10 Tips for HPLC Analysis in Pharmaceuticals

1. Choose the Right Column

   • Select the correct stationary phase (C18, C8, phenyl, etc.) based on analyte polarity.

   • Consider particle size, column length, and pore size for optimal separation.

2. Prepare Mobile Phase Properly

   • Use HPLC-grade solvents and degas them (ultrasonication, helium sparging, or vacuum filtration) to prevent bubble formation.

   • Maintain pH within the column’s recommended range.

3. Ensure Sample Cleanliness

   • Filter samples (0.22 or 0.45 µm) before injection to avoid clogging the column.

   • Avoid injecting particulate matter.

4. Maintain Column Temperature

   • Use a column oven to keep temperature constant, as it affects retention time and peak shape.

5. Use Proper Flow Rate

   • Stay within the manufacturer’s recommended flow rate range to avoid excessive backpressure or poor separation.

6. Calibrate and Validate the System

   • Perform system suitability tests (SST) before analysis: check parameters like resolution, tailing factor, and theoretical plates.

7. Optimize Injection Volume

   • Avoid overloading the column — high injection volumes can distort peaks.

8. Regular Column Care

   • Flush the column with suitable solvents after use.

   • Store columns in the manufacturer-recommended storage solution.

9. Record and Monitor Data

   • Follow ALCOA+ principles for data integrity: ensure all chromatograms and results are attributable, legible, contemporaneous, original, and accurate.

10. Follow Regulatory Guidelines

    • Adhere to ICH, USP, and pharmacopeial methods when applicable.

    • Document every step for GMP compliance.