Ghost peaks of API

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

5 posts Page 1 of 1
Dear colleagues,
Has anyone experienced significant API peak showing up in blanks?
Neat methanol and MilliQ water give same area of API peak.
This has not been solved by
1) needle , buffer loop wash
2) replacing vial - even 1st injection in fresh vial gives the peak
3)washing of column
4) replacing the column
5) not actually injecting from vial, but recording chromatogram of mobile phase. Peak still there.
Sounds like you are running a gradient, right?

Also sounds like you're not actually seeing an API peak at all.

Is this a new-to-you assay, or one with which you've had success in the past?
Yes, this is a gradient method!
Method has been used successfully before by other analysts on the same system.

I didn't seem to have any problems with it for around 3 weeks of running validation samples, the problem with blanks has just occurred very recently.
Any help would be much appreciated.

Also, the retention time matches one of the APIs very precisely.
Do you see that peak also if you perform a run without injecting anything? Does the peak increase if you increase the reequillibration time at the end of the gradient?
If the answers are yes, then you're probably looking at a gradient ghost peak caused by impurities in your mobile phase which, just by chance, elutes at the same retention time as your analyte.

If the answer is no, there's probably contamination somewehere in the system. First culprit I'd look at is the autosampler rotor seal. If it's worn and started to build up additional grooves where the analyte sticks, you may see all sorts of carryover effects.

That said, we once had a case like this where nothing seemded to help. In the end, washing and passivating the whole system with nitric acid did the job.
HPLCaddict wrote:
Do you see that peak also if you perform a run without injecting anything?


I think original poster already did this (below) but this wasn't that clear to me either.
laime. wrote:
5) not actually injecting from vial, but recording chromatogram of mobile phase. Peak still there.


This didn't make clear whether the autosampler valve positions were involved or not. We had a couple of instances where the autosampler valve rotor seal needed to be replaced due to contamination.
5 posts Page 1 of 1

Who is online

In total there are 19 users online :: 0 registered, 0 hidden and 19 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 19 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry