Peak integration is a critical component of HPLC and GC methods. It involves determining area, height, and retention time—data points that are then used in subsequent analysis, for example, concentration calculation and calibration curve construction.
Peaks are typically integrated via automated methods using the instrument's built-in software. Automated integration is preferred as it is typically efficient and not very labor-intensive, and yields consistent results. However, certain scenarios do call for manual peak integration, but as noted in a recent Chromatography Forum discussion, it is not always clear which integration method is optimal.
We explore the key considerations, criteria, and best practices associated with manual integration in chromatographic analysis.
Determining the Need for Manual Integration
The need for manual integration typically arises when coeluting peaks, software limitations, or specific method intricacies challenge the automated integration process. Some laboratories set specific criteria outlining when manual integration should be employed, but in many cases, it is at the discretion of the individual or team conducting the analysis.
The discussion surrounding the criteria for manual integration often centers on the percentage difference in peak areas. While there is no universal standard, many labs set thresholds based on practical considerations. The decision may hinge on factors such as:
- Proximity to the reporting limit: Peaks near the reporting limit, such as those of some fluorides and chlorides, may require closer scrutiny. However, those reporting limits fluctuate depending on the method.
- Coelution challenges: Certain compounds can pose challenges for automated systems. "Acetate is the problem when quantifying fluoride at low levels," explains forum user, Anionman. "I set a low shoulder threshold, but sometimes the instrument will not skim it, no matter how low I go on the shoulder threshold or how high I go on sensitivity."
- Audit requirements: Certain applications may be bound by rules around manual integration. Auditors may want to know about the frequency of manual integration, why it was performed, and the methods used. However, the extent of these inquiries can depend on the individual auditor.
As mentioned, the choice to manually integrate is subjective, so it's important that decisions are backed by sound reasoning. As one user, Chromavore, highlights, analysts must always consider if actions can be justified and defended on the basis of good chromatographic science.
Best Practices for Manual Integration
If manual integration is considered necessary (or preferable), there are some key best practices to follow:
- Clear SOPs: Well-defined standard operating procedures (SOPs) for manual integration are crucial. SOPs should consider the specificities of the chromatographic method and be regularly updated to reflect advancements in technology and industry guidelines.
- Documentation: Proper documentation of manual integrations is vital for traceability and audit purposes. Analysts should clearly justify the need for manual intervention, providing insights into the decision-making process. Some chromatography data system (CDS) software offers functionality to help with manual peak integration, such as the ability to tag manually integrated peaks and the option to add comments justifying the method.
- Training and culture: Analysts should undergo comprehensive training on the principles of peak integration and be encouraged to adopt a standard approach. Fostering a culture of continuous improvement and knowledge sharing is essential to ensuring consistent results.
A key theme throughout the forum discussion centers around the need for assessing method development. Best practices for manual integration aside, the robustness of the original method must also be considered. If manual integration is required often, it may be a signal to revisit the method development process.
Conclusion
Manual integration remains an invaluable tool in the chromatographer's arsenal, allowing for precision in challenging situations. Laboratories must strike a balance between automation and manual intervention, understanding that the selective use of manual integration contributes to the overall reliability of analytical results. As technology evolves, so too should the practices and SOPs surrounding manual integration.