How to Extract a UV Background Spectrum in OpenLab CDS

This Information Applies To: Agilent OpenLab CDS (Rev. 2.x)

Issue

This article provides the instruction on how to extract a UV background spectrum. 

Background

If your data contains UV spectral data, you can extract UV background spectra from your chromatogram.

Requirements

• Acquisition method in DAD is set to store All spectra see Figure 1. You can read more about parameter settings to store spectra here.

  
  Figure 1: Setting in acquisition method for DAD
  
  

• The injection is linked to a processing method that supports manual UV spectra extraction 3D UV Quantitative (Figure 2) or LC/MS Sample Purity (Figure 3).

  Figure 2: 3D UV Quantitative Processing Method
  
  

  Figure 3: LC/MS Sample Purity Processing Method

• To be able to carry out the procedure as described, the user needs the privileges: Data Processing > Do manual spectrum extraction; Processing Method > Edit spectrum extraction parameters and Processing Method > Edit spectra parameters. Privileges are configured in the Control Panel.

Steps to Follow

You can either

• Automatically subtract background spectra by editing the settings in Processing Method or,
• Manually subtract background spectra using the spectrum extraction tool.
  
  

Automatically subtract background spectra

1. In the Processing Method, select the Extraction > Spectrum node (UV tab), and adjust the Background mode settings.
   
   The background mode under Arbitrary spectra affects spectra manually extracted outside of an integrated peak. Under Arbitrary spectra, if you check Use external time range, the spectra from the time range specified will be subtracted to the arbitrary spectra you will extract. If the check box is cleared, no background spectra subtraction occurs. (Figure 4)
   
   
   
   Figure 4: Background mode for Arbitrary spectra
   
   
   The background mode under Peak Spectra affects spectra extracted between peak start and peak end. (Figure 5)
   
   • None: Do not subtract a background spectrum from peak spectra.
   • Spectrum at baseline start: Use the spectrum at the baseline start as a background for peak spectra.
   • Spectrum at baseline end: Use the spectrum at the baseline end as a background for peak spectra.
   • Spectrum at peak start and end: Use the average spectrum from peak start and peak end as a background for peak spectra.
   • External background time range: Use a time range as a background for peak spectra.

   
   Figure 5: Background mode for Peak spectra
   
   

   External background time range: If you use an external time for specific peak spectra or for arbitrary spectra: The average UV spectrum of the given time range is used as the background spectrum. 
   
   When you extract other UV spectra, the extracted background spectrum will automatically be subtracted. The time point or time range used for subtraction is shown in the spectrum name.
   
   
2. Save the data after the peak spectra extraction.

Manually subtract background spectra

1. (Optional) Click  icon to open Chromatogram Properties and go to Spectrum Extraction. The settings on this page control the behavior of the spectrum extraction tool. For more details, see Chromatogram Properties for Spectrum Extraction. (Figure 6)
   
   
   Figure 6: Chromatogram Properties for Spectrum Extraction
   
   
2. Click OK to apply any changes and exit this dialog box.
3. In the Chromatograms window, click icon in the toolbar to start the spectrum extraction tool.
4. To extract a background spectrum, hover the mouse directly over the signal so that the Background hint is shown. (Figure 7)
   
   
   Figure 7: Background hint
   
   
   
5. Click at the required position in the chromatogram or click and drag the cursor over the required range to extract an average spectrum over a time range.
6. When asked for confirmation, click  icon or press [ENTER] to confirm the background spectrum extraction. The spectrum extraction will occur without confirmation when you clear the Confirm extraction check box in the Chromatogram Properties. (Figure 8)
   
   
   Figure 8: Confirmation to extract background spectrum
   
   
   The extracted background spectrum is shown in the UV Spectrum window and listed under the details for the individual injection in the injection tree. (Figure 9)
   
   
   
   Figure 9: Extracted spectra under injection tree
   
   Extracting a background spectrum changes the Processing method settings in the Extraction > Spectrum section in the UV tab: The time point or time range used for the background spectrum extraction is set as External time range.
   
   When you extract other UV spectra, the extracted background spectrum will automatically be subtracted. The time point or time range used for subtraction is shown in the spectrum name. See How to Extract a UV Spectrum via a Right Click on a Peak in OpenLab 2.x, How to Extract a UV Spectrum with the Spectrum Extraction Tool in OpenLab 2.x, How to Extract Multiple UV Spectra in One Step. 

Figure 10 shows how to manually extract UV background spectra (using either time point or time range).

Figure 10: Extract UV Background Spectra 

Learn how to effectively operate your Agilent OpenLab CDS Software: HPLC-OLII-1121e - Agilent Infinity LC Systems with OpenLab CDS – Acquisition Method e-learning course available from Agilent Education