How do I create an RT to RI calibration?

Hi there!

I'm just starting to use masshunter, and I would like to use retention indices rather than retention times in my analyses, however, try as I may, I can't seem to find how I create the RT to RI calibration file that I am prompted for.

In my exhaustive Google searches, I can only find references to a file included on a demo disk, though nothing on how to actually create these files.

Does anyone have an example RT to RI calibration file that I may edit, or a means in the software to create one automatically?

Thank you all for your time,


  • Hi JamesRS,

    It sounds like you are looking for the Quant Tool "Retention Time Calibration," which is located in the Windows start menu under Agilent MassHunter Quantitative folder -> Quantitative Analysis Tools. 

    Additionally, there are some videos about using Refractive Index that are included on Quant media (either the USB thumb drive or the downloaded .UFD.) Specifically, the "Library - Retention Index - RI RT CAL map - Advanced" video goes over how to use the tool and create the calibration file. If you registered the software in, you can re-download Quant to access these videos if they aren't already installed on the PC.  The videos are located at \Supplemental\MassHunter\Videos\Quant on the media. 

  • As @brittpeterson referenced, you will need to use the "RT Calibration" program to convert a set of results into a calibration file.
    To create the calibration file, you will need to create a batch in Masshunter Quant with just those samples and create a library with only the alkanes/calibration compounds. The batch and library will run automatically to create an output, there is no method involvement there. When creating our RT file, I found it best to create a custom library with only the relevant alkanes and verifying the matches myself in Quant before running through the program.

  • Hey msweber,

    can you possibly explain how you do this in detail? Do I have to create a library using the Library Editor or can I do it more quickly? Is it not possible to use the NIST database?

    Thank you in advance

  • It is absolutely possible using the NIST database, the reason I suggest creating a separate library is so you can control various parameters, mainly:

    • specific compound name
    • eliminate possibility of incorrect or undesired matches

    When you've created a custom library in library editor with 4 compounds with very clear peak and retention times, there is no chance of a "Dodecane" being identified as "Undecane"

    It is possible that you can do this more quickly using PCDL Manager, however I haven't had the opportunity to test that software. 

    I'm not sure which step you would like more in depth and it can easily become a multiple page post to detail each individual step in depth.

    Step by step:

    1. Create a custom library using Library Editor and a higher concentration sample for target spectra.
    2. Verify the accuracy of the library vs samples by trying to run through Quant with your new custom library as the only listing in "Library Search" of library method.
    3. Create batch file in Quant with the file(s).
    4. Use the "Retention Time Calibration" tool to create an RTC file.
  • The RTC file has been created so far. I would like to use the RT Calibration File at Masshunter Qualitative Analysis, but a CSV file is required there? What do I have to do to make a CSV from the RTC file?

Reply Children
  • The method editor should accept either a CSV or an RTC file

  • I am using v7 - this version requires a csv file
  • Hi ,

    From what I can find the RTC file is a CSV file. I believe this extension was created to help with file organization in later versions of MassHunter. So you should be able to change the extension and then use it in Qual.

    Another alternative, at least in newer versions of the RT Calibration tool, is to specify the extension as .csv when creating the calibration file. 

  • Hi, can you shed some light over how to RTC creation was achieved? I have created custom library, using NIST as reference source to identify the RI-alkanes in my pure RI sample. Created a batch file - however here in Quant it wants me to 'analyse' the file before saving it. To analyse it wants me to 'calibrate' the file. I am stuck here, I don't do quantification stuff so not sure what I am doing here. I just want to create an RTC for Qual and Unknowns analysis. The videos weren't much help as they said, "usual analysis was performed using 0.1 calibration curve"...

  • I'll start with a summary of the required steps:

    1: Acquire the reference standard alkane data using your normal method for MS, should be done using EI and at least cover 40-300 AMU.

    2: Use MassHunter unknowns analysis to analyze the alkane data and only use NIST library to search. Manually set correct compound if needed.

    To do this, create a "New Analysis" then use "Add Samples..." in the File menu. Then go to Method -> Edit Method -> Library Search and set a NIST or custom library only for "Libraries". For "Compound Identification" set max hit count to 1 if custom alkane only library or set to 2 or 3 for NIST library.

    3: Create the reference library by exporting as an xml file. Alternatively, create a new library in Library Editor and copy and paste the spectra to create the library. Make sure to add the column for Retention Index.

    Now it's time to switch to MassHunter Quant (MS)...

    4: Open up a new batch and add the alkane run. Use "New Method from Acquired Scan Data with Library Search..." and set the alkane reference library you just created in Library Method. It is possible you may have to edit the deconvolution settings if all the peaks are not being marked (if you do need to do this, consider if your MS is functioning properly as the response should be clear). Once the identifications are correctly showing, save the batch. 

    In the "Library Method" window under Library Search tab, you should see a button near bottom that says "New..." under the "RT calibration file:" line. You can select that which should open another subwindow guiding you through creation of calibration file.

    Alternatively: After identifying the library, save and exit the library method window. Analyze batch, verify correct identifications, then save and close Quant. Then open an app named "Retention Time Calibration". It will be under Agilent Masshunter Quantitative -> Quantitative Analysis Tools. This presents essentially the same window as following above method so either works.

    5: To fill out the retention time calibration info:

    Batch: browse to the batch file

    Library: name the library (use the alkane reference library you created)

    Output: name the output path (it actually wants you to set the name for the calibration file, it'll add extension of "rtc" automatically).

    Congratulations, you've created your calibration file!

    6: In any methods you create for Qual or Quant and want to utilize calculation of retention indices you will "Choose..." the rtc file you just created. If you want to look at the file yourself, just change the extension to csv and/or open in excel (or similar spreadsheet program).

    There was a document that I had found a while back from 2017, document# 5991-8221EN which did a good job of providing a detailed walkthrough.

    Hopefully this will help you out!

    Edit: Even though you aren't planning to do any quantitative work, to create the file you will still need to go through quant, library editor and RT calibration app are both linked to MH Quant.

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