32 Buyers’ Guide 2021 Table 1. The physiochemical properties of wide pore and ultra-wide pore FPP and SPP particles
Particle type
Competitor 400A
FPP 400Å SPP 500Å
Particle size (µm)
1.7 1.7
FPP 1000Å 1.7 2.6
SPP 1000Å 2.6
Surface area (m2
g-1 65
41 22 15 9
)
Average pore
volume (cm3
g-1 0.6
0.2 0.1
0.05 0.05
the FPP and SPP is shown on the size distribution graph Figure 4. The data obtained from this technique can also be used to calculate the mean, median and particle distribution ratio (d90/d10).
Table 1 summarises the physiochemical properties of a range of 1.7µm FPP and 2.6µm SPP synthesised with wide (400-500Å) and ultra-wide pores (1000Å) respectively. The table also lists the physical properties of a competitor brand of large pore silicas. For the efficient separation of macromolecules, it is suggested that the particle pore size for optimal chromatographic separation should be in the order of at least 10 times the effective hydrodynamic diameter of an analyte. All particles have monodispersity (d90/d10) values ≤ 1.3 indicating they are monodispersed and do not aggregate as evidence from SEM and FIB micrographs, Figures 1a, 2 and 3.
) 300
430 950 430
1200
Average pore size (Å)
%C Surface coverage (α) (µmol/ nm2
) n/d n/d
0.69 2.2 0.33 2.1 0.32 2.9 0.15 2.3
1.7
1.3 1.3 1.2 1.2
Figure 5 illustrates the pore size distribution profiles for FPP 1.7µm and SPP 2.6µm with the numerical values for FPP 1.7µm and SPP 2.6µm shown in Table 1. The two SPP 2.6µm particles (500Å and 1000Å) have the same low average pore volume of 0.05cm3
g-1 g-1 ,
while the average pore volume of the FPP 1.7µm 400Å particle (0.20 cm3
that for FPP 1.7µm 1000Å particle (0.10 cm3
g-1 ).
For the wide-pore particles (i.e. FPP 1.7µm 400Å and SPP 2.6µm 500Å) the pore volume increases to maximum pore expansion and then begin to decrease as pore size continue to increase. For ultra-wide pore particles (i.e. FPP 1.7µm 1000Å and SPP 2.6µm 1000Å), there is steady increase in pore volume with increase in pore size to maximum pore expansion. The average pore size distribution was calculated using the Kelvin equation derived from the Barrett- Joyner-Halenda (BJH) model [13].
Application evaluation for SOLASTM
FPP and EIROSHELL™ SPP columns
Figure 4. Particle size distribution curve for an ultra-wide pore 1.7µm particle.
Gradient separation of a mixture of six standard protein compounds (in eluting order: 1. ribonuclease A (14.70kDa) 80µg/ ml, 2. cytochrome C (12.33kDa) 110µg/ ml, 3. BSA (66.43kDa) 400µg/ml, 4.
) is double
Monodispersivity (d90/d10)
myoglobin (16.71kDa) 250µg/ml, 5. enolase (67.00kDa) 430µg/ml, and 6. phosphorylase B (97.20kDa) 1.18µg/ml are compared in Figure 6 for wide-pore FPP 1.7µm C4-400Å and SPP 2.6µm C4-500Å columns, plus ultra-wide pore FPP 1.7µm C4-1000Å, and SPP 2.6µm C4-1000Å columns as shown in Figure 6.
All peaks for the wide-pore FPP 1.7µm C4-400Å column (Fig. 6A) show a slightly longer retention time (Table 3a) compared to the ultra-wide FPP 1.7µm C4-1000Å column (Figure 6B & Table 3b), suggesting some restricted diffusion of these large protein molecules in the relatively smaller pore particles. The protein macromolecules ribonuclease A and cytochrome C have better separation efficiencies (based on the number of theoretical plates, NTP) on FPP 1.7µm C4-400Å than on FPP 1.7µmC4-1000Å columns (Tables 3a and 3b). This can be attributed to the larger particle surface area 41 m2
, and subsequently higher %C 0.69 of FPP 1.7µm C4-400Å compared to 22 m2
g-1 g-1
and %C 0.33 for FPP 1.7µm C4-1000Å (Table 1). FPP 1.7µm C4-1000Å shows a better efficiency for phosphorylase B compared to FPP 1.7µm C4-400Å as seen by the number of theoretical plates in Tables 3a & 3b. This is due to the restricted diffusion of the large molecular weight phosphorylase B (97.20kDa) through the relatively smaller pore size FPP 1.7µm C4-400Å compared to the ultra-wide FPP 1.7µm C4-1000Å particle.
For SPP columns, compared to SPP 2.6µm C4-500Å (Figure 8C), SPP 2.6µm C4-1000Å (Figure 6D) show higher retention times and efficiency in terms of number of theoretical plates (Tables 3c & 3d) for ribonuclease A, cytochrome C, BSA and myoglobin. This is due to the larger pore size for SPP 2.6µm C4-1000Å which causes the unrestricted diffusion of these protein macromolecules. The separation efficiency (NTP) of the protein macromolecule, enolase in both columns (Tables 3c and 3d) are close, suggesting that the diffusion restriction due to pore size does not have an adverse band broadening effect for this analyte. SPP 2.6µm C4-500Å shows a better efficiency for phosphorylase b compared to SPP 2.6µm C4-1000Å indicating an added benefit of SPP 2.6µm C4-500Å with a larger surface area (15m2 1000Å (9m2
g-1 g-1
) compared to SPP 2.6µm C4- ) as seen in Table 1.
Figure 5. BJH pore size distribution curves. It should be noted that the pore volume decreases as pore size increases after pore expansion.
Effect of Large Pore Monodense Particles on Separation of Biomolecules
Figure 7 below illustrates the resolving
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