Chromatography Forum

Hi guys, Good morning to all

I required to find a fast method for the separation of tocotrienol and tocopherol in oil using HPLC.
As concern, tocols can be separated using reverse and normal phase. People tend to use normal phase over reverse phase due to complete separation of 8 isomers. Normally use column is silica 5um, however I recenty found this amino column

http://www.imtaktusa.com/site_media/fil ... TI559E.pdf
The spec
Unison UK-Amino, hexane / tetrahydrofuran / acetic acid = 91 / 9 / 0.1, 4 MPa
150 x 4.6 mm, 1 mL/min , 37 deg.C, 295 nm, 2 uL (2ug)

My question
1) Which is better column between silica and amino in terms of reproducibility,stability, constant retention time, duration and etc?
2) Why beta and gamma form cannot separate in C18 column??, can any1 explain me what the mechanism and principle underlying? Ist possible using any way to separate it??
3) also normal vs reverse- which is preferred??

Hope will get a reply from you all very soon.
Thanks for your replys in advance.
Merry Christmas

We use http://www.ymc.co.jp/en/columns/ymc_pack_diol_np/ in a normal phase HPLC assay

HI thanks for ur reply.
The column u suggested is diol column and the available column in lab is silica column.

Recently i found the tocols separated very fast, all 8 isomers within 6 minutes.
The author used Zorbox silica 250 x 4.6mm, id-not mentioned at 2.0ml/min with mobile phase hexane:2-propanol (99:1)

You can found the info in

ANALYTICAL BIOCHEMISTRY 180,368-373 (1989)
Separation of Tocopherol and Tocotrienol Isomers Using Normal- and Reverse-Phase Liquid Chromatography.

I am wondering why no other researchers used this method to separate the tocol isomers in their journal. Current Tocols chromatography analysis duration is about more than 15min and i never found any method that separate within 6min. May i know why???

thanks

ANALYTICAL BIOCHEMISTRY 180,368-373 (1989)
Separation of Tocopherol and Tocotrienol Isomers Using Normal- and Reverse-Phase Liquid Chromatography.

I am wondering why no other researchers used this method to separate the tocol isomers in their journal. Current Tocols chromatography analysis duration is about more than 15min and i never found any method that separate within 6min. May i know why???

thanks

I do not know the correct answer to your question. However, as the manager of a contract Analytical laboratory performing this type of analysis, I would offer the following 2 reasons:

1. we have no need of a "rapid" analysis sample numbers are simply not large enough to require a dramatically shortened run time.

2. separating 8 peaks in 6 minutes is quite a feat. However, if any part of the method is sensitive to variation, resolution could easily be lost and the analysis rendered useless. Therefore using a method where variations have much less impact is prudent.

That sound smart. thank you