Calculating signal to noise ratios for electropherogram peaks using 32 karat software

Why should I calculate signal-to-noise ratios for my electropherogram peaks?

Calculating the signal-to-noise ratios of electropherogram peaks is a way of showing how clearly the measures of our analytes stand out from background noise. This ratio helps to determine whether a peak is reliable enough to be used as a real estimate of analyte identity and quantity. This ratio is often used to validate limit of detection (LOD) and limit of quantification (LOD). Typically, the higher the signal-to-noise ratio for a given peak, the more we can trust that it is a reliable signal, and not just background noise. A low signal-to-noise ratio may indicate a poor analyte separation, low sample concentration or high instrument noise.

How does the software calculate the signal-to-noise value?

1. The baseline file acquired during the sequence is overlaid onto the electropherogram of interest.
2. The width at 50% height is calculated for each integrated peak.
3. The calculated peak width is multiplied by 20 to define the time interval for each peak.
4. The software then uses this time interval to locate and measure noise in the baseline file.
5. The measured noise is then used to calculate the signal-to-noise value for each peak.

If you have smaller intensity peaks, these peaks may not be integrated and so the signal-to-noise value will not be calculated for these peaks. In these cases, we can manually define a time interval in the baseline file that should be used for the signal-to-noise calculation.

Steps to calculate signal-to-noise values

1. Open the data file that you want to calculate signal-to-noise values for. 
2. Overlay the baseline file onto your electropherogram trace.
   1. Right-click within the electropherogram, select Annotate and select Baseline in the Other section.
   2. Click Apply to All and click OK.
   3. Right-click within the electropherogram, select Add Trace, click Open data file, locate the baseline file acquired during the sequence and click Apply.
3. Offset the baseline file trace to improve readability of the electropherograms
   1. Right-click and select Operations > Stack Traces.
   2. Enter 0.01 for y-axis and click OK.
4. Enable performance parameters to be calculated for the data file
   1. Select Method > Advanced.
   2. Click the Capillary/Performance tab, enable Calculate performance parameters for this channel with USP selected as the calculation method and close the window.
5. Annotate peaks with their width at 50% height
   1. Right-click within the electropherogram, select Annotate and specify Width at 50% height to be shown.
   2. Click Apply to All and click OK.
   3. From the toolbar, select the Analyze data icon to label integrated peaks with their width at 50% height.
      • Some smaller intensity peaks may not be integrated and will not be labelled. This is covered later in this article. 

6. Annotate peaks with their signal-to-noise ratio.
   1. Select Method > Advanced and click the Custom Parameters tab.
   2. Enter a parameter name for your new custom parameter e.g. S/N
   3. Select Per-peak for Type and Number for Returns.
   4. For source, browse to the 32Karat folder and select the SignalNoise.dll file.
      • Do not use the SignalNoiseUSP.dll file
   5. For Additional Parameters, enter the full datafile path for your baseline file.
   6. From the toolbar, select the Analyze data icon to perform the calculation using the new parameter.
   7. Right-click within the electropherogram, select Annotate and specify your custom parameter to be shown.
   8. Click Apply to All and click OK.
      • Peaks that were originally labelled with width at 50% height will be now labelled with their signal-to-noise.

7. (Optional) Annotate smaller intensity peaks with a signal-to-noise ratio calculated using a specified time interval from the baseline file.
   1. Select Method > Advanced and click the Custom Parameters tab.
   2. Make a duplicate of your previous custom parameter and modify the parameter name to include Segment e.g. S/N_segment.
   3. For Additional Parameters, modify the data file path to include the time interval in the baseline file to use for the calculation using this format: datafile path;start time,end time;  as in the example shown below:
      • C:\Data\S-N\blank.dat;13.0,16.0;
   4. From the toolbar, select the Analyze data icon to perform the calculation using the new parameter.
   5. Right-click within the electropherogram, select Annotate and specify your new segment custom parameter to be shown.
   6. Click Apply to All and click OK.
      • Peaks will be now labelled with their signal-to-noise calculated using the specified time interval.

8. (Optional) Annotate peaks with the noise value used for the S/N calculation. This can be calculated for the entire baseline data file and a specified time interval.
   1. Select Method > Advanced and click the Custom Parameters tab.
   2. Make a duplicate of your two previous custom S/N parameters and modify the parameter name to include NV e.g. S/N_NV and S/N_segment_NV.
   3. For Additional Parameters, modify the data file path to add ;NV; at the end for both custom parameters as in the examples shown below:
      • C:\Data\S-N\blank.dat;NV;
      • C:\Data\S-N\blank.dat;13.0,16.0;NV;
   4. From the toolbar, select the Analyze data icon to perform the calculation using the two new parameters.
   5. Right-click within the electropherogram, select Annotate and specify the two new custom parameters to be shown.
   6. Click Apply to All and click OK.
      • Peaks will be now labelled with the noise values for both methods.