Professional Documents
Culture Documents
Peak Integration
Shaun Quinn,1 Peter Sauter,1 Andreas Brunner,1 Shawn Anderson,2 Fraser McLeod1
1
Dionex Corporation, Germering, Germany; 2Dionex Corporation, Sunnyvale, CA, USA
INTRODUCTION
Peak detection and integration are fundamental tasks in chromatography, most often done using chromatography software. Enabling
software to detect and integrate the peaks as desired (or as required by
laboratory rules) is challenging and time-consuming. Common challenges in peak detection include:
3.5
1.0
0.50
0.121
0.18
620
630
640
650
660
670
678
1.28
2.0
0.20
0.1
Identifying Peaks
In the second derivative of the chromatogram, noise thresholds are automatically determined (as represented by the red dashed lines in Figures 7
and 8 below). The local minimum below the thresholds lower limit is the
peak apex. The points of inflection or local maxima above the thresholds
are peak start and end (Figure 7). Baseline is interpolated between points
where curvature crosses the upper noise threshold limit (Figure 8).
0.35
209
300
400
500
600
700 730
However, the baseline profile may not be a direct point to point interpolation if more than one peak is eluted on top of it. In a situation with an
unresolved peak group, analysts have several choices for determining
the location of the chromatograms baseline profile. The most common
options for drawing the baseline between two peaks: drop, valley, tangential skim, exponential skim, and Gaussian skim.
The drop method involves the addition of a vertical line from the valley
between the peaks to the horizontal baseline. The vertical line is drawn
between the start and stop points of the peak group. The valley method
sets start and stop points at the valley between the peaks, thus integrating
each peak separately. Skim procedures separate the small peak from the
larger parent with separate baselines. The parent peak is integrated from
its starting point to the apparent end of the peak group. The small peaks
baseline starts at the valley between the peaks and ends when the signal
nears the baseline. The area under the skimmed peak is added to the
parent peak, not the skimmed peak. This approach has been described
also as a tangent integration method and the small peak variously labeled
a skim, shoulder, or rider peak.
Several variations of the skim procedure are possible. Tangential draws
a straight line from the valley to the end of the peak. Figure 9 shows an
exponential skim baseline. An exponential function is used to create curvature in the skim line in an attempt to approximate the underlying baseline of
the parent peak. Using an exponential function a curved baseline is drawn
under the skimmed peak. Alternatively Gaussian skim tends to more
accurately reproduce the Gaussian shape of the parent peak.
The complexity and errors increase as resolution decreases, and the valley
of unresolved peaks shifts adding to a chromatographers woes.
Understanding these methods and techniques and being able to assign
the correct integration parameters are extremely difficult tasks, even for the
most experienced chromatographer.
Conclusion
Figure 11. Minimum Relative Area. Even though area of main peak drops below level
of non-detected peaks in previous chromatogram, the main peak is still integrated.
North America
Europe
Asia Pacific
Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294
Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0
Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380
Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722
Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
Taiwan (886) 2 8751 6655
South America
www.dionex.com