Proper integration events minimize any manual integration for UV, TIC, and EIC in the oligo analysis. The following settings are recommended. Adjust them based on your chromatographic performance.
To update integration events, we recommend using the Integration Optimizer. Alternatively, you can edit the integration events directly in the integration events table.
For information on the Integration Optimizer, see Integration Optimizer.
Prerequisites
To be able to carry out the procedure as described, you need the following privileges. Privileges are configured in the Control Panel.
Project Management > Edit content of project
Data Processing > Reprocess data
Processing Methods > Edit general parameters
Data Processing > Use Integration Optimizer
Processing Methods > Edit general parameters
Processing Methods > Edit integration parameters
Processing Methods > Save master method
Processing Methods > Save result set method
In Data Analysis, load the result set with your acquired oligo data.
Edit the processing method linked to your results.
Under General > Properties, on the Global tab:
Select ChemStation integrator.
Prepare signal-specific integration.
Under Integration Events ChemStation > Standard: Select Use signal specific events for your UV and TIC SCAN signals. For EIC signals, keep the global settings.
Extract chromatograms to generate EIC signals.
As a preparation for optimizing integration, extract a few chromatograms from the TIC of your smallest calibration standard. From your list of known ions, select a few ions that you know have a very low response.
Optimize the UV signal integration.
Select the UV signal of the smallest calibration standard.
In general, UV traces should establish baselines for peak splitting of early eluting and late eluting impurities. It is common for sample baselines to start at 9 minutes to 24 minutes to accommodate for all shortmer, early eluting impurities. Therefore, set integration events such as peak widths as broad as possible, e.g. to a value ≥ 1.0. Additionally, the UV baseline can be manually set using Baseline integration events.
Add Area sum (On) in front of your peak, and Area sum (Off) after the peak.
Add Baseline now at the start of Area sum.
Tune Peak reject so that the base line is correctly applied.
Ensure that you update only the events for the UV signal. In Integration Optimizer, do not use the global update.
Optimize TIC integration.
Select the TIC signal of the smallest calibration standard.
Adjust integration events so that your FLP peak is integrated.
Ensure that you update only the events for the TIC signal. In Integration Optimizer, do not use the global update.
Optimize EIC signals integration.
Select an EIC signal of the smallest calibration standard.
Set up a very sensitive integration to accommodate for the variation in impurity. For example, choose low values for Area reject or Area% reject
Update your method using the Update Method - Global function. This updates integration parameters for all EIC signals. Settings for UV and TIC signals remain unchanged as you set them to be signal-specific.
To check the updated parameters with another EIC signal, start the Integration Optimizer again and select Use parameters from method.