Taking the Pain Out of Chromatographic
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
Chromeleon 7 Introduces Cobra
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:
Cobra™ is Chromeleon® 7’s new peak detection algorithm that in
essence simplifies integration for all chromatographers. In designing
Chromeleon 7, Dionex introduced the concept of Operational SimplicityTM .
• Distinguishing peaks from noise
• Correctly identifying the underlying baseline
• Maintaining correct peak and baseline detection throughout a
sequence of chromatograms
• Correctly handling rider peaks and other unresolved peaks
Ideally, these challenges should be addressed using detection parameters so that the same treatment can be applied across multiple
chromatograms automatically, thereby minimizing variations introduced
by different operators. However, finding parameter combinations that
produce the desired results has often been a tedious process, causing
many chromatographers to give up and resort to manual integration,
which is subjective and labor-intensive.
In this presentation we look at a new peak detection algorithm that
addresses these issues by separating peak detection from baseline
determination. It uses second-derivative signal analysis, providing
reliable and consistent peak detection even if the underlying baselineshape changes. An easy to use interface that guides the user through the
correct set-up of peak detection parameters is also shown. Examples of
how the tools quickly solve problems with real-world chromatograms
will be given.
With the new algorithm and assistant, chromatographers can get more
accurate, reproducible results much faster and more reliably.
The principles of which are to:
• Minimize the number of steps needed to perform any task
• Make all steps easy to understand and easy to use
• Minimize time needed perform any task
Cobra is the new algortihm that adheres to these principles providing
consistent and reliable peak detection across multiple chromatograms.
With the introduction of Cobra, Chromeleon 7 provides the Cobra
Wizard; an easy to use interface that guides the user through the correct
set-up of peak detection parameters required by Cobra.
Define the integration range, specify the smoothing width, and then
identify the smallest peak to be integrated. With these settings the
Cobra peak detection algorithm instantly integrates the peaks of each
chromatogram within a sequence accurately and concisely (Figure 1). In
three easy steps Cobra has enough detail to integrate all chromatograms
within a sequence.
In reality, integration is extremely complex and diverse. Chromatographic
data systems have to decipher many different variants and effects such as
peaks of varying symmetries, overlapping peaks, valleys, varying sizes
of peaks and size ratios, shallow peak rises and declines: extreme fronting
or tailing, shifting apexes and valleys of unresolved peaks, baselines
with large sloping background absorption and background noise, etc.
(Figure 3).
1. Define the integration range.
2. Select the narrowest peak.
Cobra is a new peak detection algorithm. It uses advanced signal processing
to distinguish true peaks from noise and sophisticated curve fitting techniques
to accurately locate peak maxima and inflection points.
3. Select the smallest peak to be integrated.
Figure 1. Cobra Wizard.
Cobra Peak Detection Algorithm
Reliable chromatographic quantification depends upon accurate and
reproducible peak integration. Integration of chromatographic peaks
determines the area under the peak, the height of the peak and the peak’s
retention time. This information is used for all subsequent calculations,
such as calibration or analysis of unknowns.
In simplistic terms, integration involves summing the detector output from
peak start to peak end. To achieve this, integration is composed of two
distinct events: it is started and then terminated. Peak start and end points
define where integration commences and terminates and are identified
by significant changes in detector output or by the rate of change of the
detector output. Defining peak boundaries however is extremely challenging. In an ideal world chromatographic peaks (Figure 2) would be perfectly
symmetrical with pronounced detector signal changes that clearly identify
peak start and end.
Figure 3. Typical chromatography.
Cobra correctly integrates all types of chromatograms using just a few
parameters.
A fundamental requirement of Cobra is having an optimal number of data
points of the order of 10 to 40 to sufficiently characterize the chromatography detection signal of a component peak.
Cobra then uses an adaptive Savitsky-Golay smoothing function followed
by transformation into a 2nd derivative, assessing the 2nd derivative against
automatic thresholds based on signal characteristics.
Since the noise is amplified when differences are calculated, Cobra adopts
an adaptive Savitsky-Golay smoother to remove the noise without losing
valuable information.
The second derivative of a chromatographic signal can recognize composite peaks. Assessing the second derivative of a peak also reduces the effects of background and ensures that points of inflection and peak maxima
are accurately and consistently identified if the background absorption
has lower curvature than the analyte peak.
For example, the following two chromatograms show a peak of the same
analyte, but one has a large sloping background absorption (Figure 4).
The first derivative of these two signals is shown in the center (Figure 5).
You can see that the difference between the pure analyte chromatogram
(grey) and the chromatogram with interference (blue) is reduced. This effect is considerably enhanced in the second derivative (Figure 6). In this
case, the chromatogram of the pure analyte and the chromatogram with
interference are almost identical.
Figure 2. Idyllic chromatographic peak.
2
Taking the Pain Out of Chromatographic Peak Integration
3.5
1.0
Figure 4. Overlayed chromatograms.
–0.50
0.121
0
–0.18
620
630
640
650
660
670
678
Figure 7. Local minimum and maximum.
Figure 5. First derivative.
7.0
1.28
–2.0
0.20
Figure 6. Second derivative.
–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
Figure 8. Baseline interpolation.
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 chromatogram’s baseline profile. The most common
options for drawing the baseline between two peaks: drop, valley, tangential skim, exponential skim, and Gaussian skim.
3
SmartPeaks Integration Assistant
Chromeleon 7 addresses these issues, providing chromatographers
with the simplest of interfaces designed in accordance with Dionex’s
Operational Simplicity principles. SmartPeaks™ integration assistant
produces desired results quickly and intuitively. The user simply selects
a region of the chromatogram and SmartPeaks graphically displays
available integration options, such as valley-to-valley baselines or
exponential skims (Figure 10).
When the user selects an option, SmartPeaks adds the corresponding
parameters and values to the processing method so that the desired integration is automatically applied to all chromatograms in the sequence.
This process takes just a few seconds eliminating the trial-and-error
process of adjusting integration.
Figure 9. Baseline Profiles: 1. Drop Perpendicular, 2. Valley to Valley, 3. Tangential Skim,
4. Exponential skim, 5. 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 peak’s
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 chromatographer’s 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.
4
Taking the Pain Out of Chromatographic Peak Integration
Figure 10: SmartPeaks
New Intelligent Parameters
Conclusion
Integration is extremely complex and challenging but critical to accurate
quantitative analysis. Chromeleon 7’s new detection algorithm Cobra
not only simplifies the peak detection and integration process but also
provides new adaptable and flexible parameters to assist chromatographers achieve the best results.
Chromeleon 7 is a next generation chromatography data system that
delivers intelligent functionality while maintaining operational simplicity.
Critical data analysis is achieved accurately and reliably using powerful
features of Cobra. The Cobra Wizard and SmartPeaks integration assistant
aid and guide the chromatographer to perform the required data manipulations quickly, easily and reproducibly. Using Chromeleon 7, laboratories
gain a significant boost in overall efficiency and productivity. Chromeleon
7 is Simply Intelligent.
Minimum Relative Area is one such parameter. Minimum Relative Area
rejects peaks, if their minimum area is below a given threshold. The
minimum area is measured, on a first pass basis, relative to the sum of
all peaks (total area) in a chromatogram.
An example of its use is for an analytical method that generates a
chromatogram with peaks of interest and other smaller non-related
peaks. The requirement is to total the peak response throughout a
chromatogram. Typically to ensure that the smaller unwanted peaks are
not included the minimum area detection parameter is set to exclude
non-related peaks from being integrated. However within a sequence
the analyte concentration can vary and the main analyte peaks may drop
below the prescribed minimum area threshold. Using the Minimum
Relative Area parameter peaks of interest that drop below the prescribed
mimimum area will still be detected and integrated because they are
greater than the minimum threshold of the current chromatograms total
area (Figure 11).
Minimum Relative Area
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.
Chromeleon is a registered trademark and Operational Simplicity,
Cobra, and SmartPeaks are trademarks
of Dionex Corporation.
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