I don't have a lot of experience with HPLC, but recently I have become invovled in a project where my job is to separate certain components in a reaction mixture.
The mixture should contain QSY7 quencher, Gly-Phe di-peptide and a QSY7 conjugated Gly-Phe dipeptide plus a few insignificant by-products.

We expected to see 3 significant Peaks, but instead we see a very large and broadened peak and a nicer looking peak after this.

In the top chromatogram in the Picture a non-reacted mix of QSY7 and Gly-Phe di-peptide has been analyzed.
in the bottom chromatogram, our sample mixture has been analyzed


We would suspect the QSY7 conjugated di-peptide would elute either in between the peaks seen in the top chromatogram, or maybe after the di-peptide peak. We now suspect that the QSY7 and the QSY7 conjugated di-peptide are imbeded in the large peak.
I have changed some of the different parameters of the method in order to try an separate this large peak, like changing the column temperature and the solvent temperature, the gradient, and the amount of acetonitrile, flow-rate but I keep seeing this almost identical chromatogram.
The system is an Agilent 1100 series, and the column is the Agilent zorbax eclipse xdb-c18.

In this particular run the paramters where:
Flow rate: 1 ml/min
Gradient: 0-3% Acetonitrile (100-97% Water)
Column temp: 55 C
Water temp: 55 C
injection vol: 5 ul
run time: 6 min

I did notice the retention time on the Gly-Phe dipeptide peak was not completely the same in this run, but it has been in the runs before this.

I have not been able to find a description of a peak like this anywhere so far.
So I want to know if anyone has seen something like this before? And what the cause could be?
And if you have any ideas or advice on how to get this peak separated, if it is two Peaks in one, as we currently suspect?

thank you