Chromatographic peak integration

Chromatographic peaks are generally integrated by using chromatographic operating software. Empower, Chromeleon, OpenLab are few examples. Analyst must prefer automated integration and make adjustments manually, if necessary. The analyst is expected to use proper judgment in selecting methodology for peak integration.

Peak integration is important as end result is highly influenced by the way it happens. Incorrect results may be obtained due to improper peak integration.

The failure of the software to appropriately integrate a peak is usually obvious from visual inspection of the chromatogram (at an appropriate scale). Various errors can occur which include, but are not limited to, peak splitting, adding area due to a co-eluting peak, failure to detect a peak, excessive peak tailing,   failure of the instrument response to return to baseline or a rise in the baseline, and failure to separate peaks.

Manual integration may be employed to correct an improper integration performed by the auto-integration method and must always include documentation clearly stating the reason the manual integration was performed, the name of the analyst, and the initials of the supervisor approving the manual integration.

Unusual baseline characteristics, unidentified peaks, splitting peaks, excessive tailing or other problems may indicate a need for instrument maintenance or other corrective action. All chromatographic data shall be integrated consistently for all standards and samples. Integration parameters, both automated and manual, must adhere to valid scientific chromatographic principles. Manual integration shall be performed to accurately measure the area under the peak and shall not be performed solely for the purpose of meeting quality control criteria.

Let us understand the peak integration with the help of few examples.

Proper Integration of Chromatographic Peaks

Following examples provide an overview on properly integrated peaks.

• Figure 1 is a properly integrated single peak. The peak is symmetrically shaped and exhibits no indication of co-elution. The baseline is stable and returns to the same level (i.e., the baseline is flat). This is an example of baseline-to-baseline integration. Peaks of this nature are usually appropriately integrated automatically by the software.

Figure 1: Properly integrated single peak

• Figure 2 is a proper integration of several peaks which are not completely resolved (i.e., the response does not return to the baseline between peaks). In this instance the lowest point between the two peaks, the valley, is selected as the appropriate end point for the peaks.

Figure 2: Properly integrated unresolved peaks

• Figure 3 provides several examples of peaks with slight interferences either just prior to or immediately after the target peak. These interfering peaks are not resolved and may be included in the automatic integration. Figure 3 demonstrates examples of proper integration of these peaks.

Figure 3: Proper integration to remove interfering peaks

• Figure 4 is an example of a peak, which requires the use of sophisticated software to remove the area due to a co eluting peak. Depending on the operating software, it may be possible to remove the additional area. It is necessary that the resulting integration area preserve the Gaussian peak shape.

Figure 4: Peak shape requiring use of peak skimming

Improper Integration of Chromatographic Peaks

Inappropriate integration is any integration, either automated or manual, which excludes or adds area. The following example illustrates improper peak integration.

• Figure 5 provides an example of an improperly integrated peak. The tailing side of the peak has been removed eliminating significant area, which otherwise should be included in the peak.

Figure 5: Peak shaving by removing tail.

• Figure 6 is an example of an improperly elevated baseline. This clearly excludes a large area of the peak, which a baseline-to-baseline integration would correct.

Figure 6: Peak shaving through elevating the baseline

• Figure 7 is an improperly integrated peak, which includes both elevating the baseline and eliminating the leading and tailing edge of the peak.

Figure 7 Gross peak shaving.

Manual correction of erroneous automated data system integrations

• Figure 8 is an example of a noisy baseline resulting in poor integration by the operating software that is attempting to integrate using a valley-to-valley integration procedure. The appropriate integration of the peak eliminates area associated with baseline changes and integrates only the target peak.

Figure 8: Noisy baseline

• Figure 9 is an example of co-elution of the tailing edge of a peak resulting in additional area being included in the automated integration. The manual integration is performed to preserve the peak shape and eliminate the additional area.

Figure 9: Addition of co-eluting peak.

I hope above information must have given you an overview on peak must be integrated. Revert me in case if you any query on peak integration. I will be happy to assist you further.

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