Vegetable Oil Analysis

Vegetable Oil Analysis

Quality control and quality assurance testing are increasingly important at every point in the food supply chain, from manufacturing and packaging to distribution and retail sale. The focus on efficient and reliable food analysis has become more acute over the past few years, as high-profile cases of food fraud and adulteration have come to light.

Food fraud is a decades-old problem. According to researchers at the U.S. Pharmacopeial Convention (USP), an independent scientific non-profit organization, vegetable oils—especially olive oil—have a high vulnerability to adulteration and represent the most documented cases of food fraud, with dilution being the most common cause of problems. Over the past 30 years, more than 270 studies and articles have been published on the adulteration of olive oil alone.

In recent years, food analysis has improved dramatically and many types of adulterated food are now unlikely to escape detection. Extensive research has been done in the field of vegetable oil analysis to test for authenticity and chemical properties. For example, gas chromatography (GC) and high-performance liquid chromatography (HPLC) have frequently been used to evaluate triglyceride content in vegetable oil samples. The physical and chemical properties of vegetable oils are closely related to the type and relative amount of each constituent triglyceride in the sample.

A New Technique

Supercritical fluid chromatography (SFC) in combination with evaporative light scattering detection (ELSD) is a valuable technique for the determination of triglyceride composition of vegetable oils. Compared to GC, SFC separates triglycerides at much lower temperatures; compared to HPLC, SFC permits greater selectivity with shorter analysis times.

The potential of SFC for the separation of triglycerides has been demonstrated for many years. Using a reversed stationary phase, the separation is similar to that obtained in reversed phase HPLC. Separation is based on carbon number (total number of carbons in fatty acids) and on the total number of double bonds. Using a silver-loaded column, separation is primarily based on the degree of unsaturation (total number of double bonds). These two separation mechanisms are complementary.

This technical article demonstrates the SFC separation of triglycerides in three vegetable oil samples.

Experimental Methods

Sunflower seed oil, peanut oil, soybean oil reference oils, tripalmitin (PPP), triolein (OOO), and trilinolein (LLL) standards were purchased from Sigma-Aldrich (Bornem, Belgium). The oils were dissolved in chloroform at the 5 percent (50 mg/mL) level.

Analyses were performed on an Agilent 1260 Infinity Analytical SFC System combined with an Agilent 1260 Infinity ELSD Evaporative Light Scattering Detector. The ELSD was coupled to the SFC module using a procedure similar to the one used for SFC-MS 4.

The addition of a make-up flow before the backpressure regulator, together with additional heating at the entrance of the ELSD, was found necessary to obtain good sensitivity, reproducibility, and avoid solute deposition in the transfer capillary. Experiments show that switching off make-flow or heating immediately results in low sensitivity and unstable baseline in ELSD detection.

Analyses were performed on two different stationary phases: ZORBAX SB-C18 and Chromspher 5 Lipids silver loaded column. For the reversed phase separation, three ZORBAX SB-C18 columns were coupled in series.

Reversed Phase Separation

Figure 1 shows the UV and ELSD chromatograms for the separation of triglycerides in sunflower seed oil. As seen, the ELSD detector is more sensitive than UV detection, giving S/N ratios approximately five times better than in UV detection. In addition, the baseline is more stable than in the UV signal at this low wavelength. Moreover, the response in ELSD is more universal and less dependent on the number of double bonds in the lipid molecule.

In SFC, using a reversed phase C18 column, triglycerides are separated according to the carbon number and the total number of double bonds. By approximation, the elution order is set according to:

  • PN = CN – NDB


  • PN = partition number
  • CN = carbon number (sum of carbons in fatty acid chains)
  • NDB = sum of number of double bonds

Therefore, the PN for OOO is (18+18+18)–(1+1+1) = 51, and this compound elutes later than OLO with a PN = (18+18+18)-(1+2+1) = 50. Within a group of triglycerides with an equal PN number, additional separation can be obtained. For example, LLL and PLL (PN = 48), OLL and PLO (PN = 49), and OLO and POO (PN = 50) are separated.


  1. Qadriyyah says

    How do I get in touch with food quality and safety to request a test be done on an oil that I suspect isn’t 100% pure?

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