Challenging Chromatographic Peak Integration

Integration is a fundamental task that all chromatographers perform on a daily basis. Due to faster chromatography, which is creating more data every day, the amount of time spent on integration is ever increasing.

Peaks of varying symmetries and size, overlapping peaks, valleys, shallow peak rises, and declines are just some of the different variants and effects that a chromatographer has to face. Other considerations include peak splitting, extreme fronting or tailing, shifting apexes and valleys of unresolved peaks, and baselines with large sloping background absorption and background noise.

Considerable time and effort are expended to ensure the accuracy and appropriateness of integration because this information is used for all subsequent calculations, such as calibrations, determination of the purity of drug substances, and analysis of unknowns. To help perform integration faster, analysts work with chromatographic data systems, many of which are on the market and which all adopt a similar work flow. A complex mathematical algorithm generates a first pass attempt at integration. The software packages then provide a procedure through which the analyst can visually inspect and review the resulting chromatogram integration. Setting the view at an appropriate scale, the analyst applies peak specific instructions on integration to correct any problems. However, the integration events provided by the software packages are often extensive and nonintuitive.

The above-mentioned software functionality does not help analysts who use trial-and-error methods to establish which parameters are applicable to the problem situation while ensuring that any alterations applied adhere to the integration principles and requirements of their laboratory. When analysts are unable to manipulate chromatograms through automation, they often have to resort to manual integration, which relies solely on the experience of the individual analyst to determine correct integration for each peak.

If manual integration is employed to correct an improper integration performed by the data system, then there can be further implications for laboratories that require them to commit time and resources to establishing procedures for completing and documenting corrections to analytical results. Optimizing a chromatogram’s integration is therefore a time-consuming skill even for the most experienced chromatographer, and a significant resource drain for laboratories.

Figure 1 - Cobra wizard and SmartPeaks integration assistant tool.

The Chromeleon® version 6 Chromatography Data System (CDS) (Dionex Corp., Sunnyvale, CA) introduced Dynamic Data Processing and automatic integration updates, which reduced the analyst’s effort and displayed the results immediately. Chromeleon version 7 substantially advances that user experience by providing two chromatographic integration tools: Cobra™ and Smart- Peaks™ (Figure 1). Developed in accordance with the guiding design principle of Operational Simplicity™, these two features significantly enhance software ease of use and simplify chromatographic peak integration. 

Most people would agree that integration is subjective, and laboratories have different criteria and treatments that include but are not limited to applied levels of thresholds, determination of unresolved peaks, rider peak interpretation, and so forth. To meet the needs of all chromatographers, the level of complexity and sophistication of chromatography software cannot be diminished; however, the overlying user experience can be made easy and pain free.

Figure 2 - Complex chromatogram.

The Cobra and SmartPeaks tools intelligently improve the analyst’s experience with minimal user input. A wizard is provided to guide the operator in correctly assigning initial peak detection parameters to help refine the integration if required. Integration is more constructive and intuitive, and can be performed with confidence, thus eliminating the trial-and-error methods and manual integration. The operator can, with only a few clicks of the mouse, quickly update discreet areas of the chromatogram without impacting other correctly integrated regions.

Below is an example of a typical issue faced by chromatographers. The chromatogram has been automatically integrated; however, on the first pass, the shoulder on the peak at 10 min has not been identified (Figure 2).

Understanding which parameters to apply in such a situation is completely dependent on the knowledge and experience of the analyst, including interpreting which peak events, such as baseline level, slope thresholds, and peak width and sensitivity, are applicable.

Figure 3 - Optimizing integration using Chromeleon software version 7 Cobra wizard.

The Cobra wizard guide was developed to allow the operator to assign further integration parameters correctly. By simply activating the Cobra wizard, the operator can follow the on-screen instructions to optimize a minimal number of initial detection parameters. With Chromeleon software’s dynamic data processing, the operator can view the effects graphically in real time to obtain the desired chromatogram integration. In the example given, the shoulder of the peak at 10 min has been identified by a few clicks of the mouse (Figure 3).

The peak at 10 min is now an unresolved peak group with a drop-perpendicular interpolation of the baseline profile. Not all chromatographers adopt this approach, and some may want to apply alternatives, such as valley-to-valley or exponential skim.

Figure 4 - SmartPeaks baseline profiles: 1) drop perpendicular, 2) valley-to-valley, 3) exponential skim, and 4) tangential skim.

The SmartPeaks integration tool permits the operator to quickly and easily highlight problematic peaks, and then the software automatically displays common options for drawing the baseline between two peaks (Figure 4). The operator does not require any specific knowledge of the software parameters needed to achieve the correct integration.

An operator can use both of these tools to optimize integration for the entire chromatogram, for a small segment, or use the tools multiple times for various scenarios. Chromeleon CDS software version 7 has the power to successfully integrate a chromatographic run; it also simplifies the task and improves the user experience. Such advancements are expected to increase laboratory productivity, saving time and money, and taking the pain out of chromatographic peak integration. 

The Chromeleon CDS software version 7 next-generation chromatography data system delivers rich, intelligent functionality with Operational Simplicity, which provides the user with everything needed within easy reach. Its thoughtfully designed user interface guides chromatographers effectively toward their goals. The eWorkflows feature enables anyone to start chromatographic analysis and obtain good results with just a few selections. The powerful data analysis tools ensure that any required data manipulation can be done efficiently and accurately. Chromeleon software moves chromatographers from samples to results quickly and easily—boosting overall laboratory productivity.

Mr. Quinn is Product Specialist, Chromatography Software, Dionex Corp., 1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088-3603, U.S.A.; tel.: 408-737- 0700; e-mail: shaun.quinn@dionex.com.

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