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When i work in HPLC i usually find a peak with aS lying down shape on the base line like the one in the image, can anyone explain why does this happens and what can i infer from it.
Thanks.
Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.
D.Roesch wrote:
Hello, thanks for the feed back. here is the image i tried to upload before:
and for the general information:
I am a recent graduate from college and working with HPLC, so in general ,yes I am new at this. Here are all of the details from the method I am working on , I don’t have an image of the chromatogram right now since the laboratory is closed during this week so i´m working on my own so I can get everything ready when I get back.
• Column: Lichrosphere RP Select B 250 mm x 4 mm (5µ)
• HPLC-DAD Chromaster Hitachi
• Wavelength: 270 nm, plot speed 400(2.5 Hz)
• Flow: 1 mL/min
• Average preassure: 125 BAR
• Injection Volume: 15 µL
• Run time: 45 minutes
• Objective sample: Niacinamide, Pyridoxine HCl, Thiamine Mononitrate and base riboflavin in Soft jelly capsules
• Mobile Phase: 0.008M sodium hexanesulphonate with TEA 0.1% adjusted to pH 2.5 with phosphoric acid and Acetonitrile (91: 9)
• Sample Solvent: Sodium Hydroxide 0.1 N pH 8 with acetonitrile (90:10)
• Retention times:
o Niacinamide: 4.1 minutes
o Pyridoxine: 6.2 minutes
o Thiamine: 15 minutes
o Riboflavin: 20 minutes
I am trying to quantify all of the four vitamins in one run, and I manage to get a decent resolution and K prime on all of them.
When i tested for recovery i didn´t get the results i was hoping for so i had to change the solvent for sample extraction from water to the one with NaOH and ACN, with this change i detected a peak like the one on the picture that is like a lying down S shape peak.
Usually for me (please correct me if i am wrong) this kind of peak appears because of the change on detection or diffraction index that occurs when you injec something different than the mobile phase, i have seen this kind of peaks at the beginning of the chromatogram and since i thought they were normal didn’t called my attention, but I saw that peak again after the pyridoxine peak,i think it is because of the sample solvent but since it shows a bit later on the run not sure if its affecting the pyridoxine detection specially now since i'm getting interference from the placebo and sample solvent when i didnt before that change, is there any posibility this has something to do with it?
Thanks for any advice.
Cheers.
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