Get Empowered: Tip #207 | The Importance of Sampling Rate and Time Constant for Chromatography
Tip #207: Setting the Sampling Rate and Time Constant in Empower
In this week’s tip, we will learn why Sampling Rate and Time Constant are important for Chromatography.
Setting the Sampling Rate (or Data Acquisition Rate) for the detector is important for reproducible retention time and area calculation for peaks. And for calculating Peak Purity with 3D photodiode array data. Setting the Time Constant (or filter Time Constant) for the detector is important for achieving a good signal-to-noise ratio. Both parameters are set in the Instrument Method.
Let’s get started.
The Sampling Rate is the user-specified rate at which data for a channel is to be collected. The Sampling Rate must be high enough to ensure that collected raw data points provide a good representation of the peaks in the chromatogram. A suitable Sampling Rate produces at least 15 data points from peak start to peak end for traditional HPLC and at least 25 data points for UPLC or UHPLC.
Let’s illustrate this by collecting at different Sampling Rates. Too few data points result in inconsistency in retention time determination and area calculation for a peak from injection to injection (figure 1).
The Time Constant (or Filter Time Constant) is a noise filter which helps achieve good signal-to-noise ratio by filtering out high frequency noise. A Time Constant filters data points across the entire chromatogram, so you need to be careful not to use too high of Time Constant or it will result in distorted peaks. Use the following formula to determine Time Constant: 1/Sampling Rate (figure 2).
Let’s first look at 2D data.
Both parameters can be found in the detector portion of the instrument method. Here are examples of both HPLC and UPLC detectors (figure 3).
Adding the Points Across Peak field in the Peaks table within Review will display the number of points for each peak from start to end. Looking at the first peak, you see a width of 0.092 minutes or approximately 5.5 seconds. With a Sampling Rate of 10 points per second there are 55 points across the peak, more than enough (figure 4).
Now let’s look at 3D data.
The same rules apply for extracted chromatograms from 3D PDA data, however, if you want to calculate Peak Purity, you also need a minimum of 12 spectra from peak start to peak end. The Sampling Rate in the PDA instrument method on the General tab will affect both the number of points across the peaks in the extracted 2D chromatograms and the number of spectra across the peak used to calculate Peak Purity. Here is an example of a PDA Instrument Method (figure 5).
Looking at the first peak, you see a width of 0.047 minutes or approximately 2.8 seconds. With a Sampling Rate of 20 points per second there are 56 points across the peak, more than enough (figure 6).
Mass Detectors do adjust the Sampling Rate in the instrument method based on the mass range that you enter. That means for large mass ranges the software automatically adjusts the Sampling Rate to a lower value – and the reverse is true, i.e. for a smaller range it will calculate a higher Sampling Rate. Here is an example of data collected with an ACQUITY™ QDa™ Mass Detector and its associated Instrument Method (figure 7).
It’s that easy!
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Next week in Empower Tip #208 – We will discuss using in-needle auto additions.
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