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How do you separate tocotrienols Reverse Phase HPLC???
Posted: Thu Nov 10, 2005 9:50 am
by dan42
HI
Ive isolated some tocotrienols (predominantly d-tocotrienols) Im trying to purify these compounds using semi-prep HPLC. The solvents im using are Acetonitrile and Water. Im having trouble separating these compounds, they seem to coelute at the start, ive tried many solvent systems???
Even at 5% ACN they seem to come off fairly quickly?
Any suggestions? What method would be ideal to separate these compounds (baseline resolved)?
This would greatly be appreciated.
CHeers
Dan
Posted: Thu Nov 10, 2005 7:18 pm
by tom jupille
A Google search on "tocotrienols" "HPLC" turned up >9,000 hits. There should be something in there to get you started.
Posted: Thu Nov 10, 2005 10:02 pm
by Uwe Neue
These compounds are VERY hydrophobic. With 5% acetonitrile in water, they should not even dream to come off the column.
What is your sample solvent. If you dissolve the compound in a strong sample solvent and inject a large amount, you can get breakthrough of the compound, since the sample solvent mixes only little with the mobile phase. We will need to work on the details of sample dissolution and injection technique to get around this problem.
Posted: Fri Nov 11, 2005 1:02 am
by james little
we do a lot of tocopherol work.
usually employ a C8 4.6 x 150 column.
Either use 1% HOAc in water as solvent A or 2.5 mmolar ammonium acetate in water. We are doing LCMS and need a compatible solvent system.
Solvent B is a 50:50 mixture of isopropanol and acetonitrile. Takes very high organic to get many of the alpha tocopherol derivatives off the column.
I suspect you are not seeing the tocotrienols, but instead some impurity in the mixture at 5% organic.
Try injecting and running your gradient from 40% organic to 100% organic and see what you find over a period of 20-25 minutes with a hold of 10-15 minutes at 100%. See if you see some additional peaks.