[cholecalciferol]

I have 2 ergosterol isomers: (isomer A) 22,23-epoxide-20-hydroxyergosterol and (isomer B) 22,23-epoxide-22-hydroxyergosterol. I have used a few different brands of reverse-phase C18 HPLC columns: brownlee aquapore ODS, phenomenex Kinetek C18 and Grace Smart C18. These all separate the ergosterol isomers using acetonitrile, where isomer A elutes before isomer B.
The problem was that when I used another RP-C18 column (Grace Alltima C18), using acetonitrile, isomer B eluted before isomer A. I have used this column on other similar compounds, such as vitamin D3 derivatives, and have never encountered such a mysterious problem. I use these protocols to purify my compounds, which in this case, made me think I mixed up the samples...but I have checked and triple checked...
PLEASE HELP: I need an explanation as to why my compounds have eluted differently in this case. I look forward to getting this mystery solved!

[Ligte]

Polar compounds elute before less polar compounds, which also depends on the pH of your solution and organic solvents used on reverse phase columns. If isomer A elutes before B i would guess the hydroxy group of isomer A is separated by a single bond from the double bond. If this is the case you would have conjugation between the hydroxy group and the double bond and this would increase the polarity of compound.

i couldnt find the structure. and it would be helpfull to know what detection you are using. UV or MS