Simultaneous quantification of vitamin A,E and D

[Lorespain]

Hi everybody! I am trying the simultaneous quantification of these vitamins and I have a problem: beta and gamma tocopherol overlap in the range of 4,5-5 min. I've tried changing flow rate, sample size, mobile phase polarity to improve resolution and it has been imposible. The work conditions were: mobil phase: Hexane:isopropanol and 280nm. Now I am trying gradient elution but I can't separated the peaks. Can you give me some advices to choose the gradient properly? I don't know how to do. Thank you

[Lorespain]

I forgot something: I saw that after gradient elution you have to equlibrate the column. How do you do this? Thanks

[unmgvar]

Lorespain

here are two links for you
http://www.nacalai.co.jp/cosmosil/data/Web/AP-0060.htm
http://www.dionex.com/en-us/webdocs/821 ... 2310-1.pdf

If you are doing this for a food application because the vitamins are food supplements then expect a lot of disturbance from your product, that is generally the case.
one example i remember best is when i was helping out out find a method for different types of baby powder milk.
then sample preparation will be really the heart of your problem.

[Lorespain]

Thank you for your information but the problem I have to solve now is these two overlaped peaks of tocopherol. Have you got any idea?

[unmgvar]

try a column that will be capable of handling subtle differences in chemistry,

phenyl type columns should be best to try out.
I would use a special one called piNAP with a double ring from Nacalai.
they have applications for tocopherol in their website
the chemistry of the phase allows for simple method and great separation results

[krisradh1432]

have you tried piNAP with a double ring from Nacalai column. How the performance and reproducibility. Ist ok? worth to buy?