Chromatography Forum

Dear colleagues,
We are doing pharmacutical analysis with HPLC-UV (agilent 1100). Usualy analysis series consists 30 to 80 samples. My problem is: analysis results increasing during the series. Results growth is around 2 % per 20 samples. The issue occures in all large series (samples 30+) runs with all type of analytes.. The run is controlled with pooled sample solution. Control sample is placed in after every 20 samples. Analytes are psychoactive and anticonvulsant drugs. Eluent solution is ACN ~25% - 38 mM sodium phosphate buffer (or water). Column(s): Reverse phase.

I hope someone understand my problem (and my english ) and have solution for this matter

With best regards

-Jussi

A simple root cause could be that the ACN evaporates from your vial over the time.
Most complex would be that you get an enrichment on column or within the injection system.
Please give more details about your gradient, please post a chromatogram, injection volume?

Hello

Few questions:
-What quantitation method you're using? External or Internal std?
-do you run recalibration in the middle of sequence?
-are you using calibration curve or just one standard with concentration close to your real sample?
-do you fill vials to the top or halfway? (ACN can evaporate and concentration will be higher)
-please attach chromatograms (overlaid would be the best )

Regards

Tomasz Kubowicz

Hello

Maybe the substances act psychoactive to your detector and it shows wrong values. Or it acts anticonvulsant on the flow cell, it relaxes slightly, prolonging the light path which will result in increased values as well...
No, just kidding

Do you inject your QC sample from the same vial every time? Then surely the concentration will increase because the solvent evaporates through the pierced septum. Be sure to use individual vials for each injection. (Duplicate injection from one vial is OK if your run time is short).

Jörg

Thanks for quick answers!


Specs of the method:
After SPE and evaporation, residue is dissolved in 10 % ACN/water solution. 200 µl insert are used inside vials, it is filled halfway (100µl). Injection volume 10µl. Run time 5 min. ISTD for Quantification and with calibration curve. Calibration at the begining of the batch (not in the middle). Equipment is maintained properly.

I think it is not caused By evaporation of ACN. Enrichment sounds more probable. Or maybe something UV-lamp (saturation)?

Hello

My first issue is integration...I think it can be a reason. In my opinion all peaks are integrated in very bad way without any integration parameters adjustment.

Regards

Tomasz Kubowicz

Do you also see a similar trend in retention time. If yes, then you pumping pressure is falling slowly over time. Try to see if you have a micro leak some where or if your degassed is working properly.

If you don't then this is really strange !