How do you detect the base peak and the fragment peaks of a purified single molecule? Likewise how do you detect the
In case you have a known purified molecule, you know its molar mass. The mass of base peak is in always M+1 (if you acquire data in positive mode). You have to record your data in scan mode, for example from 20 - 1000 amu and then just open the file. You will see complete spectra. In case the base peak is missing, you have to adjust some parameters, which are instrument-dependant. Which instrument do you use?
The answer to this isn't going to be simple. There is a lot written about this, and this is just a "quick" overview.
Spela assumed you are running ESI or APCI, which give an [M+H]+ peak if run in positive mode. If in negative mode, they will give an [M-H]- peak, if the molecule is able to accept or lose a proton. It is possible you are using EI ionization which doesn't add or remove a proton.
From here, I'll assume a soft ionization such as ESI or APCI becaise they are most common on LC-MS. If you know the molecular weight of your molecule, look for the following:
· Adducts such as [M+H +Solvent]+ where the solvent can be methanol or acetonitrile (from your mobile phase)
· Another type of adduct are metals such as sodium or potassium from glassware which appear as [M+Na]+ or [M+K]+. I prefer to call these "charge carriers" since they act like the proton by conferring the charge to the ion. However, the common usage for these is "adduct". They can also be from your reaction (sodium borohydride)
· Loss of functional groups. In ESI+, these could be NH2 or OH, which leave as HNH2 (ammonia) or HOH (water). Looks for groups that could form a stable molecule if a H were added to it. Look around heteroatoms. For ESI-, look for loss of carboxyl group (CO2).
· Some compounds may not ionize given the ion source used. For example, steroids generally don't show with ESI, but appear with APCI. The example in this link also shows loss of water: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Mass-directed%20Purification%20of%20Steroids%20with%20APCI%20and%20CombiFlash%20PurIon%20App%20Note.pdf
· The ionization conditions and pH may also play a role, as does sample loading (too much sample leads to ion suppression and clusters of ions).
· Peptides and some other molecules may also carry multiple charges, so you may see your peptide at [M+2]/2 daltons, and even higher multiples.
Hi dear
Simply you can use xcaliper software for your interpretation of Base peak and it's fragments
@ Prof. Jack,
What if it is an unknown compound? how do you arrive at the molecular ion peak if using ESI.
Thank you
@Ayorinde
You need to use supplementary information, in this case. If it is a synthesized compound, there are a few reaction pathways to the product and side reactions, so those can be used to confirm the mass. If a natural product, a literature search will suggest compounds from that species or biology. Proton and carbon NMR will also suggest information about the number of H and C. Some of the halogens also provide unique MS due to their isotopic abundance. There is Also information from the H and C isotope peaks in the MS that suggest the number of H and C.
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