Over 3000 E-Learning topics / 5000 Articles & Applications
with the
Research Institute for Chromatography

Particle Size and Morphology

The benefits of employing small, spherical fully porous particles, in terms of chromatographic efficiency and the ability to operate at higher volumetric flow rates, was first proposed by Martin and Synge in their pivotal paper on partition chromatography back in 1941 [3]. It is now widely accepted and commonly exploited for many separations.

The evolution through the 10 μm irregular particles of the 1980s, to the spherical 5 μm particles in the 1990s, with more recent 3 μm and now sub-2 μm particles is well documented in various CHROMacademy modules, webcasts and tutorials; the interested reader is encouraged to review these [4-5].

The associated efficiency benefits of reducing particle size can be highlighted by van Deemter curves for a selection of different particle sizes (Figure 1). The shaded areas are the practical flow rate operating zones.

van Deemter curves for differing fully porous particle sizes

Figure 1: van Deemter curves for different fully porous particle sizes.

Another leap forward in terms of efficiency gains has been the reintroduction of superficially porous particles (SPPs), specifically sub-3 μm superficially porous particles. The concept of solid particles covered with a thin film (pellicular) that is then coated with stationary phase was first described by Hovarth et al. in the late 1960s [6].

They consisted of a solid spherical glass bead onto which a thin film, in the region of a few µm, of styrene divinylbenzene and benzoyl peroxide was coated. Either a strong cation exchanger (sulfonic acid), or strong anion exchanger (quaternary ammonium), was bound onto this layer as the stationary phase. Their primary use was in the analysis of large macro and biomolecules.

Modern superficially porous particles (schematic in Figure 2 and SEM image in Figure 3), are analogous to their forebears with a porous skin coated onto a solid bead. The stationary phase typically employed on the modern variants is that of a reversed phase nature, identical to those used on their fully porous cousins.

Schematic plots of SPP and FPP

Figure 2: Schematic plots of SPP and FPPs.

SEM images of modern SPPs

Figure 3: SEM images of modern SPPs.
(Fabrice Gritti, Georges Guiochon, Journal of Chromatography A, Volume 1228, 2012, Pages 2-19.)

The benefit superficially porous particles (SPP) offer over their fully porous counterparts (FPP) is that they offer similar efficiencies to those seen for sub-2 μm FPPs, but as they are 50% larger, they operate at much reduced back pressures. The relative performances of a fully porous 3.5 μm particle, a fully porous 1.8 μm particle, and various commercially available modern sub-3 μm particles are shown in Figure 4.

van Deemter curves for differing FPPs and SPPs

Figure 4: van Deemter curves for differing FPPs and SPPs.

The pressure profiles for a 150 mm x 2.1 mm column packed with 1.7 μm fully porous particles is plotted in comparison with a 250 mm x 2.1 mm column with 2.7 μm superficially porous particles for standard trastuzumab peptide map separations (Figure 5). Even with a 40% longer column, providing a significant increase in chromatographic efficiency as previously described, the resulting operating back pressure is still significantly lower for the SSP column compared to the FPP packed column.

Back pressure profiles

Figure 5: Back pressure profiles of 150 mm FPP packed column and a 250 mm SPP packed column.

The benefits of these more modern particles for traditional small molecule type pharmaceutical analysis are predominately due to the more mono-dispersive nature of the particles and the associated benefit of the A-term, eddy diffusion, from the van Deemter equation – often referred to as the packing term.

However, their true benefit is afforded to larger molecules, including peptides, but in particular proteins, due to the dramatic reduction in pore-depth. This reduces the diffusion path, as highlighted in Figure 2, and therefore solutes with hindered mass transfer kinetics are not as compromised as they are with more traditional >3 μm fully porous particles.

These benefits are optimally enjoyed at higher than standard volumetric flowrates/linear velocities and allow the flow rate to be continually ramped with only minor impacts on efficiency.

loading data
loading data
loading data
loading data
loading data
loading data
loading data
 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data


loading data

loading data