Phase/Column suggestions for hydrophobic plant waxes

[tmaxwell]

Hello, I recently brought a waters 2695 back to life and am trying to decide on what column to buy for the separation of plant waxes. My compounds of interest are long chain (C24-C30) aliphatic primary alcohols. I have a preparative procedure that could feasibly bring my soil/plant extracts to contain only hydrophobic alcohols, and using urea adduction and silver nitrate TLC to aliphatic hydrophobic alcohols. Although it would be nice if it were possible to separate by degree of unsaturation and linearity online instead of manually, my main goal is to separate to get specific compounds by chain length. After doing some reading it seems like reverse phase makes more sense because this way the hydrophobic chains will interact with the column, perhaps allowing me to get away with an isocratic elution to elute by order of polarity/chain length. If I chose normal phase it would be the polar head groups interacting with the column, and the mobile phase would be the primary mode of separation. To do this I will need to use some mixture of chloroform or DCM/hexanes/ACN, as the analytes dissolve best in chloroform, weakly in hexanes, and not at all in ACN.

So I suppose what I am asking is the following: does it seem like a better idea to do more prep work before loading extracts on to the hplc, or is it reasonable to try to separate by chain length, saturation, and linearity all in one method?

Second, I am asking if my logic for choosing reverse phase is reasonable, a Waters rep. suggested their general C8 column, suggesting a C18 may be too sticky to my compounds, and if you have any specific column suggestions it'd be appreciated!

Thanks for the help,

Toby

[JMB]

Some years ago I worked on the LC-MS analysis of vitamin D3 (a steroid alcohol & very hydrophobic) and esters. The method was developed by chromatographers, who tried MANY phases/columns.
col: Platinum EPS C-18 (150 x 4.6 mm; 3um particles)
sample diluent: MeOH/H2O (95:5, v/v)
[A] 0.1% phosphoric acid in water*
[B] MeCN
Gradient (several stages) from [B] 4% to 95% over 50 min, then hold.
Flow Rate: 1.0 mL/min
This gave,
Vit. D3 RT 42 min
VD3 esters RT 58 & 62 min

NB * H3PO4 was found to improve peak shape

JMB

[Peter Apps]

Hi Toby

Sorry, I can't help with your column enquiry but I have been looking at the possibility that volatile compounds in urine form urea complexes when the urine dries, in connection with scent marking in African wild dogs. I have not had much success with finding useful literature; nearly everything that comes up is narrowly specific applications for fractionation and purification.

Do you perhaps know of a general review that I could look at ?

Thanks

Peter