Many high-performance liquid chromatography (HPLC) methods rely on retention time alone, assuming that compounds are fully separated. However, when coelution occurs, quantification can be inaccurate, leading to misleading results. Without an additional assessment of HPLC peak purity, undetected impurities may compromise data quality.
Peak purity is especially important in impurity profiling and pharmaceutical quality control, where even minor coelution can affect results. Understanding how purity is assessed and recognizing the limitations of common techniques can improve confidence in HPLC data.
This article explains how to assess peak purity in HPLC, avoid common pitfalls, and apply best practices to improve impurity detection.
How Peak Purity is Assessed
A photodiode array (PDA) detector is the most common tool for evaluating peak purity. It measures ultraviolet (UV) absorbance across a peak and identifies spectral variations that may indicate coelution. HPLC software assigns metrics such as purity angle and purity threshold based on these spectral comparisons, but these values should always be interpreted carefully.
Liquid chromatography-mass spectrometry (LC-MS) provides a more definitive assessment by detecting coelution based on mass differences rather than UV absorbance. It is particularly useful for identifying low-level contaminants.
Limitations of Peak Purity Measurements
UV-based peak purity analysis is a qualitative tool that indicates spectral consistency but does not confirm whether a peak represents a single compound. A Chromatography Forum discussion highlighted this limitation when an analyst found that their calculated purity angle was below the threshold, suggesting a pure peak. However, a closer look at the spectral data revealed small variations at the peak edges, indicating possible coelution. This reinforces why purity scores should never be used alone—manual review of spectral plots is essential.
Baseline noise and detector sensitivity add further challenges. One analyst on Chromatography Forum observed that widening the scan range to 190–400 nm caused the software to flag a peak as impure, while restricting it to 210–400 nm produced a pure result. Other users noted that low-wavelength noise can distort purity calculations, leading to false positives. Selecting appropriate scan parameters reduces these errors and improves reliability.
Troubleshooting and Best Practices
Improving peak purity starts with optimizing separation. Adjusting the mobile phase composition, gradient, and column selection can reduce coelution and improve resolution. Small changes in flow rate or temperature can also sharpen peaks and minimize spectral overlap.
Interpreting purity data can be challenging, especially when relying on software-generated metrics. Forum user jcmc questioned why their peak purity match value exceeded 900 on a 1–1,000 scale, despite expectations for a lower score. Other users explained that different software platforms calculate purity differently, emphasizing the need for cross-validation. Examining spectral overlays and peak shapes helps identify coelution that automated metrics may miss.
Conclusion
Retention time alone does not confirm purity. A peak may appear well-resolved, yet hidden coelution can compromise accuracy. Instead of relying on a single metric, use retention time as a starting point for deeper investigation. Integrating orthogonal detection methods, such as LC-MS alongside PDA, provides a more complete picture. Complementary techniques and manual spectral review help ensure confidence in your results.
For more insights on troubleshooting peak purity and optimizing HPLC methods, visit Chromatography Forum, where analysts share real-world challenges and solutions. Engaging with the community can help refine your approach and improve your chromatography workflows.
