Chromatography Forum

Dear all,

I have tried to identify some impurities from the stability test of Piv-glucose-Br (glucose with Br and four OPiv substituted), for all the impurities and this compound, in their mass spectra, I could see two prominent peaks correponding to [M+Na]+ and [M+2Na]+. I also searched some literatures, in one literature, these guys also find these two prominent peaks for the glucose using different LC-MS (Agilent MSD, but ours are Thero LTQ). Obviously, the dimers are not formed because of the high concentration since they are all just some impurities in my sample.

Could anybody tell me that why in the sugar compounds, the sodium dimer ions are so easily formed? Also, why the [M+Na]+ is the prominent peak?

Thank you so much!

I can't help a lot, but I'll try:
(1) Why prominent sodium adducts? Answer: some compounds just have a very high affinity for sodium, and will form a sodium adduct in the presence of even the most minute concentrations of sodium. Others prefer hydrogen or ammonium. You can try to get rid of sodium adducts by adding lots of acid, or an ammonium buffer, but if a compound is really determined to be a sodium adduct, it probably will be. Many people maintain that sodium adducts are a disaster because they don't fragment easily. Fortunately on Thermo ion-traps they frequently fragment quite well. They often fragment differently to hydrogen adducts. This is very much the case for sugars, and there's something about it in one of the chapters of the Encyclopedia of Mass Spectrometry; I don't have it to hand, but vaguely remember that sodium can sit between the sugars spanning two sugar monomers, or something. The other thing to note is that if you have a compound that has a high-ish affinity for sodium, but is actually borderline, you might get hydrogen adducts if you make up aqueous buffers fresh, and sodium adducts the next day after the buffer has sat in a glass bottle, gradually leaching sodium, overnight. It is also partly instrument-dependent but I have no idea why.

(2) Why dimers? I assume you meant [2M+Na] rather than the doubly-charged [M+2Na]? Thermo instruments have a very low-energy ionisation source, which means they work well with fragile molecules, but also unfortunately means they easily form cluster ions of the sort [nM+Na], and you may even find clusters with multiple sodiums. You can distinguish them from real molecules because they're hard to trap (need wider isolation widths) and they frequently fragment to the monomer at very low collision energy (15-20% in Thermo-world). They're a feature of electrospray and you shouldn't worry unduly that they happen. Incidentally, they are of some physical interest too. They do, to some extent, reflect a molecule's affinity for itself. You might get rid of them by increasing the energies of early stages of the instrument (for example a bit of excessive tube-lens voltage in a Thermo instrument) but I've never tried, and don't know much about this.

Dear all,

I have tried to identify some impurities from the stability test of Piv-glucose-Br (glucose with Br and four OPiv substituted), for all the impurities and this compound, in their mass spectra, I could see two prominent peaks correponding to [M+Na]+ and [M+2Na]+. I also searched some literatures, in one literature, these guys also find these two prominent peaks for the glucose using different LC-MS (Agilent MSD, but ours are Thero LTQ). Obviously, the dimers are not formed because of the high concentration since they are all just some impurities in my sample.

Could anybody tell me that why in the sugar compounds, the sodium dimer ions are so easily formed? Also, why the [M+Na]+ is the prominent peak?

Thank you so much!

Just to be clear, an impurity means that your compound contains something that is not supposed to be there. If the adducts and/or dimers you are seeing are only formed in the ion source, then they are not actually impurities - just part of a diverse group of ions that is formed when your compound is run through the LC/MS.

To be sure, if you're actually injecting on an LC column and you see the sodium adducts come out *at a different retention time* than your target compound, now it's something you can call an impurity. But if you just see these signals coming out at the same time as your compound after LC separation, it's just a matter of which ions are formed in-source.

What is your mobile phase? Definitely try having some ammonium acetate or ammonium formate in there. If you see [M+NH4]+ or [M+H] start to appear at the expense of the sodium adduct, then the sodium was probably just an in-source effect.

Even when sodium hasn't been added to the mobile phase, trace amounts (as well as potassium) leach out from glass and other contaminant sources, and can have a very high affinity for certain molecules during ionization. Since these are created as a result of *uncontrolled* sodium levels, you're best to use a dilute ammonium buffer to flood the mobile phase with desired ions in order to get molecular (or ammoniated) ions that come about as a result of controlled conditions.