High-Performance Liquid Chromatography (HPLC) is a very delicate and useful process for your compound identification needs. It can also be a pain to execute if you don’t have the right tools or proper optimization.
Here are tips that you may or may not already know. Keep in mind that you’ll probably need to adjust for what molecules you’re analyzing for as well as solvent(s) being used.
- Your choice of glass or plastic containers should be carefully considered for storage and/or mixing of solvents and test compounds. It may affect retention times, integrity of your equipment, but most of all, the quality and output of your data.
- HPLC grade reagents should always be used and, when applicable, filtered before use.
- Ensure proper degassing of HPLC system to ensure removal of dissolved gases.
- Confirm that your choice of solvent(s) is optimized for your HPLC system’s tubing and column materials, peptides being assayed for, and other solvent(s) being used that may possess incompatible properties.
Mobile Phase Tips
- Always use purified water (not deionized). It will affect absorption levels and ghost peaks may result from poor quality water.
- Always use HPLC grade solvents. Solvents that are analytical grade may contain oxidation inhibitors or ethanol that will affect UV absorption and column retention times, respectively.
Stationary Phase Tips
- Ensure proper (and constant) cleansing of your column to avoid particulates leading to crystallization or bacterial accumulation.
- Maintain pH range of 2 to 8 unless otherwise specified by the manufacturer.
- Don’t store columns with buffers inside or else precipitates and/or bacteria may accumulate leading to ghost peaks in your chromatogram or misrepresented analytes.
- Storing columns in solvents may facilitate column degradation. Store in the last used eluent for short-term storage or flush with purified water (not deionized) for longer term (silica based columns should be stored in acetone or acetonitrile).
Overall, your choice of materials and proper care of them will help you achieve results that are accurate and reliable. You just need to remember to customize these choices based on what you’re trying to assay for.