This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Some classes of lipids are large molecules that are not sufficiently volatile to allow gas-phase analysis by means such as gas chromatography (GC). Often, non-volatile lipids can be saponified to remove the fatty acid chains and these can be derivatized to yield volatile molecules, such as fatty acid methyl esters (FAME), pyrolidides, picolinyl esters, and others. These derivatives may be readily analyzed by gas chromatography in combination with a variety of detectors, including mass spectrometry. FAME analysis by GC with flame ionization detection (FID) is a common analytical method that provides valuable information regarding the net fatty acid chain composition of a sample. However, GC of FA derivatives can provide only an approximation of the composition of the original intact molecules. If there is non-random distribution of the FA chains in the parent molecules, FAME analysis can provide no indication of the distribution of those chains on the original molecules. It is preferable to analyze the large lipid molecules intact. Liquid chromatography has become the method of choice for separation of many classes of large lipid molecules. But the choice of liquid chromatography for separation inherently implies limitations in the choices available for detection. The flame ionization detector for liquid chromatography, refractive index and ultraviolet/visible (UV/vis) detectors, and more recently the evaporative light-scattering detector (ELSD) have all been employed for detection of lipids. But these two-dimensional detectors require complete chromatographic resolution to allow all molecular species to be identified. Unfortunately, even simple natural samples of lipids often contain species with the same equivalent carbon number (ECN) that overlap partially or entirely. The ECN is a measure of the overall non-polar characteristic of a fatty acid chain or molecule, given as the number of carbon atoms minus two times the number of double bonds (sites of unsaturation), ECN = Ncarb (2 U). For example, a 16:0 (palmitic) acyl chain has the same ECN (16 0 =16) as an 18:1 acyl chain (18 2 = 16). On reversed-phase chromatographic systems, molecules containing FA chains with the same ECN elute with similar retention times.
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