46 February / March 2021
120 Å pores is the ideal choice for peptides, peptide mapping or oligonucleotide analysis. C8 columns (120 Å) allow shorter run times especially in oligonucleotide separations. Widepore Bio C18 is dedicated to larger peptides or proteins, while Bio C4 (both 300 Å) not only allows separations of large proteins but also intact MAbs (Figure 1).
In addition to the very inert base particles which don’t contain any metal impurities, inertness of the column housing can be optimised as well. For that purpose, a bioinert, metal-free hardware is available. By using a PEEK-lined stainless steel column body together with PEEK frits, any interaction with the hardware is eliminated while simultaneously maintaining the pressure stability of the column.
Figure 2: All three particle sizes show identical separation patterns for the analysis of IgG1. Using UHPLC particle size greatly improves the resolution (A). Use of 2 µm particles further allows higher throughput of analyses by using shorter columns (B). Eluent: 0.1 M KH2
PO4
improved resolution or increased peak capacity. The higher column temperature reduces the viscosity of the mobile phase allowing compounds to enter the pores and interact with the particle surface more easily and reduces adsorption onto the particle surface. Temperature can affect configuration of a protein as well as the diffusion rate and therefore effectively increases the performance.
Sub-2 µm UHPLC particles are difficult to produce, since the particles encounter higher backpressures and consequently have to be more rugged than their larger particle counterparts. One possible approach to ensure particles can tolerate this high- pressure is to reduce the porosity of the silica particles, which in turn results in very low surface areas and which comes at the cost of lower resolution [3]. In addition to the pressure related particle strength challenge, the thermal stability of the stationary phase also must be addressed.
A different solution is offered here. Instead of conventional silica-based particles, organic/ inorganic hybrid silica particles are produced that contain ethylene bridges in their silica backbone. This provides high mechanical strength with greater chemical stability, resulting in high temperature tolerance (up to 90 °C) and a working pH range from 1 to 12. These high surface area particles (standard ≥300 m²/g for 120 Å) are produced with standard pore sizes of 120 Å for peptides or
-K2 HPO4 rate: 0.2 mL/min; temperature: ambient; detection: UV at 280 nm.
oligonucleotides and with large pores of 300 Å for larger peptides or antibodies. Additionally, the particle surface possesses increased inertness due to the multi-step endcapping process employed.
Currently four different modifications for BioLC are available in these organic/inorganic hybrid silica columns (YMC-Triart/YMC-Triart Bio, YMC Co., Ltd., Kyoto, Japan). These stationary phases are available for UHPLC use with the 1.9 µm particle size and are fully scalable to 3 or 5 µm as well. C18 with
Columns for SEC with special requirements towards inertness
(pH 7.0) containing 0.2 M NaCl; flow
The separation principle in SEC is purely based on MW, more specifically, hydrodynamic volume, and the accessibility of the pores with no actual interaction with the stationary phase. Any secondary interaction has to be avoided so as not to influence the SEC separation. Consequently, the more inert the SEC stationary phase, the more reproducible the corresponding results will be. Due to the separation mechanism, larger column volumes and lower flow rates are required which results in longer analysis times. Reducing the particle size allows shorter columns and smaller column internal diameters resulting in reduced run times while maintaining or even improving resolution.
Figure 3: DAR determination of the ADC Brentuximab vedotin using BioPro HIC HT (2.3 µm, 100 x 4.6 mm ID) and a competitor column. BioPro HIC HT allows higher flow rates, while the competitor column can only be used at standard flow rates to operate below its pressure limit. Eluents: A) 20 mM NaH2 (pH 7.0) containing 1.0 M (NH4 SO4, B) 20 mM NaH2PO4-Na2
PO4-Na2 )2 25 °C; detection: UV at 280 nm; injection: 10 µL (2.5 mg/mL). HPO4 HPO4 (pH 7.0)/2-propanol (85/15); temperature:
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