Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, August 3, 2013

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a k prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference” wavelength are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post).

Step 3. Edit the method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelength far away from or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Learning how to correctly use and set up the detector parameters and options is the second most important thing as incorrect settings can cause false or misleading results. Only after you have developed a reliable and scientific method with good sample retention can you begin to provide accurate integration and concentration values and/or make UV/VIS "purity" determinations.


  1. What is we have two samples which use the same wavelength. How do we detect one from the other?

  2. Use good chromatography fundamentals and resolve the two peaks apart first (e.g. baseline resolution > 1.5). This is the goal of good method development. Additionally, be sure and look at the spectral data obtained for each peak. They may be different, and if so, can aid in identification.

  3. On selecting the proper wavelength can I first make A1% conc. by spectrophotometer and determine the lambda max and take this as my wavelength

    1. IF your compound is a 100% chemically pure standard, then yes. However, we are normally running HPLC methods because a sample contains a mixture of components. Placing a sample containing a mixture of compounds into the spectrophotometer will result in a mixed spectra, not a pure spectra of one compound. So that would not provide the lamdba max for the sample. As stated above; "To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains the compound(s) of interest on the column".

      Analyzing a mixture in a spectrophotometer cell before developing an HPLC method (or Scanning Diode-Array-Detector...) can provide some useful information. You can read about that right here in a related article, "Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer"; Link: [ ].

  4. Replies
    1. Run the detector's Operational Qualification (OQ).