Translator for HPLC HINTS and TIPS for Chromatographers

Tuesday, March 1, 2011


One of the most common problems that I see as a consultant for 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 for a multi-wavelength or Diode Array Scanning Detector (DAD or PDA). 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. As with all advanced features, proper training is required to understand and use them successfully. Using advanced features without proper training can result in analysis errors, invalid methods and very expensive product recalls. Allow me to provide a brief explanation of the “Reference Wavelength” feature as used with DAD and/or PDA detectors.

If you are running a gradient analysis, then the change in solvent properties (RI) 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 and accurately integrated than sloped ones). 

Two clever ways were developed to deal with this slippery slope of a problem:  

(Method # 1) run the same method again, but with no sample (a blank) and subtract it to produce a "blank subtracted run". This preserves the original data, but due to the time difference between injections, you are unable to confirm if anything has changed between the time of the first and second injection.

(Method # 2) collect a second channel of data (2nd wavelength signal) at the exact same time that is close to the original wavelength, BUT will spectrally not overlap with any of the peaks of interest or other compounds in the sample. As such, it can be used as a blank run for post-run 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. With this method, two separate signals are collected at the same time. A third, baseline subtracted signal can be generated from them. This method preserves the raw data obtained from all three signals (i.e. Original, Secondary, Subtracted).

Using the concept of Method # 2 described above, many HPLC manufacturers’s added a feature known as a the ‘Reference Wavelength’ to their systems. This feature allowed a chromatographer to input a second wavelength value (and bandwidth) into the method which would be used to subtract out raw data from the primary wavelength during the analysis. 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. You will never know what the original data looked like before the reference wavelength was subtracted (it has been destroyed). Only the manipulated (subtracted) result is provided. If any sample peak(s) or impurities appeared in the region (depending on the wavelength and bandwidth specified) where you selected a reference wavelength, 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. 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 (Method # 2). IOW: Turn 'OFF' the Reference Wavelength feature and record all of the signal data. The separate signals can be compared, subtracted or manipulated as needed for integration and reporting purposes, but the original signal data is left unchanged and secure. This allows you to monitor for contamination, impurities, problems or changes during the run. It also allows others to verify your method for accuracy.

Observational Notes: A common reason we observe this problem in the lab is that I am called in to diagnose a strange problem where the area of a known sample peak changes in an unexpected way. That "way" often includes going NEGATIVE, increasing in mass or decreasing in mass. *This is due to the reference wavelength feature being turned 'ON' and another peak absorbing in the region selected which contributes to the final signal. If the data collected (area) for the 'reference peak' is larger than the sample peak the resulting chromatogram will show a negative peak (this tends to be noticed!), whereas if the reference peak is smaller than the sample peak, the resulting signal decreases, which may or may not be noticed (incorrectly interpreted as a lower concentration sample). You can see the obvious danger posed by this situation. Companies can be put in a situation where all of their past data is found to be invalid and product recalls can result from this finding. The cause was lack of training in the use of the software and/or HPLC system.

How to Solve The Problem: The reason we see this feature cause so many problems in laboratories appears to be due to the fact that the Reference Wavelength feature is being turned 'ON' by default in the software for most DAD/ PDA modules (The real default value for "Reference Wavelength" should always be: 'OFF').  To make matters worse, the default values for the wavelength and bandwidths 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? Why would you use them?). We suggest using a ‘canned’ method template in most laboratories which includes a new default value for this feature... 'OFF' for all analysis methods. Most importantly of all, please obtain formal training in the use of a specialty detector such as a diode array before using one for sample analysis.

Note: The bandwidth chosen for each wavelength is also very important and if chosen poorly, can result in adding noise to your signal, reducing it or even enhancing it. Please refer to this article for more info:


  1. I just came across this while trying trying to understand exactly what the reference spectrum is for. Thank you for posting this, it will undoubtably help with my current analysis and many future analyses. I'm even considering giving a technical presentation on this very topic so others in my company who take reference spectra for granted will be enlightened. Thank you thank you thank you!

  2. Glad to hear you found it useful. The reference wavelength feature (not reference spectrum which is something else) results in a lot of collected data which is of no scientific value. Always turn the feature 'OFF' and consider using a separate channel of data to collect the 'reference' run for subtraction after the analysis is complete.

  3. My mistake, I meant reference wavelength. Thanks again.

  4. THANK YOU, thank you for this information! For many weeks our API std has shown too low a concentration/area and we could not figure out why this was happening. We even brought in an "expert" in method development from the instrument manufacturer to help us. They told us to always set the Reference Wavelength to 'ON' and use 360nm (100) for all samples. We modified the settings as they suggested and things have gotten even worse. Now our peaks sometimes go negative. THEN I found your great post which explains how this feature works and why we should not use it at all. Our problem dissappeared once we turned OFF the Reference wavelength feature and ignored the very bad advice given to us by the "expert". You clearly are the real expert and now that we understand how this feature works, we will never use it for method development again. Thank you as your great blog has saved our company and made us all aware of how important it is to ask questions and understand feature before we use them.

  5. I am having a problem which is making me feel very stupid..... I have 2 peaks partly coeluting that have sort of similar UV spectra. When I try to 'subtract out' one of the peak's UV to get a better match for the other peak, the other peak's UV is inverted (upside down) compared to the library match. Why is this and how do I stop it?

    1. This does not sound like a Reference wavelength issue. You can not perform scientifically correct Library peak matching comparisons on HPLC peaks which are not first, fully resolved apart. By definition, co-eluting peaks will have mixed spectra and the computer software used to report this data is not capable of accurately "separating" the two apart. It will add/subtract spectral data which may or may not be part of the desired peak. The result will be unpredictable and of little value chromatographically.

      The correct solution to the problem described is to develop an HPLC method which results in Gaussian peak shapes which have at least an R of 1.5. Proper method development and related techniques will allow you to obtain reliable and reproducible data.

  6. how do you turn off the reference wavelength in the software?

    1. The answer depends on which CDS software you are using.

      For ChemStation/OpenLAB, just click on the down arrow next to 'nm' where is says "REFERENCE" and it will change to 'OFF' for the wavelength selected. For many EMPOWER systems, just click on 'OFF' or leave the Reference Wavelength field empty. Make sure you select 'OFF' for all wavelengths.

  7. Thanks for this post. it not only gave me idea what the reference wavelength is for but how this actually work.