This document investigates the effect of mobile-phase ionic strength on neutral and strongly basic antibodies. The results show that strongly basic molecules interact with silica-based column packings to a much greater extent than neutral molecules. Additionally, the concentration of phosphate buffer has an impact on peak shape and retention time.
This document discusses the separation of antibodies from antibody conjugants and reaction components using size-exclusion chromatography (SEC). It highlights that mobile-phase ionic strength and pH significantly influence the proper elution of antibodies and other biomolecules.
The note presents chromatogram data showing the effect of varying phosphate buffer concentrations (20 mM to 1000 mM) on peak shape and retention for neutral and strongly basic antibodies. It notes that strongly basic molecules interact more extensively with silica-based column packings than neutral molecules, leading to adsorbance or peak tailing at low ionic strengths.
| Column | ZORBAX GF-250 (4.6 x 250 mm) |
|---|---|
| Column Part Number | 884973-701 |
| Mobile Phase | Sodium Phosphate, pH 7.0 |
| Injection Volume | 2pL |
| Flow Rate | 0.96 mL/min |
| Detection | UV (230 nm) |
Mobile-phase ionic strength and pH have a strong influence over the proper elution of antibodies and other biomolecules.
The document explains how mobile-phase ionic strength and pH influence the elution of antibodies and other biomolecules using size-exclusion chromatography.
Strongly basic molecules interact with silica-based column packings to a much greater extent than neutral molecules.