I purified my Boc-protected molecule using HPLC. The eluent is ACN/H2O/(0.05% TFA) . After I evaporated the solvent (65 oC), the product was collect as the MS result showed ~20% of the product was boc-deprotected. Why could this happen? And how to avoid this? I need your help!
Is it possible the BOC group left the molecule within the mass spectrometer during/after ionization? run an analytical HPLC on the purified material. If your HPLC still shows a pure compound, try adjusting your MS parameters, such as ESI ionization voltage (work with someone who knows your mass spectrometer on this, you won't damage the MS, but you may make it difficult to detect compounds if you change the wrong things).
It is possible, that the TFA is deprotecting some or your sample:
http://www.commonorganicchemistry.com/Rxn_Pages/Boc_Protection/Boc_Protection_TFA_Mech.htm
When you say "evaporating at 65°C" - how are you doing that? If you evaporate under vacuum then the sample will boil at a lower temperature that the heat bath / lamps / source. That said, when TFA tends to hold on to the sample so when it's dry, the sample warms up, with some TFA still present and ... deprotects.
Evaporating at a lower temperature will help also. Many users in the life sciences evaporate at a maximum of +40°C. Use a vacuum evaporator, particularly one that is suitable for use with your solvents.
Alternatively, use a different acid modifier in the HPLC buffer, formic acid or HCl perhaps? Yes, it's not as good for peak shape.
Kind regards,
Rob
TFA is a deprotecting agent for boc-protecting compounds at room temperature. Since you evaporated your sample at 65C, it is most likely that compound is deprotected. Its better to choose another reagent instead of TFA. (HCl, TMSI, ZnCl2 also boc-deprotection reagents) Qiang Peng
Rob Darrington Mehwish Iftikhar Udaya Jayasundara Jack Silver thank you all for your answers. I used TFA because it's usually used in HPLC for better seperation. Usually such a low concentration of TFA shouldn't cause Boc-deprotection. I guess I have to remove TFA before evaporation under vacuum. So is there any method to remove TFA from H2O/MeCN mixture?
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