Chromatography Forum

Hello,

I am running a LC-MS experiment on a LCQ advance Thermo finnigan.
I have got an adduct of 41 Da to the molecular weight of the compound I am analysing. I am not sure that it is an adduct or a co-eluting compound.
How can I know if it is a different molecule?
Thank you in advance for your help.

Fabien.

You can try to separate it chromatographically. Or you can do MS/MS (or MS^n, since you have a trap) and see if the fragmentation makes sense based on the structure of your known.

I don't recall ever seeing an adduct of 41, but it does correspond to water plus sodium. If you think it's an adduct or cluster, you could also try adjusting the source CID voltage and see if you can get the M+H instead.

Thank you for your reply.
I did not manage to separate it chromatographically (seems impossible). I believe that I formed an imine with the amino function of the molecule I am analysing.
MS/MS of the supposed imine molecule gave the fragmentation (162.1, 150.1 Da + (122.0 + 110.0 Da)) of the supposed imine molecule and the fragmentation pattern of the molecule itself (120.0 + 110 Da). I am not sure I can be sure if it is a different molecule.
I hope that it is understandable.
Thank you in advance.

Fabien

I'm not sure if I understand. What was the precursor ion, and what were the observed product ions, for each MS/MS experiment?

Based on your compound, it may be an ACN adduct although it's rarely observed in most current LC/MS systems. Have you tried to break this particular one and look at the mass spectrum?

Sorry, the precursor ion is 230Da (or +41Da).
I tried direct injection in methanol and IPA and I still have the 41Da adduct. So I do not think that it is a MeCN adduct.
Thank you.

Fabien.

The 271.1Da ion gave 162.1+ 150.1 Da + (122.0 + 110.0 Da)
The 231.0Da ion gave (120.0 + 110.0 Da)

I'm still a little confused. From your last post, it looks like 231 and 271 are your precursors. 231 is your M+H, and 271 is your mystery ion. Is that right?

Since I don't know the structure of your known, it's difficult to interpret the results. Oftentimes a solvent cluster (such as an ACN adduct) will be broken-up and yield the M+H upon MS/MS. Plus you have injected your sample in an environment free of ACN (presumably your sample was not dissolved in ACN, nor did your mobile phase contain any) and you still see the mystery ion. Right now I would lean toward this being a separate compound. If your current chromatography can't separate it, you could try a different stationary phase (e.g. phenyl in place of C18) or different organic modifier (e.g. methanol in place of ACN).

Thank you for your reply.
I was away so I could not answer before.
The compound is Bis-(5-amino-2-hydroxyphenyl)-methane dihydrochloride, I believe that one amino function had reacted with acetone, but i would like to be sure. (231 is the M+H) (271 matches the imine formed from the reaction between acetone and the compound)
We tried various methods and columns but we did not manage to get a LC separation.
Thank you.

Fabien.

Fabienco,

I have doubts that you adduct is imine. The imine formation is a reversible reaction and anlines and acetone are not very reactive towards each other. Imine formation is going through either aminal or through iminium ion-both not stable in you case. In LC were you use water in the mobile phase the equilibrium is shifted away from imine. Imine is going to be less polar and more hydrophobic due to the addition of 3 carbons. I am going to order your compound and see if we can do some experiments on Primesep columns. You can use formic acid, ammonium formate, ammonium acetate to elute week amines from the column. Combination of ion-excahnge and reverse phase will allow you to separate amine and amine without any efforts due to difference in basicity and hydrophobicity. I'll check if we have any substituted anilines in the lab and try to make "imine". I wil update if we make any progress (no obligation from you to buy a column if we demonstrate the applicability of Primesep).

Hi,

Sorry, I was not talking about an imine adduct but a chemical reaction during the synthesis of the compound. I do not know where you want to order the chemical? It will be difficult for you to find it commercially specially with the potential imine. Can you give me more information on the columns you are using?
Thank you.

Fabienco,

Another possibility is reaction of two hydroxy groups with acetone to form 1,3-dioxane type of compound, which might be more stable. In order to confirm that your impurity is imine I would try to hydrolyze it and try to see if you have acetone. Is there any way you can send me experimental description- I am curious about possibility of imine (mail@sielc.com). I spend a few years doing imine chemistry. Are you located in US?
To learn more about Primesep columns please visit our website at www.primesep.com

Thank you for your reply.
I do not think that I can send you details, I have got to ask, and it will not be before Monday.
I am not based in the US and never tried one of your products yet.
I will probably do so, it looks like that you have interesting products.
Your idea of the 1,3-dioxane is very interesting and seems to fit better with my experiments. Thank you very much.