Chromatography Forum

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 elutes 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 Neel,

First things first, this is a UPLC column so the dead-time/dead-volume is going to be tiny. Compounds eluting at 3 minutes is not unheard of on these columns that frequently have methods made between the 6-10 minute marks (not including eq, just post inj). Which is also interesting because your method is over an hour long with a tiny column. I'd say that if you'll probably have to re-do some method development. Your column in particular is a SCX which stands for strong cation exchange, thusly your analytes that are strong cations in the MP will retain the best on this column. This effectively means that you want all of your bases protonated and your acids should at least be neutralized when injected and while within the mobile-phase (which you have yet to describe yet other than the copy-paste from waters website). Speaking of such website, it details this combination as a special mixture to buffer pH between 5-8... will your compounds be cationic in this mobile-phase combination? I'm sure all of your acids will not be and would probably elute off the column immediately as there is no ion to "catch" these negative compounds on the SCX column. However, if you used this MP on the same column prior, like you said, and simply adjusted the methods, I'd go back to that working method and use it as a starting point to figure out which fractions run well at what pH and then make another method for whatever fractions do not meet that requirement.

However, I am a little confused what you mean by the fractions. Are these fractions taken from the original method to be re-run on a new method for better separation on the same column? Or is this fraction consistent of one of the prior fractions that had poor elution on the primary method and you're attempting to develop a method to better resolve these analytes?

Anyway, think about that and come back to us!