Chromatography Forum

Hi,
we are developping an LC method for QC of pharma substance. Validation of this method is beeing performing (PhEur) but we are encounting some problems for the detection/quantification of the impuryties. Some peaks are detected in the blank run :on a LUNA CF8(2) 100x4.6-5µm A: TFA aq. 01%v/v B: ACN starting at A/B 85/15 gradient to A/B 20/80. Some people told us it could be some contamination of the A solvent (present in commercial HPLC grade water), stocked at the head of the column until %of B is enought to elute thoose compounds ?
Do you agree ? what could be the reason of thoose peaks ?

Rq: S/N for thoose peaks are about 10.

Thanks

Some people told us it could be some contamination of the A solvent (present in commercial HPLC grade water), stocked at the head of the column until %of B is enought to elute thoose compounds ?
Do you agree?

Very much so. This is one of the most frequently occurring reasons for unexpected peaks in gradient elutions.

what could be the reason of thoose peaks ?

Apart from the above mentioned possibility, you could have troubles with “carry-overâ€

Thanks for this quick answer !

do you think thoos peaks can be disregarded when i integrate chromatogram's peak for a Pharmacopea method ? i.e performing a blank run, then the sample's one and identify which peak is due to blank ?
If one of the peak is to much closer to an impurity but R>1.5, can it be ignored for the noise calculation (for the 20 times 1/2 height width bandwidth around the peak: S/N>10) ?

Thanks

Actually, I would rather find and eliminate the source of contamination, if I were you. You can never be sure that you’ll always see the same peaks and the same peak sizes (height and area). In your case the possibilities are not that numerous – just the water and the TFA. So if you’d like to take my advice then check those two.

Best Regards

The bottom line is that for some gradient analyses it is often very difficult to rid yourself of all aberrant gradient peaks in your blanks or samples. So the approach you dictate i.e. ignoring peaks in samples that are also present in blank injections is appropriate in many cases (depending on the extent of the problem).

For the best possible baseline gradient response I have found it is best to prepare fresh your respective mobile phase on the day of analysis. However this will vary enormously from method to method but it might be worth trying for your application.

PS. For a comprehensive text on the causes of these problem peaks, I urge you to get hold of a copy of Dolan and Snyder's High Performance Gradient Elution - http://www.amazon.co.uk/High-performanc ... 012&sr=1-2