Hi,
I am wondering if anyone could help me out of the following problem.

I am running a peptide (MW 1500 Da) and a protein (MW 66kDa)on a C8, 100x4.6mm, 3u, 100A column. I just use this column to estimate the retention of the peptide and protein on a c8 column. Gradient condition: 0 min 85% 0.1% TFA/H2O; 15% TFA/ACN; 16 min 100% TFA/ACN. The RT of peptide is around 6 min, and RT of protein is about 9.5 min.

No matter the injection is blank (PBS buffer) or peptide/PBS buffer (without protein), it always gives a peak around 9.5 min, much smaller than the protein peak, though.

I have a feeling that the ghost peak is given by the protein blocked in the column due to the relatively small pore size of the column. actually, I have order a 300 A column, but it has not arrived yet. But if it's true, then why does not it come out anywhere after 9.5 min?

Any comment? Thanks in advance for any input!

Noname,
What is the shape of the peak, narrow or broad?

Try injecting the mobile phase, start, finish, and a guess at the eluting concentration. Also try the eluting guess with degased and vigorously shaken (to get air in the fluid) or a buffer degassed and gassed. Sometimes the gas causes a peak like you describe if broad.
Jim

Inject blanks multiple times. If the blanks decline, it is junk either left on the column or in the injector. If it stays the same, it is not the protein nor the peptide.