Difficulties with wavelength accuracy readings while performing a PQ of an HPLC - Tips & Suggestions
7-APRIL-2012 Last Updated: 5-JULY-2025
When performing wavelength accuracy checks during a Performance Qualification (PQ) of an HPLC system, the following tips can help mitigate common issues—especially with older detectors:
1. Preventing Bubbles in the Flow Cell
Bubbles can significantly disrupt wavelength accuracy readings. To avoid this:- Draw the Wavelength Calibration Solution through the flow cell using a syringe.
- Disconnect the syringe and allow the system to rest.
- Keep both the inlet and outlet tubing ends elevated above the flow cell to prevent siphoning.
- Once the system stabilizes, auto-zero the detector in a region with minimal absorbance (around 590 nm is recommended), then proceed with measurements.
2. Understanding Detector Limitations
Older detectors often have:- Lower sensitivity
- Wider bandwidths (typically 5–8 nm)
3. Recommended Wavelengths for Reliable Results
Given these limitations, focus on stronger, more isolated bands:- Holmium Oxide: 361, 451, 537, and 641 nm
- Caffeine: 204 nm (resolvable on most instruments), 273 nm (should be clearly visible)
4. Best Practices for Peak Identification
- Stay close to the expected peak maxima during scans.
- Avoid scanning too broadly, as this may introduce false signal direction changes.
5. Qualification Strategy
These challenges are instrument-related, not due to the calibration solutions. If your detector cannot resolve finer band structures:- Base your qualification on the clearly resolved peaks (e.g., 273, 451, 537 nm).
- Document that the instrument is not capable of resolving narrower bands like 278 or 287 nm.
- This approach still maintains NIST traceability through the holmium oxide solution.