Rapid analysis and quantification of major neutral lipid species and free fatty acids by HPLC‐ELSD from microalgae

Abstract

A method based on normal-phase high performance liquid chromatography combined with evaporative light-scattering detector was developed to quantify major neutral lipids from microalgae. A binary gradient enabled a good resolution of hydrocarbons and major neutral lipids by classes in less than 13 min. A good linearity was observed from 0.9 to 30 mg. Limits of detection (LOD) and quantification (LOQ) was from 3 to 64 ng depending on the analyte. The intra-and inter-run precision (% RSD) of analysis was found less than 10%. The method was successfully applied to microalgae lipid extracts from Botryococcus braunii, Chlorella vulgaris, Spirulina platensis, and Porphyridium cruentum. B. braunii was distinguished by the highest levels of lipids, triacylglycerol (TAGs), and diacylglycerol (DAGs) being the major compounds.Practical applications: Neutral lipids produced by the microalgae are an interesting way for the development of biofuel from renewable carbon. A simple quantification of these lipids is important in the implementation of oleaginous microalgae screening as well as in the bioprocess optimization of algal lipid production. Besides, determining quantitatively the major lipid classes provides information in the understanding of the biosynthetic pathways and the accumulation of these lipids in relation with algal culture conditions.

Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours

“…In line with earlier findings [ 24 , 29 , 31 ], the ELSD response (i.e., peak area/lipid mass) varied with the lipid class. In the present case, as also observed by Jones and coworkers [ 22 ] and Donot and coworkers [ 21 ], the ELSD responses decreased in the order FFAs, DAGs and MAGs and thus were not related to lipid molecular weight.…”

“…In line with earlier findings [24,29,31], the ELSD response (i.e., peak area/lipid mass) varied with the lipid class. In the present case, as also observed by Jones and coworkers [22] and Donot and coworkers [21], the ELSD responses decreased in the order FFAs, DAGs and MAGs and thus were not related to lipid molecular weight. As reported earlier [26,27], the lipid response curves were sigmoidal when plotted on linear axes ( Figure 1a) making it possible to define a range over which the investigated lipids exert a linear response.…”

“…For example, MAGs have a low ELSD response and therefore, the range of linear behavior is observed at higher lipid masses. Linear fits have been applied before for modeling ELSD responses of lipids within a particular lipid mass range [ 21 , 32 ] and responses at lower lipid levels have been reported not to be linear [ 22 , 29 ]. When plotted on a logarithmic scale, the responses are fairly linear with some flattening at high lipid masses ( Figure 1 e).…”

“…Furthermore, the response depends on the refractive index and density of the solute and may vary twofold as these properties change [28]. Different lipid classes indeed give very different ELSD responses [21,22,24,[29][30][31]. Strangely enough, it is often reported that the chain length and degree of unsaturation of the acyl constituents do not appear to have a significant effect on ELSD response and, thus, that different lipid species of the same lipid class generate identical responses [29,32].…”

“…Despite the emerging success of mass spectrometry (as an important tool in lipidomics) [ 5 , 6 , 7 , 8 , 9 , 10 ], traditional chromatographic techniques remain widely used for food lipid analysis [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Normal-phase high-performance liquid chromatography (HPLC) coupled to evaporative light scattering detection (ELSD) is particularly useful for separating lipids into individual classes according to the number and type of polar functional groups and has successfully been used to study the lipid composition of samples of various origin [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. In ELSD, the HPLC column effluent is nebulized in a stream of air or nitrogen to form an aerosol.…”