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Several use problems of high performance liquid chromatography
High-performance liquid chromatography (HPLC) is a widely used technique in analytical chemistry, but it comes with several common operational issues that users should be aware of. Here are some key points to consider:
1. **Does the Pump Drain the Mobile Phase from the Column?**
Many HPLC users have faced situations where the solvent bottle runs dry, causing the pump to stop working. This often raises concerns about whether the column has been damaged or if the pump has drained the mobile phase from the column. In reality, the pump is designed to deliver only liquid, not air. So, even if the solvent is exhausted, the pump will not push air into the column. However, there's a more likely cause: improper sealing of the column ends. If the caps are loose or missing, the column may dry out at the ends, but this typically affects only a small portion of the column. Drying out the entire column is rare and usually takes a long time due to solvent evaporation. If it does happen, you can try re-wetting the column by flushing it with degassed, low-surface-tension solvents like helium-degassed methanol. This helps wet the stationary phase and remove trapped gases. After a few hours of slow flow, the column can often be restored to normal operation.
2. **What Should You Know When Using PEEK Tubing and Fittings?**
PEEK (polyetheretherketone) tubing and fittings are popular for their flexibility and ease of use, especially when changing flow paths or columns from different manufacturers. These components can be connected without tools, making them convenient for frequent adjustments. However, it's important to note that PEEK is not compatible with certain solvents, such as halogenated alkanes and tetrahydrofuran, which can make it brittle over time. Also, PEEK tubing has a lower pressure limit compared to stainless steel—around 4000 psi versus 6000 psi for stainless steel. Most HPLC applications operate below 3000 psi, so this is usually not an issue. When using hand-tightened PEEK fittings, be cautious of potential leakage or slippage under high pressure, which could lead to dead volume or system failure.
3. **How to Prevent Pump Failure?**
To ensure reliable pump performance, regular maintenance and proper handling of the mobile phase are essential. Key steps include using high-quality HPLC-grade solvents, filtering and degassing the mobile phase, and venting air before each run. After finishing the analysis, always flush the pump with water or buffer solutions to prevent residue buildup. Avoid leaving water or corrosive solvents in the pump for extended periods. Replace seals and gaskets regularly, and apply lubricant when necessary. Refer to the pump’s operating manual for additional guidelines. A well-maintained pump should show a stable baseline on the chromatogram, consistent retention times, and minimal pressure fluctuations (less than 2% during isocratic elution). For quick troubleshooting, keep spare parts like seals, check valves, and fuses on hand.
By following these best practices, you can extend the life of your HPLC system and maintain accurate, reproducible results.