Complexities in lipid quantitation using thin layer chromatography for separation and flame ionization for detection.
The use of thin layer chromatography (TLC) for separation (using silica gel coated quartz rods) and subsequent flame ionization for detection (FID) was examined to determine whether this method could be used for the quantitation of lipids. However, response factors (RF) for various lipids were different and depended upon several variables including the amount of material analyzed. For example, RF were 3-fold greater when 10 micrograms of tripalmitin was analyzed as compared to 1 microgram of the same material. The amount of lipid detected by FID was also dependent upon the rate at which it passed through the flame. During analysis of methylpentadecanoate, detector response increased with scan speed, while at all speeds it was completely removed from the rod. On the other hand, depending upon the amount of cholesterol or phospholipid analyzed, the response either increased, remained unchanged or decreased with scan speed. During a fast scan, detector response was reduced because some material remained on the rod. Thus, the detector response is influenced by sample volatility. In conclusion, there appears to be a complex relationship between detector response and the amount of heat available per microgram of sample. Since we could not find a direct correlation between detector response and sample quantity, it would be difficult to use TLC-FID as a tool for quantitating the components of a lipid mixture.
D002784:Cholesterol / Q000032:analysis; D002788:Cholesterol Esters / Q000032:analysis; D002849:Chromatography, Gas / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D005230:Fatty Acids, Nonesterified / Q000032:analysis; D008055:Lipids / Q000032:analysis*; D010743:Phospholipids / Q000032:analysis; D014280:Triglycerides / Q000032:analysis
D002788:Cholesterol Esters; D005230:Fatty Acids, Nonesterified; D008055:Lipids; D010743:Phospholipids; D014280:Triglycerides; D002784:Cholesterol