil, whereas the poorly performing candidates are either excessively hydrophilic (DBE‐311, DBE‐621), sterically hindered by extensive non‐siloxane substitution (CMS‐626), or exhibit insufficiently favourable interfacial packing (CMS‐222). Based on this screen, the four surfactants that produced stable emulsions were selected for systematic evaluation of concentration and salinity effects.

FIGURE 3.

Application of the vertical holder configuration to study emulsion stability. (a) Screening of siloxane‐based surfactants for water‐in‐oil emulsions in 1 cSt silicone oil. Representative bright‐field images are shown for the eight tested surfactants, with emulsion quality classified as good (blue), poor (yellow), or no emulsion (red). The remaining emulsion images are provided in Supplementary Note S7. (b) Representative stability assays for CMS‐221. Left panel: emulsions prepared with increasing surfactant concentrations (1, 3, and 5% – water as dispersed phase). Right panel: emulsions prepared with 3% CMS‐221 and aqueous phases containing different NaCl concentrations (5–25 g/100 mL). Raw bright‐field images were collected immediately after emulsification (0) and after 1, 3, 5, and 10 days. The full dataset for CMS‐221 is available in Supplementary Notes S8.1–S8.5, while raw images of emulsions for the remaining surfactants (ABE‐3642, DBE‐224, and ABP‐263) are provided in Supplementary Notes S8.6–S8.20. (c) Heatmap of apparent destabilization rates, $k_{\mathrm{app}}$, for CMS‐221‐stabilized emulsions as a function of CMS‐221 concentration and NaCl concentration. Rates were calculated from normalized droplet‐survival fractions over 10 days. Lower $k_{\mathrm{app}}$ values indicate slower droplet loss and higher apparent stability. Red crosses indicate formulations for which stable emulsions were not formed and no apparent rate was extracted.

For these follow‐up studies, emulsions were prepared across five surfactant concentrations (1%–5%) and six NaCl concentrations (0–25 g/100 mL), yielding 30 unique formulations per surfactant. Each formulation was tested in triplicate (90 wells total), while the remaining six wells served as controls (oil with water or oil with 25 g/100 mL NaCl solution). The number of replicates was reduced from 12 to 3, as holder validation (Section 2.2) had already established $>$90% reproducibility. Plates were sealed with antifoaming tape and stored for 10 days at room temperature under static conditions to monitor droplet coalescence over time.

Raw bright‐field images acquired immediately after emulsification and after 1, 3, 5, and 10 days revealed distinct stability trends. The strongest effects of surfactant concentration and salinity were observed for CMS‐221, whose emulsions displayed pronounced sensitivity to both parameters. In contrast, emulsions stabilized by ABE‐3642, DBE‐224, and ABP‐263 were less affected and are therefore shown only in Supplementary Notes S8.6–S8.20. Figure 3b presents representative stability assays for CMS‐221, illustrating the effect of surfactant concentration (1%, 3%, and 5%; left panel) and NaCl concentration (5–25 g/100 mL; right panel). The remaining images for all CMS‐221 formulations are provided in Supplementary Notes S8.1–S8.5. Figure 3c complements these images with a quantitative stability map for CMS‐221, showing the apparent destabilization rate, $k_{\mathrm{app}}$, as a function of surfactant concentration and NaCl concentration. The rates were extracted from the time‐dependent droplet survival fraction in the first droplet layer using an apparent first‐order loss model, as described in Supplementary Note S9. Lower $k_{\mathrm{app}}$ values indicate slower droplet loss and therefore higher apparent stability, whereas higher values indicate faster destabilization.

At 1%–2% CMS‐221, most formulations showed low apparent destabilization rates. At higher surfactant concentrations, stability became strongly non‐monotonic with respect to NaCl concentration. The most stable region occurred at intermediate salinity, particularly around 10–15 g/100 mL NaCl, where $k_{\mathrm{app}}$ remained close to zero. In contrast, low‐salinity conditions at 0–5 g/100 mL NaCl destabilized more rapidly at 3%–5% CMS‐221, and destabilization increased again at high salinity. Stable emulsions were not formed for 5% CMS‐221 at 20 and 25 g/100 mL NaCl. These trends suggest that salt modulates the interfacial organization of the ethylene‐oxide‐containing surfactant layer. At very low salinity, the ethylene‐oxide headgroups are expected to remain highly hydrated and flexible, producing a soft, loosely packed interfacial layer with limited resistance to coalescence. In the intermediate salinity regime, partial dehydration of the ethylene‐oxide chains likely promotes tighter packing and a more cohesive interfacial film, yielding the highest emulsion stability. At very high salinity, extensive dehydration may instead weaken interfacial stabilization, for example by promoting surfactant desorption or less favourable interfacial packing, leading to faster emulsion collapse. The full survival curves and analogous apparent‐rate maps for ABE‐3642, ABP‐263, and DBE‐224 are provided in Supplementary Note S9.

2.3.2. Application of the Tilted Configuration to Fluorinated‐Based Emulsion Stability Studies

To demonstrate the use of the tilted configuration, we investigated water‐in–TMC 7500 emulsions stabilized with binary mixtures of two PFPE–PEG–PFPE triblock surfactants differing only in PEG block length: surfactant A (PEG 1500) and surfactant B (PEG 300). Emulsions were prepared at a fixed total surfactant concentration of 0.5% using five mixing ratios (A:B = 1:0, 3:1, 1:1, 1:3, 0:1) across a salinity series (0–25 g/100 mL NaCl; 30 different compositions in total), with each condition tested in triplicate to account for occasional needle clogging. Figure 4 summarizes the stability behaviour with water by plotting the fraction of droplets remaining in the first layer of the emulsion over 10 days for each surfactant ratio. Droplets in the first layer were counted for all three replicates at each time point, normalized to the time 0 value for that replicate, and averaged to obtain the mean fraction remaining with standard deviation. The bright‐field images of emulsions with water and the remaining studied NaCl solutions are provided in Supplementary Note S10.

FIGURE 4.

Quantitative stability profile of emulsions prepared with water and binary mixtures of PFPE–PEG–PFPE surfactants in TMC 7500, showing how the fraction of droplets remaining in the first droplet layer of emulsion evolves over 10 days for five A:B mixing ratios. A: PFPE–PEG‐1500–PFPE, B: PFPE–PEG‐300–PFPE. Data are shown as mean fractions $\pm$ standard deviation from three replicates.

Emulsions stabilized solely by the short‐PEG surfactant B destabilized rapidly, with the droplet fraction falling below 0.1 within the first day. Introducing the long‐PEG surfactant A increased stability in a composition‐dependent manner: the 1:3 and 1:1 (A:B) mixtures displayed only partial droplet retention, whereas the 3:1 mixture and the A‐only formulation maintained $>$95% of droplets throughout the experiment. This trend reflects the well‐established influence of PEG length in fluorinated triblocks, where longer chains generate thicker and more cohesive steric barriers, while shorter PEG segments yield interfacial films that lack sufficient mechanical resistance to coalescence [25].

2.4. Outlook and Limitations

A key advantage of the present design is its compatibility with closed‐loop optimization. Because the holder requires no clamps, seals, or precision alignment, it can be shuttled by a simple robotic gripper between a pipetting robot and an imaging station or plate reader. Automated image analysis (e.g., Hough‐based or machine‐learning segmentation) could then provide droplet size distributions, polydispersity, and coalescence indices at t = 0 and at scheduled time points. Such metrics would define objective functions for iterative, data‐driven optimization. In this envisioned workflow, one could execute automated “generate–image–decide” cycles: dispense a plate of formulations, centrifuge‐emulsify, acquire images immediately and after incubation, and allow an optimizer to propose the next 96‐condition batch. Closed‐loop strategies of this kind, already transformative in high‐throughput chemistry and materials science, are well suited here because each spin yields a full plate of internally consistent data, enabling multi‐objective targets (e.g., maximize stability while minimizing polydispersity) to be addressed without leaving the robotic workcell.

In this workflow, the stability readout provides a standardized basis for comparing many formulations under identical preparation, storage, and imaging conditions. Each well contains 200 $\mu \mathrm{L}$ of liquid while the dispersed phase corresponds to several thousand droplets under the conditions used here, so the assay captures collective droplet survival and coalescence trends rather than isolated single‐droplet events. Because confinement, meniscus, and evaporation effects are shared across wells, the platform is well suited for relative ranking of oil/surfactant/salt compositions. However, promising candidates identified in the plate format should be validated at larger volumes when bulk storage behaviour, product‐specific rheology, or long‐term macroscopic stability is relevant.

Other limitations are related to the current hardware implementation. The most frequent emulsification failures are associated with needle clogging or bending, which can reduce reproducibility across plates. Clogging can be mitigated by introducing additional cleaning or prefiltration steps, whereas bending is best addressed through an automated or guided insertion workflow that prevents operator‐induced deformation. The current implementation is specifically designed for standard 96‐well plates, and direct miniaturization to 384‐well or higher‐density formats is constrained by the geometry of commercial needles. The standard needle hubs used here have an outer diameter of approximately 8 mm, whereas 384‐well plates have substantially smaller well spacing and a typical well diameter of $\sim$3.6 mm. As a result, commercial needles with hubs cannot be arranged in a one‐needle‐per‐well configuration at this pitch, meaning that higher‐density formats would require a redesigned support in which hubless capillaries are fixed directly into a plate‐compatible array, rather than a simple geometric rescaling of the present holder. This limitation concerns format miniaturization, not the achievable sample throughput: in a typical centrifuge equipped with a multiwell‐plate rotor, four 96‐well plates can be processed in parallel, corresponding to up to 384 emulsions per centrifugation step. Since centrifugation requires only minutes, the practical throughput is limited by liquid handling, imaging, and image analysis rather than by droplet generation itself. Increasing the volume per condition would similarly require a redesigned holder compatible with larger receiving formats, such as deep‐well 96‐well plates, 48‐well plates, 24‐well plates, or tube‐rack formats. In such implementations, the centrifugal capillary‐dispensing principle would remain unchanged, but capillary length, immersion depth, loaded volume, centrifugation conditions, and imaging geometry would need to be re‐optimized.

Although this study used siloxane surfactants in silicone oil and PFPE‐based surfactants in fluorinated oil, the platform is not intrinsically limited to these oil/surfactant systems. These formulations were selected primarily to validate the two density‐dependent configurations. The underlying mechanism is independent of surfactant chemistry and relies on centrifugal forcing of a dispersed phase through a capillary into an immiscible continuous phase, followed by capillary‐controlled pinch‐off. Therefore, broader oil/surfactant systems should be compatible with the platform in principle, provided that the phases remain immiscible, the formulation does not clog the capillaries, and the surfactant adsorbs sufficiently rapidly to stabilize the newly generated interface. However, the stable pinch‐off window remains formulation specific and must be re‐established for each new oil/surfactant system. In particular, excessive centrifugal forcing can shift droplet formation from capillary‐controlled pinch‐off to jetting, while the position of this boundary depends on interfacial tension, viscosity ratio, wetting behaviour, density contrast, needle geometry, and immersion depth. To rationalize these dependencies, we provide a first‐order operating map based on capillary number in Supplementary Note S3, where the stable operating window is estimated from the balance between capillary‐controlled pinch‐off, jetting, and practical dispensing time. These maps should be interpreted as geometry‐informed guidelines for the tested holder designs and liquid systems, rather than as universal limits; future work should extend them through systematic experimental sweeps of oil viscosity, interfacial tension, needle dimensions, centrifugation speed, and immersion depth.

3. Conclusion

We developed the centrifugal emulsification platform that enables parallelized droplet generation directly in standard well plates. Within minutes, dozens of emulsions can be generated under identical conditions, and the format is inherently suited to closed‐loop optimization. This approach opens opportunities for systematic studies of emulsion stability, rapid formulation screening for biochemical assays, and the discovery of sustainable alternatives to PFAS‐based systems. Its simplicity and compatibility with standard laboratory equipment make it broadly applicable across soft‐matter research and formulation science.

4. Experimental Section

4.1. Materials

To evaluate the application of the vertical holder configuration in emulsion generation, 1 cSt poly(dimethyl‐siloxane) (ABCR GmbH, Germany) was used as the continuous phase, with Milli‐Q water or NaCl solutions of varying concentrations as the dispersed phase. Emulsions were stabilized using the following siloxane‐based surfactants: ABE‐3642, CMS‐221, CMS‐222, CMS‐626, DBE‐224, DBE‐311, DBE‐621, and ABP‐263 (Gelest, USA). For experiments involving the tilted holder configuration, the continuous phase consisted of TMC 7500 (TMC Industries, USA), while Milli‐Q water served as the dispersed phase stabilized with a fluorosurfactant (RAN Biotechnologies, USA). The stability of fluorinated emulsions was further examined in the presence of selected PFPE‐PEG‐PFPE surfactants (Creative PEGWorks, USA): (i) PFPE 5k, PEG 300, PFPE 5k, and (ii) PFPE 5k, PEG 1500, PFPE 5k. All emulsions were prepared and stored at room temperature (20).

4.2. Holder Fabrication and Assembly

The holders were designed using AutoCAD and printed using a Prusa MK4 MMU3 fused deposition modeling (FDM) printer with a gyroid infill pattern at 10% density. The printing material was Ultrafuse PLA Tough (filament diameter: 1.75 mm, natural color). The vertical configuration measured 128 $\times$ 88.3 $\times$ 32.9 mm³ (length $\times$ width $\times$ height), and the tilted configuration measured 128 $\times$ 88.3 $\times$ 29.3 mm³. Both designs contained 96 evenly spaced cylindrical bores, each 7 mm in diameter, corresponding to the layout of a standard 96‐well plate (Supplementary Note S1).

Blunt‐ended stainless‐steel needles with a length of $12.7\mathrm{mm}$, an inner diameter of $60\mu \mathrm{m}$, and an outer diameter of $190\mu \mathrm{m}$ were manually inserted into the bores and held in place by a tight frictional fit. The plate wells were cylindrical reservoirs with an internal diameter of $6\mathrm{mm}$ and a height of $11.2\mathrm{mm}$. After assembly, the needle outlet was positioned approximately $2\mathrm{mm}$ above the bottom of the well in the vertical configuration and approximately $3\mathrm{mm}$ above the bottom of the well in the tilted configuration. In the tilted design, each needle was mounted at an angle of 30 relative to vertical to facilitate smooth droplet pinch‐off in denser oil phases. No adhesives or mechanical locks were used; the holder rested on top of the plate with sufficient friction and weight to maintain alignment during centrifugation.

4.3. Emulsion Imaging

Bright‐field images were acquired using a SpectraMax i3x plate reader equipped with a MiniMax 300 imaging module (Molecular Devices, USA) at 20 . Images were recorded in transmitted light with a 5 ms exposure time. The z‐position was adjusted to capture the first droplet layer: for emulsions in oils less dense than water, imaging was performed at the droplet layer resting on the glass–emulsion interface; for emulsions in oils denser than water, imaging was performed at the droplet layer located at the emulsion–oil interface.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smtd70983‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.