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31 22m2


surface area was developed by Boyes and Kirkland in 2017 intended for reversed- phase separations of very large proteins and monoclonal antibodies (mAbs) [10,11].


g-1


Most methods for the separation of proteins by RP-HPLC use acidic mobile phase additives, e.g. formic and trifluoroacetic acid, in both the aqueous (A solvent) and organic (B solvent) components of the mobile phase system. The organic modifier frequently consists of acetonitrile, or acetonitrile with a short aliphatic alcohol (propanol, butanol or isopropanol) which is added to increase the elutropic strength of the B solvent. In addition to using an organic gradient, elevated column temperature (40-90°C) is frequently employed to deliver the desired chromatographic profile [1].


In this report two newly synthesised monodense particles are described with a wide pore structure (400-1000Å); the


SOLASTM 1.7µm (Glantreo, Cork, Ireland) fully


porous particle (FPP) and EIROSHELL™ a 2.6µm (Glantreo, Cork, Ireland) superficially porous particle (SPP). Both materials are designed specifically to separate proteins and other biomacromolecules up to approximately 150 kDa. These new materials were bonded with a short butyl silane (C4) stationary phase and demonstrated high efficiency for the separation of large protein molecules which rapidly enter the porous structure, are adsorbed on the stationary phase and then desorb resulting in reduced band broadening.


Experimental


The SOLAS™ and EIROSHELL™ particles are manufactured via a modified Stöber process [7]. The particulars of this process and subsequent bonding and packing protocols are a trade secret to the company.


• The external morphology of the particles


was examined by using scanning elec- tron microscopy (SEM) [FEI Inspect F, (Hillsboro, Oregon USA) ] operating at 10 kV. The surface area, pore size, pore size distribution and pore volume meas- urements of the particles were performed based on the nitrogen gas sorption method using a Micromeritics Tristar II (Micromeritics, Norcross, CA, USA) sur-face and pore size analyser.


• The particle size distribution was determined by an electric sensing zone technique based on the Coulter principle on a Multitier 4e Coulter counter (Fullerton, CA, USA) instrument. A CE 440 fully automated elemental analyser (Exeter Analytical Inc., North Chelmsford, MA, USA) with thermal conductivity detection was used to determine the elemental analysis of the C4 bonded FPP and SPP particles.


Using an in house packing technique, stable reversed phase C4 columns were packed that can be applicable for separating large molecular weight compounds such as proteins and peptides. HPLC performance and biomacromolecule application evaluations were carried out using an Agilent 1200 LC (Santa Clara, CA) and Jasco LC 4000 (Easton, MD) series systems. Flow rates of 0.2ml/min were used for all columns which had dimensions of 2.1mm ID x 50mm. (John Shepard, Shepherd Hardware, PA, USA)


Figure 1. SEM of FPP (left) versus Manufacturer, A (right) particle showing greater uniformity of porosity in the SOLAS spheres. Notice the fine spherical structure in (1a) compared to agglomerated, uneven particle sizes in (1b) circled in red.


Results


Fully porous and superficially porous particles synthesised for the purpose of this report are based on existing technologies to yield spherical and monodense particles with wide pores for optimum biomacromolecule chromatographic efficiency.


Scanning electron microscopy (SEM) results after the particles have been manufactured are shown in Figure (1a) for FPP 1.7µm particle and (1b) for Manufacturer A 1.7µm particle.


Figure 2. SEM images of FPP 1.7µm monodense particles.


The FPP particle in Figure 1a is more monodense than the other Manufacturer particle in Figure1b and this has been shown in previous work to give better column efficiency [6].


Figure 3. SEM and FIB images of SPP particle.


Figure 2 shows the SEM images for 1.7µm FPP while Figure 3 illustrates the SEM images of 2.6µm SPP. The new SPP prepared in this report is comprised of the solid core (1.9µm) and porous shell (350nm) structure. The SEM images show the wide-pore and ultra-wide pore structures to be just about visible and evenly distributed within the particles. The particle size distribution of


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