Chromatography Forum

I have been trying to run HPLC with electrochemical detection for dopamine and serotonin in microdialysates but have been getting a ghost peak that interferes with dopamine. I have been unable to get rid of this peak and still can't figure out where it's coming from. Details about the protocol and steps I've taken to get rid of the peak are listed below. Any help in getting rid of the ghost peak will be much appreciated!

System:
Shimadzu LC 20 AD
Rheodyne manual injector (have tried steel 7125 and PEEK 9125)
Phenomenex Kinetex C18 column (100x2.1)
BAS radial flow cell for electrochemical detection
Also have guard column and pulse damper

Protocol:
Mobile phase is 150 mM NaH2PO4, 4.8 mM Citric acid, 3mM SDS, 50 uM EDTA, 11 % MeOH, 17% ACN, pH 5.6 with NaOH
Electrode set at 550 mV
Flow rate is 0.2 mL/min

Information about Ghost Peak:
Shows up regardless of what I inject (mobile phase straight from reservoir, aCSF, HPLC grade water, etc.)

Does not appear when flipping injector to load and back to inject

Have tried 4 injectors and sample loops, rotor seals made of vespel or tefzel, many different hamilton syringes, several columns, and several guard columns but peak is still there

Peak size changes with injection volume (so larger at 20 uL injection vs. 5 uL)

Peak size changes with electrode potential suggesting it is an electroactive compound

Wow! You've already covered the "usual suspects" thoroughly. My first thought was "system peaks" (basically equilibrium upset due to injection), but those usually go away when you inject mobile phase.

The only other suggestion I can make (and it's a long shot!) is to try tweaking the mobile phase strength (change the aqueous/organic ratio) a bit and see what happens to the retention of your ghost. With any luck, you may be able to tweak the selectivity so that at least it no longer interferes.

Is it right on the dopamine? If it is, it is probably carryover, not a ghost.

I don't think that it's dopamine because it produces a response from the detector at potentials that are too low for dopamine. I'm wondering if it could be a contaminant in the mobile phase that is somehow depositing in the injector. I recently switched to a different brand of acetonitrile and the ghost peak has gotten much smaller (maybe 1/3 of its original size).

If the ACN really has something to do with it, and since you say it varies with injection amount, I would think it is a system peak. If it is, the retention of your analyte is too low.

Thanks for the feedback! Would you mind explaining what you mean by the retention of the analyte being too low? Do I need to use a "weaker" mobile phase to fix this (maybe using less solvent)?

System peaks in isocratic methods usually appear near tm (~no retention). If this interferes with your analyte then that analyte also has about no retention. There is a number of ways to try to overcome this, if you ask questions about that it indicates to me that you should do some reading before continuing.

hello, i think it's a peak from system clean your system hplc

rince your systeme with water, nitric acid 10% for 25 minute, water for 2

hours and you varie injector position.

on your mobile phase let methanol only.

if you have chromatogramme let show.

Sorry for my english.

Shows up regardless of what I inject (mobile phase straight from reservoir, aCSF, HPLC grade water, etc.)

Does not appear when flipping injector to load and back to inject

Peak size changes with injection volume (so larger at 20 uL injection vs. 5 uL)

Have you ruled out glassware such as sample vials, pipets, etc. Did you use a vial or pipet to get the sample of the mobile phase you injected? Since switching the injection valve didn't show the same problem (i.e. mobile phase injection) it sounds like it is being introduced during a transfer/prep step.