When analyzing high–molecular‑weight analytes (~10,000 Da or greater), you may observe no retention when using Cogent Diamond Hydride™ (100 Å pore size) columns.
This behavior is expected and is a direct consequence of pore accessibility and surface area utilization in silica‑based chromatographic media.
1. Why Large Molecules Do Not Retain on Diamond Hydride™ (100 Å)
The vast majority of a silica particle’s interactive surface area resides inside its pores, not on the external particle surface. Retention mechanisms—whether ANP, RP‑like, or mixed‑mode—require the analyte to enter these pores to interact with the stationary phase.
For very large molecules such as:
- Proteins
- Large peptides
- Polymers
- Other macromolecular species
…the 100 Å pores are too small for the analyte to enter. If the analyte cannot reach the internal surface where retention occurs, it will:
- Pass through the column rapidly
- Elute at or near the solvent front
- Show little to no retention despite method changes
This is exactly the behavior observed with high–molecular‑weight compounds on the Diamond Hydride™ column
2. Correct Column Choice: Cogent Bidentate C8 300 Å (Wide‑Pore)
To retain and separate larger biomolecules, you must use a wide‑pore stationary phase, such as the Cogent Bidentate C8 300Å™ HPLC column.
The 300 Å pore size allows macromolecular analytes to:
- Physically enter the pore network
- Access the full functionalized surface
- Exhibit meaningful retention and separation
The Cogent Bidentate C8 300 Å phase is therefore the recommended choice for:
- Proteins
- Peptides
- Polysaccharides
- Polymers
- Large hydrophobic or amphipathic biomolecules
3. Recommended Next Steps for Analysts
A. Choose the Correct Stationary Phase
- For MW > ~10,000 Da: Use Cogent Bidentate C8 300 Å
- For mid‑size polar biomolecules (<10 kDa): Diamond Hydride™ may still be appropriate, depending on shape, charge, and folding
B. Modify Mobile Phase Conditions Accordingly
- Matching 300 Å pore phases with proper organic/aqueous balances is crucial
-
Typical starting points include:
- 0.1% formic acid or 10 mM ammonium formate (LC‑MS compatible)
- 60–90% organic (for ANP‑compatible macromolecules)
- Or RP‑oriented gradients for hydrophobic peptides/proteins
C. Consider Sample Preparation
Because large biomolecules have strong nonspecific interactions, ensure:
- Adequate filtration (0.2 µm recommended)
- Avoidance of salts that precipitate in high‑organic conditions
- Use of appropriate solubility aids (e.g., low % MeOH, ACN, or pH adjustment)
4. Summary
High‑MW molecules cannot retain on Cogent Diamond Hydride™ (100 Å) because they cannot enter the pores where chromatographic interactions occur. Instead, they elute at the solvent front. The correct solution is to switch to a wide‑pore 300 Å column, specifically Cogent Bidentate C8 300™, which is engineered to provide retention for proteins, peptides, polymers, and other high‑MW species.
