[bonkers]

Hello,

Im trying to separate vitamine D2 and D3 and am using isocratic method with 93% water and 7% MeOH. The retention times are about 13 minutes and the peaks aren´t fully separated. Can anyone suggest ways to separate them more efficiently?

[Toxophilus]

See http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Application_Notes/7141.Par.0001.File.tmp/7141.pdf
Alternatively, do a trial gradient.

[bonkers]

See http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Application_Notes/7141.Par.0001.File.tmp/7141.pdf
Alternatively, do a trial gradient.

In that paper they separated D2 and D3 with 100% MeCN. Doesnt that mean that they have a really good column because, the more MeCN you use, the higher the eluetion force the mobile phase has. The higher the elueting force the faster my components exit from the column and are therefore closer to each other. So should the 93% water 7% organic mixture be the best for separating my components or is there another alternative?

I don´t get what you mean by "trial gradient"?

[Arkcon]

Because those two molecules are structurally similar, their separation will be a little difficult as you have it.  If Toxophilus: hadn't provided an app note, I might have said it was too difficult, so yes, Sigma is advertising an exceptionally good column.  It may work exactly as they advertise, or it might not.  A gradient run, where you use the HPLC pumps to mix varying concentrations of water and methanol, will help you separate these components easily.  If you still want to use isocratic, then you can still use a gradient to scout out what percentage of methanol/water works best for your column and samples.