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 100m(2)/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.

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.

Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase.

A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7µm) was developed. Its performance was evaluated and compared to fully porous silica particles with 5µm (commercially available as FRULIC-N) and 3µm diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode.

Superficially porous particles vs. fully porous particles for bonded high performance liquid chromatographic chiral stationary phases: isopropyl cyclofructan 6.

This work reports a comparison of HPLC separations of enantiomers with chiral stationary phases (CSPs) prepared by chemically bonding cyclofructan-6, functionalized with isopropyl carbamate groups on fully and superficially porous particles (SPPs). The chromatographic performance of the superficially porous CSP based column was compared with columns packed with 5 µm and 3 µm fully porous particles (FPPs). At a flow rate of 3.0 mL/min the number of plates on column afforded by the SPP column was ∼7× greater than the number of plates on column (same length) obtained when using the 5 µm FPP based column. The flow rate providing the highest efficiency separation was ∼1.0 mL/min for the SPP column while it was ∼0.5 mL/min for both FPP columns. It was found that the selectivity and resolution of the separations were comparable between fully porous and superficially porous based columns (under constant mobile phase conditions), even though the SPP column contained lower absolute amounts of chiral selector. When tested under constant retention conditions, the SPP based CSP greatly improved resolution compared to the FPP based columns. At high flow rates the efficiency gained by using superficially porous CSP was accentuated. The advantages of columns based on SPPs become more obvious from the viewpoint of plate numbers and resolution per analysis time.

Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography.

The kinetic performance of a bare silica and C18 phase prepared from the same sub-2µm and 3.5µm base materials were compared in the HILIC and RP mode using both charged and neutral solutes. The HILIC column was characterised using the neutral solute 5-hydroxymethyluridine, the weak base cytosine, and the strong base nortriptyline, the latter having sufficient retention also in the RP mode to allow comparison of performance. Naphthalene was also used as a simple neutral substance to evaluate the RP column alone. The retention factors of all substances were adjusted to give similar values (k'∼5.5) at their respective optimum linear velocities. Reduced van Deemter b-coefficients (determined by curve fitting and by the peak parking method, using a novel procedure involving switching to a dummy column) were significantly lower in HILIC for all substances compared with those found under RP conditions. Against expectation, c-coefficients were always lower in RP when compared with HILIC using sub-2µm particles. While measurement of these coefficients is complicated by retention shifts caused by the influence of high pressure and by frictional heating effects, broadly similar results were obtained on larger particle (3.5µm) phases. The mechanism of the separations was further investigated by examining the effect of buffer concentration on retention. It was concluded that HILIC can sometimes show somewhat inferior performance to RP for fast analysis at high mobile phase velocity, but clearly shows advantages when high column efficiencies, using longer columns at low flow velocity, are employed. The latter result is attributable to the lower viscosity of the mobile phase in HILIC and the reduced pressure requirement as well as the lower b-coefficients.

Applications of superficially porous particles: high speed, high efficiency or both?

The new generation of superficially porous particles (SPPs) offers impressive chromatographic efficiency compared to totally porous particles. Specifically, modern sub-3-µm SPPs generate much improved reduced plate height but at lower backpressure compared to sub-2-µm totally porous particles. This feature makes them attractive for various types of applications and SPPs are quickly being adopted in many analytical laboratories. In this review, we use optimization theory to compare the performance limit of modern SPPs and totally porous particles under optimized conditions, in order to answer the question: what are the optimal applications for modern SPPs? Are they most suitable for fast separations, or for high efficiency separations, or for both? Successful examples of using modern SPPs in different application areas are reviewed, over a wide range of sample complexity and analysis time. Practical aspects of the use of such particles and future development possibilities are also discussed.

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research.

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 microm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 microm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 degrees C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.