Hi Mehraz,
Thank you for the figure. I will try to explain what you see.
The negative jump is due to the lower salt concentration in the acetate buffer. Variations in the jump can happen due to weighing and adjusting the buffer. Next, we are looking for the pre-concentration which seems to work slightly. In general, we are looking for a slope between 10 – 50 RU/s but yours is much lower. Not sure why and it puzzles me.
One options is that such a short peptide lacks sufficient charges (despite the pI calculation) to have sufficient pre-concentration effect on a carboxylated sensor surface.
Diluting a ‘high’-pH solution in a ‘low’-pH solution has definitely some effect but is dependent on both solution strengths and dilution factor. You can try some (larger volume) dummy dilutions and check with pH-paper what the effect is. I can remember diluting a ligand in pH 3.0 just to reach pH 4.5 because of the buffer.