In the first two parts of this series (HPLC Solutions #127 and #128), we looked at how HPLC data systems integrate chromatograms and some of the adjustments that can be made if you don’t like the way the default settings perform. Even with proper adjustment of the settings, you may still observe occasional or regular problems with integration. Here we’ll look at several common problems and how to correct them.
Figure 1 shows three examples of problems that are commonly encountered in integrated chromatograms. In each case, the solid red line shows how the data system selected the baseline for integration and the dashed lines are the corrected baseline. Normally the baseline is determined by drawing a straight line connecting the baseline before the peak to that after the peak. In example (a) the baseline has a negative peak or dip before the peak of interest is eluted. The data system mistakenly identified this as the low point in the baseline ahead of the peak with an obvious error in determining the peak area. The problem was corrected by moving the baseline to the position of the dashed red line.
The second chromatogram, (b), shows a dilemma that is often encountered. The default integration assigned a perpendicular drop from the valley between the two peaks, so the peak areas for the first and second peaks are divided as shown. In this case, the proper integration is to skim the smaller peak off the tail of the larger one. You can see that this treatment may not make much difference in the area of the larger peak, but the original integration assigns more than twice the appropriate area to the minor peak. This is especially critical in reporting pharmaceutical impurities, where a batch of product may fail quality testing if the impurity levels are reported to be higher than they actually are. In making a decision on this, I use what I call the “10% Rule,” which tells me that if the minor peak is less than 10% of the peak height of the major one, it should be skimmed (dashed line), whereas if it is more than 10% of the major peak, a perpendicular drop should be used (solid line). You can see that, even though the top of the larger peak is not visible, the larger peak is at least ten times as tall as the minor one. Another example of the application of the 10% Rule was given in HPLC Solutions #115.
In the last example, the data system determined that the peak in chromatogram (c) returned to the baseline too early. This is a particularly common problem with small peaks on a noisy or drifting baseline. In this case, the correct baseline was drawn with the dashed line to the point where the peak actually returned to the true baseline.
In each of these cases, manual integration was required. I regularly get questions in the training classes I teach about this subject. In some laboratories, workers are not allowed to manually integrate peaks because management is afraid of negative feedback from regulatory authorities. This is being falsely conservative. There are clear guidelines for this published in the US Code of Federal Regulations, CFR 21 Part 11. The essential rules for manual reintegration are (a) the person doing the integration must be identified, (b) the date and time must be noted, (c) a copy of the original (raw) data must be preserved, and (d) a reason for the change must be recorded. Most modern data systems include an “audit” function that can be turned on; this forces compliance with these requirements.
Yes, it may be possible to have every chromatogram integrated properly, but this is only likely when all the peaks are large and the baseline noise is minimal. With small peaks on a noisy or drifting baseline, it is very likely that manual integration will be required to get high quality results.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com