Ultra high efficiency/low pressure supercritical fluid chromatography (UHE/LP-SFC) for triglyceride analysis: Identification, quantification, and classification of vegetable oils

Abstract

Triglycerides of vegetable oils have been extensively studied. Non-aqueous reversed-phase liquid chromatography and silver-ion chromatography are most frequently used to achieve their separation. In previous works, we presented the use of supercritical fluid chromatography with long columns (75 cm) packed with fused-core particles to provide ultra-high-performance separations, with a low-toxicity fluid (carbon dioxide) compared to the usual liquid-phase methods. In the present paper, we describe the quantification of triglycerides with supercritical fluid chromatography and evaporative light-scattering detection. Thanks to the isocratic elution mode, this quantification can be simplified, assuming (a) identical response coefficients for compounds having a close structure, (as only triglycerides are quantified), and (b) constancy of the response coefficient along the analysis (no elution gradient). Therefore, the relative concentrations of triglycerides were easily assessed. Only one calibration curve for one reference compound (in this case triolein) was required. The resulting relative concentrations are in good accordance with the numerous publications available. Relative quantification with UV detection at 210 nm is also proposed, facilitated by the very low UV absorption of carbon dioxide and with a calibration curve taking account of the variation of UV response according to double bond number. Nineteen vegetable oils are compared. The identification of triglycerides was carried out based on previous knowledge of these oils, but also with the help of a Goiffon retention diagram, based on the relationship between the logarithm of retention factor and the total double bond number. Finally, cluster analyses were computed, based on evaporative light-scattering detection or UV quantification data. They allow a quick comparison of the triglyceride content between the oils, in the goal to exchange one by the other for certain applications, or to compare a new oil to well-known ones.

Keywords: evaporative light‐scattering detection (ELSD); lipids; supercritical fluid chromatography (SFC); triacylgylcerols; vegetable oils.

FIGURE 1

Calibration curve for SFC‐ELSD quantification: logarithm of peak area obtained with OOO standard solutions, versus the logarithm of OOO concentration. Full data are presented in Table S2 (supplementary information) and the graph of standardized residuals in Figure S1

FIGURE 2

UV response coefficient (A) versus the total number of double bonds in 12 TG (based on data from ref. presented in Table S3). UV response coefficient for any TG was calculated from this calibration curve. The graph of standardized residuals is available in Figure S2

FIGURE 3

Goiffon diagram of the logarithm of retention factor vs. the total number of double bonds, for the TG observed in the 19 vegetable oils. Analytical conditions: see details in experimental part. For the sake of clarity, not all useful lines have been drawn in this figure

FIGURE 4

Hierarchical cluster analysis from (A) ELSD data (in Table S4) and (B) UV 210 nm (in Table S5) to classify the 19 vegetable oils. Identical colors of the oil name indicate belonging to the same cluster, defined by the cutting lines (interrupted grey lines)

FIGURE 5

ELSD chromatograms of Almond (red lower trace), Apricot Kernel (blue medium trace) and Rapeseed (green upper trace), belonging to the same HCA cluster. Apricot kernel and Rapeseed traces were shifted (dotted blue lines) to facilitate comparison of the overlaid chromatograms

FIGURE 6

ELSD chromatograms of Pistachio (lower green trace), Argan (second blue trace), Peanut (third red trace) and Sesame (upper orange trace). The chromatographic traces were shifted (see interrupted grey lines) to facilitate comparison of the overlaid chromatograms

FIGURE 7

ELSD chromatograms of Macadamia (lower blue trace) and Avocado (upper red trace)

FIGURE 8

ELSD chromatograms of Knifophia Uvaria (lower blue trace), Kahai nut (medium red trace), and Safflower (green upper trace). Safflower and Kahai nut were shifted (dotted blue lines) to facilitate comparison of the overlaid chromatograms