Set up a Processing Method

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
Processing Method > Create processing method
Data Processing > Reprocess data
Processing Method > Edit signal parameters
Processing Method > Edit chromatogram extraction parameters
Processing Method > Edit spectrum extraction parameters
Processing Method > Edit identification parameters
Processing Method > Edit calibration parameters
Processing Method > Save master method
Processing Method > Save result set method

In Data Analysis, load the result set with your acquired oligo data.
Create a new method with method configuration MS, and save it.
Link the new method to the result set.
Set the method parameters as specified below.
Reprocess and save your results.

Method section	Settings
General > Signals, Blank Subtraction tab	Blank subtraction applied on: Select All injections Use specific blank: Select the third blank in your current result set. Ensure that the third blank injection baseline closely resembles the baseline of the 25 µL sample You have to set this parameter individually for each new result set.
Extraction > Chromatogram, MS tab	Single m/z expansion chromatograms: This is typically set to either 0.3, 0.7 asymmetrical, or ±0.5 symmetrical. Determine this parameter empirically.
Extraction > Spectrum MS tab	Arbitrary spectra: Select Use external time range Peak spectra: For spectrum type, select Average peak spectrum. This is needed to identify impurities across the entire TIC. For background mode, select External background time range External background time range (recommendation): Start time: 0 min End time: 3.0 min Automatic spectrum extraction: Select the check box, and choose Identified Peaks. Spectral smoothing: Select the check box. The width is typically 1 or 2, but this is method-specific. Determine this parameter empirically.
Compounds > Calibration, General Table tab	Set Number of levels to 4.
Compounds > Identification, Compound Table tab	Add a compound for your full-length product (FLP). Use exactly the same name and spelling as in the ion list. Set Signal for this compound to the UV signal. Set the expected retention time as appropriate to our analysis. Set Peak match for this compound to Largest area.
Compounds > Calibration, Compound Table tab	Set Amount unit for your FLP compound to µg. Keep the Level 1 to Level 4 fields empty. The different amounts are defined by the injection volumes of calibration standards in the sequence (see Set up a Sequence). Set Origin for your FLP compound to Ignore.

Method section

Settings

General > Signals,

Blank Subtraction tab

Blank subtraction applied on: Select All injections

Use specific blank: Select the third blank in your current result set.

Ensure that the third blank injection baseline closely resembles the baseline of the 25 µL sample

You have to set this parameter individually for each new result set.

Extraction > Chromatogram,

MS tab

Single m/z expansion chromatograms: This is typically set to either 0.3, 0.7 asymmetrical, or ±0.5 symmetrical. Determine this parameter empirically.

Extraction > Spectrum

MS tab

Arbitrary spectra: Select Use external time range
Peak spectra:
For spectrum type, select Average peak spectrum. This is needed to identify impurities across the entire TIC.
For background mode, select External background time range
External background time range (recommendation):
Start time: 0 min

End time: 3.0 min
Automatic spectrum extraction: Select the check box, and choose Identified Peaks.
Spectral smoothing: Select the check box. The width is typically 1 or 2, but this is method-specific. Determine this parameter empirically.

Compounds > Calibration,

General Table tab

Set Number of levels to 4.

Compounds > Identification,

Compound Table tab

Add a compound for your full-length product (FLP). Use exactly the same name and spelling as in the ion list.

Set Signal for this compound to the UV signal.
Set the expected retention time as appropriate to our analysis.
Set Peak match for this compound to Largest area.

Compounds > Calibration,

Compound Table tab

Set Amount unit for your FLP compound to µg.
Keep the Level 1 to Level 4 fields empty. The different amounts are defined by the injection volumes of calibration standards in the sequence (see Set up a Sequence).
Set Origin for your FLP compound to Ignore.

12-Sep-2024