Chromatography Forum

Hi all

I have a Perkinelmer series 200 HPLC with autosampler and FLD detector.
My column is a shodex KW-803 SEC column.
Flow rate of 1 ml/min with an isocratic method of sodium citrate buffer.

When i inject a our protein sample, main protein peak detect at about 9 min and unknown peak detect at about 22 min.
So i try to inject same concentration and different volume 10, 20, 40, 80 ul. Then unknown peak increases relatively sample injection volume.
And i try to inject different concentration and same volume. Then unknown peak dose not increase.

And when i inject mobile phase, it dose not detect any peak.

I tried to find the source of the unknown peak but in vain.

Where else could i have missed or what else can i try?

Your suggestions would be a great help.

In what do you dissolve the sample?

I dissolved the sample with mobile phase.

Your description is consistent with a "system peak" resulting from the equilibrium upset caused by injection. These are usually minimized when the sample is dissolved in the mobile phase, but can still sometimes occur as a consequence of pressure changes. If it''s not interfering with anything, I wouldn't worry about it.

A pressure change peak that increases with increasing inj. volume, and that does not appear when injecting only mobile phase? I think Bionano is not telling us something about his method.