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Stationary Phase

The final parameter, is ironically the least commonly varied when analyzing biomolecules, but probably the most commonly varied when analyzing traditional small molecule pharmaceuticals; the stationary phase.

The chromatographic selectivity of a biomolecule is predominately driven by its solubility in the mobile phase, s value, rather than its propensity to interact with the stationary phase. Therefore, selectivity is typically controlled and optimized by adjusting the mobile phase gradient, rather than changing the type of stationary phase ligand. Straight alkyl chain stationary phase ligands are by far the most common with shorter, C4, chains preferred for intact mAbs and medium sized, C8, chains the preference for mAb fragments.

Both variants operate by exploiting only hydrophobicity as the retention mechanism. However, there are a few alternatives available, one of which is a diphenyl stationary phase; this contains a short alkyl chain, still facilitating hydrophobic retention, but the main mechanism of retention is via the phenyl groups, specifically π-π interactions between aromatic and dipole containing moieties. This enables enhanced retention of polar and aromatic containing residues.

However, it is not possible to predict retention and the best approach, as is always, is to empirically trial the different stationary phases when the desired chromatographic separation cannot be achieved via the previously discussed parameters. An example of the different selectivity of a degraded Herceptin sample on a traditional C4 and alternative diphenyl stationary phase is shown in Figure 1.

Effect of stationary phase selectivity on degraded Herceptin samples

Figure 1: Effect of stationary phase selectivity on degraded Herceptin samples.

 
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