What Should You Flow, and What Shouldn’t in HPLC? It Depends on Your Column!
What you flow, what you avoid flowing, and in what order, all depend on your column’s stationary phase and physical parameters.
Let’s break it down scientifically:
Stationary Phase Chemistry
1. Reversed-phase columns (like C18):
- Prefer polar or aqueous-organic solvents (like water/acetonitrile or water/methanol).
- Avoid 100% water for long periods (it can “collapse” the hydrophobic phase).
- Avoid highly nonpolar solvents; they are incompatible.
2. Normal-phase columns (like silica):
- Prefer nonpolar solvents (like hexane, ethyl acetate).
- Avoid polar solvents or high water content; they can deactivate the silica surface.
3. Ion-exchange columns:
- Need buffered aqueous phases with controlled pH.
- Avoid purely organic solvents or pure water unless specifically designed.
4. HILIC columns:
- Need high organic content (acetonitrile-rich) with small amounts of water.
Column Dimensions and Particle Size also Play a Role
Small particle size (≤2 µm, UHPLC columns):
- Require low viscosity mobile phases for high pressure systems.
Short columns:
- Need well-matched solvent strength to avoid overwhelming the stationary phase.
Before You Flow Anything, Check the compatibility of your:
- Mobile phases
- Injection solvent
With the stationary phase of your column to avoid phase collapse, silica dissolution, or resin damage.
Pro Tip:
Know your column first, its material, particle size, bonded phase, then choose what flows through it!
If you don’t know your stationary phase or what is compatible with it, check the column user manual provided with your column by manufacturer.
Saving your column’s life (and your chromatography quality) starts with respecting what it’s built to handle.
Summary:
- Not every column loves the same solvents.
- Not every method can tolerate every sample or every mobile phase.
- A good run starts with matching your mobile phase and its flow to your column.
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Resource Person: Abanoub Efraim