Most HPLC (UHPLC) instrument manufacturer’s provide default sampling rate values within their software packages. Please do not use them as they were just put there to fill in the entry field. Many chromatographer's use these values without first understanding if they are appropriate for their own methods. This is a common mistake. Just as the manufacturer does not know what wavelength, flow rate or mobile phase you will use, they also do not know what sample(s), method and/or conditions you need. As such, they provide numerous default values in these data entry fields to satisfy the software's requirement. Just as you select an appropriate wavelength and bandwidth, you should always calculate and enter the correct data acquisition rate value yourself to make sure it is appropriate for your specific application, detector type and method.
The Peak shape's role during integration: For each chromatographic analysis you must determine the optimum sampling rate for the chosen detector. An accurate value is critical for proper instrument set-up, quantification and integration of your sample(s) peaks. In the most basic sense, the area under a perfectly Gaussian peak requires at least ten points to describe it with some accuracy. Ten points will provide basic data about the shape of an ideal peak to the computer. Since peaks are rarely perfectly symmetrical, a larger number of points will provide more accurate integration of the peak’s actual shape and total area. This will improve run-to-run reproducibility and quantification. We suggest you include twenty to thirty data points to allow for a more detailed fit to the peak. Too few points across a peak and you lose detail and sacrifice reproducibility. Too many points and you start to introduce noise into the system.
With these facts in mind we can next think about the detector’s data acquisition rate. You must select a data rate (sampling rate) that is sure to provide the recommended 20 to 30 data points across the peak width (we use the commonly calculated peak width at half height as the time measurement). Select a detector sampling rate that will provide you with this degree of detail and resolution. This is best accomplished by initially looking at an actual chromatogram of your sample. Look at the chromatogram and use the narrowest sample or standard peak past the void time, with good retention as an example to determine the best acquisition rate. The narrowest peak will be the worst-case scenario and will insure that you have enough points across all of the remaining peaks in the sample. It's width is often measured in units of time (seconds/minutes). This data can often be read directly off of a generated data acquisition report.
(a) If your narrowest peak has a peak width of 1.00 minute (60 seconds), then divide 30 points into 60 seconds for a result of 2 seconds per data point. The preferred sampling rate would be 2 seconds, 0.03 minutes or 0.5 Hz (depending on the units used by your detector).
(b) If your narrowest peak has a peak width of 0.20 minutes (12 seconds), then divide 30 points into 12 seconds for a result of 0.4 seconds per data point. This equals a sampling rate of 2.5 samples per second or 2.5 Hz.
To Determine the Data Acquisition Rate For Your Detector You Need To:
- Calculate the best data rate for each method and not use a generalized value (though similar methods will often use the same rate).
- Use your existing sample integration data results to identify the narrowest chromatographic peak in your analysis (at half-height).
- Record the width value of this peak (usually in units of time).
- Divide this number by thirty (30) to determine the preferred sampling rate.
- Use this value, or a value close to it, for your detector’s sampling rate.