Determining cause of random peak

Agilent 1260 infinity II HPLC, BioRad column  using 0.0025 M sulfuric acid isocratic run. DAD detector.  Been using the method for a while and recently a random peak has shown up at 8 minutes. Have tried rinsing the system with ACN, Meoh, Water. Three different sources of water and sulfuric acid. Tried several different vials and have removed septum. See peak with both needle rinse  and without. Tried using different channels. Replaced lamp due to hours. Pressure is consistent. Peak shows up when injecting MP removed from the MP being used. It is not a huge peak about 33 to 100 area units. If I inject Meoh or acn the peak will increase. I tried a blank run and the peak is gone but a blank run does not activate Rheodyne valve. This one has me stumped. Open for any suggestions.

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  • Hi  , the two most likely candidates would be the rotor seal of the Autosampler injection valve, or a buildup of contamination on the column itself.
    If you have a zero dead volume union (ZDV), you could replace the column with a ZDV and then do a series of injections of your mobile phase and see if any peaks come through after injection. If no peaks then suspect column as the source of the ghost peak.

    If you have a spare new-ish or known good LC column alternative then put that on the system and check with injections of Mobile Phase, ACN or MeOH (if compatible with the column used). If you are still getting the same peak then it probably else where in the system (Needle seat, capillaries, rotor seal of injection valve, any column switching valves). If no peak, suspect the Bio-Rad column is contaminated.

    Conversely you can disassemble and sonicate your Rheodyne valve rotor seal (or replace seal if required), then re-assemble, run a pressure test to makes sure no leaks and then re-test with blank runs and injections of mobile phase or MeOH or ACN to see if the ghost peak is still present.

    I hope that gets you somewhere. Please let me know how you get on.

    Regards,

    Damian

  • Thanks Damian

    I removed column and added a zero union fitting and peak showed up right after injection. Removed Rheodyne valve seal and sonicated that and head in Meoh for 20 minutes. Still have peak. Bypassed all column heater valves and peak still exist. Bypassed needle, seat and injection pump valve, still have peak. Replaced needle and seat with new and rebuilt metering valve, peak still there. Replaced rheodyne valve seal with another good used one, still a peak. If I inject 1ul I get the same peak area as a 10ul injection but if I inject 20ul it does double. Any other advise would be helpfull.

    A little more data. If I cycle the bypass valve no peak. If I do a needle rinse 100s I see the peak. Rinse is DI water and the lines have all been rinsed with Meoh. I turned off the needle rinse on the method earlier as I thought it might be the problem with no change but something is being brought into the equation that the needle rinse procedure does.

    Thanks

    Chris

  • Hi Chris,

    You have certainly done an extensive effort at troubleshooting this issue.

    Ok so to summarize it looks like the peak only occurs which the injection valve is actuated correct? If you just run your gradient without injection and with a ZDV you get no signs of a peak? If so, then one of the only candidates remains the rotor seal of the injection valve. If sonication hasn't helped it may need to be replaced.

    Agilent Autosamplesr are a flow-through design which means in mainpass mode the needle, needle seat and sample loop are included in the flow path, except in bypass mode. So, if those elements were contaminated you should see you contamination peak even without injection.


    Point of clarification - "I removed column and added a zero-union fitting and peak showed up right after injection" - are you sure this is the same peak? Was this peak reproducible over multiple injections?

     I would connect the pump straight to the detector, run a normal gradient and confirm no peaks. If no peaks then the pump and the detector can be eliminated from t-shooting. Then connect the autosampler into the flow path (Pump_>ALS->Detector) and run a gradient again (no injection) and confirm no peaks. Then repeat with MP, ACN or MeOH injections to see if a peak occurs. If it does then it really has to be to be the ports of the rotor seal that are introduced into the flow path when the valve switched to bypass mode during injection, and that is literally the only point of difference in these two flow path comparisons. Or it is coming from your MP, think Glassware, pippettes etc. that you use to prepare your mobile phase, not just the solvent itself.

    If that doesn't shed some light on the issue then it's time for an engineer visit I think Chris. Based on the info you have already provided I'm out of ideas other than rotor seal replacement, which is an expensive part.

    Regards,

    Damian 

  • No it has not. Everything has been rebuilt with no change. I have done more testing and have determined that the peak is air. I also believe that this is an engineering issue in the design of the injection system specifically the needle to seat system allowing air to be pushed into the system when the needle drops down onto the seat. I did some online hunting and found other people with this same system and they also have this mystery peak and have done all the rebuilding process I have gone through. I did some back data reviews and this has been an issue since day one of the install. I can show this same peak since install in 2018. It should have been caught by the initial instrument check with the agilent install column as it will appear on any column or eluent. I am talking to the one of the sales people now trying to get me in contact with someone who might have enough honest knowledge on the design to figure out a fix as just replacing the module or having a service engineer come in and do everything I have done is a waste of money.  The instrument was on contract for several years so if it was a one off bad part the problem would have disappeared at some point but it has not. This is very frustrating.

    Thanks

    Chris

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  • No it has not. Everything has been rebuilt with no change. I have done more testing and have determined that the peak is air. I also believe that this is an engineering issue in the design of the injection system specifically the needle to seat system allowing air to be pushed into the system when the needle drops down onto the seat. I did some online hunting and found other people with this same system and they also have this mystery peak and have done all the rebuilding process I have gone through. I did some back data reviews and this has been an issue since day one of the install. I can show this same peak since install in 2018. It should have been caught by the initial instrument check with the agilent install column as it will appear on any column or eluent. I am talking to the one of the sales people now trying to get me in contact with someone who might have enough honest knowledge on the design to figure out a fix as just replacing the module or having a service engineer come in and do everything I have done is a waste of money.  The instrument was on contract for several years so if it was a one off bad part the problem would have disappeared at some point but it has not. This is very frustrating.

    Thanks

    Chris

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