Superficially Porous Particles Research Articles

A stochastic view on column efficiency

A stochastic model of transcolumn eddy dispersion along packed beds was derived. It was based on the calculation of the mean travel time of a single analyte molecule from one radial position to another. The exchange mechanism between two radial positions was governed by the transverse dispersion of the analyte across the column. The radial velocity distribution was obtained by flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 μm BEH-C18 particles. Accordingly, the packed bed was divided into three coaxial and uniform zones: (1) a 1.4 particle diameter wide, ordered, and loose packing at the column wall (velocity uw), (2) an intermediate 130 μm wide, random, and dense packing (velocity ui), and (3) the bulk packing in the center of the column (velocity uc).First, the validity of this proposed stochastic model was tested by adjusting the predicted to the observed reduced van Deemter plots of a 2.1 mm × 50 mm column packed with 2 μm BEH-C18 fully porous particles (FPPs). An excellent agreement was found for ui = 0.93uc, a result fully consistent with the FIB-SEM observation (ui = 0.95uc). Next, the model was used to measure ui = 0.94uc for 2.1 mm × 100 mm column packed with 1.6 μm Cortecs-C18 superficially porous particles (SPPs). The relative velocity bias across columns packed with SPPs is then barely smaller than that observed in columns packed with FPPs (+6% versus + 7%). uw=1.8ui is measured for a 75 μm × 1 m capillary column packed with 2 μm BEH-C18 particles. Despite this large wall-to-center velocity bias (+80%), the presence of the thin and ordered wall packing layer has no negative impact on the kinetic performance of capillary columns. Finally, the stochastic model of long-range eddy dispersion explains why analytical (2.1–4.6 mm i.d.) and capillary (<400 μm i.d.) columns can all be packed efficiently (1 <hmin< 2) with particles of size larger than 2 μm. In contrast, the model predicts that 0.4–1.2 mm i.d. columns and 2.1 mm i.d. columns cannot be packed well (hmin>3) with sub-2 μm particles and with 1 μm particles, respectively.

New frontiers and cutting edge applications in ultra high performance liquid chromatography through latest generation superficially porous particles with particular emphasis to the field of chiral separations.

About ten years after their introduction to the market (happened in 2006), the so-called second generation superficially porous particles (SPPs) have undoubtedly become the benchmark as well as, very often, the preferred choice for many applications in liquid chromatography (LC), when high efficiency and fast separations are required. This trend has interested practically all kinds of separations, with the only exception of chiral chromatography (at least so far). The technology of production of base SPPs is advanced, relatively simple and widely available. The deep investigation of mass transfer mechanisms under reversed-phase (RP) and normal-phase (NP) conditions for achiral separations has shown the advantages in the use of these particles over their fully porous counterparts. In addition, it has been demonstrated that SPPs are extremely suitable for the preparation of efficient packed beds through slurry packing techniques. However, the research in this field is in continual evolution. In this article, some of the most advanced concepts and modern applications based on the use of SPPs, embracing in particular ultrafast chiral chromatography and the design of SPPs with engineered pore structures or very reduced particle diameter, are revised. We describe modern trends in these fields and focus on those aspect where further innovation and research will be required. Graphical Abstract Word cloud of cutting edge applications of superficially porous particles in liquid chromatography.

On the relationship between radial structure heterogeneities and efficiency of chromatographic columns

The general dispersion theory of Aris is applied to predict the virtual asymptotic dispersion behavior of packed columns. The derived model is also used to estimate the actual pre-asymptotic dispersion behavior of modern 2.1 mm × 50 mm columns packed with sub-2 μm fully porous particles (FPPs) during the transient dispersion regime. The model accounts for the actual radial distribution of the flow velocity across the column diameter. From the wall to the center of the column, focused-ion-beam scanning electron microscopy (FIB-SEM) experiments were recently performed to reveal the existence of a thin (0.15dp wide, dp is the average particle diameter) hydrodynamic boundary layer (THBL), a thin (3dp wide) and loose orderly packed layer (TLOPL), a 60dp wide and dense randomly packed layer (WDRPL), and a large (≃460dp) randomly packed bulk central region [1].The theoretical calculations of the actual pre-asymptotic reduced van Deemter curves (2.1 mm × 50 mm column, sub-2 μm BEH-C18 FPPs, n-hexanophenone analyte, acetonitrile/water eluent, 80/20, v/v, flow rate from 0.05 to 0.35 mL/min) confirm that the impact of the sole THBL on column dispersion can be neglected. In contrast, the contribution of the TLOPL to the reduced plate height (RPH) is about 0.2 h unit at optimum reduced velocity. Most remarkably, the negative impact of the TLOPL on column performance may be fully compensated by the presence of the adjacent WDRPL if the depth of the velocity well were to be 5% of the bulk velocity. In actual 2.1 mm × 50 mm columns packed with sub-2 μm FPPs, this velocity depth is as large as 25% of the bulk velocity causing a significant RPH deviation of 0.7 h unit from the RPH of the bulk packing free from wall effects. Maximum column performance is expected for a reduction of WDRPL density. This suggests optimizing the packing process by finding the proper balance between the stress gradient across the WDRPL (responsible for the deep velocity well) and the friction forces between the packed particles (responsible for the rearrangement of the particles during bed consolidation). Past and recently reported RPH data support the theoretical insights: the stress gradient/particle friction balance in the WDRPL is better realized when packing superficially porous particles (SPPs) rather than FPPs in 2.1–4.6 mm i.d. columns (the RPH deviation is reduced to 0.4 h unit) or sub-2 μm particles in 100 cm × 75 μm i.d. capillaries combining high slurry concentrations and sonication (the RPH deviation is reduced to only 0.15 h unit).

Reversed-phase chromatography with large pore superficially porous particles for high throughput immunoglobulin G2 disulfide isoform separation

Reversed-phase liquid chromatography (RPLC) has been commonly used in IgG2 disulfide isoforms analysis. Recently, the columns packed with large pore superficially porous particles (SPP) have become available commercially. This work explores the application of this SPP technology in IgG2 disulfide isoforms separation. A high throughput and improved resolution RPLC method is developed with the optimization of column selection, gradient, temperature and flow rate. Compared with the small particles RP-UHPLC columns, large pore SPP columns provide unique selectivity and several new peaks were resolved and identified to be the free thiol variants of the IgG2 disulfide isoforms. The optimized method enables the detailed characterization of cysteines related variants in a single and fast method.

Future perspectives in high efficient and ultrafast chiral liquid chromatography through zwitterionic teicoplanin-based 2-μm superficially porous particles

With the aim of pushing forward the limits of high efficient and ultrafast chiral liquid chromatography, a new Chiral Stationary Phase (CSP) has been prepared by covalently bonding the teicoplanin selector on 2.0μm Superficially Porous Particles (SPPs). An already validated bonding protocol, which permits to achieve teicoplanin-based CSPs exhibiting zwitterionic behaviour, has been employed to prepare not only the 2.0μm version of the CSP but also two other analogous CSPs based, respectively, on 2.7μm SPPs and 1.9μm Fully Porous Particles (FPPs). The kinetic performance of these CSPs has been compared through the analysis of both van Deemter curves and kinetic plots by employing in-house packed columns of 4.6mm internal diameter and different lengths (20, 50 and 100mm). In particular on the columns packed with 2.0μm SPPs, extremely large efficiencies were observed for both achiral (>310,000 theoretical plates/meter, N/m; hr: 1.61) and chiral compounds (>290,000 N/m; hr: 1.72) in HILIC conditions. Thanks to their efficiency and enantioselectivity, these CSPs were successfully employed in ultrafast chiral separations. As an example, the enantiomers of haloxyfop were baseline resolved in about 3s, with a resolution higher than 2.0, (flow rate: 8mL/min) on a 2cm long column packed with the 2.0μm chiral SPPs.

Intraparticle and interstitial flow in wide-pore superficially porous and fully porous particles

Using a synthetic particle model, viscous fluid flow through packed beds of porous particles is directly simulated at the pore scale (1000Å) using the lattice Boltzmann method to characterize intraparticle and interstitial flow. Synthetic particle models are derived from synthesis conditions, scanning electron and focused ion-beam microscopy. A fully porous particle (FPP) and a superficially porous particle (SPP), derived from the FPP, both with the same external surface, are studied. Packed beds of random packings and body-centered cubic packings of the SPP and FPP models were generated by a Monte Carlo procedure that employs random translation and rigid-body rotation of the particles.Detailed velocity distributions are presented for the interstitial and intraparticle regions of the packed beds and within the particles. These results confirm that porous particle packed beds are heterogeneous systems which require extensions to classical theory for correctly predicting the resistance to flow. It is shown that SPPs require less pressure than FPPs to maintain the same flow velocity. For the random SPP and FPP packed beds, the particle hull mass flux is ≈10% of the interstitial flux and ≈3% of the total volumetric flux in the flow direction through the SPP hull. The calculated intraparticle pore velocities confirm that an internal flow, characteristic of “perfusion” chromatography, exists within the porous shell that can enhance biomolecular separations.

New anthracenyl polar embedded stationary phases with enhanced aromatic selectivity, a combined experimental and theoretical study: Part 1-experimental study

New polar embedded aromatic stationary phases of different functionalities (mono- and trifunctional) and on different silica supports (Fully Porous Particle (FPP) and Superficially Porous Particle (SPP)) were synthesized to determine the impact of the functionality on the retention process and the selectivity towards aromatic compounds. A full experimental characterization was performed using a combination of techniques (elemental analysis, thermogravimetric measurements, infrared spectroscopy and solid-state NMR) to differentiate unambiguously the mono- and trifunctional Stationary Phases (SP). Commercially available columns with either an aromatic group or a polar embedded group were compared to the new stationary phases. The latter presented enhanced affinity for polycyclic aromatic hydrocarbons (PAH) structures compared to alkylbenzenes, especially when using methanol instead of acetonitrile as the organic modifier.

Chromatographic separation of racemic praziquantel and its residual determination in perch by LC-MS/MS

An enantioselective depletion study of praziquantel (PZQ) in perch muscle was done using a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Two cyclic polypeptides and two cyclic saccharide chromatographic columns were evaluated. The new hydroxypropyl β-cyclodextrin (HP-RSP) superficially porous particle (SPP) column produced the optimum separation and was selected for the study. Deuterium labeled PZQ-d11 was used as the internal standard. The method was linear over concentration ranges of 5.00–5.00×102μgL−1 (r2≥0.99) for (-)-R-PZQ and (+)-S-PZQ. The average recoveries of R-PZQ and S-PZQ at three spiked levels of 5.00, 50.00 and 5.00×102μgkg−1 ranged from 86.1% to 98.2%, and the intra-day and inter-day relative standard deviations were less than 5%. The decision limit (CCα) and detection capability (CCβ) of R-PZQ and S-PZQ in perch muscle matrices were all 1.0μgkg−1 and 5.0μgkg−1, respectively. The method was successfully applied in monitoring the depletion of praziquantel enantiomers in perch muscle following oral administration. The elimination rate of R-PZQ and S-PZQ in perch muscle tissue is equivalent.

Quinine bonded to superficially porous particles for high-efficiency and ultrafast liquid and supercritical fluid chromatography

Two new anion-exchange columns were prepared by bonding tert-butyl carbamoylated quinine to 2.7 μm superficially porous particle (SPP) silica to create chiral stationary phases for high-efficiency and ultrafast chromatography. Performance and retention parameters of these new columns are compared with an analogous 5 μm fully porous particle (FPP) based Chiralpak QNAX column and a 3–4 fold increase in efficiency was observed. Ultrafast separations ranging from 12 s down to sub-second are shown using 2.7 μm SPPs bonded via hydrosilation to the selector. Potential benefits of 2.7 μm SPP based columns for increased LC-MS compatibility were investigated. A van Deemter plot comparison showed 2.7 μm SPP based columns provided a lower reduced plate height and a higher optimal linear velocity compared to the 5 μm FPP based column. With geometry-independent kinetic plots, 2.7 μm SPP and 5 μm FPP based columns were assessed for their kinetic performance and the maximal number of plates each column can generate in a given analysis time. The 2.7 μm SPP based column showed remarkable performance improvements in speed and efficiency as indicated by the kinetic plots.

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Impact of straight, unconnected, radially-oriented, and tapered mesopores on column efficiency: A theoretical investigation

Superficially porous particles (SPPs) can be prepared from a pseudomorphic transformation (PMT) which produces straight, unconnected, and radially-oriented mesopores (ROMs). ROMs can be either both ends open in fully porous particles (FPPs) or one-end-closed in SPPs. The impact of ROMs on the longitudinal diffusion (B/u), solid–liquid mass transfer resistance (Csu), and on the eddy dispersion (A(u)) height equivalent to a theoretical plate (HETP) of 3D randomly packed columns was investigated based on theoretical viewpoints. Torquato's theory of effective diffusion in packed beds (B term), Giddings’ coupling theory of eddy dispersion (A term), and Giddings’ generalized nonequilibrium theory (Cs term) are applied to make predictions. First, it is found that the A term is nearly independent on the internal structure of the particle. Secondly, in the absence of flow, infinitely narrow and both ends open (no constriction effect) ROMs induce an internal hindrance factor of 23 regarding diffusion along the axial direction. Experimental data reveal that one-end-closed and 80Å wide ROMs in SPPs lead to a measurable internal hindrance factor of 27 regarding diffusion in the porous shell. Thirdly, above the optimum speed, the Cs coefficient is dependent on the geometry (cylinders, cones, etc.) of the ROMs: when ROMs are conical in SPPs, Cs is expected to decrease by 80% with respect to cylindrical ROMs. From an application perspective, PMT-SPPs prepared with narrow ROMs are well suited for the analysis of small molecules at or below optimum speed (lowest B term) while PMT-SPPs made of wide and conical ROMs are ideal for the analysis of large molecules above optimum speed (smallest Cs term).

Recent advancements and future directions of superficially porous chiral stationary phases for ultrafast high-performance enantioseparations.

This review focuses on the use of superficially porous particles (SPPs) as chiral stationary phases for ultra-high performance liquid enantioseparations. In contrast to what happened in achiral separations where core-shell particles invaded the market, the introduction of SPPs in chiral liquid chromatography (LC) has been relatively recent. This is due in part to the technical difficulties in the preparation of these phases, and in part to scarce understanding of mass transfer phenomena in chiral chromatography. As a matter of fact, nowadays, the development of superficially porous CSPs is still in its infancy. This paper covers the most recent advancements in the field of core-shell technology applied to chiral separations. We review the kinds of chiral selectors that have been used for the preparation of these phases, by discussing the advantages of chiral SPPs over their fully-porous counterparts for high efficient high throughput enantioseparations. Notwithstanding the apparently obvious advantages in terms of the mass transfer of chiral SPPs, some critical aspects that could impact their development are presented.

Size exclusion chromatography with superficially porous particles

A comparison is made using size-exclusion chromatography (SEC) of synthetic polymers between fully porous particles (FPPs) and superficially porous particles (SPPs) with similar particle diameters, pore sizes and equal flow rates. Polystyrene molecular weight standards with a mobile phase of tetrahydrofuran are utilized for all measurements conducted with standard HPLC equipment.Although it is traditionally thought that larger pore volume is thermodynamically advantageous in SEC for better separations, SPPs have kinetic advantages and these will be shown to compensate for the loss in pore volume compared to FPPs. The comparison metrics include the elution range (smaller with SPPs), the plate count (larger for SPPs), the rate production of theoretical plates (larger for SPPs) and the specific resolution (larger with FPPs). Advantages to using SPPs for SEC are discussed such that similar separations can be conducted faster using SPPs.SEC using SPPs offers similar peak capacities to that using FPPs but with faster operation. This also suggests that SEC conducted in the second dimension of a two-dimensional liquid chromatograph may benefit with reduced run time and with equivalently reduced peak width making SPPs advantageous for sampling the first dimension by the second dimension separator. Additional advantages are discussed for biomolecules along with a discussion of optimization criteria for size-based separations.

Pirkle-type chiral stationary phase on core–shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations?

Pirkle-type Whelk-O1 chiral stationary phase (CSP) was prepared on 2.6μm superficially porous particles (SPPs). The chromatographic behavior of columns packed with this new CSP was compared with that of columns packed respectively with 1.8 and 2.5μm Whelk-O1 fully porous particles (FPPs). In the comparison, both thermodynamic and kinetic aspects were considered. Contrary to initial expectations, chiral columns packed with 2.6μm SPPs were quasi-comparable to those packed with 2.5μm FPPs, apparently due to larger contributions to band broadening from both eddy dispersion and, especially for the second eluted enantiomer, adsorption–desorption kinetics. These findings raise the question if SPPs, in spite of the undeniable advantages of their morphology to speed up mass transfer, are always the best choice for high-efficient ultrafast chiral separations. The last part of the work focuses on the use of short columns (10mm long) and very high flow rates to realize the separation of the enantiomers of trans-stilbene oxide (TSO) in normal phase mode in less than 1s.

Comprehensive two-dimensional liquid chromatography–tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants

A novel system for comprehensive two-dimensional liquid chromatography coupled to a triple quadrupole mass spectrometer is described for the analysis of wine components. The first dimension consisted of a 250-mm microbore cyano column utilizing 5μm diameter particles, interfaced to a 50-mm superficially-porous particle C18 column with 2.7μm diameter particles. Both columns were operated under reversed-phase conditions. Correlation between the two chromatographic separation modes was decreased by designing a 60-s shift gradient program in the second dimension, and the increase in orthogonality was evaluated quantitatively utilizing a number of orthogonality metrics. The system was employed for the analysis of a red wine sample, without preliminary clean-up procedures, and a total of 43 polyphenols were separated and identified. Comparison with a one-dimensional LC system showed a large increase in the number of identified components with the two-dimensional system. Optimized multiple reaction monitoring experiments allowed for the determination of trans-resveratrol, which is one of the most active antioxidant component of wine, and for monuron, a plant protection product (herbicide) of interest to regulatory agencies. The estimated limits of detection and of quantification were 0.3μgL−1 and 1μgL−1, respectively, well below the minimum detection limit (10μgL−1) set by current regulation.

Hydroxypropyl beta cyclodextrin bonded superficially porous particle-based HILIC stationary phases

ABSTRACT(R,S)-Hydroxypropyl-modified β-cyclodextrin (RSP-CD) is a well-known chiral stationary phase. In this work, hydrophilic interaction liquid chromatographic (HILIC) selectivities of RSP-CD was demonstrated. Further, an evaluation of chromatographic performances of fully porous particles (FPPs)- and superficially porous particles (SPPs)-based RSP-CD stationary phases was performed. The RSP-CD-bonded SPP-based stationary phase showed faster and more efficient HILIC separations compared to the FPP-based stationary phases. In addition, the SPP-based RSP-CD stationary phase showed excellent selectivities for many classes of small polar molecules. Since the SPP-based stationary phase is allowed for separations performed at high flow rates without significant loss of efficiency, ultrafast separations (analysis times under 1 min) also was accomplished utilizing SPP-based RSP-CD stationary phase.

Synthesis, characterization, and evaluation of a superficially porous particle with unique, elongated pore channels normal to the surface

In recent years, superficially porous particles (SPPs) have drawn great interest because of their special particle characteristics and improvement in separation efficiency. Superficially porous particles are currently manufactured by adding silica nanoparticles onto solid cores using either a multistep multilayer process or one-step coacervation process. The pore size is mainly controlled by the size of the silica nanoparticles and the tortuous pore channel geometry is determined by how those nanoparticles randomly aggregate. Such tortuous pore structure is also similar to that of all totally porous particles used in HPLC today. In this article, we report on the development of a next generation superficially porous particle with a unique pore structure that includes a thinner shell thickness and ordered pore channels oriented normal to the particle surface.The method of making the new superficially porous particles is a process called pseudomorphic transformation (PMT), which is a form of micelle templating. Porosity is no longer controlled by randomly aggregated nanoparticles but rather by micelles that have an ordered liquid crystal structure. The new particle possesses many advantages such as a narrower particle size distribution, thinner porous layer with high surface area and, most importantly, highly ordered, non-tortuous pore channels oriented normal to the particle surface. This PMT process has been applied to make 1.8–5.1μm SPPs with pore size controlled around 75Å and surface area around 100m2/g. All particles with different sizes show the same unique pore structure with tunable pore size and shell thickness. The impact of the novel pore structure on the performance of these particles is characterized by measuring van Deemter curves and constructing kinetic plots. Reduced plate heights as low as 1.0 have been achieved on conventional LC instruments. This indicates higher efficiency of such particles compared to conventional totally porous and superficially porous particles.

Ultrafast separation of fluorinated and desfluorinated pharmaceuticals using highly efficient and selective chiral selectors bonded to superficially porous particles

The separation of fluorinated active pharmaceutical ingredients (APIs) from their desfluoro analogs is a challenging analytical task due to their structural similarity. In this work, fluorine containing APIs and their corresponding desfluorinated impurities were separated on five new 2.7μm superficially porous particles (SPPs) functionalized with bonded chiral selectors. The unique shape selectivity of bonded macrocyclic glycopeptides and oligosaccharides was utilized to separate seven pairs of fluoro/desfluoro APIs resulting in some unprecedented selectivity values. For example, SPP bonded isopropyl cyclofructan 6 yielded a selectivity of 2.73 for voriconazole and desfluoro voriconazole. Further, the SPP based columns allowed for rapid separations ranging from 9 to 55s with very high efficiencies ranging from 45,000 to 70,000plates/m (at high flow rates) in both reversed phase and polar organic modes. Chromatographic separation and detection by HPLC-ESI-MS was demonstrated using ezetimibe and voriconazole and their desfluorinated impurities. Among the tested phases, SPP hydroxypropyl-β-cyclodextrin separated the most fluorinated and desfluorinated analogs with baseline resolution.

Superficially Porous Particle Based Hydroxypropyl-β-cyclodextrin Stationary Phase for High-Efficiency Enantiomeric Separations.

A superficially porous particle (SPP)-based hydroxypropyl-β-cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)-based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP-based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation.

Synthesis and optimization of wide pore superficially porous particles by a one-step coating process for separation of proteins and monoclonal antibodies

Superficially porous particles (SPPs) with pore size ranging from 90Å to 120Å have been a great success for the fast separation of small molecules over totally porous particles in recent years. However, for the separation of large biomolecules such as proteins, particles with large pore size (e.g. ≥ 300Å) are needed to allow unrestricted diffusion inside the pores. One early example is the commercial wide pore (300Å) SPPs in 5μm size introduced in 2001. More recently, wide pore SPPs (200Å and 400Å) in smaller particle sizes (3.5–3.6μm) have been developed to meet the need of increasing interest in doing faster analysis of larger therapeutic molecules by biopharmaceutical companies. Those SSPs in the market are mostly synthesized by the laborious layer-by-layer (LBL) method. A one step coating approach would be highly advantageous, offering potential benefits on process time, easier quality control, materials cost, and process simplicity for facile scale-up. A unique one-step coating process for the synthesis of SPPs called the “coacervation method” was developed by Chen and Wei as an improved and optimized process, and has been successfully applied to synthesis of a commercial product, Poroshell 120 particles, for small molecule separation.In this report, we would like to report on the most recent development of the one step coating coacervation method for the synthesis of a series of wide pore SPPs of different particle size, pore size, and shell thickness. The one step coating coacervation method was proven to be a universal method to synthesize SPPs of any particle size and pore size. The effects of pore size (300Å vs. 450Å), shell thickness (0.25μm vs. 0.50μm), and particle size (2.7μm and 3.5μm) on the separation of large proteins, intact and fragmented monoclonal antibodies (mAbs) were studied. Van Deemter studies using proteins were also conducted to compare the mass transfer properties of these particles. It was found that the larger pore size actually had more impact on the performance of mAbs than particle size and shell thickness. The SPPs with larger 3.5μm particle size and larger 450Å pore size showed the best resolution of mAbs and the lowest back pressure. To the best of our knowledge, this is the largest pore size made on SPPs. These results led to the optimal particle design with a particle size of 3.5μm, a thin shell of 0.25μm and a larger pore size of 450Å.