Quantitative Densitometry in Situ of Lipids Separated by thin Layer Chromatography

Abstract

Operating parameters are described for a densitometric method to determine in situ eight lipid classes separated by thin layer chromatography. The separated lipids, visualized on the TLC plate by a cupric acetate-phosphoric acid charring method, were quantitatively determined by spectrodensitometry using the Shimadzu CS-910 Dual Wavelength TLC Scanner. Plates were scanned in either a linear scanning mode or in a zigzag scanning mode (flying spot).

Reproducibility of a) sample application (spotting) and b) the lipid separation procedure was determined by scanning. Transmittance measurements yielded response areas that were 2.8 X higher than reflectance measurements. Operating parameters such as scanning direction, wavelength, single and dual-wavelength measurement, scanning speed, and slit geometry were studied. Optimal conditions were established for quantitative densitometry of lipids on thin layer plates.