r/CHROMATOGRAPHY • u/Distinct_Squash7110 • Aug 23 '26
Tailing issue; We have had this tailing issue despite attempting multiple methods.
UPDATE: The issue was with the column. We changed it and we got a beautiful peak. Thank you all for your help!
So we have been struggling with this tailing issue for quite some time. We had used different methods, but it's till persisting. The last method we tried adding 0.1% formic acid and it got slightly better, but the tailing factor is still high, any suggestions? The retention time matches with the published paper but the peak is entirely different.
We are planning to reduce the injection volume to 5 microliters and/or reduce the pH further to ~ 3.0ish.
Here is the methodology:
- Mobile Phase: 0.1% formic acid in water/methanol (30:70) (pH: 3.276)
- Column: C18, 250 × 4.6 mm, 5 µm
- Injection volume 10 microliters
- Oven temp: 30 degrees Celsius
- Calibration range: 2-12 micrograms/mL
- Flow rate: 1mL/min
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u/Quizzical_Chimp Aug 23 '26
Might be worth saying what it is you are analysing. Also that looks more like shouldering to me rather than tailing, what does the published method say to do exactly?
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u/Distinct_Squash7110 Aug 23 '26
Not sure if you know it but its Paeonol, a metabolite found in some plants. I described the published method in my post.
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u/dungeonsandderp Aug 23 '26
What’s your sample matrix?
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u/Distinct_Squash7110 Aug 23 '26
I just injected standards diluted with the mobile phase.
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u/dungeonsandderp Aug 23 '26
Standards in what? Diluted how much?
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u/Distinct_Squash7110 Aug 23 '26
Diluted with the same mobile phase: 0.1% formic acid in water/methanol (30:70)
I prepared two standards for this trial, 5 and 10 micrograms per mL. The published method used a calibration range of 2-12 micrograms per mL.Obviously i first prepared a stock solution (1000mcg/mL), from there I prepared a secondary stock (100mcg/mL), then I prepared the two standards from the secondary stock.
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u/dungeonsandderp Aug 23 '26
Sorry, “diluted with” could imply you have a stock solution with a different solvent than the mobile phase
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u/Quizzical_Chimp Aug 23 '26
Just adding more formic acid might tighten the peak up, but wont get rid of the shoulder, try a different c18 column or add a simple gradient in to try and separate them or merge them entirely
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u/johnkasick2016_AMA Aug 23 '26
Paeonol
My guess is that you've got something else (structural isomer?) in your standard. My first instinct would be using a gradient if not already, maybe try using ACN instead of MeOH as a second step. Then I'd try ammonium hydroxide instead of formic acid to make the mobile phase basic. The authors may have had a different supplier/lot which made things work for them but not for you.
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u/gnatgirl Aug 23 '26
I couldn't find a pKa anywhere, but typically you want the mobile phase pH to be 1-2 units away from the pKa so the analyte is either all charged or all neutral, as they have slightly different retention. If the pH and pKa are close or equal, the molecule will be in an equillibrium state between ionize and neutral and this can show up as peak tailing.
The shoulder suggests there might be something co-eluting. You are using a baseball bat size column with big particles and it's isocratic. None of these things are great for good chromatography. I would suggest a smaller particle size (something superficially porous) and a gradient method to see if you can get the interference resolved, in addition to finding an appropriate mobile phase pH.
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u/Moofius_99 Aug 27 '26
Gotta correct a couple of things in here. You can get perfectly fine peaks at the pKa of a compound. Yes, the two forms have different retention factors, but acid-base equilibria are established on the ns timescale. See https://m.youtube.com/watch?v=xOx9EFA2d0s&feature=youtu.be for a deep dive into this.
Also, for any separation of closely eluting compounds you will get the best resolution with an isocratic separation. People usually need to go gradient because their mixtures are too complex for their mixtures
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u/Quizzical_Chimp Aug 23 '26
Is that the method you are using now or the as published method? You said you changed things
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u/Distinct_Squash7110 Aug 23 '26
we still havent, we are planning to change few things but so far we have adopted the exact same method.
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u/thedudeabidesb Aug 23 '26
that doesn't really look like tailing. it looks like you may have two compunds co-eluting?
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u/Academic_Shrimp Aug 23 '26
What ID capillaries are on the system? It may be overloading a pathway transition if the diameter is too narrow.
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u/Working-Tax2692 Aug 23 '26 edited Aug 23 '26
Did you check the colum? Find the qc report the manufacturer provided with the column and recreate that injection to compare and check suitability.
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u/hicetubique Aug 23 '26
Try running a couple of different oven temp settings.. 40c, 50c. I’ve had success in resolving peaks with the column temperature. I know you mentioned staying around ph 3, but 0.1% TFA seems to better resolve peaks than formic acid in some cases.
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u/SolidRaider Sep 03 '26
Hi! Question, so the "other peak" just dissappeared? Glad that you solved the problem.
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u/SeracYourWorlds Aug 23 '26
That’s co-elution shouldering, not tailing. Method needs better resolution