Chromatography Forum

Hi all, Good Morning,

Just now I come across with one journal that managed to separate all tocols in RP-HPLC within 18 minutes using caratenoid column C30 250x4.6mm, 3um.

More detail in
Influence of Growth Temperature on the Amounts of Tocopherols, Tocotrienols, and ç-Oryzanol in Brown Rice
J. Agric. Food Chem., Vol. 55, No. 18, 2007 pg 7561

100% solvent B (acetonitrile/tetrahydrofuran/water 70:25:10, v/v/v) between 0 and 25 min
linear gradient to 100% solvent A (acetonitrile/tetrahydrofuran, 50:50, v/v) from 25 to 35 min
100% solvent A from 35 to 45 min
ending with 100% solvent B from 45 to 60 min
25 °C ,1.0 mL min-1

However, the peak separation is not good with low resolution specially beta and gamma peaks.
What can I do to increase the resolution of these peaks to get better separation.
I am scare to upload the chromatogram as it is from journal.
And what the disadvantages of C30 column over C18 column

Hope will get good reply from you all.
Best regards.

Myy understanding of the C30 column's unique resolution of these compounds is that it's a shape selectivity effect due to the longer chains. Given that they had to use a 250mm lomg column packed with 3 micron material, not to mention a ternary gradient, I doubt that there are any easy ways to improve the resolution. Temperature might be the major candidate.

I don't know of any major problems with the C30 outside of the obvious dewetting issues with high aqueous mobile phases.

Mike do you know these papers:

Afaf Kamal-Eldin, Stefanie Görgen, Jan Pettersson and Anna-Maija Lampi, Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols: Comparison of different chromatographic columns, Journal of Chromatography A Volume 881, Issues 1-2, 9 June 2000, Pages 217-227

Gianfranco Panfili, Alessandra Fratianni, and Mario Irano, Normal Phase High-Performance Liquid Chromatography Method for the Determination of Tocopherols and Tocotrienols in Cereals, J Agric Food Chem. 2003 Jul 2;51(14):3940-4

Merete Møller Nielsen and Åse Hansen, Rapid High-Performance Liquid Chromatography Determination of Tocopherols and Tocotrienols in Cereals, Cereal Chemistry, 2008, Volume 85, Number 2 Pages 248-251

Yes I read these papers before.
Some used silica column (separated all 8 peaks nicely) while some used C18 column (only separated 6 peaks. beta and gamma tocols co-elute together) disadvantages of C18.
Alternative RP column is C30 which able to separate 8 peaks yet need to improve to get nice peaks.
My senior said changing Reverse phase to normal phase oftentimes could decrease the sensitivity of detector and HPlC, so she prefer me to use reverse phase. That the reasons I terribly looking fro RP-HPLC that can separate all 8 peaks.
research works really sicken me even I just started. Still long way to go

If the resolution problem is at least partially due to peak broadening (rather than just peak spacing) then the following may help. I have worked with a group that included 0.1% phosphoric acid in the aq. mobile phase to sharpen the peaks of vitamin D3, its homologs and precursors.

You could add it to reservoirs [A] and ---very easy to run a trial, BUT, as always, ensure that column is thoroughly equilibrated with the phosphoric acid phases, before running samples.

Please let us know if this does/does not help.

This application should probably help you

http://www.nacalai.co.jp/cosmosil/data/Web/AP-1071.htm

it is a naphtylethyl group (kind of a double phenyl) and the application is very simple isocratic and done in 20 minutes
a simple gradient would probably improve it further and make the peaks sharper

Thanks JMB. Tocols are instable with the presence of acid however I not sure how this small amount of phophorus acid can effect on tocols.Need to run the experiment.And unmgvar - couldnt open the link.It comes with blank page.TQ

This application should probably help you

http://www.nacalai.co.jp/cosmosil/data/Web/AP-1071.htm

it is a naphtylethyl group (kind of a double phenyl) and the application is very simple isocratic and done in 20 minutes
a simple gradient would probably improve it further and make the peaks sharper

Have you tried this column to separate tocols. How the stability, reproducibility of retention time and etc of this column? So far, I never read any journal that used this column and how much it cost roughly? I am interest on it -can performed like silica column in reverse phase condition.

Hi all,
Good Evening.
I bought this column from nacalai (http://www.nacalai.co.jp/cosmosil/data/Web/AP-1071.htm) which the spec is 250 x 46mm, 5um. Tne packing material is naphtylethyl group (kind of a double phenyl).
I used it for tocopherols separation using isocratic methanol and water (90:10), Flowrate-1ml/min, temp-30C. The injection amount is 20ul of 50ug/ml.

The problems is the peaks are too broad and short.
I even tried injecting small amount of standards and results was even shorter with broader. The condition was given by the supllier and it is the optimum as I changed the flow, temp and proportion, the separation was not good. I aso tried using isopropanol but the results are same.

This are the chromatogram 1) tocopherols
http://www.imagespace.us/images/6bzwoeuzy5pz850j2c1.jpg

How can I increase the peaks sharpnes to get better separation. You can see the second and third peak does not touch the base. Can you experts give solution to this.
Waiting for your replies.
Thank you

Hello krisradh,
http://orgchem.colorado.edu/hndbksuppor ... chrom.html

This site might help you