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HPLC HINTS & TIPS for Chromatographers


Modern UV/VIS detectors offer the operator a choice of one to several hundred different signal wavelength choices (as is the case for Diode Array Detectors). Besides being able to specify a single wavelength, you often can choose a signal  bandwidth (bw) to associate with each wavelength [e.g. for a 280 nm signal with 10 nm bandwidth this is often written as: 280 (10) or 280:10]. Signal Bandwidth is the total number of nanometers across the specific signal value chosen. For example: If you selected a signal wavelength at 280 nm and chose a bandwidth value of 10 nm, then you are actually gathering all signal data from 275 nm to 285 nm (5 nm to the left of the apex and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of increasing the signal selectivity of the detector as you are only collecting data within a tight window. If you were to increase the bandwidth to 60 nm in the same example you would now be collecting data starting at 250 nm and continuing through to 310 nm. The additional data collected over this wide range can reduce the total noise (by averaging it over a wide range), improve the S/N ratio (increases sensitivity), but it also reduces the selectivity. Large bandwidths also increase the chance you may include peak signal data from other co-eluting components into your signal data. You must select a bandwidth range which is always 'safe' from any other potential peak. As with many things in life there is a balance between selectivity and sensitivity.

  • When developing methods we recommend that you initially choose a bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity and sensitivity. It is also a common bandwidth value used on detectors which have a fixed signal bandwidth (such as many single or variable wavelength detectors).

  • If you have determined the exact signal maximum for your sample and you would like to gain additional sensitivity for your sample (and thus decrease selectivity), re-run the analysis using several different increasing signal bandwidth values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range of the detector and also away from any potential co-eluting peaks. *Be sure and calculate the actual signal to noise ratio of the peak of interest after each analysis. This is a critical step! Do not be fooled by increases in the peak height or area alone as these changes are not always synonymous with better signal to noise ratios. Only by measuring the actual baseline noise level for each run and comparing it with the actual peak signal obtained will you be able to determine if increasing the bandwidth has provided you with better noise reduction and signal strength.

  • To increase spectral signal selectivity choose a bw value that is very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal data that is at or near the apex of your selected wavelength. This can be very useful when trying to discriminate your signal from nearby signal peaks, especially at low wavelengths such as 210nm. > Bill Letter, 12/06/10.

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