Chromatography Forum

Hello,

Would anyone be able to tell me the best way to approach seperating an amino acid from an almost identical compound. The interfering amino acid has one NH2. My compound has an OH in place of the NH2. My compound is 1 amu greater. The interfering amino acid is endogenous in my samples.

Using LC/MS/MS
NH2 column
MPA -AmmBicarb:H2O pH10
MPB ACN

thanks

If you are within about 1.5 pH units or so of the the pKa of the amino group on your AA of interest, tweaking the pH should make it move relative to the interference.

Thank you,

I have only tried pH10 and 7. Should I try testing in these increments. 8, 8.5, 9.0, 9.5?

Is there anything I could do in the extraction to remove or minimize the AA.

I am doing a protein crash with 100%ACN.

You may be able to separate the interference by use of an ion-exchange type SPE (solid phase extraction).
If it has an amino group you can protonate it with the proper pH buffer. You did not state whether the analyte will hold onto a proton or not, but if it does you may be able to have it remain uncharged but have the interference charged by proper choice of pH buffer. The interference will then retain on the ion exchange material and you can elute your analyte off.

For example, you might look into SCX, PRS and CBA SPE (Varian) other manufacturers offer similar packings.

Alp

Thanks for the explanation.

I will try SPE if changing the pH of the MP doesn't work.