Problem with TFA reaction and separation

[meshohel]

I prepared tripeptide Boc-Pro-Pro-phe-OBn, then I did hydrogenolysis to deprotect OBn than I ran Normal phase column and I saw quite good separation in the column with small impurities. Then I ran TFA reaction to deprotect Boc group, during the reaction I checked with TLC and it looks single spot with little more polar than the starting material. When I tried to rotavap the reaction and washed with ether and finally After TLC I found the compound becomes more polar and it has more than one spots. It moves slowly in 90% Methanol in DCM. the reaction mixture turned yellow when I stopped the reaction. Although I purified the starting material but I got more than one spots. Strange!!! Can anyone suggest me why it happened??

Did I run over reaction? I ran about 8 hours.
What are possible side reactions?
is there any way to separate tripeptides in normal phase silica gel.

Thank you.

[Don_Hilton]

A bit more detail might help. You say everything was fine until you stopped the reaction and worked it up. It may be that the important information is in how you worked up the reaction. It has been 30 years since I did some peptide synthesis, but I do remember proline giving me difficulty. From memories of my difficulties, you might see if you can find a sample of the pro-pro and pro-phe diketopiperazines and see how they behave on TLC.

On sillica jel chromatography of peptides, it may be worth looking in some of the literature to see what others have done. If you can make peptides move in well on a slillica gel TLC plate, they should move through a column. But if they do not move well on the TLC plate, they are going to take forever on the column. I have memories of going through lots of sillica jel, back years ago. But I don't remember which compunds I was running through them.