What Is Sample Degradation by Hydrolysis in HPLC Columns? (and How TYPE‑C™ Columns Mitigate It)
Hydrolysis is a common degradation pathway for many analytes during HPLC analysis; it is facilitated by acids or bases in solution and by reactive surfaces of solids that contact the liquid phase—including the stationary phase inside a column.
In several application areas (e.g., natural products, bio‑actives, forensic analyses), 10–20% of bio‑active compounds have been shown to hydrolyze “on‑column” under typical chromatographic conditions, which can compromise accuracy, quantitation, and impurity profiling.
Why Hydrolysis Happens Inside Conventional Silica Columns
- High surface area intensifies contact: Both irregular and spherical silica phases are engineered with very high surface‑to‑volume ratios. Greater surface area → more surface interactions → higher likelihood of on‑column chemical change.
- Silanol (Si‑OH) activity persists even when “fully end‑capped”: Conventional phases can still present ~30–50% free silanols, which can act as acidic/interactive sites that facilitate analyte hydrolysis or secondary retention.
- You see it in your data: The prevalence of on‑column hydrolysis has been highlighted by PDA (photodiode array) and LC‑MS measurements, where degradation products appear and increase over repeated injections or at certain pH conditions.
How Cogent TYPE‑C™ (Silica Hydride) Columns Change the Equation
Cogent TYPE‑C™ materials replace the conventional hydroxylated silica surface with a silica‑hydride (Si–H) surface. As a result, the packing is much less hydrolytically active than ordinary silica bearing Si‑OH groups.
In many cases, the parent compound does not hydrolyze on the column, enabling you to track injected quantities rather than degradation products—a major advantage for non‑targeted or stability‑sensitive analyses.
Key takeaway: By minimizing acidic/interactive surface sites, TYPE‑C™ columns can reduce on‑column hydrolysis and improve fidelity between what you inject and what you detect—especially valuable when screening unknowns or labile bio‑actives.
What This Means for Method Developers
- If you observe unexpected degradants or mass balances that don’t add up, consider on‑column hydrolysis as a root cause—especially with acid/base mobile phases and silica phases with appreciable silanol activity.
- For sensitive compounds (esters, lactones, some pro‑drugs, acylated natural products, etc.), migrating from conventional silica to Cogent TYPE‑C™ can help preserve the native molecule through the separation.
At‑a‑Glance: Conventional Silica vs. TYPE‑C™ for Hydrolysis‑Prone Analytes
- Conventional silica (Si‑OH rich) → high surface area + residual silanols (even after end‑capping) → greater risk of on‑column hydrolysis; degradants frequently confirmed by PDA/LC‑MS.
- Cogent TYPE‑C™ (Si–H surface) → much lower hydrolytic activity; in many cases no on‑column hydrolysis observed, enabling more faithful quantitation of the parent.
