Relationship between densitometric peak area and concentration of lipid in the quantitative analysis of a lipid mixture separated by thin-layer chromatography

Abstract

A mixture of the neutral lipids squalene, cetyl stearate, triolein, oleic acid and cholesterol was separated by thin-layer chromatography and the components were measured quantitatively by photodensitometry. Several empirical expressions relating densitometric peak area ( A) and lipid concentration ( C) were subjected to statistical analysis, and the relationship A = k· C B is linear in the logarithmic form log A = B·log C + log k within 95% confidence limits on the observed results. Exponent B, representing the proportionality factor in the expression relating the relative differential changes in peak area and concentration, it constant for lipids studied on the same chromatoplate, whereas the constant k is lipid dependent. All standard measurements per plate are therefore utilized for estimating a common B value, which gives greater accuracy than in the individual cases. The plate capacity for measurements of unknown lipid concentrations may consequently be increased, as a common B value, based on a reduced number of standard measurements, may be estimated with the same accuracy as an individual B value, based upon the non-reduced number of standard measurements. Equations are given for the calculation of B and k and for the determination of unknown concentrations with appropriate confidence intervals.