Chromatography Forum

I have been working with a method for a few years now and recently i have been getting a ghost peak appearing in my sample chromatograms.it shows at the same retention time (~17mins) everytime but not in every sample. It doesnt show in my diluent and mobile phase ever, but when it appears it shows up in my placebo blank as well as my samples and standards(rarely). The method is poor but to date has at least been consistent. The method isocratic, with a m.p. of 60:40 Buffer(0.05M K2HPO4): ACN, 210nm, @1.5ml/min, using a kromasil C18 4.6x150mm silica column. The product is an anti-biotic. Lately also we have been using HPMC solutions on these columns for trials and are suspsicious that this might be causing the peaks even though the blanks are clean. we have changed the systems, columns, filters and reagents without consistent success. we have also seen strange results with different filters and on one occasion a sample filtered with a millex 0.45um filter contained the peak on every system and column it was tried on consistently however when the same sample was refiltered with millex no peak appeared...??? we have run a sample on our PDA and the peak is 99.4% pure and shows a very different specrum to the active and its impurities....we are starting to pull our hair out here, so if any one has experienced anything such as this before your insight would be greatly appreciated.
thanks in advance
Aidan

First, your PDA data shows that the problem is contamination, not carryover. There is nothing wrong with your column, mobile phase, or HPLC hardware. The problem is most likely dirty glassware, pipets, vials, filters, syringes, etc. used in sample prep. Another possibility is your autosampler is is dirty; how do you wash the needle?

We have considered contamination, as the wavelength is @ 210nm it would detect any sort of contamination. it just seems very difficult to narrow it down as it is so random....as you said it is most likely either the vial, syringe or filter as these were the only variables in contact with the contaminated sample i mentioned earlier. We generally use methanol:DH2O or ACN:DH2O (80:20) as our needle wash.
Thanks for your time
Kind Regards
Aidan

Dear Aidan:

Your problem might have come from the syringe filter (in sample prep). My story: sometime ago, we did method development using one brand of syringe filter, but when we started the validation, we ran out of it, so we tried to switch to another brand (I believe it’s a Nylon, 0.45um, 13cm). When we ran sample/standard, we observed two extraneous small peaks! We decided to wait until we got more of the original filter to resume validation.

To avoid possible contamination, you may try to use dedicated LC systems for R/D trials. I have also ran samples with HPMC, but we did not observe ghost peak afterward. We use dedicated columns and tubings (the section that we could switch easily) for this thing.

Alfred.

I would like to agree it may be the filter. YOu may design a systematic test: e.g. 1) different section of the filtrate; 2) different filters and the same sample; 3) the same filtrate different column; 4) the same filtrate different LC; 5) centrifuge instead of filtering; 6) different sample preparation, esepcially different ratio of organic; 7) look into anything that may be very nonpolar.
SO how did you prepare the sample, what is the diluent ? concentration? do you know any that may not very soluble in your Diluent?
Good luck

What makes me wonder here is how your PDA knows this unknown is 99.4% pure when you don´t even know what it is?

Maybe is because it is called Alladin and has a magic lamp!

(Sorry, it just came to my mind and I couldn't resist to write this joke - no offense to anyone, just a humour break!)

he is perhaps talking about the "Peak-Purity" ( Peak Homogeneity ) by DAD.

JM

Dad knows best? (Another joke, it´s hot-humid here in Germany, a joke helps)

No joke here: sample vials as well as septa can cause strange peaks. Most vial manufacturers do not know what you are talking about, but you can purchase certified clean vials from reputable suppliers like Waters.