Preparation of Silica‑Based Superficially Porous Silica and its Application in Enantiomer Separations: a Review

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https://doi.org/10.1007/s41664-021-00155-2 REVIEW

Preparation of Silica‑Based Superficially Porous Silica and its Application in Enantiomer Separations: a Review

Zhongshan Liu¹ · Kaijun Quan² · Hui Li² · Jia Chen² · Ming Guan¹ · Hongdeng Qiu^2,3

Received: 26 October 2020 / Accepted: 7 December 2020 / Published online: 10 March 2021

Abstract

Chromatographic substrate material, as a carrier of chromatographic stationary phases, plays a vital role in defining its chromatographic characteristics, including column efficiency, stability, peak capacity and so on. In recent years, superficially porous silica (SPS) was widely valued for its excellent performance in improving the column efficiency due to its special structural properties, which was considered as a real substitute for fully porous SiO₂ (FPS) and had been used to develop a new generation of highly efficient stationary phase. To help researchers better understand the SPS and further promote its application and development in the field of chromatography, the rapid separation mechanism, preparation method and its applications in the rapid separation and analysis of enantiomers were systematically introduced, and the development prospects of SPS were also prospected in this paper.

Keywords Superficially porous SiO₂ (SPS) · Stationary phase · Enantiomers separation · Chromatographic base Abbreviations

SPS Superficially porous silica FPS Fully porous SiO₂

UPLC Ultra-performance liquid chromatography TEOS Tetraethyl orthosilicate

HPLC High-performance liquid chromatography LBL Layer-by-layer

PICA Polymerization-induced colloidal aggregation UPS Ureidopropyltrimethoxysilane

CTAB Hexadecyl trimethyl ammonium bromide TOMAB Trioctylmethylammonium bromide ODA Octadecylamine

DDA Dodecylamine

CTAC Cetyltrimethylammonium chloride DMA N,N-dimethyldecylamine

SOS Sphere-on-sphere

MPTMS 3-Mercaptopropyltrimethoxysilane PVA Polyvinyl alcohol

DMDA N,N-dimethyldecylamine TMB 1,3,5-Trimethylbenzene CSP Chiral stationary phase LOD Limit of detection PIM Polar ion mode POM Polar organic mode RP Reverse phase NP Normal phase TFA Trifluoroacetic acid TEA Triethylamine MeOH Methanol 2-PrOH Propan-2-ol CAN Acetonitrile Ipam Isopropylamine DEA Diethylamine AA Acetic acid

NH₄TFA Ammonium trifluoroacetate NH₄HCO₂ Ammonium formate NH₄OH Ammonium hydroxide TEAAc Triethylammonium acetate NH₄Ac Ammonium acetate

Zhongshan Liu and Kaijun Quan contributed equally to this work.

* Ming Guan guanm@xjnu.edu.cn

* Hongdeng Qiu hdqiu@licp.cas.cn

1 College of Chemistry and Chemical Engineering, Xinjiang Normal University, Urumqi 830054, China

2 CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China

3 College of Chemistry, Zhengzhou University, Zhengzhou 450001, China

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1 Introduction

Currently, chromatographic technology is one of the most important methods of separation and analysis, which is widely used in many fields such as pharmaceutical, food, environment, life science [1–6]. Chromatographic packing material as the “heart” of the chromatographic column has a significant role in defining its separation efficiency and application range [7–10]. Therefore, the development of high-performance chromatographic packing material has always been one of the most potential and creative research fields in the chromatographic rapid separation and analysis of complex systems. Silica (SiO₂) is one of the most important chromatographic packing substrates due to its excellent physical and chemical properties. After nearly 50 years of development, the preparation technology of size and shape-controlled SiO₂ microspheres have achieved great development, which makes the chromatographic performance of SiO₂-based stationary phase get a rapid development. However, it is still difficult to meet the increasingly high requirements of separation and analysis technology when human society analyzes the complex unknown world. Thus, the development of chromatographic packing with better performance has always been the goal pursued by chromatographers. In recent years, various types of smaller size SiO₂ packing materials have been developed on the basis of traditional 5 μm SiO₂ stationary phase. For example, Ultra-Performance Liquid Chromatography (UPLC) technology based on sub-2 μm stationary phase has greatly advanced the development of chromatographic technology [11–14]. However, other studies have found that although reducing the particle size of the packing can improve the efficiency of the separation column, it will lead to a sharp increase in the column pressure, which not only causes difficulty in filling the packing but also requires the use of expensive UPLC equipment [15]. In addition, the radial thermal gradient generated by the frictional heat between the mobile phase and the stationary phase driven by the high pressure will cause

chromatographic peak broadening. These factors limit the further improvement of column efficiency by reducing the SiO₂ packing diameter [16].

SPS is a new type of core–shell type chromatographic packing composed of a solid core and a porous shell layer surrounding the solid core (Fig. 1). The concept of core–shell type packing materials was proposed by Horvath in the late 1960s [17]. Unlike FPS packing, SPS is a porous shell wrapped around a solid core. Its original design is to shorten the path of solute diffusion and maintain a low back pressure at a certain flow rate (Fig. 2). Studies have shown that this new type of SPS packing can not only maintain the advantages of the existing small size packing materials’ high efficiency and rapid separation but also could avoid excessive back pressure due to its special structure [18]. As the carrier of chromatographic stationary phase, the SPS packing materials can have the bigger effective surface area that interacts with the separation substance to improve separation efficiency, while the presence of a nonporous core can reduce the central porous regions where cannot effectively participate in the separation process, which can help shorten the analysis time and reduce peak broadening [19]. That is to say, the superficially porous packing can improve the separation efficiency by reducing the pore depth to allow a shorter residence time of the solute in the particle. The higher efficiency of the SPS packing relative to the fully porous material allows the use of a shorter column for the same efficiency, which has a smaller backpressure. Therefore, SPS can make up for the deficiencies of the FPS and have become a new star in the development of high-performance chromatographic stationary phases. To further promote the development and application of this packing material, this article takes SPS packing as the core to review its separation mechanism, preparation method and its application in the separation of enantiomers samples.

2 Advantage of SPS

The unique structure of SPS is the main reason for its efficient separation. Gumustas and co-workers have systematically discussed the effect of A, B and C of SPS packing on column efficiency based on van Demeter Eq. (1) combined with related literature [20]. It was found that the SPS usually have a narrower particle size distribution and shorter solute diffusion path than the conventional FPS packing [21, 22], which can reduce the A item and the B item and effectively reduce the theoretical plate’s height (H) to improve column efficiency [23, 24]. The SPS can reduce C item due to shorter solute diffusion path, which has an obvious advantage for improving the separation efficiency of larger solutes with slow diffusion rates [25].

Fig. 1 SPS with different pore structures. Reprinted with permission from [77]

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Where A, B, and C are constants, and μ is the linear velocity of the mobile phase.

As the carrier of the stationary phase, SiO₂ microspheres mainly provide an interface for interaction with analytes.

The specific surface area of the stationary phase has a significant impact on its column capacity. The SPS packing has one more porous shell than nonporous SiO₂ packing, which can increase its capacity and functional area. Since the FPS microspheres are distributed throughout the pores, the specific surface area is significantly higher than that of the SPS microspheres. However, the mobile phase will still be diffused into the central porous region of FPS microspheres during the separation process, which would prolong the analysis time. On the contrary, the SPS uses a nonporous inner core to occupy the center zone, which greatly reduces the pore depth to allow shorter residence time of the solute in the particle thus improving the separation efficiency. It has been found that core–shell columns filled with particles of 2.6–2.7 μm SPS microspheres show better or the same efficiency compared with columns (BEH C18) which are filled with 1.7 μm FPS, but with much lower backpressure [26]. In the given column efficiency, the SPS for its higher efficiency relative to the FPS material can use a shorter column or bigger size SiO₂ material to avoid the excessive backpressure. That is the reason why a superficially porous particle generates less back pressure than a fully porous particle. In addition, because the porous layer of SPS is located on the surface, the pore size can be regulated easily. The above factors all play a role in improving the analysis speed significantly and reducing the operating pressure limits.

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H=A+B∕𝜇 +C𝜇

3 Preparation Method of SPS Microspheres

The SPS microspheres are composed of a nonporous inner core and a surface porous layer, so the preparation method usually includes two steps that prepare a mono-disperse nonporous SiO₂ inner core and construct a porous shell on the surface of the inner core.

3.1 Preparation of Monodisperse Nonporous SiO₂ Core

SPS is generally obtained by constructing a porous layer on the surface with mono-disperse nonporous SiO₂ as the core. Thus, the morphology of nonporous SiO₂ usually has a significant influence on the morphology of final SPS [27]. At present, the nonporous SiO₂ preparation methods mainly include the sol–gel method [28], seed growth method [29, 30], semi-batch preparation method [31], emulsion method [32] and spray drying method [33], among which the seed growth method and the semi-batch method are the most commonly used, because the SiO₂ prepared by them have the better monodispersity. Both of them are derived from the Stöber method which refers to the preparation of SiO₂ by the hydrolysis of silicon alkox- ide under ammonia catalysis and the subsequent silanol condensation. The size of mono-disperse nonporous SiO₂ can be adjusted from 10 to 500 nm by changing the concentration of ammonia using the Stöber method. However, even SiO₂ with a diameter of 500 nm is still too small to use for liquid chromatography packing. Chen et al. proposed a model formula for the particle size growth of nonporous SiO₂ microspheres, which verified the relationship between the particle size and the total surface area of the microspheres and the new nucleation, by studying the SiO₂ growth process in the Stöber method [34]. On this basis,

Fig. 2 Schematic diagram of mass transfer; a SPS; b FPS

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the researchers developed the so-called “seed growth method”, by means of constantly diluting the concentration of the reactants and prolonging reaction time, while adding new tetraethyl orthosilicate (TEOS) and obtained SiO₂ microspheres with a diameter of 2.7 μm [35]. Zhao et al. obtained a large particle size monodisperse nonporous SiO₂ with 4.7 μm by continuously adding precursor solutions (including hydrolysis solution and TEOS) to the seed suspension Silica microspheres [36]. However, the seed growth method requires multiple steps and a long reaction time. Thus, the researcher developed the “Semi- batch preparation method” by means of continuously adding an ethanol solution of silicon source (TEOS) with a small flow rate to the reaction solution, which can greatly simplify the operation steps and save reaction time. Naka- bayashi group found that the particles surface potential can be reduced by the adsorption of cations with a large ionic radius in the early reaction stage that accelerate particle coagulation process and the size of SiO₂ particles could be effectively increased in a micrometer range by adding KCl at a low water concentration [31]. Use this strategy, monodisperse SiO₂ particles with an average size of 6.6 μm were successfully obtained by adding 3 mol·m⁻³ KCl to the Stöber reaction solution. Zhang et al. solved the particle size dispersion by adding an appropriate amount of TEOS to the reaction solution in advance and investigated the effects of ammonia concentration and reaction temperature on the particle size of the SiO₂. And this method was used to prepare monodisperse nonporous silica microspheres with a particle size of 2.0–4.5 μm [37]. Thus, the

“seed growth method” and the “Semi-batch preparation method” are commonly used for core preparation, because they can prepare 1–5 μm nonporous SiO₂ core stably and have small particle size distribution, which basically meets the requirements of chromatographic packing.

3.2 Construction of Superficially Porous Layer The surface porous layer is the main platform for chromatographic separation by high-performance liquid chromatography (HPLC), and its shell thickness, pore size and pore structure all have significant effects on the separation [38].

As shown in Table 1, the structure parameter and chromatographic performance of some of the existing SPS-based stationary phase were summarized. At present, the construction of the surface porous layer on the nonporous SiO₂ core surface mainly includes the “bottom-up method” and

“top-down method”. The “bottom-up method” refers to the further growth of a porous layer composed of nanoparticles, nanofibers or nanorods SiO₂ on the surface of the nonporous SiO₂ core, such as layer-by-layer (LBL) method, polymerization-induced colloidal aggregation method (PICA) and template method. The first two methods are porous layers

formed by continuously stacking nanoscale SiO₂ and the template method is that the surfactant micelle (served as pore template) is co-grown with the silicon source. Then, the surfactant is removed by calcination to obtain a porous layer.

The “top-down method” refers to etching and dissolving the nonporous SiO₂ core surface layer under alkaline conditions and then re-growing with the surfactant. The porous layer was obtained by calcination to remove the surfactant, such as the template dissolution and deposition method. Those different methods will be discussed in details below.

3.2.1 The LBL Method

The LBL method utilizes electrostatic interactions and other forces including hydrogen bonds, covalent bonds, and van der Waals forces to realize the nanoscale SiO₂ layer-by-layer stacking [39]. As shown in Fig. 3, the LBL method first needs deposits charged polymer and oppositely charged nanoscale SiO₂ alternately around the nonporous SiO₂ core and repeats this process until the desired shell thickness is obtained (up to 50 times) [19]. The LBL method is the earli- est preparation method used in commercial production and is currently the main method used in commercial production of chromatography columns [22, 23, 40]. The 2.7 µm Halo core–shell packing prepared by LBL method was regarded as milestones work and received great attention from researchers because of its excellent chromatographic performance.

Subsequently, researchers have successively developed SPS with smaller particle size for the chromatographic stationary phase, such as Phenomenex 2.6 µm, Kinetex 1.7 µm, which also show good performance [41, 42]. The advantage of the LBL method is that it has a mature process to adjust the thickness of the shell and the aperture of the shell. However, the disadvantages of the LBL method are also very obvious time-consuming. Although there was a multilayer-by-multilayer method developed by Dong group which can obtain 6–7 layers at a time, it still cannot solve the time-consuming problem and high cost [43].

3.2.2 The PICA Method

To solve the time-consuming and laborious problem of LBL method, Kirkland et al. first reported the polymerization- induced colloidal aggregation (PICA) synthesis of SPS in 2000 year[44]. Chen and Wei [45] further optimized this method. As shown in Fig. 4, surface-modified solid SiO₂ were suspended in the coagulation reaction mixture of urea and formaldehyde under acidic conditions then formed the condensation layer of urea–formaldehyde polymer and ultrapure silica sol particles which would coat on the surface of the solid core. The SPS was obtained after remov- ing the urea–formaldehyde polymer by calcining at 600 °C and then sintering at a high temperature of 1000 °C. This

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Table 1 The structure parameters and chromatographic performance of some SPS-based stationary phases Particle size/μmThickness of the shell/nmMain pore size/nmBET surface area/ (m2·g−1)Pore volume/ (cm3·g−1)Plate numberModifierColumn/mmReferences 2.71608.4,14137–225,000 (naphthalene)C18100 × 2.1[27] 2.2 ~ 10022690.29219,789 (dimethyl- naphthalene)C18–[62] 2.4 ~ 20037610.22265,000 (fluorene)C18100 × 4.6[63] 2.49198.310.685.135,000 (toluene)C1850 × 2.1[68] 1.8–6.73300.74–C187.5 × 2.1[69] 1.8–8.92230.6–C187.5 × 2.1 2.750010.41140.34156,000 (phenan- threne)C18100 × 2.1[21] 1.8–7.51010.1920,900C1850 × 2.1[71] 2.6–7.81070.216,490C1850 × 4.6 5.1–8.31000.29551C1850 × 4.6 1.9 ± 0.0618010145.60.3211,300 (naphthalene)C18–[72] 1.9 ± 0.0618015105.80.26–C18– 5.7–0.91,1.54204–73,080 ( p-nitroani- line)Diol‐modified50 × 2.1[74] ––72,000 (lysozyme)C450 × 2.1 6.3–6.9337–68,000 (p-nitroani- line)C850 × 2.1[75] 6.5–2.610330.5255,381 (dimethyl- naphthalene)C18250 × 4.6[76] 4.825030––6200C1875 × 2.1[44] 2.7251025.3–0.19 ~ 17,000 (naphthalene)C18100 × 2.1[46] 2.723028.1–0.07 ~ 17,000 (naphthalene)C18100 × 2.1 2.6420026.5–0.0716,300 (naphthalene)C4100 × 2.1 2.6721045.2–0.1118,900 (naphthalene)C4100 × 2.1 3.4626045.4–0.1515,200 (naphthalene)C4100 × 2.1 2.9 ± 0.12300 ± 1.2–––50,442 (propylben- zene)C1850 × 2.1[48]

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method has greatly improved the batch-to-batch repeatability and Chen et al. prepared a SPS with a pore size of 45 nm using this strategy [46]. The method was further improved via modifying the surface of solid SiO₂ core with urea-pro- pyltrimethoxysilane in neutral condition [47]_. Because the solid SiO₂ core was modified under neutral conditions which were conducive to inhibit the hydrolysis of ureidopropyltrimethoxysilane, thus a highly monodisperse solid SiO₂ core was obtained. Meanwhile, the thickness of the shell layer can be controlled by adjusting the weight ratio of silica core/

colloidal silica sol (up to 500 nm) and the pore size can be adjusted using silica sols of different particle sizes (up to 40 nm). Yang et al. utilized the refluxing method to con- trol the hydrolysis rate of the ureidopropyltrimethoxysilane (UPS) serving as the solid SiO₂ core modifier and to avoid the agglomeration of SiO₂ core while optimizing the reaction conditions (pH, temperature, colloidal silica concentration and reaction time) to inhibit secondary nucleation.

Under optimized conditions, the shell thickness and pore size of SPS can achieve up to 300 nm and 50 nm, respectively, by adjusting the weight ratio of silica core/colloidal silica sol and the particle size of colloid silica sol [48].

3.2.3 Template Method

The template method is evolved from the modified Stöber method. In this method, the surfactant (such as alkyl quater- nary ammonium salt [49, 50] or alkyl amine [51]) was added to the sol–gel system, after the surfactant would form the micelle template and co-grow with the silicon source in the shell during the growth stage, then through the subsequent calcination treatment, the surfactant in the shell layer will be removed to produce the pores required for separation. In the past, some mechanisms have been proposed to explain the growth process of mesoporous silica particles [52–60].

But, no growth mechanism has pointed out how mesoporous silica particles grow up from the microstructure units and produce the final macrostructure. Recently, Qu et al. found that the porous shell structure was not assembled linearly, but in layers under the conditions of ethanol and water as the reaction solvent and hexadecyl trimethyl ammonium bromide(CTAB) as the template agent [61]. During the reaction, a layer of basic skeleton will be preferentially formed, and then the next layer will be assembled after the skeleton was assembled. This assembly mechanism was confirmed by the relationship between the shell thickness and reaction time. As shown in Fig. 5, it is obvious that the shell layer thickness is stepped increased with the reaction time.

On that basis, researchers use this method to prepare the SPS packing and have found that the pore size can be adjusted in a wider range by appropriately changing the reaction conditions. For example, the pore size can be adjusted between 5 and 19 nm by adjusting the stirring speed of the

reaction [27]. When using the two surfactants as the template, the pore size can be adjusted between 7 and 22 nm [62]. The pore size also can be adjusted between 7 and 37 nm by adjusting the polarity of the organic phase in the oil–water biphase [63]. Qu et al. further studied the for- mation mechanism of porous shell layers, which provided more data support for refined preparation of SPS [64]. And some SPS with different pore structure characteristics such as radial and dendritic channels were prepared by adjusting the reaction conditions [65]. However, the pore size of almost SPS prepared by the template method was relatively small when the shell thickness was high, which couldn’t obtain satisfactory results when used for separating mac- romolecular substances. To solve this problem, researchers studied the effects of surfactants on the aperture structure and found that the aperture structure could be adjusted using different surfactants [66]. For example, for the first time, Bai et al. proposed a new method for the synthesis of monodisperse porous silica microspheres with mesoporous channels perpendicular on the nonporous SiO₂ core surface using the trioctylmethylammonium bromide (TOMAB) as the assistant reagent of CTAB which served as the main template to expand the size of the CTAB micelles. And the result showed that the pore size can be expanded from 2.6 to 10.6 nm, while the shell thickness reached 198.3 nm [67].

In subsequent studies, the pore size distribution is more con- centrated adjusted by optimizing the experiment conditions [68]. Although the pore size has been extended to 10.6 nm, it is still theoretically unable to satisfy the rapid separation of large molecules. Qu’s group further used CTAB and octadecylamine (ODA) as structure-directing agents to prepare SPS with fibrous pores [62]. The pore diameter could be adjusted between 7.1 and 22.3 nm. By adjusting the amount of ODA used as a co-surfactant, it was successfully applied to the separation of proteins from 12 to 66 kD. Cheng et al.

also reported a sub-2 μm SPS microspheres prepared using dodecylamine (DDA) as a catalyst, template and porogen [69]. And the pore diameter could be adjusted between 3.5 and 16 nm by changing the concentration of DDA and the temperature of hydrothermal treatment. It was also found that the shell thickness of 300–700 nm could be obtained by adjusting the ultrasonic treatment time and the volume ratio of methanol to water when using the ultrasonic-assisted method, but the pore diameter could only be adjustable between 3.4 and 8.5 nm [21].

3.2.4 Template Dissolution and Redeposition Method The template method can be called a “bottom-up method”, whereas the template dissolution and re-precipitation method is a “top-down method”. This method, as shown in Fig. 6, generally refers to partial dissolution of the nonporous SiO₂ surface under acidic or alkaline conditions, the

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surfactant serving as templated and coprecipitating with the silicon source on the SiO₂ microspheres surface. Finally, the surfactant was removed by calcination to obtain the required porous layer. The cetyltrimethylammonium chloride (CTAC) was used as a template in the presence of NH₄F, and surface pores with two channel orientations (wormhole-like and radial oriented pores) were obtained. The thickness of the porous shell could be adjusted from 10 nm up to the radius of the nonporous SiO₂ microspheres [70]. Although the main pore diameter is less than 10 nm, the limitation of the conventional method requiring an autoclave is removed, which brings hope for the large-scale preparation. Wei et al. proposed a new swelling strategy to prepare the SPS microspheres [71]. The effect of surfactants with different alkyl lengths and different swelling agents on pore size was studied and the results showed that the swelling expansion method could maintain the ordered pore structure of the

material more than acid or alkali corrosion. Some researchers used the etching method under acidic or alkaline conditions to further enlarge the aperture. For example, Min et al. enlarged the SPS pore size from 4.9 nm to 10 nm and 15 nm with the help of reflux operation in different acids (hydrochloric acid and hydrofluoric acid), respectively [72]. Besides, a dandelion-like SPS microspheres with pore diameter 7 nm was obtained with N,N-dimethyldecylamine (DMA) and CTAC as double templates, and ammonia and ammonium fluoride as etching reagent [73]. The spherical morphology and monodispersity of nonporous SiO₂ can be well maintained by this method. Although it can increase the pore size of SPS packing by etching method and improve its ability to separate macromolecules, the expense is its mechanical strength. The chemical etching process also

Fig. 3 Schematic diagram of layer-by-layer method to prepare the SPS. Reprinted with permission from [46]

Fig. 4 Schematic diagram of polymerization-induced colloidal aggregation method. Reprinted with permission from [46]

Fig. 5 Relationship between the thickness of the porous shell layer and the reaction time. Reprinted with permission from [61]

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should be controlled very carefully, because the adhesion and agglomeration of particles can easily occur during the etching process.

In addition, there are also studies on the preparation of SPS materials by the “one-pot method”. The sphere-on- sphere (SOS) (Fig. 7) particles prepared by this method can be considered as an alternative SPS material. Adham et al.

synthesized SOS SPS microspheres in one pot by adding methanol, ammonia solution and 3-mercaptopropyltrimethoxysilane (MPTMS) to the aqueous solution of pre-formu- lated polyvinyl alcohol (PVA) and CTAB at room temperature for 24 h [74]. The pores of the SOS SPS material mainly come from the gap formed by the disorderly accumulation of nanometer nonporous SiO₂ microspheres, and the pore diameter is usually less than 2 nm. The Adham team then used N,N-dimethyldecylamine(DMDA) and 1,3,5-trimethylbenzene (TMB) as swelling agents to expand the pore size to 6.9 nm [75]. Qu’s group introduced CTAC into the reaction system, which reacted in the presence of sodium meta- silicate and formamide in a pot for 3 h, dried and calcined to obtain a stick-ball SPS material with a pore diameter of about 2.5 nm [76]. The material is modified with n-octade- cyltrichlorosilane and then packed into a column, with the characteristics of low back pressure. It is worth noting that the reaction time of this method is usually only 3 h, very fast for the synthesis of SPS material.

4 Application of SPS in Enantiomer Separation

The SPS is widely concerned by researchers due to its unique structure and excellent chromatographic performance, and gradually used as a substitute for the FPS packing for the rapid separation and analysis of different substances. Bai’s group systematically discussed its application in the rapid separation of small molecules, peptides and biological macromolecules [77]. The chromatographic separation and analysis of enantiomer are very challenging and important. The unique structural advantages of SPS packing have brought

new opportunities for the separation of such substances.

Therefore, this review will focus on the application of SPS packing in the separation of enantiomer which provides a reference for developing the high-performance chiral chromatographic packing. As shown in Table 2, the structural information of SPS-based chiral stationary phase (CSP) and chromatographic condition have been summarized to show its potential advantage in enantiomeric separation. The SPS-based CSP can reduce analysis time while maintaining high column efficiency and sensitivity. Some researchers have demonstrated the advantages of SPS-based CSP over FPS based in chiral separation [78–81]. Armstrong and co- workers undoubtedly offered a wide comprehensive work in the evaluation of SPSs for chiral separations [82–84].

They bonded hydroxypropyl-β-cyclodextrin, cyclofructan-6 based selectors, teicoplaninand vancomycin on 2.7 μm SPSs, respectively. The result focusing on their kinetic behavior showed that the performance of all chiral SPSs outperform their FPSs counterparts in multiple working conditions including normal phase, polar organic mode and HILIC mode. In the study of Schmitt et al., the chiral selector tert- butylcarbamoylquinine (tBuCQN) was immobilized on sub-2 μm FPS and 2.7 μm SPP and their column performance in enantioseparation was evaluated in comparison to 5 μm FPS [85]. The results showed that new support materials for tBuCQN-based CSP to provide highly efficient and fast enantioseparations for their relatively lower mass transfer resistance, meanwhile the 2.7 μm SPP-based CPS outper- formed their fully porous sub-2 μm counterpart due to various parameters affording reduced plate height. Lomsadze et al. gave a contribution to the fundamental analysis of SPS for ultra-fast chiral separations [86]. They developed different polysaccharide-based CSPs by coating the polymeric selector on 2.6 μm fused-core particles. Compared with the FPS-based CSPs, the SPS-based CPS with similar content of chiral selector have higher enantioselectivity and undegraded efficiency at higher flow rates. In addition, several separations were carried out in 15–30 s with the plate counts over 10,000 plate/m even at very high flow rates. Geibel et al.

prepared three different CSP by modifying the zwitterionic

Fig. 6 Schematic diagram of template dissolution and re-precipitation method. Reprinted with permission from [73]

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chiral ion exchange selector obtained by conjugating quinine and 2-aminocyclohexanesulfonic acid through a urethane bond on three different types of SiO₂ materials including the 3 μm FPS (120 Å), 3 μm FPS (200 Å) and 2.7 μm SPS (160 Å), respectively [87]. All of them were used to separate the same enantiomers and the results indicated that SPS showing better separation efficiency than FPS because of lower vortex diffusion and mass transfer resistance (Fig. 8).

Bezhitashvili et al. used a CSP prepared by covalent immo- bilization cellulose on the surface of SPS particles (Phenom- enex, diameter 3.6 μm, pore diameter 50 nm) to separate the enantiomers on the HPLC platform (Fig. 9). The results indicate that the combination of polysaccharide-based chiral selectors bonded on SPS is a very useful approach to prepare the CPS for fast and highly efficient separations of enantiomers in HPLC. With the assistance of appropriate instru- ments, the highly efficient separation of enantiomers can be achieved even at higher flow rates and the sample analysis can be completed in 15 s[88, 89]. The results show that the enantiomers including chiral sulfoxide, trans-stilbene and ethazoline can achieve baseline separation in 30 s. Min et al.

prepared a CSP using a SPS ( diameter 1.49 ± 0.04 μm, shell thickness 206 nm, specific surface area 213.6 m²·g⁻¹and pore diameter 9 nm) by the combination of modified seed growth method and template dissolution and re-deposition method. Then, it was modified with teicoplanin for rapid separation of enantiomers of natural amino acids. The results show that the obtained chiral stationary phase has excellent chiral separation performance and 6 enantiomers of natural amino acids (valine, methionine, norleucine, alanine, leucine, norvaline) can be baseline separated with the resolution ranging from 1.9 to 5.0 in less than 7 min [90]. Guo et al.

modified vancomycin on a 2.7 μm SPS to prepare a chiral stationary phase which was successfully used to analyze the degradation products of racemic citalopram with the limit of

detection(LOD) as low as pg level [91]. Folprechtova et al.

applied the SPS modified with teicoplanin and vancomycin at the same time as the chiral stationary phase, which was used in supercritical fluid chromatography system to realize the efficient separation of tryptophan and ketamine deriva- tives by means of complementation of two chiral ligands [92]. Ismail et al. have prepared a CPS by modifying the teicoplanin selector on 2.0 μm SPPs and it was successfully employed in ultra-fast chiral separations due to their efficiency and enantioselectivity [93]. As an example, the enantiomers of haloxyfop were baseline resolved in about 3 s with a resolution higher than 2.0. Roy et al. used teicoplan- inchemically modified macrocyclic glycopeptide and isopropyl-derived 6-cyclofructan as the chiral selector to prepare three CSPs, respectively. And all of them realized the efficient separation of 100 chiral analytes in 0.2 min (13 s) on supercritical fluid chromatography system [94]. Their results further indicated that SPS-based CSP have shown advantages in enantiomeric separations in HPLC by con- serving selectivity while providing higher efficiency separations with significantly reduced analysis time. Hellinghausen et al. prepared a CSP with hydroxypropyl-β-cyclodextrin as the chiral selector. Then, it was used to separate 100 kinds of pesticide enantiomers under different modes including polar ion mode (PIM), polar organic mode (POM), reverse phase model(RP) and normal phase (NP) model. Finally, 74 kinds of pesticide enantiomers of them achieved baseline separation [95]. The team combined multiple CSPs including macrocyclic glycopeptide-based CSP, cyclodextrin-based CSP and cyclofructan-based CSP worked in a complementary manner to separate chiral amines and the results showed that 150 kinds of chiral amines were mostly separated within 5 min [96]. Barhate et al. prepared a CSP by bonding the macrocyclic glycopeptide chiral selector to a 2.7 μm SPS, which was used to separate the intermediate enantiomers during Verubecestat synthesis [97]. And the purity analysis of the enantiomers in the entire synthesis process of verubecestat can be achieved by only combining two CSP columns.

From the above, it can be concluded that the SPS packing has great potential in developing chiral stationary phases with better performance and it should be regarded as an ideal substitute to the FPS packing.

5 Perspectives

SPS as the support material in HPLC has been showing intrinsic advantages in rapid and efficient separation of substances in complex systems because of its unique structural advantages. The most significant design of SPS is that the solid SiO₂ microsphere was applied as the core to cover

Fig. 7 SEM image of SOS particles. Reprinted with permission from [74]

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Table 2 Summary otableormations of SPS-based CSP and its applications in enantiomeric separation Nominal particle diameter /μm

ModifierFlow

rate/(mL/ min) Column/ (mm

× mm)Chiral compoundComposition of mobile phase1 tR1tR2αRsReference3 2.7Tert-butylcarbamoylquinine0.550 × 4.6Racemic Fmoc-PheMethanol/glacial acetic acid/ ammonium acetate (98/2/0.5, v/v/w)

1.492.031.516.0285a 21.502.021.536.21 3.6Cellulose-(3,5-dichlorophenyl- carbamate)1100 × 4.6EtozolineMethanol1.111.412.33.388a 2-Benzylsulfinyl benzamide0.961.4277.9 2-(3-Bromobenzylsulfinyl) benzamide0.971.466.57.2 2-(4-Methylbenzylsulfinyl) benzamide0.982.3815.311 2-(Benzylsulfinyl)-N,N-dimethyl benzamide1.071.292.13.7 2-(Benzylsulfinyl) N-methyl benzamide11.162.32 2-(2-Methylbenzylsulfinyl) benzamide0.961.116.94.9 2-(3-Methylbenzylsulfinyl) benzamide0.971.829.97 1.49Teicoplanin0.1150 × 2.1AlanineEthanol/water (80:20, v/v)2.13.832.390a Valine22.92.31.9 Methionine1.24.23.75 Leucine1.72.62.72.1 2.7Vancomycin1100 × 2.1Tolperisone2.0 mM TEAAc in methanol0.70.81.251.491a 2.7Vancomycin0.8Mianserin0.50.71.81.4 2.7Vancomycin1.2Promethazine0.60.81.631.4 2.7(R,S)-Hydroxypropyl-β- cyclodextrin0.8150 × 4.6Cycloprothrin

MeOH/16 mM NH

4HCO2(60:40,v / v), pH 3.65.15.41.071.496a 5.96.41.132 0.5TriadimenolCAN/16 mM NH4HCO2(20: 80,v/v), pH 3.67.59.51.445.7 1010.51.061.6 1EPNCAN/16 mM NH4HCO2 (30:70,v / v); pH 3.66.67.41.092.1 1BenalaxylCAN/16 mM NH4HCO2(30:70,v / v); pH 3.61.61.91.362.4 Teicoplanin1100 × 4.6HaloxyfopMeOH/NH4HCO2(100:0.2,v / w)1.11.44.032.4 Quinine0.5100 × 4.6BrodifacoumMeOH/NH4HCO2 (100: 0.3,v / w)671.172.5 11171.789

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Table 2 (continued) Nominal particle diameter /μm

ModifierFlow

× mm)Chiral compoundComposition of mobile phase1tR1tR2αRsReference3 2.7Vancomycin2100 × 4.6AlprenololMeOH/NH4HCO2 (100: 0.2, v/w)2.83.11.424.797a Modified macrocyclic glyco- peptide0.8100 × 4.6ssMeOH/AA/NH4OH (100: 0.2: 0.05, v/v/v)1.21.51.152.3 Vancomycin0.3150 × 4.6TramadolMeOH/NH4HCO2 (pH 3.6; 48 mM) (30: 70, v / v)6.781.211.5 2.7Teicoplanin2100 × 4.6( ±)-2′-(4-bromophenylamino) spiro{indoline-3,5′-[4′,5′]- dihydrothiazole}-2-one

CO2/MeOH/TFA (80/20/0.1,v / v / v)1.34.313.464.2592b 5-hydroxy-DL-tryptophanCO2/MeOH/DEA/TFA (60/40/0.05/0.05,v / v / v / v)1.072.282.232.32 2-(2-methoxyphenyl)-2-methyl- amino-cyclohexanonCO2/2-PrOH/ACN/TEA/TFA

(90/5/5/0.05/0.05,v / v / v / v / v)

2.4131.262.68 Vancomycin2100 × 4.64-chloro-N-butylcathinoneCO2-MeOH (80/20,v / v)0.850.981.161.2 NicardipineCO2-MeOH-Ipam-TFA (80/20/0.025/0.025,v / v / v / v)0.891.521.783.14 2.7Vancomycin4100 × 4.6FluoxetineCO2/MeOH- 3% (w/v)water- 0.1%(v/v) TEA- 0.1% (v/v) TFA (75/25)

0.760.881.191.9495b Modified macrocyclic glyco- peptide4100 × 4.6NicotineCO2/MeOH- 0.1% (v/v)TEA (60/40)1.852.351.363.4 100 × 4.6TramadolCO2/MeOH- 0.2%(v/v) TEA- 0.3%(v/v) TFA (60/40)0.080.251.411.9 Isopropyl derivatized cyclofructan-64100 × 4.6NorephedrineCO2/MeOH- 0.2%(v/v) TEA- 0.3% (v/v)TFA (80/20)2.12.41.142.26 4100 × 4.61-(1-Naphthyl)ethylamineCO2/MeOH- 0.2%(v/v) TEA- 0.3% (v/v)TFA (80/20)1.51.81.243.06 Teicoplanin4100 × 4.6DichloropropCO2/MeOH- 0.1%(w/v) ammonium formate (60/40)0.91.41.74.18 2.7Vancomycin4.9530 × 4.6ThaildomideMethanol0.080.12.778c Teicoplanin aglycone4.730 × 4.65-Methyl-5-phenylhydantoin0.080.12.4 Hydroxylpropyl-β-cyclodextrin4.7550 × 4.6Jacobsen’s catalystAcetonitrile/methanol/TFA/TEA (97:3:0.3:0.2)0.130.151.8 Cyclofructan-7 dimethylphenyl carbamate4.830 × 4.6(R)-( +)-2,2′-Diamino-1,1′- binaphthaleneHeptane/ethanol(90:10)0.160.21.9

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Table 2 (continued) Nominal particle diameter /μm

ModifierFlow

× mm)Chiral compoundComposition of mobile phase1tR1tR2αRsReference3 2.7Quinine-hydrosilated150 × 4.6Dichlorprop80/20 MeOH/100 mM NH4OAc (pH 6.0)1.21.42.681 c Quinine-mercapto4.75.53.5 2.7ZWIX20.550 × 32-amino-2-phenylbutyric acidMethanol containing 50 mM for- mic acid and 25 mM ammonia0.60.71.452.5487c Mefloquine0.20.46.338.79 2Teicoplanin1100 × 4.6D,L-ProglumideACN/H2O (85:15) + 20 mM HCOONH4(pH 7.5)2.032.271.203.9993 c Dansyl-D, L-Methionine1.922.681.7311.09 Fmoc-D, L-Glutamine4.054.91.276.68 Z-D, L-Methionine2.273.361.7713.9 2.7D, L-Proglumide1.922.101.162.41 Dansyl-D, L-Methionine1.782.501.778.72 Fmoc-D, L-Glutamine3.74.521.295.24 Z-D, L-Methionine2.132.191.8311.53 1 The abbreviation of solvent: trifluoroacetic acid (TFA), triethylamine (TEA), methanol (MeOH), propan-2-ol (2-PrOH), acetonitrile (CAN), isopropylamine (Ipam), diethylamine (DEA), acetic acid (AA), ammonium trifluoroacetate (NH4TFA), ammonium formate (NH4HCO2), ammonium hydroxide (NH4OH), triethylammonium acetate (TEAAc), ammonium acetate (NH4Ac) 2 ZWIX obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond 3 a, b and c represent the results obtained on different chromatographic platforms. a. HPLC; b. supercritical fluid chromatography(SFC); c UPLC

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the useless microporous separation region of the stationary phase, which can effectively reduce the mass transfer path and the column pressure. Numerous studies have confirmed

the SPS packing compared with the FPS packing have the obvious advantages in the separation of various substances including the enantiomer, biomacromolecule, small organic molecules and so on. SPS has been regarded as a prefer- able substitute of FPS to develop the stationary phase with better performance. And there are two expectations for the application of superficially porous packing in developing the higher performance stationary phase. One is to develop small particle size stationary phase with high column efficiency, which can be used in conventional HPLC system, using the characteristics of low back pressure. The other is to further optimize the mass transfer behavior of analytes on the SPS by adjusting the pore structure and size of the surface layer. Additionally, SPS also has the limitation and the insufficiency, such as the preparation method is time- consuming and the yield is low, researchers should make further study in later research.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21822407, 22074154), Xinjiang “Tianshan Youth Plan” Project (No. 2017Q027), the “Light of West China” Program from Chinese Academy of Science, and the Gansu Province Basic Research Innovation Group Program (20JR5RA573).

Fig. 8 Exemplary chromatograms obtained on the different columns showing enantiomer separations of acidic analyte Fmoc-Phe.

Reprinted with permission from [87]

Fig. 9 Fast separation of enantiomers of 2-benzylsulfinyl benzamide (a), 2-(3-bromobenzylsulfinyl) benzamide (b), 2-(benzylsulfinyl)-N- methyl benzamide (c),

2-(benzylsulfinyl)-N,N-dimethyl benzamide (d), 2-(2-methylbenzylsulfinyl) benzamide (e), 2-(3-methylbenzylsulfinyl) benzamide (f), etozoline (g) and trans-stilbene oxide (i). The mobile phase was methanol in the cases a-g and n-hexane/2- propanol = 98/2 (V/V) in the case (i) at the flow rate 5 mL/

min in the cases a–f and i and 5.5 mL/min in the case g.

Chromatograms were recorded at 220 nm and 160 Hz detec- tor frequency. Reprinted with permission from [88]