This allows you to monitor for contamination, impurities, problems or changes during the run. The separate signals can be compared, subtracted or manipulated as needed for integration and reporting purposes, but the original signal sample data, 'A', is left unchanged and secure.
IOW: To acquire scientifically useful data, turn 'OFF' the Reference Wavelength software feature and record all of the signal data. 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 (Method # 2). It is for this reason alone that we teach chromatographers to always turn this feature 'OFF' by default. If any sample peak(s) or impurities appeared in the region where you selected a reference wavelength/bandwidth, then the resulting data would have been subtracted from your actual sample and you would never know it happened or have any 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.
Only the newly manipulated (subtracted) result is provided, 'C'. You will never know what the original data looked like before the reference wavelength was subtracted from it (it has been destroyed). This subtraction occurs in real-time, on your raw data gathered from the detector and the resulting data reported to the user is in fact the result of the subtraction only. This feature allowed a chromatographer to include with each signal choice, 'A', a second wavelength value, 'B', (and bandwidth) as part of the method which would be used to subtract out raw data from the primary wavelength during the analysis. Using the concept of Method # 2 described above, many HPLC manufactures added a software feature known as a the ‘Reference Wavelength’ to their systems. The benefit of this method is that the signals are all acquired using the same time base (unlike Method #1). Original, Secondary, and Subtracted signals). This method preserves the raw data obtained from all three signals (i.e. A third, baseline subtracted signal, 'C', can be generated from them. With this method, two separate signals, 'A' and 'B', are collected at the same time (this is the key). You can then subtract the second acquired ‘blank’ signal run from your original signal run and the resulting chromatogram should have a flatter baseline (less drift) for quantification purposes. If selected carefully, it can be used as a pseudo blank run for post-run baseline subtraction. This is tricky as you want it close enough to show the drift, but far enough away to not show any sample signal.
(Method # 2) Set up the detector to collect a second channel of data (2nd wavelength signal) that is close to the original wavelength selection, BUT far enough away from the original signal such that it will not overlap any of the peak spectra of interest or other compounds in the sample. In analysis errors, invalid methods and perhaps very expensive product recalls.Īllow me to provide a brief explanation of the “Reference Wavelength” software feature as seen and used with many DADĪnd/or PDA detectors (e.g. Using advanced features without proper training can result With all advanced features, proper training is required to understand and use Software, but its use and function are a mystery to most chromatographers. DAD/PDA) detectors provide this extra software feature in their chromatography Most manufacturers of advanced HPLC UV/VIS This is a one-time zero of the signal and has nothing to do with the special software feature we discuss in this article. When you manually press the 'Auto-zero', you are adjusting the displayed signal plot to a know reference point (often 0.0 volts). This is usually known as "zeroing" the detector and occurs just once, at the start of each run.
How to choose appropriate settings for the modern UV/VISĭetectors. One of the most common problems that I see as a consultant forĬhromatography laboratories today relates to