HALO BioClass columns are currently comprised of the HALO Peptide and HALO Protein phases for reversed-phase separations of peptides, proteins, and antibodies. These columns are made using superficially porous, Fused-Core® particles, which were first introduced, for small molecule separations, in 2006. The advantages of superficially porous particles for improved speed, efficiency and peak capacity have been well demonstrated and documented in the literature over the last 7+ years. The benefits of Fused-Core particles are also pronounced for RPLC separations of larger molecules such as peptides, polypeptides, and proteins, which have much smaller diffusion coefficients and have much slower mass transfer than low MW analytes. A larger average pore size and a thinner porous layer are also important for improved peak shape, efficiency, and recovery of large biomolecules.
In the last two years, the HALO BioClass portfolio has been expanded with the introduction of the new 400-Ångstrom pore size HALO Protein C4 and ES-C18 phases. HALO Protein 400 Å columns employ a new particle size and geometry, using a 3.4 µm particle with a very thin 0.2 µm porous shell. The combination of the wide pore diameter and thin shell allow very fast separations of a wide range of polypeptides and proteins up to 500 kDa in molecular weight. In Table 1 the characteristics and properties of the current portfolio of columns in the HALO BioClass family are shown.
| Characteristic | Peptide ES-C18 | Peptide ES-CN | Protein C4 | Protein ES-C18 |
| Pore Size (Å) | 160 | 160 | 400 | 400 |
| Particle Size(s) (µm) | 2.7 and 5 | 2.7 and 5 | 3.4 | 3.4 |
| Surface Area (m2/g) | 90 and 60 | 90 and 60 | 15 | 15 |
| Low pH Limit | 1 | 1 | 2 | 2 |
| High pH Limit | 8 | 8 | 9 | 9 |
| Maximum Temperature (°C) | 90 | 90 | 90 | 90 |
| Endcapped | No | Yes | Yes | Yes |
| Ideal MW Range Operable MW Range | MW ≤ 10kDa MW ≤ 15-20 kDa | MW ≤ 10kDa MW ≤ 15-20 kDa | 2 kDa < MW < 500 kDa | 2 kDa < MW < 500 kDa |
| Target Analytes | Peptides and polypeptides | Peptides and polypeptides | Monoclonal antibodies, polypeptides and proteins | Polypeptides and |
Elevated column temperature decreases mobile phase viscosity and improves mass transfer for these larger molecules. It also improves recovery and reduces sample carryover and memory effects—particularly for hydrophobic biomolecules (e.g., amyloid peptides, membrane proteins). The resistance to hydrolysis of the HALO Peptide and HALO Protein phases at low pH with high temperatures is derived from sterically protected bonding technology (Peptide ES-C18 and ES-CN; Protein ES-C18) or from very dense bonding and exhaustive endcapping (Protein C4). This enhanced stability and excellent column lifetime translates to stable and reproducible retention times and selectivity. This stability permits reproducible qualitative and quantitative results, which are critically important for peptide mapping, biomarker quantitation, and intact protein analysis. Some example separations using the HALO Peptide and Protein columns at elevated temperatures are shown below.
In this first example (Figure 1) a very fast separation of various peptides and small proteins ranging in molecular weight from 238 to 13,700 Da is presented. A complete separation of these 11 analytes is performed in less than one minute at 60 °C using a 50-mm column on a conventional HPLC system (delay volume > 1.0 mL) at a maximum pressure of 330 bar. A 2.1 x 50 mm, 1.7 µm column run at an equivalent 1.0 mL/min flow rate at 60 °C would generate a maximum pressure of ~650 bar (9444 psi).
This second example demonstrates the efficient separation of a mixture of polypeptides and proteins ranging in molecular weight from ~12 to 250 kDa in less than 10 minutes (Figure 2, black trace). The second chromatogram (red trace) from a subsequent injection of the same sample, after over 14,500 column volumes of mobile phase, shows very little change in retention at 90 °C. The applicability, performance and stability of the HALO Protein C4 phase are clearly demonstrated under these conditions.
HALO BioClass columns will deliver speed, high peak capacities, and ruggedness to your HPLC and UHPLC separations of peptides, polypeptides and proteins. MAC-MOD Analytical is currently offering an excellent opportunity for you to try these columns. Please refer to our web site for more information about the HALO BioClass columns and about our current offer.
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