Artefact peak in RI chromatogram


I am working with a RI detector.(1200 agilent)

My eluant ist 800 MeOH, 100 Water, 100 0.1M ammonium acetate 

I have a problem at about 3 minutes. In some chromatograms, a peak is present but in other not. 


1: Blank

2: reference

3: sample 1 first preparation

4: sample 1. second preparation


I washed the system with IPA : no peak

I made injection of 10 blanks: no peak

10 injections of the sample: peak is present.


One of my hypothesis was a change in the eluant. The reinjection of the same vial over the time is leading to the same chromatogram.

  • A few ideas/thoughts/comments:

    - Can you clarify the "change in the eluent"?

    - I guess you´re running isocratic with premixed solvent, right?

    - Are you using an isocratic pump? If not, have you set up your bin/quat pump correctly?

    - 80% organic is a lot for a 100 mmol buffer. Can´t you lower the percentage or concentration?

    - Make sure you don´t overload the detector.

    - Can we see the entire method?

    - Can we see the chromatogram?

  • First of all, yours questions and you feedback are really stimuling. thank a lot for that. 

    As change in the eluent, I mean lost of ammonium acetate. I prepared the sample in the fresh eluent. The lost of ammonium acetate leads to a difference between the current eluent and the original eluant in the vial.


    Waht do you mean with overload the detector. The analyte is eluating later. 

  • Hello,


    glad I could give a few thought-provoking impulses.


    Since I haven´t seen a chromatogram, I did not know when your compund eluted.


    Not quite sure what you mean by "lost of ammonium acetate".


    In any case, it´s always a good idea to first go back to standard known conditions, so moving away from your application. If the hardware is proven to be working, the application can be investigated.

  • Good Morning,

    "" The reinjection of the same vial over the time is leading to the same chromatogram. ""

    Try this...

    Temporarily double your run time

    Blank (2 injections)

    Sample (2 injections)

    Blank (2 injections)


    Your first two blanks should be "clean" - if not then I would suspect that there may be issues with either your reagents, separation chemistry, and/or hardware configuration. You've already established the blanks are "clean" in your original post; thus, I don't expect anything unusual to show up - here we need a starting point for the extended runtime to compare against the sample and the final two blank injections.


    Hopefully, your two sample injection results are "duplicates" of each other and that interference peak has resolved out in to the extend runtime Given your statement that the 10x-inj are reproducible I suspect this will be the case and that the two subsequent blank injections will be "clean."


    Your last two blanks should be "clean"

    - if the first injection in the second blank has the extra peak AND the second injection has the same extra peak but at a lower response then we can start looking at things such as needle wash (make-up and timing) and/or hardware configurations (poor fitting makes, worn injection valve parts, etc...)


    -- If the 2x-Sample-Inj are duplicates and the 2x-2nd-Blank-inj are both clean then one should suspect that there is an unknown constituent in the sample and/or introduced during any sample preparation steps. Easiest way to solve this for an isocratic method, IMHO, is to extend the runtime to allow this constituent to elute from column (or if the CDS allows add a delay before next injection); however, this may not be desirable from a sample workflow requirement. If increasing the runtime isn't desirable then we get into the preverbal hell of either sample cleanup or modifying the separation chemistry ((from your original post - it seems that has already been done and may be the root cause of this event)). If you have a gradient system then you might look into changing the gradient to either resolve the peak out of the column within the runtime and/or strong flush the column at the end of the runtime and then setup an T0-equilibrium before the next injection.


    One of the most valuable things I learned from my Analytical Chemistry Professor - if you are beating your head against a wall, step back, you've missed something simple - good Chemistry is easy Chemistry! How I miss that man and my old advisor - two brilliant Analytical and Physical Chemists, RIP Dr. P and Dr. W.

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