Late Night’s in the Laboratory

By October 11, 2020

Late nights in the laboratory seem to be the only thing I can really count on when I am trying to get reproducible chromatography for certain compounds. But what I really want, is to go home knowing my chromatography is reproducible and my method is GOOD!

Allow me to explain…this doesn’t always happen. Sometimes I can develop a method, achieve great resolution between critical peaks, great linearity, great sensitivity…then test the robustness with three different column lots and call it a success – DONE. Other times, it’s like I am cursed by the chromatography wizards.  I make up my sample and inject it into a column and see…NOTHING. I inject it again, NOTHING. What is going on? I’ll tell you. The steel in my column is EATING my compound!

I have learned, through experience, that I may need to passivate my column and maybe even my system.  One strategy that has worked is making lots of injections of my sample at higher concentrations. This can “satiate” the hunger of my column and stop it from eating my compound. However, this process can take HOURS.  In addition, this can also cause me to have carryover from injection to injection and therefore lead to reproducibility problems. Poor reproducibility is NOT the cornerstone of a good method.

Another challenge is sensitivity and peak shape. These are related. Sometimes, I can’t see my compound very well, if at all!  A sensitivity challenge to be sure.  In other cases, I can’t see it because it is eluting on the tail of a broader peak. If only I could figure out a way to sharpen that peak up a bit and move it away from my compound.  Maybe I can try a different column chemistry? An additive? I need to pick one carefully though, making sure it does not cause ion suppression or solubility issues. AND, I’m supposed to keep the method simple for transfer to other labs.  Another big set of method evaluations on the horizon…

What I can tell you is, I have noticed this problem seems to relate to compounds containing acidic function (polar acids, peptides with acidic residues), phosphate groups (more phosphates = bigger problems), or any other functional group that likes to bind to metal. These compounds interact and adsorb to any metal surface they come in contact with and columns sure do have a lot of metal.  I’ve tried PEEK and PEEK lined steel, but high efficiency separations need higher performance than these can deliver. Titanium? It’s a metal. Same problem.

While that is great information to have, how many of us have the time to predict and plan around such issues? The complex samples and chromatographic challenges we face today do not provide us with the time for endless experimentation. And this leads to late nights in the lab.  All, so we can create methods that REDUCE RISK and PROVIDE CONFINDENCE in our results. The results everyone in my organization depends on.

When will there be a solution? Stay tuned…

Additional resources:

Blog: Addressing the Mystery of Sample Loss

Blog: In need of some Reversed Phase Polar Acid Relief

Video: Get Ready to See What You’ve Been Missing