Chromatography Forum

Hi,

I have been trying to develop a LC-MS method for anionic compounds on a hypercarb column.

I tried to set up the optimal conditions on HPLC with UV detection before moving to LC-MS. I have found that, in order to insure reproducible RTs across runs, I have to use as buffer B a mixture 75:25 of 50mM NH4HCO3 pH 9.3:AcCN.

The actual Ammonium bicarbonate concentration in this mix is 37.5 mM, but I am still afraid of shifting the method to LC-MS.

Did anybody ever worked at such concentration of buffer on LC-MS? Do you know which precautions and/or post column tricks that should be used with these buffer concentrations?

Thank you very much

Have a great day

Just try it... The main thing is to solve analytical problem.

I'm successfully developed several HILIC-HPLC-ESI-MS/MS methods with 50mM NH4HCO3aq. : MeCN 1:1 %v. for anionic organic compounds.

Suppression of ionization in ESI often increases with increase of concentration of salt in eluent.

Optimization of LC-MS parameters is more reasonable, because the limitation of the eluent compositions is very different for UV and API MS (ESI, APCI, APPI etc.).

Best wishes, Alex.

I've never seen optimal signal levels anywhere above 5-10 mM of anything. so I always try to stay on the low side. I presume you have tried weaker buffer levels and you're not getting the reproducibility you desire?

Sure, try it, but if the signal is lousy, try again with a weaker buffer and see if at least the signal is good. Consider that with better detection limits, you're going to be injecting less of the compounds on your column than you did with UV method development, which means that a weaker buffer may suffice to stabilize the RTs as long as the concentrations are not too high. You can always set an upper limit on how much you are willing to inject on the column, and if a sample just has too much, then you try it again with dilution.

Also keep in mind that aside from the chemistry you require to get the separation you want - negative ESI-MS *does* work with acidic or neutral additives as well, sometimes even better than with basic additives.

But yeah, sometimes when a method just doesn't translate over, one has to try different column chemistry or different separation tricks.

Typically I find it easier to infuse the compounds of interest in a selection of buffer matrix's before I start any chromatography. What I find is there is usually a range of aq - solvent compositions which give a decent signal at the desired mass. Outside of this range the signal decreases often significantly. Addition of differing types and concentrations of buffers also effect the strength of the signal you see. Armed with this information you can predict what is going to happen to your signal if you elute your compound under different conditions, which can dictate the choice of column you use.
In the transfer of chromatography to the LCMS sometimes you find that the compound elutes in a mobile phase composition which reduces the signal to zero, hence you can be waiting for your forever without seeing it, frustrating! The simplest way is to plan out what is entering the source at any one time and how this will effect you signal, now I have never used 50 mM bicarbonate before so can't tell one way or the other whether it is possible or not. However, 30 minutes with an infusion pump, and 4 - 5 different aq - solvent mixtures + compounds will tell you everything you need to know about what will happen in the source when you run your chromatography.

best regards

RJH

Sigh, on a tangent, I miss having infusion pumps available... my current lab has an Agilent 6460 MS with no integrated syringe pump, it's a brand-new facility, and I have yet to successfully make a case that having a syringe pump around would save loads of time in method development. So there are some optimizations I would prefer to be doing more frequently (modifier concentration, source parameters etc) that I've been skipping unless I'm having trouble reaching the target detection limit.