Fractions and IEX-UPLC

Hello, seeking some help here. 

I want to separate and analyse (on Mass Spec) acidic and basic species from a mAb. We have an IEX method, where we have decent separation, we tweaked the method to collect acidic, main and basic species in fractions. In this first instance, I want to confirm if this fraction overlay with the peaks prior to collection. So I have concentrated the fraction using a speed vac, enough to inject onto UPLC with the IEX method. It looks like none of the fractions like the column and elute within 3 mins of the run, which I find very surprising. So I was hoping if anyone can suggest any improvement here. 

Column - Bioresolve SCX mAb column 4.6mm x 100mm
Mobile Phase - IonHance CX-MS A and B
Flow rate - 0.2mL/min
Run time is 65 mins 

Happy to provide more information, if needed


  • Hi Neel22,

    What is the diluent and injection volume of the concentrated fraction.  Is there any chance that the peak is being eluted in the "dead volume"?

  • lizh
    edited July 2023
    I asked a SME on this and this is what they recommended.

    From the question below about IEX fractions. Even though the concentrates are derived from ammonium acetate and some acetonitrile, It sounds like the Neel22 is possibly concentrating the sample matrix which is interfering with adsorption to the IEX stationary phase and/or denaturing the mAb fraction which is changing the surface charge profile of the protein. My suggestion would be to try buffer exchanging the fraction into the original sample matrix using a low volume spin filter with reservoir rather than speedvac.


    If the protein is denatured from the ammonium acetate then it is not likely to look the same in re-injection on the same IEX column. fraction comparison of profiles with proteins like they want to do is typically better with SEC. Even then if the protein is denatured it can change the hydrodynamic volume of the protein.


    If the method is reproducible run-to-run and the eluent is flowing through an optical detector you should see a negative space in the chromatogram where the flow is diverted. If it’s 1 fraction this is comparable. However if your doing multiple fractions in 1 run, then the detector response gets a bit distorted as the flow is re-initiated in the original path and the detector responds to a shift in the mobile phase composition.  

    Hope this is helpful!