We want to use the Supelco NH2 SPE columns to separate our fractions for plant, animal, and bacterial projects. The regimen separates the
Conditioning Step: Condition ALL DSC-NH2 tubes with 1-3 mL hexane (use 1 mL for 1 mL DSC-NH2 cartridge, 3 mL for 3 mL
DSC-NH2 cartridge)
Apply Sample : 1-3 mL of lipids in chloroform to DSC-NH2 Tube #1
Elution Steps:
1. LC-NH2 Tube #1:
a. 2:1 chloroform:isopropanol elutes neutral lipids like triglycerides, diglycerides, monoglycerides, cholesteryl esters, and
cholesterol; collect, evaporate solvent, reconstitute in hexane, and go to elution step 2 to fractionate the neutral lipids
b. 2% acetic acid in diethyl ether elutes fatty acids
c. methanol elutes phospholipids
2. LC-NH2 Tube #2 stacked on top of LC-NH2 Tube #3: pour reconstituted sample from step 1a into top of Tube #2
a. hexane elutes cholesteryl esters through the tubes
b. hexane containing 1% diethyl ether and 10% methylene chloride elutes triglycerides through the tubes
3. Separate Tube #2 and Tube #3 and elute separately
a. 5% ethyl acetate in hexane elutes cholesterol from Tube #2 and also the residual cholesterol in Tube #3
b. 15% ethyl acetate in hexane elutes diglycerides from Tube #2
c. 2:1 chloroform:methanol elutes monoglycerides from Tube #2

We found out that we do not get all the fatty acids out in step "1.b." above. About half comes out in the phospholipid fraction (1.c.).

If we react the fatty acid and phospholipid fractions with methanol/sulfuric acid , we will get the methyl ester of the fatty acids that come out in these two fractions. When the phospholipids in 1.c. above are reacted with the methanol/sulfuric acid mix, will the phosphate group be replaced and a methyl ester result in a straight chain fatty acid methyl ester? If I take the results of theset wo fractions, will I be "over reporting? my fatty acid concentration?

It could be that I just need to do a serial spike recovery assessment and not worry about the chemistry. But I would like to know.
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