Separation of Nile Blue‐labelled fatty acids by CE with absorbance detection using a red light‐emitting diode

Abstract

The separation of fatty acids derivatised with Nile Blue (NB) by CE with detection using a red light-emitting diode (LED) was examined. NB was selected as the derivatisation agent due to its high molar absorption coefficient of 76,000 M(-1) cm(-1) at 633 nm, making it well suited for sensitive absorbance detection using a red 635 nm LED. NB-labelled fatty acids were separated by both MEKC using SDS micelles, i-PrOH and n-BuOH and by NACE in a number of solvents including MeOH, EtOH and ACN. The sensitivity of NACE was superior to MEKC, with detection limits of 5x10(-7)-7x10(-7) M obtained for each acid, approximately 20 times lower than the MEKC method. The NACE detection limits are approximately 100 times lower than previous reports on the separation of fatty acids by CE using indirect absorbance detection, ten times lower than using indirect fluorescence detection and are inferior only to those obtained using precapillary derivatisation and direct fluorescence detection. The efficiency of the NACE method was also superior to MEKC and allowed the separation of unsaturated fatty acids to be examined, although it was not possible to baseline-resolve linoleic (C18:2) and linolenic (C18:3) acids in a reasonable time. The method was used to analyse the fatty acid profile of two edible oils, namely sunflower and sesame oils, after alkali hydrolysis, where it was possible to identify both the saturated and unsaturated fatty acids in each sample.