1. Start Clean, Stay Clean
- Filter all mobile phases and samples (0.22 µm preferred).- Never let salts or particulates enter the column.
- Use fresh solvents — no old mixtures sitting for days.
2. Respect the Limits
- Stay within the column’s specified pH, temperature, pressure, and flow rate.- Make flow changes gradually to avoid bed disturbance.
3. Treat Buffers Carefully
- Avoid high-strength buffers (>50 mM).- Flush with water before switching to high organic.
- Never store columns in buffered solutions.
4. Storage Made Easy
- Reversed-phase columns: store in 100% ACN (or as per manufacturer’s note).- Always cap tightly to prevent solvent evaporation and microbial growth.
5. Smart Equilibration
- Equilibrate with 10–20 column volumes when changing mobile phase composition.- Ensure stable baseline, stable pressure, and consistent retention time before analysis.
6. Injection Discipline
- Use sample diluent weaker (more aqueous) than mobile phase starting conditions.- Avoid injections with high viscosity or high organic content.
- Filter → degas → inject.
7. Cleaning that Actually Works
- Try a gradient flushing approach when performance declines:1. Water
2. Water: ACN (50:50)
3. 100% ACN
4. Strong wash (IPA or THF if allowed)
- Reverse flow only when approved by manufacturer.
8. Document Everything
- Adopt ALCOA+ behavior in column usage logs:- Record in real time, keep data accurate, ensure traceability, and maintain column
Tags:
Pharmacy