Ghost Peaks/ Artifact Peaks Removed by MP Filtration

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

6 posts Page 1 of 1
Curious if anyone has faced a similar problem.

I believe that I have a ghost peak in an assay / related substances method from contamination in my aqueous mobile phase. Normally I'd just redevelop the method, but we're committed to this. What is truly odd is that this ghost peak appears regardless of the mobile phase vendor, grade, instrumentation used, meticulousness of glassware washing, eluent additive vendor, etc.

The reasoning behind my supposition that the ghost peak is from mobile phase A is that:
1.) I can increase the size of the ghost peak in a proportional manner with the length of equilibration time at initial conditions (at 5%B)
2.) If (using a binary pump) a C18 column (Small, wide bore and >5um particle size so as not to increase pressure unnecessarily) is placed after pump A and before the mixing T the ghost peak disappears.
3.) If Mobile Phase A is filtered through a divinyl benzene or polystyrene solid phase extraction cartridge the ghost peak area decreases about 90%

Method Fundamentals:
1.) Gradient Method: MPA is 0.1%FA in Water; MPB is 0.1% FA in ACN
2.) Stationary Phase: Phenyl-Hexyl on positively charged hybrid silica particles, sub 2 micron particle size
3.) Ghost peak elutes at about 43% ACN (co-eluting with a specified impurity)

ID by LC/MS?
1.) Ghost Peak analyte does not seem to ionize, work ongoing to concentrate on solid phase extraction cartridge and then analyze.

ID by Spectra?
1.) Looks like a phthalate, but if it was, it should easily ionize in LC/MS

One last piece of data:
In the mobile phase A filtered through the divinyl benzene or polystyrene solid phase extraction cartridge, on day one the ghost peak is not detectable, but by 48 hours the peak has grown to a detectable level.
I was thinking, do you have any contact with gloves when preparing the mobile phases? We had the issue of artifact peaks leaching from the gloves when preparing mobile phases here at my site.
However, your last bit of info doesn't seem to match that case. Might be something to consider though.
While it's tempting (and scientificslly fulfilling) to pursue identifying the ghost, it may be that the cost-effective solution is simply to prep and filter the A mobile phase daily.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Hi Tom,
Thanks for your suggestion. I am also leaning in this direction; being agnostic of the ID of the contaminant behind the ghost peak and just applying a daily preparation requirement.

Curious, has anyone seen this increase in ghost peak area over time? For example, at 48hours the ghost peak appears in filtered mobile phases that it was not present in at T0. Just completed an evaluation after allowing the mobile phases to stand for five days. The Ghost peak area has increased from the 48 hour results. Anyhow, experiments performed have pretty much ruled out that the source is either the sinkers or system tubing.

What I am getting at is, has anyone ran into air contamination in their laboratories?
What I am getting at is, has anyone ran into air contamination in their laboratories?
Unless you are in a "Level 3" containment facility, it's almost impossible to avoid. Fortunately, most of the contamination floating around doesn't affect our methods. That said, four decades ago I ran into a situation where a customer had to temporarily relocate to a university lab several miles from the manufacturing site in order to analyze meat samples for residues of an antibiotic added to animal feed at the time.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thank you Tom. Always a pleasure to learn and hear anecdotes from an expert. Thanks for your replies :)
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