Template-in-template assembly nanostructured microspheres for high performance chromatography

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: minor_revision

Abstract
Concurrent precision architecture of morphology and nanostructure in mesoporous microspheres is pivotal for high performance separations. Herein, we develop a template-in-template assembly nanostructuring (TiTAN) strategy to precisely synthesize monodisperse microspheres with ordered mesoporous nanostructure. Microfluidic droplet templating ensures uniform particle morphology (CV=3%), while structure-directing agents within droplets enable tailored pore configurations, including 2D hexagonal, body-centered cubic, face-centered cubic, and cubic double gyroidal mesostructures. Through regulating hydrothermal conditions, structural parameters can be fine-tuned with 2 Å spatial resolution. By extending this manufacturing capability to a variety of material chemistries, chromatographic materials can now be de novo architectured with rationales, with the performance demonstrated by the solution of a classical separation challenge: resolving critical pairs (whose selectivity, α, infinitely approaching to 1), and with the shortest possible time. Beyond separation medium, the TiTAN strategy also suggests a route towards general synthesis of porous material with precision macroscopic morphology and microscopic nanostructure.

Introduction
Mesoporous materials, known for their suitable pore size for mass transfer and large surface area for molecular adsorption/partitioning, have emerged as an important separation medium and been widely used in the fields of chemical separation, environmental analysis, and pharmaceutical purification. Given the essential demands of separation performance in terms of resolution, efficiency and speed, it is of great significance to precise design the structure of these separation medium, which involves the simultaneous control of both the external morphology and internal mesoporous nanostructure of microspheres. The morphology of microspheres (including shape, size, and size distribution) determines the laminar flow field, which affects eddy dispersion of the fluidic transportation process. On the other hand, the mesoporous structure, including pore size, surface area, pore volume and pore configuration, is highly relevant to the intraparticle mass transfer process, which collectively contributes to the separation kinetics. Therefore, the development of a robust synthesis strategy enabling precise control over both the microsphere’s morphology and mesopore’s structure is of paramount significance for high-performance chromatography.

Current solution-phase synthetic methodologies can afford fine control of the morphology and dispersity of mesoporous nanoparticles, however, pushing this level of controllability up to micrometer-scale remains challenging. During the formation of microspheres, the heterogeneous nucleation process and growth kinetics, as well as insufficient colloidal stabilization, result in a broad size distribution and non-uniform morphology. To address these issues, strategies such as polymer-seed swelling, core-shell self-assembly, hard templating, and emulsion-based synthesis have been developed to produce mesoporous microspheres with tailored shape and narrow dispersity. Nevertheless, the pore networks in these materials typically emerge from stochastic processes—phase separation or random condensation of precursors—which fundamentally restricts precise control of mesoporous nanostructure, thereby compromising chromatographic performance and applicability.

Over the years, various strategies have been introduced to precise control the orderliness of pore structure of microspheres. The development of ordered mesoporous silica (e.g., SBA-15 and MCM-41) has realized uniform nanostructure and long-range periodicity through cooperative assembly of surfactant templates and guest species, or by iterative nanocasting of mesoporous solids. Crystalline frameworks, i.e., metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), exploit reversible bonding (metal-ligand coordination in MOFs; dynamic covalent bonds in COFs) to facilitate error correction and network rearrangement, thereby affording thermodynamically stable, highly ordered pore lattices. Nevertheless, the formation of these ordered nanoporous materials is based on the orientational growth of the nanostructure units, which inevitably results in irregular or crystalline morphologies rather than “real spheres”. The difficulty, if not completely infeasible, of assembling such non-spherical materials into uniform and permeable beds, as required to achieve satisfactory separation kinetics, limit their chromatography applicability. Consequently, the development of methodologies which can enable precise tuning of ordered mesoporous structures while maintaining spherical and uniform morphology, remains an unmet synthetic challenge.

Herein, we report a template-in-template assembly nanostructuring (TiTAN) strategy based on a microfluidic platform, in order to realize precision synthesis of microspheres with uniform spherical morphology and well-defined mesoporous structure. The sphere morphology and monodispersity are precisely modulated by microdroplet templating, while the ordered mesopores inside are independently structured via colloidal templating. By extending this manufacturing capability to a variety of spherical sizes, pore configurations and material chemistries, chromatographic media can now be de novo architectured with rationales, with separation performance realized by design. These advantages have been demonstrated by the solution of an old, but long-pursued separation challenge: resolving critical pairs (whose selectivity factor, α, infinitely approaching to unit), and with the shortest possible time.

Results
Monodisperse microspheres with ordered mesoporous structure were synthesized via a TiTAN strategy based on droplet microfluidic technology. Specifically, the dispersed phase consisted of an acidic volatile organic solvent containing amphiphilic surfactant (e.g., Pluronic PEO-b-PPO-b-PEO) as structure-directing agent (SDA) and hydrolysate. Using the previously developed microfluidic platform, this phase was emulsified into uniform micro-sized droplets with fluorinated oil (HFE-7500) containing fluorosurfactants (e.g., perfluoropolyether-b-polyethylene, glycol-b-perfluoropolyether) as the continuous phase. Following collection in FC-40 carrier oil (ρ=1.85 g/cm3), the generated droplets floated to the liquid-air interface, enabling fast solvent evaporation. During evaporation, the progressive concentration of amphiphilic surfactants and precursors induced the formation of liquid-crystalline phase, while simultaneously promoting the condensation of surrounding silica species. In this process, the droplets served both as microscale reaction vessels and morphology templates for microsphere formation. Ultimately, monodisperse microspheres with well-defined mesoporosity and regular spherical morphology were prepared, followed by subsequent hydrothermal treatment and calcination to remove organic residues and enhance framework crosslinking.

As an example, silica microspheres with 2D hexagonal (2D-hex) mesostructure were prepared using P123 triblock copolymer as SDA and tetraethoxysilane (TEOS) as reaction precursor. As a result of emulsification in microchannels, monodisperse droplets were generated with a diameter of 12.8 µm (CV=2.6%), and the corresponding silica microspheres were obtained following solidification after solvent evaporation. Large-scale field emission scanning electron microscopy (FE-SEM) revealed that the silica microspheres have an average diameter of 3.9 µm with a narrow size distribution (CV=2.4%). Following hydrothermal treatment at 130 °C for 24 h, the microsphere size slightly increased to 4.2 µm, and finally decreased to 4.1 µm after calcination at 550 °C for 6 h. The microsphere size distribution remained monodisperse throughout the post-treatment process (CV=2.8% after hydrothermal treatment and 3.3% after calcination). Instead of appearing as a smooth spherical surface, the calcined silica microspheres exhibited as sphere-like polyhedrons composed of multiple planes, which were found to consist of parallel arrangements of cylindrical silica rods. Characterized by transmission electron microscopy (TEM), highly ordered mesoporous frameworks with pore size of approximately 8 nm were observed along the [110] facet. Small-angle X-ray scattering (SAXS) analysis showed that the silica microspheres have three distinct and sharp diffraction peaks indexed to the 100, 110, and 200 reflections, respectively, demonstrating the formation of an ordered 2D-hex mesoporous configuration (p6mm space group). The nitrogen adsorption-desorption isotherm of the silica microspheres exhibited a representative type IV curve with a sharp hysteresis loop at P/P0=0.6−0.7. The pore size has a narrow distribution at 7.7 nm, further confirmed the well-defined mesoporosity of the material.

While various types of ordered mesoporous structured materials have been designed and synthesized, hexagonal and cubic symmetries represent the most prominent and well-studied classes. By adjusting the SDAs and post-treatment conditions, the TiTAN strategy enables the precision construction of these typical mesostructures in monodisperse spherical materials. Common mesostructures with cubic symmetry include body centered cubic (bcc), face centered cubic (fcc) and cubic double gyroidal (cdg) structures. Bcc configuration can be formed by SDAs with a high hydrophilic/hydrophobic (VH/VL) volume ratio as templates. The silica microspheres with a Im3m cubic configuration were synthesized by using F127 as SDA (Im3m space group). The microspheres thus synthesized exhibited a classical type IV nitrogen adsorption-desorption isotherm with an H2-type hysteresis loop, characteristic of cage-like mesostructure. The pore size calculated by BJH model showed a spherical cage diameter of approximately 13.9 nm, with the window size between cages measured less than 4 nm.

Using high-molecular-weight block copolymer, PEO-b-PMMA as the SDA, fcc configuration can be synthesized with Fm3m mesostructure and large unit cell, as well as uniform and periodic cage-like pore frameworks. The nitrogen adsorption-desorption isotherm demonstrated a steep capillary condensation in the relative pressure from 0.89 to 0.95 and an H1-type hysteresis loop, indicating the generation of uniform large mesopores. Calculated from adsorption and desorption branches based on BJH model, the pore size reached as high as 20.9 nm, while the window size was approximately 12 nm.

One-step microfluidic synthesis of cdg configuration, which is one of the most complex mesostructures, can be challenging. Nevertheless, by first synthesizing silica microspheres with a 2D-hex configuration, followed by a solvothermal post-treatment (80 °C for 72 h in hexane), the mesostructure can be converted into cubic double gyroid (Ia3d space group). After calcination, the silica microspheres exhibited long-range ordered pore structures, distinct from the original 2D-hex mesostructure, with a pore size of 8.4 nm.

Apart from pore configurations, the pore structure parameters of silica microspheres—including pore size, specific surface area, and pore volume—can be finely tuned by adjusting the hydrothermal conditions and the volume of SDA introduced. With silica microspheres templated by P123 as an example, the nitrogen adsorption-desorption experiment revealed that the pore size can be tuned from 4.4 to 8.4 nm with the increase of hydrothermal temperature and treatment time, at a pore size adjustment precision of 0.2 nm. This angstrom-level pore size was found reproducible across three independent batches, with a CV of 2.1%, demonstrating good reproducibility of pore size tuning via hydrothermal treatment. The full width at half maximum (FWHM) of the mesopores varied from 1.1 to 1.5 nm, showing consistent narrow size distribution across different hydrothermal conditions. The tunability of mesopore size is based on a general property of non-ionic surfactants: the PEO units undergo partial dehydration at higher temperature, which leads to the volume reduction of the hydrophilic corona and the increase in the micelle size. The enlargement of micelle size manifested in two aspects: on one hand, scattering peaks shifted to the larger d-spacing, as observed in SAXS spectra, accompanied by an increase in the unit cell parameter from 8.72 to 10.26 nm; on the other hand, the specific surface area and pore volume were proportionally increased by the micelle size. As presented in Supplementary Table 1, the specific mesopore surface area increased from 410 to 596 m2/g with prolonged hydrothermal treatment time and elevated temperature, meanwhile the pore volume increased significantly from 0.37 to 0.97 cm3/g. The pore structure parameters of large mesoporous cubic Fm3m silica microspheres, templated by PEO-b-PMMA, exhibited similar hydrothermal regulation effects.

The effect of SDA volume on pore structure parameters has also been investigated. With the mass ratio of P123/TEOS (marked as P/T) raised from 29.5 to 46.3 wt%, the silica microspheres’ isotherms increased in uptake capacity in a stepwise manner. Meanwhile, the pore volume resulted from mesopores showed a linear increase from 0.65 to 1.08 cm3/g, while the pore size expanded from 6.1 to 9.2 nm accordingly. Calculated from d-spacing, the unit cell parameter increased from 9.9 to 10.5 nm when P/T increased from 29.5 to 33.7 wt%, and maintained constant between 33.7 to 46.3 wt% P/T. Based on the observed changes in lattice parameters and pore size, it is hypothesized that with the increase of template amount, the micelle size increased first and quickly reached an equilibrium. Further increasing the template amount only changed the hydrophobic portion of micelles, which consequently led to a gradual increase in pore diameter and pore volume, and a reduction in pore wall thickness (from 3.8 to 1.3 nm). Excessive amounts of templates can lead to phase transition or disordered micelle assembling, resulting in significant deviations in pore structure parameters from the above-mentioned trends.

It needs to be highlighted that, during fine-tuning of the internal mesoporous structure, the external morphology of microspheres remained precisely regulated by the droplet templates. While tuning pore structures, all silica microspheres exhibited both monodisperse size distribution and well-defined spherical morphology. Besides, the effect of SDA volume on the microspheres’ sphericity is strongly correlated with the symmetry dimensionality of the ordered mesoporous structure. For instance, in microspheres with 3D symmetric mesoporous configurations—such as fcc templated by PEO-b-PMMA—the external morphology remained unchanged while increasing SDA volume, maintaining a good sphericity. In contrast, microspheres with 2D symmetric mesoporous configurations—such as the 2D-hex mesostructure—underwent a significant morphology transition from spherical to polyhedral, and eventually spindle-like, as the introducing amount of P123 increased. On the other hand, the internal mesoporous structure also remained independently regulated by the colloidal templates. While the silica microsphere size was tuned from 3 to 5 µm using droplets of different sizes as templates, the pore configuration and pore size, pore volume, and specific surface area all remained consistent, which were independent of the outer morphology. These results demonstrated that the template-in-template assembly nanostructuring strategy can enable precision and independent regulation of external morphology and internal pore structure, providing a versatile platform for rational design and manufacture of mesoporous microspheres.

In the development of separation methods for multicomponent molecular systems, resolving critical pairs—the two components in a mixture with the lowest resolution—is always a challenging task. The core parameter for evaluating the separation performance of a critical pair is resolution (Rs), as defined by Eqs. 1–3. Baseline separation is quantitatively defined as Rs ≥ 1.5. While increasing the selectivity factor (α) by stationary phase modification may be an effective way to improve resolution, it involves stationary phase chemistry development, optimization and screening, and inevitably accompanied by changes in the retention behavior of other components and/or the introduction of unexpected secondary interactions. Therefore, increasing the retention factor (k) and separation efficiency (N) becomes crucial for pushing limit of the resolution of the critical pairs. According to separation chemistry theory, both k and N are highly associated with the structure of the packing material, including particle size, size distribution, surface area and pore architecture. As demonstrated above, the TiTAN strategy makes it possible to rationally design and precision manufacture of the nanostructured microsphere materials, which provides an enabling way to simultaneously optimize k and N to reach an ideal resolution for critical pairs and within a reasonable time frame. For this purpose, we developed monodisperse silica microspheres with 2D-hex mesoporous structure as a separation medium. Such a nanostructured material possesses straight pore channels and uniform pore size due to the 2D-hex configuration, which confers a synergetic advantage: unified diffusion pathways enhancing mass transfer of both ligand molecules during chemical bonding, and analyte molecules during chemical separation. The performance comparison of different ordered mesoporous structures demonstrated that, as expected, the 2D-hex structure indeed exhibited high retentivity and efficiency.

The TiTAN synthesized silica microspheres exhibited a monodisperse size distribution with a mean diameter of 5.0 µm (CV=4.7%). After functionalization with monomeric C18 ligands, the microspheres were packed into a 100 µm i.d. column (denoted as OMP, for ordered mesoporous particle). The OMP column demonstrated high mechanical stability under prolonged high-pressure conditions (65 MPa), maintaining structural integrity without particle fragmentation or bed deformation. To evaluate the chromatographic performance of the OMP column, a reference column packed with conventional porous particles (denoted as TPP, for totally porous particle) of identical size (5.0 µm mean diameter, CV=11.8%), column dimensions and bonding stationary phase was used as a benchmark for comparison.

Separation performance was evaluated under isocratic conditions (60:40 v/v ACN/H2O) using a standard mixture of thiourea (dead-time marker) and alkylbenzenes (methyl-, ethyl-, propyl-, and butylbenzene). As shown in Fig. 4a, the OMP column exhibited significantly stronger retention than the TPP column: butylbenzene eluted with ~40% longer retention time on OMP. The retention factor on OMP increased more steeply with analytes’ hydrophobicity, reaching k=19.2 (butylbenzene) on the OMP column versus k=7.8 on the TPP column. Elemental analysis and nitrogen adsorption-desorption experiment revealed that OMP supports a higher C18 ligand density (2.65 μmol/m²) compared to TPP (<2.30 μmol/m²), and offers a larger specific surface area (500 m²/g versus 350 m²/g), respectively. The combination of increased ligand coverage and accessible surface area concordantly raised the phase ratio, enhancing the retentivity of the ordered mesoporous separation material. The raised phase ratio also resulted in a high loading capacity of the OMP material.

Figure 4c presented the reduced plate height (h) versus reduced linear velocity (v) for butylbenzene on the OMP and TPP columns. Without correction for extra-column band broadening, the minimum reduced plate height (hmin) is 2.78 for the OMP column—only 68% of that of the TPP column (hmin=4.08), representing almost 50% enhancement of efficiency. The coefficients a, b, and c (reduced form of A, B, and C, respectively) are listed in Supplementary Table 7, by fitting the data to Knox equation. The a-term for the OMP column is merely 62% of that for TPP column, indicating that the narrowed particle size distribution (due to the monodisperse spherical morphology) significantly reduces eddy diffusion, the major contribution of band broadening. Despite a smaller average pore diameter, the OMP column still exhibited a reduced c-term (by 56%) than the TPP column. This demonstrated that the ordered 2D-hex pore configuration effectively reduced intraparticle mass-transfer resistance and accelerated partition equilibration of the analytes between the mobile and stationary phases, leading to the pronounced enhancement of separation efficiencies.

An additional set of totally porous particles (denoted as hTPP, for homemade totally porous particle) was intentionally synthesized to closely matched to OMP in terms of particle size, monodispersity, and pore size. As shown in Supplementary Table 7 and Supplementary Fig. 21, OMP exhibited pronouncedly higher efficiency (2.78 vs. 3.82 for hmin) and higher retentivity (19.2 vs. 4.0 for k) than that of hTPP, both outperforming the conventional totally porous counterparts.

Benefiting from monodisperse particle size distribution, higher accessible surface area, and straight and uniform pore configuration, both k and N of the OMP column increased, and the Rs for critical pairs can be improved as a natural result. To quantitatively evaluate the resolution enhancement, a modified kinetic plot was constructed. A hypothetical critical pair whose selectivity factor set as low as 1.01 was chosen as probing compounds, while the column efficiency was kept same. As shown in Fig. 4d, calculations show that the resolution achievable on the OMP column is higher than that on the TPP column over the entire time range. For short time runs (<100 min), the OMP column’s resolution is slightly higher; however, as the analysis time increases and more theoretical plates are generated, the advantage of the OMP column becomes increasingly significant. Specifically, the OMP column achieves a 14% higher Rs than TPP column at 1000 min, and the Rs enhancement becomes 33% at 10,000 min. From a separation speed point of view, in order to reach the same Rs (e.g., Rs=1.1), the conventional TPP column needs 4380 min, whilst the OMP column needs only 1178 min, ~3/4 shortened runtime, indicating a significantly faster separation speed while realizing the same level of resolution, by using ordered mesoporous separation materials.

To demonstrate the potential of the precisely constructed ordered mesoporous separation media for high performance chromatography, a series of challenging critical pairs was chosen for investigations. Polycyclic aromatic hydrocarbons (PAHs) are a class of homologous compounds composed of multiple benzene rings with similar molecular weights. Due to the absence of π–π interaction or spatial stereoselectivity of the classical monomeric C18 phase, achieving effective resolution of PAHs solely based on the hydrophobicity of C18 chemistry remains a significant challenge. On the OMP column, sixteen standard PAHs were effectively separated under isocratic conditions. The critical pair, benzo(b)fluoranthene and benzo(k)fluoranthene, realized ideal separation with Rs=1.77, while the TPP column only reached a low resolution of Rs=0.68. Impressively, indeno(1,2,3-cd)pyrene and benzo(g,h,i)perylene have been resolved on the OMP column with Rs=0.92, while on the TPP column the critical pair cannot be distinguished at all.

Isomers, such as xylene, possess identical molecular weights and almost identical hydrophobic characteristics (Δlog P≈0.08 for xylene), with merely slight difference in dipole moment and spatial configuration. In previous studies, efficient separation of xylene isomers has required the incorporation of additional interactions to improve selectivity, or the use of materials with pore sizes matching isomers dimensions, such as molecular sieves or MOFs, for spatial resolution. In this study, under isocratic elution (50:50 v/v ACN/H2O) on the OMP column, o-xylene was effectively baseline-separated from p- and m-xylene with Rs=1.78 and 2.45, respectively, while p- and m-xylene also realized resolution (Rs=0.96). In contrast, running the mixture on the TPP column, only o-xylene can be resolved, while no resolution was identified for the p- and m-xylene.

Isotopologue separations—where analytes differ only in constituent element weight, and their dipole moment and hydrophobicity are virtually indistinguishable—represent one of the most stringent tests of chromatographic resolution. Typically, efforts on separating such pairs have required either extraordinarily long columns or recycling chromatography in order to generate sufficiently high numbers of theoretical plates, which is a time-consuming and labor-intensive process. As shown in Fig. 5g-i, with OMP medium, however, under isocratic conditions (20:80 v/v ACN/H2O), the 15 cm short OMP column achieved a satisfactory Rs=1.51 for benzene/benzene-d6 within 55 min, whereas the TPP counterpart delivered only partial separation (Rs=0.78). For the even more challenging dibromobenzene/dibromobenzene-d4 pair, where the TPP column failed to recognize the isotopologues at all, the OMP column, within 250 min, successfully split the critical pair (Rs=0.67).

Methods
Synthesis of ordered mesoporous silica microspheres
The ordered mesoporous silica microspheres were synthesized based on the TiTAN strategy. Typically, the continuous phase was fluorinated oil-HFE-7500 with 1 wt% PFPE-b-PEG-b-PFPE as surfactant, and a sol-gel reaction precursor containing SDAs was applied as the dispersed phase. The microfluidic platform enabled droplet generation at a super throughput of 240,000 Hz per chip, resulting in a production yield of approximately 10^9 microspheres per hour.

To prepare the sol-gel reaction precursor, 400 µL TEOS was hydrolyzed in 268 µL HCl (0.1 M) under vigorous stirring at 1000 rpm for 60 min. Then, the hydrolyzed TEOS solution was mixed with a porogen solution tailored to the target pore configuration. For 2D-hexagonal configuration, the porogen solution was 1890 µL acetonitrile containing 145 mg P123. For cubic double gyroidal configuration, the porogen solution was 1822 µL acetonitrile containing 190 mg P123. For body centered cubic configuration, the porogen solution was adjusted to 1821 µL acetonitrile containing 161 mg F127. For face centered cubic configuration, the process required initial hydrolysis of 400 µL TEOS in 750 µL HCl (0.3 M) for 30 min before mixing with a porogen solution consisting of 74.4 mg PEO-PMMA and 6215 µL acetonitrile.

For droplet generation, the dispersed phase (36 µL/min) and continuous phase (120 µL/min) were injected into the microfluidic chip by syringe pumps. The generated droplets were collected in a petri dish containing FC-40 fluorinated oil, and the solvent evaporation was conducted at 25 °C. The solidified microspheres were transferred to an autoclave containing 1 M HCl solution and placed in an oven for 24 h at 130 °C. After that, the microspheres were calcined in air at a rate of 1 °C/min to 550 °C, and kept at 550 °C for 6 h to completely remove SDAs. For certain configurations, the hydrothermal treatment varied slightly – 80 °C treatment for 72 h in hexane for cubic double gyroidal configuration, and 80 °C treatment for 24 h in hexane for body centered cubic configuration.

Synthesis of ordered mesoporous microspheres with other substances
For the synthesis of ethane-bridged silica microspheres, the dispersed phase was prepared by first mixing 330 µL 1,2-bis(triethoxysilyl)ethane with 268 µL HCl (0.1 M) under stirring for 1 h, followed by addition of 1820 µL acetonitrile containing 144 mg P123. After hydrothermal treatment, the SDAs were removed by Soxhlet extraction with ethanol at 80 °C for 24 h. Phenyl-bridged silica microspheres were synthesized by mixing 153 mg of 1,4-bis(triethoxysilyl)benzene with 150 mg P123, 300 µL HCl (0.1 M), and 1350 mg methanol under stirring for 5 min, the SDAs were also removed by Soxhlet extraction with ethanol at 80 °C for 24 h. Titania microspheres were prepared by mixing 400 µL titanium(IV) butoxide, 120 µL acetylacetone, and 200 µL HCl (2 M) under stirring for 5 min, followed by addition of 2180 µL acetonitrile containing 122 mg P123. After droplet solidification, titania microspheres were calcined in air at a rate of 1 °C/min to 350 °C, and kept at 350 °C for 6 h to totally remove SDAs. Zirconia microspheres were obtained by mixing 564 mg zirconium (IV) butoxide, 120 µL acetylacetone, 800 µL ethanol, and 400 µL HCl (2 M) under stirring for 30 min, followed by addition of 2180 µL acetonitrile containing 122 mg P123, the calcination conditions were identical to that of titania microspheres.

Stationary phase bonding
The TiTAN-synthesized silica microspheres were bonded with octadecylsilyl stationary phase. Specifically, 50 mg microspheres were dried at 120 °C for 12 h. Then, 0.12 mmol dimethyloctadecylchlorosilane and 0.24 mmol 2,6-lutidine were dissolved in 5 mL dry toluene. Dry microspheres were dispersed in the mixed solution and reacted under N2 at 80 °C for 40 h. Finally, the microspheres were thoroughly washed with dichloromethane, methanol, and water.

Chromatographic column preparation
TiTAN-synthesized microspheres and commercial packing materials were chosen to pack capillary liquid chromatography columns. The capillary columns were prepared by high-pressure slurry packing method. Briefly, a single perfusive particle with ~100 μm in diameter was tapped into one end of the capillary (100 μm i.d. and 365 μm o.d.) to be packed, which was used as the outlet frit of the column. Then, from the other end of the capillary, a slurry of the packing material was introduced and packed under a high pressure up to 6000 psi. When the packing was finished, another single particle was tapped into the inlet of the capillary.

Evaluation of chromatographic performance
Chromatographic columns were evaluated on an Ultimate 3000 RSLC nano liquid chromatography system (Thermo Scientific). Alkylbenzenes were used as samples to evaluate separation performance (toluene, ethylbenzene, propylbenzene and butylbenzene), and thiourea was added to determine the column dead-time. The samples were injected by a 4 nL Valco nanovolume injector, eluted under the isocratic condition with a mobile phase of 60/40 v/v ACN/H2O, and detected by a UV detector at the wavelength of 214 nm. The Knox curve was constructed to eliminate the effect of particle size, which can be described as:

h = a v^(1/3) + b/v + c v (Eq. 4)

The reduced plate height (h), and reduced linear velocity (v), were calculated by:

h = H / dp (Eq. 5)

v = u * dp / Dm (Eq. 6)

Where dp is the particle size of the packing material, and Dm is the diffusion coefficient of the analyte. The diffusion coefficient of butylbenzene referred to the database established by the previous study. Additionally, a modified version of the kinetic plot was constructed to quantitatively evaluate the separation resolution potential. Typically, the standard kinetic plot was obtained by transforming a series of experimentally measured (u0, H)-data into a set of (t0, N)-data, using Eqs. 7–8:

N = (ΔP_max * K_v0) / (η * u0 * H) (Eq. 7)

t0 = (ΔP_max * K_v0) / (η * u0^2) (Eq. 8)

Where ΔP_max is maximal allowable instrument pressure. K_v0 is the permeability, η is mobile phase viscosity, u0 is linear velocity and H is theoretical plate height (calculated based on butylbenzene). In this work, the kinetic plot was constructed for a pressure limit of 60 Mpa. Further, the modified kinetic plot can be obtained by transforming (t0, N)-data into a set of (log(t0/Rs), Rs)-data, replacing N with Rs using Eq. 1 (as already introduced in the main text). Wherein, a hypothetical critical pair whose selectivity factor (α) set as low as 1.01 was chosen as probing compounds, while the column efficiency was kept same. The retention factor (k) of the hypothetical critical pair was equal to that of butylbenzene (19.2 for OMP and 7.8 for TPP).

Separation of critical pairs
All chromatographic separations were performed under isocratic elution conditions at a flow rate of 800 nL/min with UV detection at 210 nm. For polycyclic aromatic hydrocarbons separation, 70/30 v/v ACN/H2O were applied as the mobile phase. For xylene isomers separation, 50/50 v/v ACN/H2O were applied as the mobile phase. For isotopologues separation, 20/80 v/v ACN/H2O were applied as the mobile phase.

Data availability
The data supporting the findings of this study are available within the paper. Data is available from the corresponding author on request. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present a novel strategy termed template-in-template assembly nanostructuring (TiTAN) for the precise\nsynthesis of monodisperse microspheres with ordered mesoporous nanostructures. They further demonstrate the potential\napplication of these materials in high-performance chromatography. While the work builds upon the authors’ prior\nmicrofluidic precision manufacturing approach (Angew. Chem. 2024), its key innovation lies in the simultaneous\narchitectural control over both morphology and nanostructure in mesoporous microspheres. From a fundamental\nnanoscience perspective, this represents an elegant and technically sound advancement.\nHowever, the claimed benefits of such mesoporous nanostructures for chromatographic separation remain unconvincing for\nseveral reasons:\nLimited Separation Efficiency: The plate height values (Figure 4C) indicate suboptimal separation efficiency, suggesting that\nthe mesoporous nanostructure does not confer meaningful improvements in chromatographic kinetics.\nLack of Demonstrated Selectivity: The separations shown in Figure 5 fail to achieve baseline resolution for the tested\nanalytes, raising doubts about any substantive enhancement in selectivity.\nDurability Concerns: The mechanically fragile nature of the surface mesopores casts uncertainty on the long-term stability\nand applicability of these materials for complex or high-throughput separations.\nGiven these limitations, the work, while methodologically interesting, does not presently meet the threshold for\ntransformative impact expected by high-profile interdisciplinary journals such as Nature Communications.\nMinor Comments:\nResolution Calculation (Figure 5b2): The reported resolution value of 1.66 appears inconsistent with the lack of baseline\nseparation in the chromatogram. The authors should verify their calculations.\nImproved Data Representation: A plot of logk versus carbon number would better assess whether the separation follows a\nreversed-phase mechanism, as such a relationship should yield a linear trend.", "reviewer_2": "The manuscript by Prof. Zhang and coworkers reports the synthesis of monodisperse microparticles with a mesoporous\nnanostructure for chromatographic applications. The preparation of silica microparticles begins with the use of microfluidics\nto generate monodisperse droplets containing TEOS, hydrochloric acid, and an amphiphilic polymer surfactant.\nSubsequently, rapid solvent evaporation from the droplets induces self-assembly of the surfactant into a liquid crystalline\nphase, while simultaneously promoting the condensation of silica in the hydrophilic domains. Finally, hydrothermal\ntreatment and calcination yield monodisperse silica particles with an ordered porous structure. By adjusting the SDAs and\npost-treatment conditions, the authors demonstrate that the nanostructures of the microparticles can be finely tuned. The\nauthors further showcase the application of these particles in chromatographic separation, where they exhibit high\nperformance compared to conventional silica particles.\nIn my opinion, this work is very interesting; however, several issues still need to be addressed prior to publication:\n1. Almost all of the SEM images lack surface details, making it difficult to extract useful information beyond particle size from\nthe presented images.\n2. Why did the authors choose to use the particles mentioned in the main text (CV = 4.7%, Figure S14) for the separation\nperformance comparison, rather than the 5 µm particles shown in Figure S12, which clearly have a more spherical\nmorphology and more uniform size?\n3. In the comparison of separation performance, there are too many variables between the two types of particles, such as\nuniformity, pore structure, and morphology, making it difficult to draw accurate conclusions.\n4. The authors are encouraged to compare the influence of different nanostructures on separation performance to provide\nmore comprehensive conclusions.\n5. It would be helpful for the authors to clarify the production yield of such porous particles prepared by microfluidics, as well\nas the sample loading capacity that can currently be separated using their chromatographic system.", "reviewer_3": "The authors have presented a method for synthesizing mesoporous particles with a well-defined internal structure using a\nmicrofluidic method they refer to as TiTAN. This method is especially exciting for uses in chromatography, as the authors\nhave demonstrated in their measurements showing superior separations of small molecules using particles with different\ninternal structures and similar macroscopic (~4 µm) sizes. In my opinion, these are significant results in terms of the\nsynthesis and application of the particles in an incredibly useful way -- and likely to be highly impactful. In fact, I can think of\nseveral more applications of these particles to fundamental scientific studies that extend beyond chromatography.\nNevertheless, as I am not an expert in chromatography, my review of this manuscript focuses exclusively on the authors'\nclaims regarding the well-defined internal structure of the particles as probed using small-angle X-ray scattering (SAXS) and\ngas adsorption measurements. SAXS is one of the most appropriate techniques for measuring structure in systems such as\nthis. Gas adsorption measurements similarly provide a good estimate of the pore size, and demonstrate the authors can tune\nthis parameter with great precision. I have a few questions and concerns regarding these results, although overall I\nbelieve they strongly support the authors' conclusions.\n(1) A comment: I would appreciate if the authors would label the relative peak positions (in terms of scattering vector Q)\nrelative to the fundamental peak (Q*) in their figures, rather than indexing the reflections. While this information is redundant,\nit would help the reader interpret these results. The authors should indicate each peak perhaps with an arrow of some sort.\nI realize this request may seem odd, but indexing the peaks this way makes it much easier for the reader to assess the quality\nof the mesopore structure. This is commonly done in the block copolymer community (see for example Chapter 5 in Basic X-\nray Scattering for Soft Matter by de Jeu).\n(2) In some samples, for example the Ia-3d/gyroid structures, the higher order reflections are quite weak (see Fig. 2g). How\nare the authors identifying peaks, and can they offer some insight into the relatively weak signals for higher values of Q?\n(3) The authors show extraordinary control over the pore size (Fig. 3b). I assume these results are reproducible, but I wonder\nif the authors can comment on the variability between pore sizes between independent batches of particles? I may have\nmissed this, but I cannot find in the manuscript or SI how many independent trials were performed. Were the nitrogen\nadsorption-desorption measurements performed on independent batches? Is the high tunability of pore size that the authors\ndemonstrated within a single batch of the particles? Etc. Some clarity regarding these points is necessary."}, "author_response": "Response to Reviewer Comments\nReviewer #1:\nThe authors present a novel strategy termed template-in-template assembly nanostructuring (TiTAN)\nfor the precise synthesis of monodisperse microspheres with ordered mesoporous nanostructures. They\nfurther demonstrate the potential application of these materials in high-performance chromatography.\nWhile the work builds upon the authors’ prior microfluidic precision manufacturing approach (Angew.\nChem. 2024), its key innovation lies in the simultaneous architectural control over both morphology and\nnanostructure in mesoporous microspheres. From a fundamental nanoscience perspective, this\nrepresents an elegant and technically sound advancement.\nWe sincerely appreciate your positive and insightful comments regarding our work. Your encouraging\nremarks are a strong motivation for our research. In response to your comments, we have made the\nfollowing revisions:\n(i) We have re-optimized the methods and re-performed the separations to achieve baseline\nresolution of the critical pairs, and updated the chromatography results accordingly in Figure 5.\n(ii) We have performed ultrahigh pressure experiments to evaluate the mechanical stability of ordered\nmesoporous particles.\n(iii) We have verified the resolution calculation and clarified the definition of baseline separation in\nthe manuscript.\n(iv) We have also replaced Figure 4b with a plot of log(k) versus carbon number.\nHowever, the claimed benefits of such mesoporous nanostructures for chromatographic separation\nremain unconvincing for several reasons:\n1. Limited Separation Efficiency: The plate height values (Figure 4C) indicate suboptimal separation\nefficiency, suggesting that the mesoporous nanostructure does not confer meaningful improvements in\nchromatographic kinetics.\nResponse: Thank you for raising this point. We would like to clarify that, among the four ordered\nmesoporous nanostructures reported in our study, the 2D hexagonal configuration is indeed not the\none with the best chromatographic kinetics. Nevertheless, we consider it an ideal packing material for\nchromatographic investigations. This is because the objective of this work was to design and\nmanufacture well-structured materials towards improved chromatographic resolution; while\nchromatographic resolution, by definition, is not entirely built upon column efficiency but also\nretentivity (as shown in Equation 1). Therefore, in selecting the mesoporous configuration for\nchromatographic evaluations, both efficiency and retentivity were taken into account.\nSpecifically, as shown in Fig. A1(a), face-centered cubic mesoporous microspheres can achieve an\nexcellent reduced plate height (h = 1.9), outperforming the 2D hexagonal mesoporous microspheres\nmin\n(h = 2.7). However, the retention factor of the face-centered cubic mesoporous microspheres (k =\nmin\n1.34) is considerably lower than that of the 2D hexagonal mesoporous microspheres (k = 19.2). Taking\nboth into account, the 2D hexagonal nanostructure offers a favorable balance between efficiency and\nretentivity.\nTo further justify the selection of the 2D hexagonal mesoporous microspheres for application, we\ncompared the modified kinetic plots of face-centered cubic mesoporous microspheres (high efficiency\nbut low retentivity) and 2D hexagonal mesoporous microspheres (high retentivity with decent efficiency\ncomparable to most commercial columns). As shown in Fig. A1(b), the 2D hexagonal mesoporous\nmicrospheres realized higher resolutions in shorter time, making them ideal for separations of\nchallenging critical pairs. At the end of the day, chromatography is about resolution, while efficiency,\ncertainly a key factor, is used to support the total resolution as a collective result.\nFigure A1. Comparison of Knox curves (a) and modified kinetic plots (b) of 2D hexagonal mesoporous\nmicrospheres (2D-hex) and face-centered cubic mesoporous microspheres (fcc).\n2. Lack of Demonstrated Selectivity: The separations shown in Figure 5 fail to achieve baseline resolution\nfor the tested analytes, raising doubts about any substantive enhancement in selectivity.\nResponse: We appreciate your insightful view. In the application, we intentionally selected the critical\npairs with challenging selectivity factors, in order to demonstrate the significant resolution\nenhancement provided by the ordered mesoporous nanostructures’ enhanced retentivity and efficiency.\nAs evidenced in Figure 5d-f, even though the critical pairs are esteemed challenging to resolve\nchromatographically, the ordered mesoporous materials are still able to provide good selectivity\nallowing for distinctive recognition of the peaks.\nHowever, we agree with you that achieving baseline resolution for the tested analytes should be\nsubstantively meaningful. By definition, when resolution (Rs) is or above 1.5, the peaks are considered\nbaseline separated, allowing for clear identification and individual integration (for quantification), which\nis of paramount importance to analytical chemists. In this regard, we have re-optimized the method and\nre-performed the separation. As shown in the revised Figure 5g-i, after re-optimized the mobile phase\nfrom 30:70 v/v ACN/H O to 20:80 v/v ACN/H O, the Rs of benzene and benzene-d6 on ordered\n2 2\nmesoporous particles increased from 1.21 to 1.51, achieving the baseline separation. While under the\nsame conditions, the Rs on totally porous particles increased from 0.65 to 0.78, remained significantly\nlower than that achievable with the ordered mesoporous particles, let alone baseline separation. We\nhave also updated Figure 5a-c with the baseline-separated chromatogram of benzo(b)fluoranthene,\nbenzo(k)fluoranthene, and benzo(a)pyrene, which was originally put in the Supporting Information. In\ntotal, compared to the totally porous particles, the ordered mesoporous material demonstrated a\npronounced improvement for critical pairs from non-baseline to baseline separations.\nChanges to the Manuscript, Figure 5:\nFigure 5. Separations of critical pairs on monodisperse microspheres with ordered mesoporous\nstructure. (a-c) Chromatograms of a polycyclic aromatic hydrocarbon mixture (a) on TPP (b) and OMP\n(c) columns. Mobile phase: 70:30 v/v ACN/H O. (d-f) Chromatograms of a xylene isomer mixture (d) on\n2\nbenzene/benzene-d (g) on TPP (h) and OMP (i) columns. Mobile phase: 20:80 v/v ACN/H O. All\n6 2\nseparations were performed on 15 cm columns under isocratic elution conditions at a flow rate of 800\nnL/min with UV detection at 210 nm.\nChanges to the Manuscript, Line 326: As shown in Figure 5g-i, with OMP medium, however, under\nisocratic conditions (20:80 v/v ACN/H O), the 15 cm short OMP column achieved a satisfactory Rs = 1.51\n2\nfor benzene/benzene-d within 55 min, whereas the TPP counterpart delivered only partial separation\n6\n(Rs = 0.78).\n3. Durability Concerns: The mechanically fragile nature of the surface mesopores casts uncertainty on\nthe long-term stability and applicability of these materials for complex or high-throughput separations.\nResponse: Thank you for emphasizing the importance of mechanical stability. Figure S17 shows SEM\nimages of the ordered mesoporous particles (OMP) before and after pressure testing, demonstrating\nthat the OMP material maintains structural integrity without fragmentation up to at least 50 MPa.\nHowever, more quantitative data are needed for this concern. Therefore, we have supplemented the\nstudy with two additional experiments: (1) we tested the back pressure as a function of flow rate and\nextended the maximum pressure test to 65 MPa; and (2) we also evaluated the stability of OMP during\nlong-term operations under ultra-high pressure. The results from these experiments provide strong\nevidence for the long-term stability and applicability of OMP materials for complex or high-throughput\nseparations.\nChanges to the Supporting Information:\nFigure S18. Mechanical stability test of ordered mesoporous particles. (a) Back pressure as a function\nof flow rate; (b) Back pressure fluctuation during 24 hr running at the flow rate of 1.0 µL/min. Column:\n27 cm × 100 μm i.d.; mobile phase: 50:50 v/v MeOH/H O.\nprolonged high-pressure conditions (65 MPa), maintaining structural integrity without particle\nfragmentation or bed deformation (Figures S17 and S18).\nGiven these limitations, the work, while methodologically interesting, does not presently meet the\nthreshold for transformative impact expected by high-profile interdisciplinary journals such as Nature\nCommunications.\nResponse: Based on your insightful and constructive comments, we have performed further\nexperiments and extended the discussion in order to solve all the concerns you raised. We believe our\ndetailed responses and the revised Manuscript and Supporting Information have clarified all the\nconcerns and provided strong evidence for the claims, and the work should now meet the high standard\nas expected by Nature Communications.\nMinor Comments:\n4. Resolution Calculation (Figure 5b2): The reported resolution value of 1.66 appears inconsistent with\nthe lack of baseline separation in the chromatogram. The authors should verify their calculations.\nResponse: Thank you for this concern. The formula and calculation process for the resolution (Rs) are\nprovided below. We have double-checked our calculations and confirmed that the value of 1.66 is\ncorrect. By definition, “baseline separation” is according to the widely accepted quantitative criterion\nof Rs ≥ 1.5 (Colin F. Poole, The Essence of Chromatography, Section 1.6, 2002), which our result satisfies.\nWe understand, however, a visual evaluation alone may not always accurately indicate whether peaks\nare baseline separated. To avoid any ambiguity, we have included a clear explanation of this definition\nin the revised manuscript.\n2t t  221.619.3\nRs R2 R1  1.66\n1.7  W W  1.70.700.93\n1,h/2 2,h/2\nMeanwhile, we agree with your observation that Peaks (2+3) and Peak 1 did not appear to achieve\ncomplete baseline separation in the chromatogram (Figure 5b2). This occurred due to the overlapping\nbetween Peak 2 and Peak 3. With reference to Figure 5b3, Peak 2 eluted slightly earlier than Peak 3. As\na result, the combined Peak (2+3) exhibited fronting rather than a Gaussian peak shape.\nChanges to the Manuscript, Line 214: The core parameter for evaluating the separation performance\nof a critical pair is resolution (Rs), as defined by Equations 1-3. Baseline separation is quantitatively\ndefined as Rs ≥ 1.5.\n5. Improved Data Representation: A plot of logk versus carbon number would better assess whether\nthe separation follows a reversed-phase mechanism, as such a relationship should yield a linear trend.\nResponse: Thanks for this suggestion, Figure 4b has been replaced with a plot of log(k) versus carbon\nnumber. Updated Figure 4b shows an excellent linearity (R² > 0.998), confirming that the separation\nfollows a reversed-phase mechanism.\nChanges to the Manuscript, Figure 4:\nFigure 4b. Comparison of retention factor of ordered mesoporous particle and totally porous particle.\nReviewer #2:\nThe manuscript by Prof. Zhang and coworkers reports the synthesis of monodisperse microparticles with\na mesoporous nanostructure for chromatographic applications. The preparation of silica microparticles\nbegins with the use of microfluidics to generate monodisperse droplets containing TEOS, hydrochloric\nacid, and an amphiphilic polymer surfactant. Subsequently, rapid solvent evaporation from the droplets\ninduces self-assembly of the surfactant into a liquid crystalline phase, while simultaneously promoting\nthe condensation of silica in the hydrophilic domains. Finally, hydrothermal treatment and calcination\nyield monodisperse silica particles with an ordered porous structure. By adjusting the SDAs and post-\ntreatment conditions, the authors demonstrate that the nanostructures of the microparticles can be\nfinely tuned. The authors further showcase the application of these particles in chromatographic\nseparation, where they exhibit high performance compared to conventional silica particles.\nWe are grateful for your favorable and supportive evaluations. We also appreciate the detailed\nsuggestions you have provided. In response to your comments, we have made the following revisions\nto improve the manuscript:\n(i) We have added high-magnification SEM images for all ordered mesoporous microspheres to better\nillustrate surface details.\n(ii) We have updated the comparison between ordered mesoporous particles and totally porous\nparticles, with closely matched pore size, monodispersity, and particle size.\n(iii) We have introduced a comparative study of four different ordered mesoporous structures in the\nSupporting Information.\n(iv) We have also expanded the discussion on production yield and loading capacity of the ordered\nmesoporous material.\nIn my opinion, this work is very interesting; however, several issues still need to be addressed prior to\npublication:\n1. Almost all of the SEM images lack surface details, making it difficult to extract useful information\nbeyond particle size from the presented images.\nResponse: Thanks for this valuable suggestion. We have introduced high-magnification SEM images for\nthe ordered mesostructured microspheres. This should enable our readers to have a clear insight of the\nsurface details of these microspheres.\nChanges to the Supporting Information:\nFigure S5. SEM morphology characterization (a, c) with different magnifications and particle size\ndistribution (b) of the cubic double gyroidal mesoporous silica microspheres templated by P123.\nFigure S6. SEM morphology characterization (a, c) with different magnifications and particle size\ndistribution (b) of the body centered cubic mesoporous silica microspheres templated by F127.\nFigure S7. SEM morphology characterization (a, c) with different magnifications and particle size\ndistribution (b) of the face centered cubic mesoporous silica microspheres templated by PEO -b-\n249\n2. Why did the authors choose to use the particles mentioned in the main text (CV = 4.7%, Figure S14) for the separation\nperformance comparison, rather than the 5 µm particles shown in Figure S12, which clearly have a more spherical\nmorphology and more uniform size?\nResponse: Thank you for raising this concern. We selected the particles in Figure S14 (Figure S17 in\nrevised Manuscript) for comparison based on their high separation performance, as a result of the high\nretentivity, despite their less spherical morphology and slightly higher CV value.\nDue to a higher content of structure-directing agent used in the manufacturing, the particles in Figure\nS14 possess a larger pore volume and a higher orderliness of mesoporous structures, which inevitably\nresulting in a sphere-like polyhedron morphology composed of multiple planes. In our experiments,\nthese properties are proved more conducive to achieving high efficiency and high retentivity, making\nthe sphere-like polyhedral particles more suitable for separations of challenging critical pairs.\nOn the other hand, the actual difference in monodispersity between the two types of particles is not as\nlarge as that suggested by their CV values. Compared to the highly spherical particles, the sphere-like\npolyhedral particles cause greater error in particle size statistics, resulting in a larger apparent CV value.\nIn theory, these two particles should exhibit similar monodispersity, as they both originate from\nmonodispersed droplets (CV < 2%), which are generated from the same microfluidic chip.\n3. In the comparison of separation performance, there are too many variables between the two types\nof particles, such as uniformity, pore structure, and morphology, making it difficult to draw accurate\nconclusions.\nResponse: We appreciate your insightful view. According to your suggestion, we have redesigned\nexperiments for rigorous comparisons. Since commercially available packing particles with uniformity\nand pore structure identical to our ordered mesoporous particles (OMP) are lacking, we synthesized\nconventional totally porous particles for comparison, based on the method described in our previous\nwork (Angew. Chem. Int. Ed., 2025, 64, 202418642). The homemade totally porous particles (hTPP)\nexhibited morphological and pore structure parameters closely matching that of the OMP: in terms of\nmorphology, hTPP and OMP showed comparable monodispersity (CV = 3.6% for hTPP and 4.7% for OMP)\nand a similar particle size of 5.0 µm; regarding pore structure, hTPP had a pore size of 8.2 nm (bare\nsilica), which was reduced to 7.3 nm after C18 bonding, closely matching that of the OMP. It should be\nnoted that the specific surface area of hTPP could not reach that of OMP, as the latter benefits from the\nhighly efficient utilization of pore space afforded by its ordered mesoporous architecture, enabling an\nextraordinary high specific surface area. Nonetheless, the hTPP still possess a specific surface area of\n347 m²/g (bare silica), which, although lower than that of OMP, remains among the highest in\ncomparison with commercial totally porous particles.\nUpon controlling these parameters as closely as possible (as shown and listed in Figure S20 and Table\nS8), we further compared the chromatographic performance of the two types of particles. As shown in\nFigure S21 and Table S7, both efficiency and retentivity of hTPP are inferior to that of OMP, clearly\ndemonstrating the meaningful improvements resulting from the ordered mesoporous structure.\nChanges to the Supporting Information:\nFigure S20. SEM morphology characterization (a) and particle size distribution (b) of the homemade\ntotally porous silica microspheres.\nFigure S21. Performance comparison between monodisperse ordered mesoporous particle and\nhomemade totally porous particle. (a) Chromatograms of an alkylbenzene mixture on 15 cm long OMP\nand hTPP columns. Analytes: thiourea, methyl-, ethyl-, propyl-, and butylbenzenes (in order of elution);\nmobile phase: 60:40 v/v ACN/H O; UV detection: 214 nm. (b) Knox curves of OMP and hTPP.\nMaterial Silica C18-Silica\nSpecific surface area (m2/g) 347 208\nPore size (nm) 8.2 7.3\nPore volume (cm3/g) 0.78 0.55\nCarbon load (%) 0 10.4\nParticle size (µm) 5 5\nCoefficient variance of\n3.6 3.6\nparticle size (%)\nLigand density (μmol/m²) 0 1.58\nTable S8. Comparation of Knox coefficients and reduced plate heights obtained with TiTAN-synthesized\nordered mesoporous particles, commercial totally porous particles, and homemade totally porous\nparticles.\nh a b c\nmin\nOMP 2.78 0.65 4.83 0.14\nTPP 4.08 1.05 5.14 0.32\nhTPP 3.82 1.02 4.09 0.32\nChanges to the Manuscript, Line 271: An additional set of totally porous particles (denoted as hTPP, for\nhomemade totally porous particle) was intentionally synthesized to closely matched to OMP in terms\nof particle size, monodispersity, and pore size (Figure S20 and Table S8). As shown in Table S7 and Figure\nS21, OMP exhibited pronouncedly higher efficiency (2.78 vs. 3.82 for h ) and higher retentivity (19.2\nmin\nvs. 4.0 for k) than that of hTPP, both outperforming the conventional totally porous counterparts.\n4. The authors are encouraged to compare the influence of different nanostructures on separation\nperformance to provide more comprehensive conclusions.\nResponse: Based on your suggestion, we have introduced performance comparison of the four different\nordered mesoporous structures. Knox plots and separations of an alkylbenzene mixture were conducted,\nand the results have been added to the Supporting Information. These microspheres were controlled\nto have similar particle and pore sizes. The results show significant differences in efficiency and\nretentivity among the four ordered mesoporous structures.\nChanges to the Supporting Information:\nFigure S15. Performance comparison between microspheres with different ordered mesoporous\nstructures, i.e., 2D hexagonal (2D-hex), cubic double gyroidal (cdg), body centered cubic (bcc) and face\ncentered cubic (fcc) structures. (a) Chromatograms of an alkylbenzene mixture on 15 cm × 100 μm i.d.\ncapillary columns. Analytes: thiourea, methyl-, ethyl-, propyl-, and butylbenzenes (in order of elution);\nmobile phase: 60:40 v/v ACN/H O; flow rate: 100 nL/min; UV detection: 214 nm. (b) Knox curves of\n2\nmicrospheres with different ordered mesoporous structures.\nChanges to the Manuscript, Line 230: As shown in Figure S15, the performance comparison of different\nordered mesoporous structures demonstrated that, as expected, the 2D-hex structure indeed exhibited\nhigh retentivity and efficiency.\n5. It would be helpful for the authors to clarify the production yield of such porous particles prepared\nby microfluidics, as well as the sample loading capacity that can currently be separated using their\nchromatographic system.\nResponse: Thanks for this suggestion, we have expanded discussions on both production yield and\nloading capacity. Particle yield depends on the droplet generation throughput. In this work, we\nemployed a 120-channel array microfluidic chip based on our previous design (Angew. Chem. Int. Ed.,\n2025, 64, 202418642), which generates droplets at an overall rate of ~240,000 per second with zero\ndefective index, i.e. 100% production yield. This high-throughput process manufactures ~109\nmicrospheres per hour with a single chip, enabling the preparation of packing materials for one\nthousand 15 cm-long, 100 μm i.d. chromatographic columns (~106 particles needed for one column), or\nten 10 cm-long, 2.1 mm i.d. analytical scale columns (~108 particles needed per one).\nMeanwhile, to determine the loading capacity of the ordered mesoporous particles, we used\nmethylbenzene as a standard probe molecule and evaluated the column efficiency at different injection\nconcentrations. The plot of column efficiency versus sample mass shows that the loading capacity—\nusually defined as the sample mass at which a 10% efficiency loss is observed (David V. McCalley, J.\nChromatogr. A, 2008, 1193, 85-91) —was 0.087 μg in this study.\nChanges to the Manuscript, Line 406: The microfluidic platform enabled droplet generation at a super\nthroughput of 240,000 Hz per chip, resulting in a production yield of approximately 109 microspheres\nper hour.\nChanges to the Manuscript, Line 257: The raised phase ratio also resulted in a high loading capacity of\nthe OMP material, as demonstrated in the loading mass test (Figure S19).\nChanges to the Supporting Information:\nFigure S19. Column efficiency as the function of loaded mass for methylbenzene on 15 cm × 100 μm i.d.\ncapillary columns. Mobile phase: 60:40 v/v ACN/H O; flow rate: 800 nL/min; UV detection: 214 nm. The\n2\nefficiency loss was maintained within ~10% for solute loading mass up to 0.087 μg.\nReviewer #3:\nThe authors have presented a method for synthesizing mesoporous particles with a well-defined\ninternal structure using a microfluidic method they refer to as TiTAN. This method is especially exciting\nfor uses in chromatography, as the authors have demonstrated in their measurements showing superior\nseparations of small molecules using particles with different internal structures and similar macroscopic\n(~4 µm) sizes. In my opinion, these are significant results in terms of the synthesis and application of\nthe particles in an incredibly useful way -- and likely to be highly impactful. In fact, I can think of several\nmore applications of these particles to fundamental scientific studies that extend beyond\nchromatography.\nNevertheless, as I am not an expert in chromatography, my review of this manuscript focuses exclusively\non the authors' claims regarding the well-defined internal structure of the particles as probed using\nsmall-angle X-ray scattering (SAXS) and gas adsorption measurements. SAXS is one of the most\nappropriate techniques for measuring structure in systems such as this. Gas adsorption measurements\nsimilarly provide a good estimate of the pore size, and demonstrate the authors can tune this parameter\nwith great precision. I have a few questions and concerns regarding these results, although overall I\nbelieve they strongly support the authors' conclusions.\nWe are deeply grateful for your positive and inspiring comments on our work. Your encouraging words\nare a great motivation for our research. We have read your suggestions carefully and made the following\nrevisions to our work:\n(i) We have refined all SAXS figures to include labelling of peak positions relative to the fundamental\npeak (Q*).\n(ii) We have also expanded the discussion on pore size reproducibility, and introduced reproducibility\ndata for pore size tuning between independent batches.\n1. A comment: I would appreciate if the authors would label the relative peak positions (in terms of\nscattering vector Q) relative to the fundamental peak (Q*) in their figures, rather than indexing the\nreflections. While this information is redundant, it would help the reader interpret these results. The\nauthors should indicate each peak perhaps with an arrow of some sort. I realize this request may seem\nodd, but indexing the peaks this way makes it much easier for the reader to assess the quality of the\nmesopore structure. This is commonly done in the block copolymer community (see for example\nChapter 5 in Basic X-ray Scattering for Soft Matter by de Jeu).\nResponse: Thank you for this excellent suggestion. We have revised all SAXS figures in the Manuscript\nin line with your suggestion. These revisions should enhance the clarity of our results to readers.\nChanges to the Manuscript, Figure 1:\nFigure 1. SAXS pattern (i) of the ordered mesoporous silica microspheres.\nChanges to the Manuscript, Figure 2:\nFigure 2. SAXS patterns of body centered cubic (c), face centered cubic (g), and cubic double gyroidal\n(h) configurations, respectively.\n2. In some samples, for example the Ia-3d/gyroid structures, the higher order reflections are quite weak\n(see Fig. 2g). How are the authors identifying peaks, and can they offer some insight into the relatively\nweak signals for higher values of Q?\nResponse: Thank you for this insightful comment. In comparison with materials with 𝐼𝑎3̅𝑑 structure\nobtained through direct hydrothermal synthesis, those prepared via droplet evaporation followed by\nsolvothermal treatment are prone to have asymmetric structural distortions during the evaporation\nprocess. Such distortions can reduce structural order and broaden diffraction peaks, which are further\namplified during solvothermal treatment, ultimately leading to poor long-range order and the absence\nof distinct reflections in the higher q region.\nThe structural assignment to 𝐼𝑎3̅𝑑 was made based on: (i) In comparison with untreated or\nhydrothermally treated samples that displayed a 2D hexagonal mesostructure, the SAXS patterns of the\nsolvothermal treated samples exhibited clear differences in both peak positions and intensities,\nconfirming the occurrence of a pronounced phase transition (Figure A2); (ii) When P123 was used as\nthe structure-directing agent, the surfactants initially formed a 2D hexagonal (p6mm) mesophase\n(Figure A2b), upon solvothermal treatment in hexane at 80 °C, the hydrophilic PEO chains of P123\nundergone partial dehydration and became hydrophobic, which reduced the V /V ratio (or increased\nH L\nthe packing parameter, g), thereby driving the mesophase transition from 2D hexagonal to bi-\ncontinuous cubic 𝐼𝑎3̅𝑑 with lower interfacial curvature; and (iii) the TEM images no longer displayed\n2D hexagonal channels but instead, revealed pore architectures consistent with 𝐼𝑎3̅𝑑 as reported in\nthe literature (Facile Synthesis and Characterization of Novel Mesoporous and Mesorelief Oxides with\nGyroidal Structures, J. Am. Chem. Soc., 2004, 126, 865-875) (Figure A3).\nTaken together, these results strongly support our assignment of the 𝐼𝑎3̅𝑑 space group.\nFigure A2: SAXS patterns of (a) hydrothermally treated, (b) direct calcined, and (c) solvothermally\ntreated monodisperse microspheres prepared via TiTAN strategy.\nFigure A3: TEM images of (a) hydrothermally treated, and (b) solvothermally treated monodisperse\nmicrospheres prepared via TiTAN strategy.\n3. The authors show extraordinary control over the pore size (Fig. 3b). I assume these results are\nreproducible, but I wonder if the authors can comment on the variability between pore sizes between\nindependent batches of particles? I may have missed this, but I cannot find in the manuscript or SI how\nmany independent trials were performed. Were the nitrogen adsorption-desorption measurements\nperformed on independent batches? Is the high tunability of pore size that the authors demonstrated\nwithin a single batch of the particles? Etc. Some clarity regarding these points is necessary.\nResponse: We appreciate your comment regarding the pore size reproducibility. We have expanded the\ndiscussion on the variability in pore sizes between independent batches of particles. We intentionally\ncollected droplets over a time span of three days and to produce three independent batches,\nrespectively, with each batch divided into two groups subjected to different hydrothermal treatments.\nNitrogen adsorption-desorption experiments were performed on independent batches to measure the\npore size. As shown in Figure S8, for each batch, the two groups treated under distinct hydrothermal\nconditions exhibited angstrom-level differences in pore size, approximately 2-3 Å. Meanwhile, between\ndifferent batches, the ordered mesoporous microspheres treated under the same hydrothermal\nconditions showed consistent pore size, with CV 2.1%, demonstrating good reproducibility.\nChanges to the Supporting Information:\nFigure S8. Reproducibility of pore size tuning via hydrothermal treatment. Droplets from three\nindependent batches were collected over three days, and each batch was divided into two groups\nsubjected to different hydrothermal treatments.\nChanges to the Manuscript, Line 157: This angstrom-level pore size was found reproducible across three\nindependent batches, with a CV of 2.1%, demonstrating good reproducibility of pore size tuning via\nhydrothermal treatment (Figure S8).\nOther Revisions:\n(i) We have revised the format of the manuscript in accordance with Nature Communications\nformatting instructions.\n(ii) We have also noticed and corrected an editing error. The time unit labeled in Figure 4d should be\n“min” instead of “s”. This correction does not affect the conclusions, as the performance\nimprovement of ordered mesoporous particles over totally porous particles remains unchanged.\n(iii) Three of the authors: Hanchen Cao, Jikai Chen, and Xiangyu Huang, since August 2025 will be\ncontinuously funded for their PhD studentship by the grant NSFC 22574134. These authors have\ncontributed significantly to the revision of the current manuscript. In this regard, NSFC 22574134\nis included in the Acknowledgement list."}, {"round": 2, "reviewer_comments": {"reviewer_2": "In my opinion, the authors have addressed the concerns from Reviewer #1.", "reviewer_3": "The authors have fully addressed my comments regarding the original manuscript, and I am fully satisfied with their\nresponses. I am pleased to recommend publication of this work to the Editor."}, "author_response": ""}]

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