Baseline Noise: Manual Calculations - Tip294

OBJECTIVE or GOAL

Measure baseline noise for the signal-to-noise calculation, starting with manual calculations.

ENVIRONMENT

• Empower
• Empower Tip of the Week #294

PROCEDURE

STEP 1
Typically, noise is measured in an area of the chromatogram where the baseline is relatively flat and there are no spurious chromatographic peaks. This representative section of baseline over which noise is measured is commonly proportional to the chromatographic run time (Figure 1).

STEP 2
When noise is measured to calculate signal-to-noise, the noise region selected is commonly proportional to the peak width of the chromatographic peaks of interest (Figure 2).

STEP 3
Using the displayed section of baseline, let’s explore some of the variants that make baseline noise measurements so difficult to pin down. 

1. The use of various calculations in different chromatography software packages can be particularly frustrating if you base critical validated factors like Limit of Detection (LOD) or Limit of Quantitation (LOQ) on the noise calculation. 
2. In laboratories where the chromatography software of multiple vendors is used routinely, it is not uncommon to find chemists resorting to hand calculations of chromatographic noise to help eliminate the differences between the various software packages. 
3. In environments where a single vendor’s software is used, it is common for chemists to rely on the software calculations for noise. This can cause problems for these environments if they need to transfer methods to labs where a different software is used, or if the lab decides at some point to switch to a different vendor's software. 

Manual calculations are subjective and time consuming. Automated calculations depend on which vendor's software is used. What should laboratories do to obtain consistency? The answer is to understand the calculations used by each of the software products in your laboratory and in the laboratories that will be the recipient of the methods you develop and configure (Figure 3).

STEP 4
If you measure noise manually, you might get a room full of chemists to agree on where the bottom of the noise is in the example chromatogram. Where to draw the upper line, though, could be a subject of greater debate. Where should we measure the magnitude of the noise? Should we do it on the right side of the chromatogram or the left side of the chromatogram? To eliminate this problem, should we make it a requirement that the upper and lower lines be parallel to each other (Figure 4)?

STEP 5
Where do you place the parallel line? Do you eliminate the spike at 26.6 minutes as an outlier? If you include it, are you overstating the measured noise in this chromatogram? Maybe you should choose a different section, where you don’t have these outliers, over which to measure noise (Figure 5).

STEP 6
How about this section? We are not actually suggesting that this is where noise should be measured. This might be too extreme to be what chemists in your lab would do, but it does demonstrate the highly subjective nature of using manual calculations without proper analyst training, and without specific variables like the time region to use and the y-axis scaling to be used being written in the analytical method (Figure 6).

We've noted that manual calculations are largely subjective and may cause problems with borderline results in a validated laboratory environment. The use of automated calculations such as peak-to-peak noise will be discussed in the next Tip of the Week.

ADDITIONAL INFORMATION

This can be done with either the Pro or QuickStart interface.