Does anyone know what the best column for doing preparative HPLC of neutral and polar lipids extracted from algae and higher plants is?
The case is that we are going to buy the prep. HPLC-ELSD system and at the moment I am trying to find columns which are appropriate for my goals.
I think that the best option is SEC ( Size exclusion column), you can select for the size
If you want to separate polar and non-polar lipids from algae in prep scale (100 mg - 100 g) you do not need to use HPLC.
Dear Dmitry, we are also going to separate phospho-, sulfo- and glycolipids and so on. I know, that the two-dimensional prep. TLC is the still reliable method and I often use it to separate various classes of the neutral and polar lipids, anyway, prep HPLC is really necessary for us, since we are often deal with necessity for obtain ca 50-100 mg of individual lipid or secondary metabolite for it's further analysis via NMR and ESI\APCI-MS\MS. Of course, for NMR spectroscopy we use SHIGEMI microvials, but we do need to get an preparative HPLC system which could be easily modified by current goals, but the lipid separation and analysis is a main goal.
Roman, I would consider Daniel's idea as an option. But from my experience with prep HPLC for purification of sensitive lipids especially polar lipids it is a good idea to use a usual 250x4.6x4-5 mkm column for purification mgs amounts of the compounds for research. Various phases can be used - RP, Diol (HILIC) etc but for good recovery we should avoid to use silica gel based phases. The best technique for prep lipid separation is liquid-liquid chromatography, but even a basic set up is expensive.
Dear Daniel and Dmitry, I'm highly grateful for your opinions and tips! Also I would ask you what type of detector is better and why: refractometric, ELSD or CAD?
Roman, from my point of view RI is too cranky but for some applications like epoxy-lipids, epoxy-acetylenic compounds, polyphenolic compounds, tocotrienols etc. it is superior to ELSD and CAD which are better than RID in the 99.9% of other cases. If I were you and I were focused on a new instrument I would buy a CAD (but it is much cheaper to buy a used ELSD).
Dmitry, if I understand a chromatographic equipment correctly, we should buy a nitrogen generator to work with ELSD or CAD or bottled gas is better? Some analysts have told me what we can use purified and compressed air, but another thinks what air does not good idea since warm oxygen could lead to fire if it reacts with organic solvents, so the rare gas is safer.
Roman, nitrogen generator is the best option, but if you have a nitrogen line in your Institute or have at least 200 L liquefied nitrogen tank this will work fine. Usage of bottled nitrogen is not practical as you will need to use a lot of gas cylinders. You do not need to purify the nitrogen from oxygen or water. Of course, you can use compressed air instead (this was a common practice 20 years ago everywhere) if this passes your Institute safety regulations.
You can try our method that is used by Avanti...with ELS detection..see
2000 Stith, Bradley J., Jennifer Hall, Patrick Ayes, Leslie Waggoner, James Moore, Walt Shaw. Quantification of major classes of Xenopus phospholipids. Journal of Lipid Research 41: 1448-1454.
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