Strange Peaks on HPLC

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

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Hello everyone, i come with a basic question.

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.

Image

Image
Hi,

Welcome to the forum.

I can't click the image, but I guess you're referring to the "solvent front" peak. It's a disturbance from the injection that gives a signal and basically marks your column dead time = the time it takes for unretained material to reach the detector. See this link.
There is no image and also no chromatography data of any kind. Please tell us about your analysis and HPLC method details. Is this the first time you have used HPLC? Are you a student? You might want to ask your teachers for help as if they can see what you are doing, the question will be easy for them to answer.

If your "blip" is just the void volume peak, then this article will provide you with a simple explanation as well as why it is one of the very first things we measure and describe when we first set up any HPLC method. https://hplctips.blogspot.com/2011/05/d ... -time.html
Hello, thanks for the feed back, all of the information on the post below, cliked submit before i should had. sorry!
Hello, thanks for the feed back. here is the image i tried to upload before:
Image

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.
hi D.Roesch ,
firstly is this chromatogram you shows is your real chromatogram? i didnt get it ?
secondly , yes , as you said , this shape of noise mostly happen due to change in diffraction index , usually it happens with every injection at t0, from work experience , the water part cause the negative part of the S shape , and the organic solvent part cause the positive part of it ( in your case Acetonitrile 9% of the 15 uL injected , from your column dimentions and flow rate i expect that the dead time may be about 2.5 to 2.8 min according to your system , in this diagram it is so early which i dont understand , this issue may appear during the run adjacent to sample peaks for the same reason , this issue appears and differs with wavelength differeing , so try to change wavelength , i had worked on the water soluble vitamines , i used wavelength 210 nm and i didnt have a such problem , so use you DAD to scan your sample at wide range of wavelengths and choose the most suitable one ,this for your strange peak ,
about elution of all components in one run with suitable retention factor, it is ion pair method, elution sequence represent the degree of basicity, interaction degree with the ion pair agent , and the last one riboflavin has the high molecular wight significant effect , so it has the highest retention , by trial and error control the ion pairing type, concentration column packing type , and organic solvent concentration ,
and about acetonitril concentration in the mobile phase , note that the small change in it ration affects significantly the peak with the highest retention factor the most, good luck with your issue
Thanks Hossam Kamala for all the valuable information!

To answer your question, that image is not from my chromatogram i just wanted to show the peak shape i was getting since i cant get the chromatogram right now.


and again thanks for information!

Cheers
not at all ,

https://drive.google.com/open?id=1WrVyT ... UCXvfhQned
https://drive.google.com/open?id=1cTa3q ... KhSMboR31j

i have found this paragraph , it is related to your issue , it is from text book called, THE HPLC SOLVENT GUIDE ,
Thank you very much, it was quite useful, wich book were you reading?

Cheers!
D.Roesch wrote:
Hello, thanks for the feed back. here is the image i tried to upload before:
Image

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.


Is this peak occurring in every injection? I would suggest also recording your system pressure. That looks to me like a bubble may have gone through the system and through the flow cell. If you see a small pressure spike as well at the same time you see this phenomena in the chromatogram that could reaffirm an air bubble of some sort. If your baseline is stable just running mobile phase and you don't see this, then it could point to your syringe or something occurring during the injection process that is introducing an air bubble.
Hi D.Roesch,

The text H. Kamal refers to is:

https://www.wiley.com/en-us/The+HPLC+So ... 0471411383

The HPLC Solvent Guide, 2nd Edition by Paul C. Sadek, ISBN: 978-0-471-41138-3, Feb 2002, 664 pages.

I recommend it as well, good to read!
MattM
Hello,

This looks like a solvent front. This is normal.
Kind regards,
Ade Kujore
Marketing
Cecil Instruments
Cambridge
United Kingdom

email:- ade.kujore@cecilinstruments.com
telephone:- +44 (0) 1223 420821
web site:- www.cecilinstruments.com
Registered Number 909536
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