HPLC HINTS & TIPS for Chromatographers
- Tip# 102: REFERENCE WAVELENGTHS (as used in HPLC UV/VIS):
The number one problem that I see as a consultant in chromatography laboratories today revolves around the lack of understanding of how to choose appropriate settings for the modern UV/VIS detectors. Specifically, the optional use and configuration of a Reference Wavelength value. Most manufacturers of advanced HPLC UV/VIS (esp. DAD/PDA) detectors provide this extra feature in their chromatography software, but its use and function are a mystery to most chromatographers. Allow me to provide a very brief explanation of it here.
If you are running a gradient analysis the change in solvent properties and temperature over time can cause noticeable baseline drift during the run. This drift up or down relative to the starting baseline can cause a number of quantification problems with the analysis reporting software (as flat baselines are more easily integrated than slopes). Two clever ways to deal with this problem are: (Method # 1) run the same method again, but with no sample (a blank) and subtract it or (Method #2) collect a second channel of data (2nd signal wavelength) at the same time that is as close to the original wavelength, BUT will not detect or overlap with any of the peaks of interest or compounds in the sample. As such, it can be used as a blank run for baseline subtraction. You can then subtract the blank run from your original run and the resulting chromatogram should have a flatter baseline for quantification purposes.
Using the concept of the 2nd method described above, many manufacturerss added a feature known as a Reference wavelength to their systems. This feature allowed a chromatographer to input a second wavelength value into the method which would be used to subtract out raw data from the primary wavelength during the analysis. This occurs in real time and the resulting data reported to the user is in fact the result of the subtraction. You will never know what the original data looked like before the reference wavelength was subtracted. Only the manipulated result is provided. If any sample peak(s) appeared in the region (depending on the wavelength and bandwidth specified) where you selected a reference wavelength, then the resulting data would have been clipped off of your real sample and you would never know or have a record of it ! This brings up a serious validation issue as you are modifying the original data with no way of knowing (or documenting) how you have changed it. It is for this reason alone that we teach chromatographers to always turn this feature 'OFF' by default. If they want to make use of the feature, then we suggest that they simultaneously collect data from a second, separate wavelength channel such that the two raw data streams are preserved for validation purposes. The separate signals can be compared, subtracted or manipulated as needed for reporting purposes, but the original data is left secure. This allows you to monitor for contamination, problems or changes during the run.
Observational Opinion: The reason we see this feature cause so many problems in the field appears to be due to the Reference Wavelength feature being turned 'ON' by DEFAULT in the software (should be 'OFF') and to make matters worse, the default values often supplied by the manufacturers are actually used by most chromatographers (what are the odds that the random values placed in the system are even relevant to your analysis?)! We suggest using a canned method template in most laboratories which includes a new default value for this feature... 'OFF' for all analysis methods. > Bill Letter, 05/30/10.
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