baseline noise

<p><span>On my UPLC-TUV, I have a 100uL-mixer installed. I do see very noisy baseline thru the run. Please see the attached file. I was told to change to a 425uL mixer. Is there other way to reduce the noise?</span></p><p><span>Thank you,</span></p><p><span>Yliu</span></p>

Comments

  • Hello:

    What are you mobile phase conditions, gradient, flow rate and wavelength, detector time constant and data rate, backpressure maximum etc.. I would just like to be sure of the issues that you are encountering.

    The 425 uL mixer is designed for TFA gradients (specific to peptide anlyses), where the noise is the result of the TFA high background absorbance that removes the available light energy and increases noise and is observed at low wavelengths < 220nm in UV analyses. I just want to check that this is your case.

    Let me know,

    Liz

  • Hi Liz,

    Thank you for your quick reply.

    My mobile phase A is 0.1%TFA in the mixture of water:THF (970:30) and mobile phase B is MeCN:MeOH:IPA:TFA (850:150:10:0.85), and I am using UV=217nm. The flow rate has been changed from 0.76 to 0.4ml/min (no difference, and same noisy background obtained). The sampling rate is 10, the back pressure max is about 6000psi, filter time constant is 'normal".

    I am doing the peptide impurity profile, which is originally adopted from a HPLC method (95min). In Alliance system, we have this HPLC method (0.5ml/min) set up and use a pulse damper and a static mixer to reduce the noise. Now, we need to transfer to UPLC and to make the total run time shorter, that's how I encounter the noise issue.

    I think adding a 425uL mixer would definitely help the noise level on my UPLC. Not only we have TFA in the mobile phase but THF, which is another component with UV cut off <220nm.

    Liz, if you have looked at the chromatogram that I sent over yesterday. The synchronous noise seemed to me is more hardware related issue, isn't it? Would you recommend to use a pulse damper (operating pressure 500-6000psi) to my system? One more question, I am using a UPLC column from Phenomenex, not ACQUITY UPLC column. Would a finger tight fitting connected btw the column to the detector is an issue as well?

    Thank you,

    Regards,

    YLiu

  • Hello:

    The gradient composition is created by adjusting the individual flow rates to correspond to the desired composition, with very low dispersion pumps bandspread (10µL) and 110µL of real volume, as you know these peptide separation use Formic Acid or TFS which are UV absorbers in the low UV, nevertheless the analysis has to be run at low UV wavelength, 217nm, in this case. Therefore, the low light energy visualizes the compositional ripple and you see high noise. And so the system needs more mixing volume and therefore, the 425 µL mixer was created. I believe this will solve the issue. I just wanted to ensure I understood the details before confirming. This mixer will work for pressures up to 10,000 PSI, we have an alternate if higher pressure is required.

    I do believe that the high pressure reusable fittings will work with the column, if it convenstional fiitings. What do you think of this column in comparison with the ACQUITY peptide columns?

    Best regards,

    Liz

  • NKSNKS
    edited July 14
     Hi @mannkind , @lizh

    I am facing similar problem in Waters UPLC (quaternary pump) with TFA in both the mobile phases. Could you please let me know if changing the mixer form 100 to 425 microliter worked for you? Any other suggestions are also welcomed.

    Thank you
    NKS
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