Proper HPLC Operation: Tips and Tricks from Pros

High performance liquid chromatography (HPLC) is a versatile analytical technique used widely in chemistry and biochemistry research. However, operating HPLC equipment properly is critical for generating accurate and reliable results. Here I’ll share key learnings on good HPLC practices:

Always Purge Lines 

Before each use, purge all solvent lines to prevent cross-contamination between mobile phases. This avoids precipitation of buffers or salts which can clog flow paths.

Protect Columns 

Keep HPLC columns capped with guards when not running samples. Check the column specs match your application needs before each sequence.

Mind the Pressure 

Know the pressure and flow rate limits for your instrument configuration. If pressure fluctuates rapidly, inspect for issues like air bubbles or leaks.

Record Details
Log analytical method parameters, mobile phase composition, gradient programs, etc. Thoroughly document system details to aid reproducibility and troubleshoot suboptimal separations.

Proper Startup/Shutdown 

On startup, prime columns with mobile phase for at least 10-15 minutes before injecting samples. Always flush sufficient solvents at shutdown to eliminate sample carryover.

Be Patient 

It often takes multiple attempts to refine an HPLC method. Adjust parameters incrementally and allow sufficient column re-equilibration between runs. If peaks co-elute, try much slower gradients.

While developing HPLC expertise takes practice, applying these basic principles will lead to success. Let us know if you have other tips from your experience!