Hi everyone, when I tried to analyse my pure standard solution (Phytol), there are two peaks that emerged side by side in my HPLC analysis. I've tried with different mobile phases (acetonitrile, methanol, and mixture of both) and the peaks still show side by side (just different retention time).
Any idea how to resolve this issue? Could there be a contaminant in my standard?
Many thanks and best regards,
Method needs to be developed. Are you using an existing method? Change the mobile phase ratio and see.
Thanks for the reply Udaya. Yes I'm currently using an existing developed method. Acetonitrile:methanol at 70:30 ratio. Still the peaks remained as shown in the picture.
Hi
If it is already developed and validated too also assume you have single peak only in past. Now you have to do some troubleshoot before any change in method.
- Are you using DAD/PDA detector, if yes how different these peaks are from each other in terms of spectrum
- You have to check the integrity/ age of column , mobile phase and standard itself. If all these are confirmed then I will go further
Regards
Did you try to inject less? If the peak shape is ok, the solvent strength of your sample diluent is to high.
If you are using RP-HPLC try to decrease the amount of methanol or Acetonitrile and increase the amount of water . for example you can use 70 : 30 (meoh :H2O)
Sorry for the late responses everyone due to my other commitments.
I have tried 70:30 (ACN:MeOH) as well as with 0.1% phosphoric acid. The two peaks are still shown. My injection volume is 20 uL.
I'm sorry but my setup is only UV-VIS detector.
However, while using the same column for the detection of other compounds at different wavenumber, it clearly showed one peak (not sure if this information is necessary).
Hi Dear
Let me assume the method was showing one peak in past and you are having now two peaks. Are you using different instrument or same instrument but different model? Like some advance series equipment form the same manufacturer. With the emergence of UPLCs and still high demands of HPLCs many manufactures have come up with hybridized instruments claiming these works for both HPLC and UPLC conditions. That’s is true but, in some cases, you are forced to tweak some conditions/parameters to compensate the hardware changes.
You might be going through this issue.
If the same column, solutions and mobiles phases give two peaks on one equipment and one peak on other equipment then check the difference in both instruments but if everything is same then you must check with service engineer what has been changed recently in the instrument giving two peaks. If you are using steel tubing the little deformation in ferrule could lead to changes in peak shapes.
Apologies for my late response. I was busy organizing another conference the past month.
Zhao Weihua - I am using normal phase isocratic elution
Muhammad Akmal - For the scenario you described, I think it is not applicable in my case. I have tried with another compound alpha-tocopherol and it only yielded one peak.
You may have already resolved this, but I would suspect you are looking at cis and trans isomers that are not fully resolved. The very small peak at about 5 minutes may be isophytol. We use GC in our lab for phytol.
Dear Shae,
Yes ! It has been resolved. And you are right. I've asked the manufacturer (after countless persistence and non-replies) and that was the solution that have provided.
For your case, do you take into account for all cis/trans phytol isomers? or do you have a specific isomerism you're interested?
We are only looking at chromatographic purity and the ratio of the main peaks cis/trans. We also have a specification on the allowable amount of isophytol. Glad you were able to resolve your issue, sorry I didn't see your post earlier so I could have saved you some time!
Best regards,
Shae
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