30 Buyers’ Guide 2021
New Monodense Large Pore Silica-Based Materials for the Analysis of Biomacromolecular Compounds by Reversed-Phase Liquid Chromatography
V. K. Langsi, J. L. McGrath, J. J. Hogan, S. Analakkattillam, J. P. Hanrahan Glantreo Ltd, ERI Building, Lee Road, Cork City, T23XE10, Ireland.
www.glantreo.com
info@glantreo.com
Introduction
Over the last decade there have been significant advances in the development of mon-odisperse large pore (400-1000Å) silica particles, designed either as fully porous (FPP) or superficially porous particles (SPP), to facilitate the reversed-phase separation of bi-omacromolecules such as intact large proteins. Columns packed with such particles have demonstrated superior resolutions accompanied by lower column back pressures during these separations compared to smaller pore materials of the same particle size [1, 2, 3].
The selection of the optimal particle size, stationary phase chemistry and particle design impacting chromatographic efficiency can minimise the mass transfer effect when using large pore particles [4]. Macromolecules can move rapidly in and out of the shell layer of porous shell particles with large pore sizes, resulting in reduced band broadening at higher mobile phase velocities. SPP particles exhibit advantages with biomacromolecules for fast separations at high mobile phase velocities because of superior mass transfer (kinetic) properties (i.e. smaller van Deemter C term) [5]. The thickness of the porous layer determines the size range of analyte molecules that would enter the pore structure with minimum restriction for interaction with the appropriate stationary phase. The larger the pore size the more macromolecular molecules would access the internal pore volume with unrestricted diffusion, thereby increasing efficiency and column performance. For example a SPP particle with 160 Å pores, specifically designed for separating peptides and small proteins. This column is capable of delivering good efficient peak shapes for ribonuclease A (13.7 kDa) but yields poor peak shapes with poor efficiency for solute molecules above 15 kDa molecular weight. Whereas,
particles with pore sizes >400Å have been demonstrated to separate large proteins greater than 400 kDa [1, 6].
Monodensity in silica structure has also been demonstrated to have an effect on the mass transfer of large molecules which could otherwise lead to peak broadening, peak tailing and subsequent loss in chromatographic efficiency. The production of monodense particles has been reported in a previous publication [7] Silica particles possessing a monodense structure with a homogeneous internal pore structure would allow for more efficient and rapid diffusion of large solute molecules into and out of the silica pore structure resulting in a more efficient and effective chromatography. It has also been shown that it is easier to pack monodense silica particles compared to traditional silicas as a result of the homogeneous distribution of particles during the packing process [7]. Moreover, large pore spherical monodense particles with low pore volumes have been researched to experience reduced inter and intraparticle tortuosity which increases diffusion and permeability of solutes [8].
In addition to having a large pore size and monodense structure, the retention capacity, selectivity and efficiency of these
new particles is also influenced by the type of alkyl bonded stationary phase. Joseph K. Kirkland et al have demonstrated that different macromolecules such as peptides and proteins would have different retention and selectivity on wide-pore SPP columns containing C4, C8 and C18 stationary phases using acetonitrile/aqueous trifluoroacetic acid (TFA) mobile phase. Using a six-protein mixture comprised of ribonuclease A-(13.7kDa), cytochrome C (12.4kDa), Bovine Serum Albumin-(66.4kDa), apomyoglobin-(17kDa), enolase-(46.7kDa) and phosphorylase B-(97.2kDa), they demonstrated only a minor difference in peak shape and yield on C8 and C4 phases. However, the shorter C4 phase showed slightly greater retention due to its higher ligand density on the silica base support (4.2 µmol/m2
a bulkier C8 phase (2.0 µmol/m2 carbon) [1].
for 0.88% carbon) compared to for 0.92%
Between 2009 and 2014 Advanced Material Technology (AMT) continued to lead the development and commercialisation of HALO®
BioClass material with the release of 2.0µm particle size, 400Å pore size, material for the analysis of large molecules. The first 1000Å superficially porous 2.7µm particle with a shell thickness of 0.5µm and
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