Introduction

Separation of lipid extracts into functional compound classes is traditionally performed by silica-gel column chromatography. This method describes an alternative method using solid-phase extraction (SPE) columns containing silica-gel particles coated with an aminopropyl stationary phase. These columns can be easily and inexpensively prepared in the lab (see corresponding method), and offer several advantages over pure silica gel. One main one is improved resolution between ketone, alcohol, and carboxylic acid functional groups, so they are well suited for separating extracts of biological materials with lots of polar lipids. Pure silica gel will give better separations for nonpolar compounds (hydrocarbons and esters). The method is optimized for separations using a vacuum manifold to speed elution, but can also be carried out (slowly) at ambient pressure. Slight adjustments to the elution schedule may be required in that case.

Materials

• SPE column (precleaned and stored in drying oven at 80C)
• hexane
• 4:1 hexane/DCM
• 9:1 DCM/acetone
• 2% formic acid in DCM (note A)
• methanol
• vacuum manifold or filter dome

Warnings

• The glass SPE columns can be difficult to insert and remove from a rubber stopper. Be careful when applying pressure by hand - they can break and cause serious cuts.
• Used SPE columns, if left lying around on the floor, can cause people to slip and land on their heads.

Procedure

1. Insert a precleaned and dried (note B) SPE column into a rubber stopper and mount in the vacuum filter dome. Wetting the stopper with water makes this easier. Rinse the SPE cartridge with 4 column volumes of clean hexane.
2. Dissolve your dried sample in a minimal amount (~0.3mL) of hexane (note C) and transfer onto the SPE column using a glass pipette.
3. Elute fractionations from the column using the appropriate solvent schedule (below), which can be conveniently added to the top of the column using a graduated cylinder. Do not allow the vacuum to suck all solvent through the column - turn off the vacuum before the solvent level reaches the top filter (note D). With no vacuum, the SPE column will stop dripping with the solvent level right at the filter. Collect each fraction in a clean VOA vial.
4. Dry sample fractions in the Turbovap, and transfer to GC vials using an appropriate solvent like DCM.

Notes

1. Prepare this solvent by mixing 2% by volume formic acid (85% formic in H2O) into 98% DCM, i.e. the 2% number does not refer to the final concentration of formic acid.
2. Because silica gel is used as the support for the stationary phase, the separation is sensitive to the amount of water adsorbed onto the column. Leaving the SPE columns in an 80C drying oven for several days is an easy way to ensure reproducible separations. Do not remove the column from the drying oven until you are ready to use it, and do not leave it sitting in the hood for more than 15 minutes.
3. A common mistake is that students worry hexane will not adequately dissolve free fatty acids, and so use DCM (or other solvents) to transfer their sample onto the column. The presence of even very small amounts of DCM on the column will cause most of fraction 2 to elute with fraction 1. Several years of experience have shown that hexane is an adequate solvent for small quantities of free fatty acids.
4. Fractions will not be cleanly separated if you allow the sample to dry on the SPE column. Once you begin washing the column (step 1), keep the stationary phase wet with solvent throughout the entire procedure.
5. Rarely something goes wrong with the separation, and some compounds (mainly fatty acids) can be left on the SPE cartridge at the end of the procedure. If your sample is very precious, you can wash the SPE column with methanol as a final step. This will flush all remaining analytes from the column, and you can save this fraction as insurance until you have confirmed that the separation worked.