Chromatographic Packing Research Articles

Sustainable hydrophilic ultrasmall carbonaceous spheres modified by click reaction for high-performance polymeric ion chromatographic stationary phase

Novel poly(ethylvinylbenzene-divinylbenzene) (EVB-DVB) agglomerated with ultrasmall carbonaceous spheres (UCSs) anion-exchange packings for ion chromatography (IC) were constructed. Hydrophilic UCSs with mean sizes of 62–98 nm were synthesized in quantity by the polydiallyl dimethyl ammonium chloride aided hydrothermal carbonization of fructose. The green strategy based on the thiol-ene click reaction with cysteamine in aqueous system was first designed for the hyperbranched polyquaternary amine (HPA) grafting of UCSs with negligible damage on their monodispersity. The HPA modified UCSs were evenly distributed on sulfonated EVB-DVB substrate to form one uniform layer of functional nanospheres without observable coagulum. Seven typical anions (F−, Cl−, NO2−, Br−, NO3−, SO42- and PO43−) were baseline separated on constructed packing in 5 min with high efficiencies in the range of 44,800–71,100 plates m − 1. The rapid separation of polarizable anions, small organic acids and saccharides could be also accomplished under isocratic elution with competitive peak symmetry and efficiency. Good reproducibility was demonstrated by consecutive injection. Thiosulfate in water reducer was further detected on prepared packing in 4 min with detection limit of 0.04 mg L − 1 (S/N = 3) and good repeatability.

Fabrication of highly crosslinked and monodispersed silicon-containing polymeric microspheres via photo-initiated polymerization and their application in capillary liquid chromatography

Although inorganic silica-based and polymeric micron-sized spheres have widely been explored as column packing materials in high performance liquid chromatography (HPLC), they are still suffering the problems of either alkali corrosion of silica or polymer swelling. It is still necessary to search simple approaches for fabrication of monodisperse micron-sized hybrid particles as packing materials in HPLC. A novel kind of silicon-containing polyacrylate microspheres was designed and fabricated via two-step swelling and photo-initiated polymerization approach using 3-(allylpropylsilane) propyl acrylate (TAPA) containing both acrylate and vinyl groups and trimethylolpropane triacrylate (TRIM) as precursors. After carefully optimizing the fabrication conditions, the monodisperse micron-sized microspheres could be acquired as chromatographic packing, exhibiting excellent mechanical stability and reproducibility. Due to existence of electron-rich vinyl groups, three kinds of thiols such as octadecanethiol (ODT), dithiothreitol (DTT) and trimethylolpropane tris(3-mercaptopropionate) (TTMP) were facilely anchored onto the surface of microsphere via photo-initiated thiol-ene click reaction. They were applied in the separation of small molecules by cLC-UV and complex biosamples by cLC-MS/MS. A total of 6691 unique peptides from 1771 unique proteins was identified by ODT-modified microsphere, which was higher than those by unmodified and DTT/TTMP-modified poly(TAPA-co-TRIM) microspheres. It was expected this kind of hybrid microspheres can be further modified and widely applied in chromatographic field, offering great potential in commercialization.

The sustainable, one-pot and high-yield synthesis of ultrafine carbonaceous nanospheres with high anionic separation efficiency

The unique and tunable size-dependent properties (less than 100 nm) make ultrafine carbonaceous nanospheres (UCNSs) superior and promising in plentiful modern technological applications. However, fabricating monodiperse UCNSs in quantity was still challenging, especially via a sustainable way. This article presents a one-pot high-concentration hydrothermal carbonization (HTC) of glucose under the aid of polyquaternium-11 to fabricate monodisperse UCNSs with controllable sizes of 51–100 nm. The monodispersity of UCNSs can even be achieved at glucose concentration of 500 g L-1 and in 5-fold enlarged HTCs, with output up to 38.1 g. A novel green strategy based on the thiol-ene click chemistry with cysteine in aqueous system were further designed for the hyperbranched polyquaternary amine grafting of UCNSs without negligible damage on their monodispersity. Grafted UCNSs were applied as latices in ion chromatography packing, showing high separation efficiencies for common anions with better peak symmetry compared with reported or common commercial latex-agglomerated counterparts to the best of our knowledge.

Preparation of Micron⁃Sized Chromatographic Packing TiO2 by Titanium Organic Frameworks

Preparation of Micron⁃Sized Chromatographic Packing TiO2 by Titanium Organic Frameworks

Preparation and application of chiral silica gel spheres based on L-glutamic

无机介孔硅球因其具有足够的机械强度、热稳定性,以及适应多种流动相的优点,成为高效液相色谱(HPLC)柱填料中使用最广泛和最重要的材料。但在此研究领域中,并未见球形的全无机手性硅胶用作HPLC手性固定相。该文以无机球形介孔硅胶作为研究对象,通过堆砌硅珠法,以硅溶胶为原料,L-谷氨酸(L-Glu)为手性源,在手性环境中制造出脲醛树脂与胶体二氧化硅混合的小球,在550 ℃高温下煅烧除去树脂部分,制备基于L-Glu的无机介孔硅胶球。通过元素分析、红外光谱、扫描电镜、透射电镜和氮气吸附等表征证明这是一种具有规则球形的手性硅胶球,其手性来源于硅胶球自身的骨架和孔结构。将L-Glu手性硅胶球作为固定相制备了HPLC色谱柱,以正己烷-异丙醇(9∶1, v/v)作为流动相,流速为0.1 mL/min,考察了该手性柱对一系列外消旋化合物的拆分性能。实验表明,该手性柱拆分了15种外消旋化合物,其中特罗格尔碱、吡喹酮、3-苄氧基-1,2-丙二醇、1,2-环氧己烷、3-羟基-2-丁酮、2-甲基四氢呋喃-3-酮、异丙基缩水甘油醚达到基线分离;还分离了10种苯系位置异构体,o,m,p-氨基苯酚、o,p-氯苯酚、o,m,p-碘苯胺、o,m,p-甲苯胺、o,m,p-二硝基苯、o,m,p-氯苯胺、o,m,p-硝基苯酚、o,m,p-溴苯胺达到基线分离。实验表明,L-Glu手性硅胶球在手性分离方面具有良好的可行性,与普通硅胶相比不需要进一步修饰就可以有较好的手性分离效果,是一种低成本、制备便捷的手性无机硅胶固定相。

Non-conjugated flexible network for the functional design of silica-based stationary phase for mixed-mode liquid chromatography

The functionalized non-conjugated organic network modified silica microspheres are proposed as the stationary phase of liquid chromatography, which can effectively avoid some defects of organic framework materials in liquid chromatography. Due to the co-existing of pyridine ring, carbonyl group, amide group and triazine ring in the non-conjugated flexible organic network on the silica surface, the developed mixed-mode stationary phase exhibits multiple interactions between the stationary phase and the analytes during the separation process. A variety of nucleoside bases, organic acids, antibiotics, pesticides, alkylbenzenes, polycyclic aromatic hydrocarbons and sulfonamides achieved ideal resolution and flexible selectivity in separation. Compared with the commercial chromatographic columns under their optimized chromatographic conditions, it shows better performance for the separation of complex analytes. The influence of chromatographic conditions on retention behavior indicates that the column's multiple retention mechanisms make it suitable for mixed-mode liquid chromatography. The stationary phase prepared by the new design strategy also has excellent chromatographic reproducibility, repeatability and stability with the intraday RSD of 0.09%–0.12% (n = 10) and the interday RSD of 0.37%–1.64% (n = 5) for the retention time. The separation results of actual samples also prove its potential in the analysis of complex samples. In short, we designed and prepared the non-conjugated flexible network modified silica stationary phase material for liquid chromatography that is different from organic framework materials. Its excellent separation ability shows that we have successfully reported a new kind of liquid chromatography packing with functional design and facile preparation method.

A facile process for the preparation of organic gel-assisted silica microsphere material for multi-mode liquid chromatography.

Organic gel (OG) has excellent characteristics, including a large surface area, adjustable pore/channel size, and good chemical stability, and has attracted great attention in the field of materials. However, the OG packed column is difficult to pack due to the weak mechanical strength and poor monodispersity. Herein, 1-allyl-3-methyl imidazolium hexafluorophosphate-co-1-dodecanethiol ([AMIm]PF6-co-TDDM) was prepared on the silica microsphere for chromatographic packing available in multimode liquid chromatography (LC) mode with the good mechanical properties of silica microspheres through a simple OG synthesis method. [AMIm]PF6-co-TDDM@SiO2 hybrid microspheres with uniform particles and narrow particle size distribution are used as stationary phases of LC. These microspheres are used in anion-exchange (IEC), reversed-phase (RP), and hydrophilic interaction (HILIC) mode for the separation of different analytes. Such microspheres can also be used for the preliminary qualitative analysis of active ingredients in actual samples in addition to organic acids, alkylbenzenes, and nucleoside bases. The [AMIm]PF6-co-TDDM@SiO2 chromatography packing also has good reproducibility and stability. The adhesive properties of organogels and the adsorption properties of silica gel simplify the synthesis of stationary phase materials. This simple and effective strategy for preparing [AMIm]PF6-co-TDDM@SiO2 composite microspheres by one-pot method can expand the application of OG as a functional additive on silica microspheres in LC.

Monodisperse core–shell silica particles as a high-performance liquid chromatography packing material: Facile in situ silica sol-gel synthesis

In recent years, core–shell silica particles (CSSPs) have been increasingly used for highly efficient separation at fast flow rates and relatively low back pressures in high-performance liquid chromatography (HPLC). However, material synthesis techniques for producing CSSPs economically in batch processes remain elusive. In this report, a practical and straightforward method for the preparation of CSSPs is presented. By refluxing freshly prepared nonporous silica particles in ammonia–water solution in the presence of poly(diallyldimethylammonium chloride) at 70–100 °C, CSSPs with shell thicknesses of up to 300 nm and pore sizes from 8 to 25 nm were easily prepared. The effects of the synthetic conditions on the shell thickness, surface area, and pore size were investigated in detail, and the method reproducibility was evaluated in scale-up experiments. A mechanism of CSSP formation is also proposed. The CSSPs were characterized via scanning electron microscopy, transmission electron microscopy, laser particle size (dynamic light scattering) analysis, and nitrogen adsorption and desorption experiments. The synthesized 3.4-μm CSSPs were functionalized with dimethyloctadecylchlorosilane and used as an HPLC packing material, exhibiting excellent separation performance for both small molecules and large biomolecules. In summary, we report the simplest method developed thus far for the preparation of monodisperse core–shell silica particles suitable for HPLC column packing.

Synthesis and chromatographic evaluation of poly(pentabromostyrene)-silica composite: A versatile stationary phase for separating both polar and non-polar aromatic compounds

Polymer-silica composite materials have been witnessed as one of the most promising chromatographic packing materials. The demands of novel stationary phases are always existing due to the requirements for the separation of special samples. In our present work, the novel poly(pentabromostyrene)-silica composite stationary phases were successfully prepared by surface initial free-radical polymerization. The polymer chains bonding on the surface of the poly(pentabromostyrene) contain plenty of phenyl moiety, which provide hydrophobic and π-π interactions between the stationary phase and analytes. Besides, the bromide atoms, existing in the polymer, provide extra polar interaction with polar or easily polarized analytes. Compared with the typical C18 stationary phase, the novel stationary phase showed special separation selectivity. Samples, including polycyclic aromatic hydrocarbons, phenols, anilines, amino acids, halogenated benzenes, acetophenone derivatives, benzene homologous compounds, were all well separated by the new stationary phase. Also, the great stability (0.13% ≤ RSDs ≤ 0.98%, n = 5) of packed columns and the good synthesis repeatability were demonstrated. Finally, the new columns were applied to the separation of fullerenes. The loading ratio reached 2.67% (36 mg of fullerenes was loaded onto the column packed with 1.35 g stationary phase), with high yield (86.3%) of high pure C60 (>95%). This novel stationary phase has a great potential to be a complementary to traditional C18 column and a special column for fullerenes separation.

A multiscale modelling study on the sense and nonsense of thermal conductivity enhancement of liquid chromatography packings and other potential solutions for viscous heating effects

We report on a numerical study of the thermal conductivity and temperature distribution in analytical packed bed and monolithic HPLC columns to assess the feasibility of a number of potential solutions to the viscous heating problem that would normally impede high efficiency separations when moving to extreme operating pressures (e.g., 2500 bar). Computational fluid dynamic (CFD) simulations were employed to study heat transfer on three hierarchical levels of the column: meso‑pore level, through-pore level and column level. At the first level, realistic values were determined for the conductivity of the porous zone (kpz), depending on the internal structure of the porous zone and the mobile phase used (acetonitrile, water or a mixture of both). These kpz-values were in turn used at the second level to determine realistic values for the effective conductivity of the bed (keff). It was shown that the presence of a solid core only has a minor effect on the packed bed conductivity. Using highly conducting materials as core material can be expected to maximally lead to a 60% increase in bed conductivity. Contrarily, in monolithic beds, the presence of a core material would form one continuous phase of highly conducting material, thus greatly enhancing the conductivity of the bed. At the third level, the temperature field in the entire column (bed and column housing) was resolved for three typical boundary conditions: isothermal, adiabatic and still-air oven. The effect of different physical properties (inlet pressure, mobile phase composition, bed conductivity, wall conductivity and column ID) on these temperature fields was investigated. It was shown that, theoretically, besides 1 mm ID columns also “core-shell monoliths” can provide a solution to viscous heating (by increasing the bed conductivity). Other possible solutions are proposed and discussed.

Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions

We developed a mixed polymer brush, which consists of temperature-responsive polymer and cationic polymer modified beads as functional chromatographic matrices, for temperature-modulated multiple hydrophobic and electrostatic interactions. The mixed polymer brush was modified on silica beads through the combination of reversible addition−fragmentation chain transfer (RAFT) polymerization of N,N-dimethylaminopropyl acrylamide (DMAPAAm) and surface initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). Zeta potential measurement of the mixed polymer brush revealed that the zeta potential increased with increasing temperature, which was attributed to the exposed PDMAPAAm on the beads arising from the shrinking of PNIPAAm upon increasing the temperature. The prepared beads were used as the packing material of high performance liquid chromatography (HPLC) columns, and the elution behavior of steroids, adenosine nucleotides, and proteins through the column was observed. The retention time of steroids increased with increasing the column temperature because of the dehydration of PNIPAAm in the mixed polymer brush. Adenosine nucleotides were also retained by the columns, which was attributed to the electrostatic interaction with PDMAPAAm in the mixed polymer brush. Several proteins were adsorbed on the column at elevated temperatures because of the enhanced electrostatic interaction of exposed PDMAPAAm and the enhanced hydrophobic interaction resulting from the dehydration of PNIPAAm. By exploiting this unique property, mixtures of proteins could be separated by simply changing the column temperature. These results indicate that the developed mixed polymer brush modified beads would be useful as functional chromatographic packing matrices for thermally-modulated multiple hydrophobic and electrostatic interactions.

A novel method to purify adenovirus based on increasing salt concentrations in buffer

With the rapid development of gene therapy, gene-based medicine with adenovirus as vectors has become a new method for disease treatment. However, there are still enormous challenges in the large-scale production of adenoviruses for clinical use. Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. This method could significantly remove a large amount of serum proteins and host cell proteins, increase the amount of sample loaded on the IEC column, and improve the binding of the adenovirus samples to the packing media. A purity of > 95% could be obtained after one chromatography operation, and the number of purification steps and the amount of used packing media were reduced. The method is simple, economical, and efficient, and has excellent applications.

Poly(glycidyl methacrylate-co-1,4-dimethacryloyloxybenzene) monodisperse microspheres - synthesis, characterization and application as chromatographic packings in RP-HPLC

The study describes the synthesis of porous microspheres with glycidyl methacrylate (GMA) and 1,4-dimethacryloyloxybenzene (DMB) using the seed swelling polymerization. As a shape template the polystyrene (PS) seed obtained via the dispersion polymerization was used. Oxirane, esters and aromatic groups are present in the structure of copolymers. Changing the reaction mixture composition – the amounts of crosslinking monomer and PS seed, almost monosized poly(GMA-co-DMB) microspheres of the diameters in the range of 8.20 μm to 11.61 μm and highly developed porous structure with specific surface area (SBET) up to 353 m2/g were obtained. The pore size distribution and the volume of pores measured for the copolymer in the dry and the swollen states differ. In the dry state in the internal copolymer structure mesopores were observed whereas in the swollen state additionally micro- and macropores appeared. The copolymer obtained with the equimolar mixture of monomers was applied as the column packing material and tested in the reverse-phase HPLC. The isocratic separation of alkylbenzenes was achieved with satisfactory resolution using the 100 × 4.6 mm i.d. column with the acetonitrile/phosphate buffer mobile phase. The theoretical plate numbers up to 12,000 were obtained for benzene as the analyte. Using the retention indices for the test compounds it was found that the retention mechanism on the studied copolymer is the sum of π-π hydrophobic interactions, dipole interactions and to a smaller extent of hydrogen acceptance interactions rather than hydrogen donation interactions.

Glycopolymer Grafted Silica Gel as Chromatographic Packing Materials.

The modification of the surface of silica gel to prepare hydrophilic chromatographic fillers has recently become a research interest. Most researchers have grafted natural sugar-containing polymers onto chromatographic surfaces. The disadvantage of this approach is that the packing structure is singular and the application scope is limited. In this paper, we explore the innovative technique of grafting a sugar-containing polymer, 2-gluconamidoethyl methacrylamide (GAEMA), onto the surface of silica gel by atom transfer radical polymerization (ATRP). The SiO2-g-GAEMA with ATRP reaction time was characterized by Fourier infrared analysis, Thermogravimetric analysis (TGA), and elemental analysis. As the reaction time lengthened, the amount of GAEMA grafted on the surface of the silica gel gradually increased. The GAEMA is rich in amide bonds and hydroxyl groups and is a typical hydrophilic chromatography filler. Finally, SiO2-g-GAEMA (reaction time = 24 h) was chosen as the stationary phase of the chromatographic packing and evaluated with four polar compounds (uracil, cytosine, guanosine, and cytidine). Compared with unmodified silica gel, modified silica gel produces sharper peaks and better separation efficiency. This novel packing material may have a potential for application with highly isomerized sugar mixtures.

Analysis of microstructure–effective diffusivity relationships for the interparticle pore space in physically reconstructed packed beds

We evaluate the effective diffusion coefficient in the interparticle pore space of eight physically reconstructed chromatographic packings of fully porous and core-shell particles, employing an analytical formula that involves the three-point microstructural parameter calculated from two-point and three-point correlation functions through an approach based on sampling templates. Diffusivities calculated by the approximate analytical formula are close to those obtained from pore-scale simulations in the reconstructions using a random-walk particle-tracking technique. Diffusivities are affected, apart from the interparticle porosity, by the actual bed microstructure and packing defects like particle oligomers, spalled shells, larger voids, and debris. Importantly, the data for the microstructural parameter and effective diffusion coefficient over the porosity range spanned by the individual packings (0.363–0.444) reveals non-monotonic behavior. Our analysis demonstrates that the three-point microstructural parameter reflects this morphological specificity of the packings and, as a result, Eq. (3) provides accurate estimates of the effective diffusion coefficients. The presented numerical approach can therefore be applied to evaluate diffusivities in packed beds with all their salient features just from the geometrical information embodied in the bed porosity and the three-point microstructural parameter.

A Thioether-Embedded Mixed-Mode Cyano-Bonded Chromatographic Stationary Phase: Preparation, Characterization and Retention Mechanism

In this paper, a thioether-embedded mixed-mode cyano-bonded chromatographic packing has been prepared by using gas silylation method and thiol-ene click chemistry. The solid-state 13C CP/MAS NMR and elemental analysis have been used to characterize the synthesized materials. Detailed chromatographic characterization of the new phase was performed by alkyl benzene homologues in reversed-phase liquid chromatography (RPLC) and was characterized by nucleic acid bases and nucleosides in hydrophilic interaction liquid chromatography (HILIC). The hydrothermal stability has been studied by n-butylbenzene, and results show that the novel cyano-phase had higher stability. In addition, linear solvation energy relationships (LSERs) have been used to investigate the effect of stationary phase property on the retention factor of seven aromatic solutes. The results have shown that the Sil–S–CN phase has a pronounced strong polarity and dipole interactions between stationary phases and analytes.

Synthesis and evaluation of porous polymethylsilsesquioxane microspheres as low silanol activity chromatographic stationary phase for basic compound separation

Polymethylsilsesquioxanes (PMSQ) are potentially useful materials for liquid chromatography owing to their unique chemical, electrical and mechanical properties. Surprisingly however, no systematic studies on the use of spherical PMSQ particles as chromatographic packing have been reported. Accordingly, we present a comprehensive study aimed to characterize the chromatographic properties of this material in high performance liquid chromatography (HPLC) and to compare them with those observed on methyl (C1) bonded silica phase under comparable conditions. Porous spherical particles were synthesized by a two-step hydrolysis and condensation procedure from methyltrimethoxysilane (MTMS) as a sole precursor. The as-synthesized microspheres possess spherical shape, narrow size distribution, mesoporous structure, high surface area (817 m2 g−1) and reasonable carbon load (16.6%). They can be used directly as the HPLC stationary phase without the need for size classification. The PMSQ phase exhibits typical reversed-phase chromatographic properties with higher methylene selectivity and low silanol activity compared with the C1 column. The retention mechanism for basic compounds was systematically evaluated by studying the effect of pH, ionic and solvent strength of the mobile phase. Basic compounds displayed lower retention factor and symmetric peak shape on the PMSQ column whereas longer retention and strong tailing peaks were observed on the C1 column. The difference in retention behavior between the two columns is explained in terms of different principal retention mechanisms. Because of the low silanol activity, retention of basic compounds on the PMSQ column is governed solely by a reversed-phase mechanism. By contrast, multiple interactions including reversed-phase, cation exchange and simultaneous reversed-phase/cationic exchange interaction contribute to the retention on the C1 column, as previously observed on other silica based reversed-phases. Furthermore, the PMSQ phase exhibited significantly enhanced stability under alkaline conditions compared with its silica-based counterpart. Taken together, the favorable morphology and pore structure combined with the benefits of low silanol activity, high pH stability and prolonged column lifetime make the newly developed PMSQ phase a promising and viable alternative to silica based reversed-phase packings for separation of basic compounds.

Determination of Pharmaceuticals by Dynamic Cell Membrane Chromatography Coupled with High-Performance Liquid Chromatography

ABSTRACTAs a biological affinity chromatographic method, cell membrane chromatography (CMC) using a silica stationary phase covered with specific cell membrane has been used in screening active components. The innovation of this work is that the bioactive cell membrane and the chromatographic packing are mixed and absorbed for the first time to form the pre-column. The pre-column was placed in front of a C18 column to create dynamic CMC online high-performance liquid chromatography (HPLC) system. The retention behavior and dynamic changes of pharmaceuticals were studied for this system. The results indicate that the retention time of the drug was increased and the symmetry factor reached the analytical level after the addition of the dynamic cell membrane pre-column. Therefore, the dynamic CMC coupled with HPLC system may be a potentially rapid and efficient drug analysis approach for the interaction of drug molecule and receptor on red blood cell membranes.

Online measurement of phthalate–particulate matter interactions by membrane introduction mass spectrometry (MIMS)

ABSTRACTTo enable further study and assessment of indoor inhalation exposure risk, an online apparatus enabling measurement of semi-volatile compound partitioning on household particulates was developed. An example for use of the apparatus is described using dimethyl phthalate (DMP). The system employs direct measurement by membrane introduction mass spectrometry (MIMS). The MIMS system was calibrated using known gas phase DMP concentrations produced by gravimetrically calibrated permeation devices. The quantity of DMP sorbed by particles is described first using a model particle type, a reverse-phase liquid chromatography packing material, and then with a household dust sample. In addition, the desorption of semi-volatile compounds from a household dust sample was monitored using the apparatus, and characteristic fragment ion signals for phthalate compounds were observed.

High efficiency and fast separation of active proteins by HIC chromatographic pie with sub-2 μm polymer packings

This paper reports the development of hydrophobic interaction chromatography (HIC) by synthesizing sub-2 μm polymer packings which was packed into a chromatographic pie for fast separation of native proteins at low pressures demonstrating high efficiency. Using styrene as monomer and ethylene dimethacrylate (EDMA)as swelling agent, the polystyrene seeds with an average particle size of 0.8 μm and monodisperse polymeric microspheres with a particle size of 1.5–5.0 μm were synthesized through dispersion polymerization and one-step swelling method, respectively. In order to separate active proteins, the microspheres were modified to hydrophobic chromatographic packings through covalent bonding with benzene methanol. Compared with the traditional column chromatography, the sub-2 μm polymer packings in chromatographic pie exhibited higher column efficiency for protein separation at lower column pressures, even at higher flow rates. The van Deemter curve showed that the flow rate had insignificant effect on column efficiency of chromatographic pie. Seven example proteins were clearly separated within 3 min at a flow rate of 10 mL/min. The applicability of this method was further demonstrated by the separation of human serum samples. The results indicated that this chromatographic mode can be potentially applied for the fast separation of complex active proteins, such as protein drugs from natural products.