Chromatography Forum

These are two injections, performed straight after each other of the same sample. Why does one show clear definition of the peaks and then the other seems to be masked by a high background across the entire 45min injection? This occurs regularly throughout the run where one injection is fine and then the next is 'masked'.

If there was any over run into the next injection I could see why a part of the chromatogram would be masked, but why all of it?



I am using an Aglient 1100 with a ESA coulochem II electrochemical detector for the RP analysis of catecholamines.

Thanks

When you repeat the injections of these samples? Do the samples fail on being rererun? Do the failures occur in the same place with the entire sequence being rerun? (Can we tie the problem to specific samples which may either run bacly by thems selves or may have stuff in them that interferes with the next run?)

Is this UV? If so, what is the level of background absorbance (in mAU) being "zeroed" at the start of the run? It appears you have significant background absorbance even in the "good" chromatogram. To assess this you'll likely need need to flush the detector cell with HPLC grade water to get a reasonable zero value then flush with your mobile phase to get a value relative to water.

I found this application for catecholamines:
Separation of Seven Catecholamines by HPLC
A mixture of seven catecholamines was separated using a GROM-SIL 120 Octyl-5 CP column and detected by electrochemical detection.

Catecholamines II, Grom Application Number 01027

A mixture of seven catecholamines was separated using a GROM-SIL 120 ODS-4 HE column (150 x 4mm, Part No. GSOD40512S2504) by isocratic elution with a mixture of 6.9g sodium phosphate, 37mg EDTA, 150mg sodium octane sulfonate, 60mL acetonitrile, 5mL of THF and the rest made up to 1000mL with water in around 20 minutes and detected by electrochemical detection. The catecholamines separated were noradrenaline, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylalanine, dopamine, epinine, homovanillic acid and 3-O-methyldopa.

Beside the RP column what is the difference to your methode? Please let us know, and do after the first injection a blank run, just inject mobile phase. Than we will see. On your second chromatogram it seams to be a blank run, there is no separation, no compounds.

There is a peak on the second chromatogram. The peak at ~14.5 mins just pushes above the baseline at ~ 600mV which is where that same peak reaches on the first chromatogram.

I'm not sure what the problem is though. The only thing I can suggest is trying another detector cell if you have a spare one. Maybe you have an intermittent problem with one of the electrodes that is triggered/corrected by an auto-zero, hence it occurs for the whole of a run when it occurs.