Integration Errors in Chromatographic Analysis, Part I: Peaks of Approximately Equal Size

Apr 01, 2006
Volume 24, Issue 4, pg 402–414

Table I: Relative response for test solutions*
Thus, these two effects will tend to balance each other, with the eventual error being a complex function of relative peak size and tailing. In most cases, the tailing effects will dominate, resulting in a positive bias for the small peak. However, situations can occur where the two effects exactly balance, resulting in no error. Unfortunately, it is difficult to predict such situations. When the first peak is small, the effects of tailing are generally absent, although the influence of the larger peak produces a loss in the first peak's area at high area ratio, probably due to the change in the valley position. These results are qualitatively consistent with earlier work (3). Slight differences with other studies probably reflect the variabilities from working with real chromatographic data rather than artificial peaks.

Finally, it is important to note that integration errors for height measurements are smaller than the corresponding area measurements in almost every situation. The use of height for quantitative measurements has been abandoned by many analysts, in the belief that area measurements are either more precise or more accurate. The data presented here do not support such an argument. While area measurements for severely tailed peaks can be more reproducible than height, for most chromatographic systems with only moderate tailing, height measurements offer a clear accuracy advantage for poorly resolved peaks (resolution less than 1.5), as the overlap contribution to peak height from the tailing produces less error than the area contribution. This conclusion is general for all integration methods and relative peak sizes, and is consistent with previous studies (3–5).

Integration Errors for the Valley Method

The data in Table II indicate that the valley method produces negative errors in every resolution situation. The errors are particularly large (greater than 25%) for many combinations. For example, as the second peak gets smaller, the area errors for the large peak are reduced from –28% to –16%, while the area errors for the small peak increase from –26% to –76% at resolution equal to 1.0. Similar trends are observed at resolution equal to 1.5, but the errors are smaller. Comparable results are found when the small peak is eluted first.

It is relatively easy understand these errors by looking at Figure 2, and imagining the baselines being drawn to the valley from the start of peak 1 and from the end of peak 2. The loss of area is particularly important for the smaller peak. The height of the valley above the baseline (due to decreasing resolution) is responsible for the error. Any errors from tailing effects are considerably less important. Again, peak height measurements produce significantly less error than area measurements, especially for resolution better than 1.5, and when the peaks are of similar size. However, even it these situations the errors are often greater than 10%.

Table II: Integration errors for differing relative peak areas, resolutions, integrated method*
It is important to note that the valley method does have some validity in chromatography, although not in the situations described here. In chromatograms with multiple peaks and complex baselines, the valley method might indeed produce less error than the drop method. For example, unresolved matrix components or wandering baselines can create artificially high valleys between peaks, resulting in a significant positive error if the drop method is used. In such situations, the real baseline more closely follows a valley-to-valley path. However, identification of this phenomenon requires careful review of the baseline, and is often missed by a less experienced analyst. When peak widths are significantly different, the more narrow peak can be more accurately integrated using the valley method, although skimming would probably be a better choice (5).

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