I'm currently trying to set up a standard curve of pure phytol to compare my plant extract and other samples. I've run the HPLC with pure phytol on a C18 column with 10% acetonitrile and 90% water but the separation does not give good peaks. The area under the curve does not give a gradual increase in concentration (concentrations ranging from 0.00001M to 1M) with similar concentrations for each peak.
My solvent for the phytol was isopropenol and I am using a PDA detector to analyse the HPLC results.
If anyone has a method I could look at or suggest any changes it would be greatly appreciated.
Hi Mattew,
Is it an isocratic HPLC method? I would rather suggest a gradient method, starting from high aqueous mobile phase composition to high solvent composition. Regarding the solvents, consider the use of solvent with slightly higher strength (such acetone).
Good Luck
Hi Loai,
The HPLC is isocratic. I have tried a gradient method with the above mentioned solvents but I have not tried other solvents. I will try acetone (and maybe some others). Can you recommend how fast (or slow) the gradient should change for the method?
Thank you
I would try first a slow gradient and see if it is O.K. or need to be adjusted. I would also check if the method suits the plant extract (not only the pure phytol).
Greeting Mr. matthew, may I know if the method that you applied for HPLC analysis of phytol success? If yes, have you publish your work or can you share with me the method you used? This is my email [email protected]
Thank you so much
Check the internet for suitable methods. But I would suggest a slow ramp initially to ensure the phytol is baseline resolved. For gradient work use a 5 cm column since a 25 cm column is too 'sluggish' to changes in the mobile phase composition.
Dear Syamimi
Unfortunately I was not using a published method. Due to time constraints I was not able to continue with the work and switched to an NMR analysis to complete my project. The NMR detected the phytol but it did not appear in my plant sample. My conclusion from this is that there was no free phytol but that it was incorporated into other molecules, like chlorophyll.
I believe that the columns I was using (Sephadex if I remember correctly), were old and not suited for phytol elution, but I have no evidence of this.
Dear Matthew,
I know this post has been outdated. But do you remember what is the range of wavelength you have used to detect phytol?
Dear Chai
Its been a whole since I was on the project (and on this platform, sorry for the delay). If I remember correctly I was detecting broadly in the visible spectrum (there was a possibility I also got data from the UV range).
Another problem, that may have been fixed after I left that research group, was the age of the column I was using. There wasn't any record of past usage of the column and, in my opinion, was the cause of my troubles.
Go with 30:70 (Meoh:acn) for 20mins at 235nm. U will get your peak at RT-15min. Do not use water for separation because it may take the run bit longer since the phytol is non-polar.@ Mathew
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