Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

A laboratory study of the bleaching process in stigmasta-3,5-diene concentration in olive oils

The bleaching effect was simulated in pilot plant by measuring the influence of temperature (40, 50, 60, 70, 80, and 90°C), time (5, 10, 15, 20, 25, and 30 min), and concentration of solid adsorbent [1.5 and 8% (w/w) of Tonsil supreme NFF] on stigmasta-3,5-diene (STIG) obtained by dehydration of steroidal compounds. Conditions were chosen to simulate those used in industrial operations. The presence of refined oils in extra virgin olive oil can be detected by these newly formed steroid hydrocarbons. Experimental results indicated that STIG did not exceed an imposed limit of 0.15 mg/kg in extra virgin olive oil, when oils were bleached with 1.5% earth at temperatures ≤80°C for 30 min in admixed to oils sold as virgin. A large proportion of the adulterations were not detectable by the official methods. Color determinations (CIE-1931) chromatic coordinates) were replicated on a refined oil and in admixed extra virgin olive oil. Color of olive oil was not significantly affected by mixing with refined oil (≤20%).     

Evaluation of olive oils through the fatty alcohols,the sterols and their esters by coupled LC-GC

The analysis of the fatty alcohols, the wax esters, the free and the esterified sterols, as well as that of minor components provides a wealth of information about the quality of an oil or fat, its pretreatment and admixture with other oils. Some results obtained by an easy, nearly fully-automated method are shown for olive oils. More specific information is obtained than by the previous saponification methods.     

Analysis of the wax ester fraction of olive oil and sunflower oil by gas chromatography and gas chromatography-mass spectrometry

The wax ester fractions of solvent-extracted sunflower oil and “extra virgin” olive oil were obtained by solid-phase extraction and subsequently subjected to gas-chromatographic and gas chromatographic-mass spectrometric analysis. The comprehensive qualitative analysis of these fractions, which was carried out by the interpretation of mass spectral data, revealed several types of wax esters. In olive oil, shortchain, even-numbered wax esters, saturated and unsaturated long-chain, even-numbered wax esters, benzyl esters, and the diterpenic esters phytyl and geranylgeranyl ester (the latter as a minor component) are present. With the exception of benzyl esters, all these esters occur in sunflower oil as well, but in considerably different amounts compared to those in olive oil. Whereas unsaturated wax esters are present in a negligible amount, diterpenic esters, mainly geranylgeranyl esters, represent the major part of the wax ester fraction.     

Detection of olive oil adulteration with canola oil from triacylglycerol analysis by reversed-phase high-performance liquid chromatography

The objective of this study was to explore the use of reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to detect adulteration of olive oil with less expensive canola oil. Previously this method has been shown to be useful in the detection of some other added seed oils; however, the detection of adulteration with canola oil might be more difficult due to similarities in fatty acid composition between canola oil and olive oil. Various mixtures of canola oil with olive oils were prepared, and RP-HPLC profiles were obtained. Adulteration of olive oil samples with less than 7.5% (w/w) canola oil could not be detected.     

Evaluation of nonpolar methyl esters by column and gas chromatography for the assessment of used frying olive oils

The measurement of unaltered methyl esters separated from polar methyl esters by column chromatog-raphy was used to evaluate the alteration of an olive oil that had been used 15 times to fry potatoes. Unaltered methyl ester (the nonpolar fraction) decreased significantly (94.9 ± 0.8%vs 98.2 + 0.5%; p < 0.05), while the polar fraction increased significantly (4.0 ± 0.7%vs 2.1 ± 0.7%; p < 0.05) after 15 fryings. The unrecoverable fraction also increased. In order to avoid column contamination the gas Chromatographic analysis was only done on the nonpolar fractions. Linoleic and oleic acids showed a tendency to decrease while saturated fatty acid tended to increase. The unsaturated/saturated fatty acids ratio decreased from an initial value of 7.05 to 6.40 in the last frying. Quantitative gas Chromatographic analysis using both the percentage fatty acid composition and the relative amount of unaltered methyl esters showed a significant oleic acid decrease after 15 fryings (75.8 ± 0.6vs 78.9 ± 0.2 mg/100 mg oil; p < 0.05). To whom correspondence should be addressed     

Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography

A silver-ion high-performance liquid chromatography column (hexane/acetonitrile as solvent, ultraviolet detection) was used to analyze the fatty acid distribution (as fatty acid methyl esters) of a representative sample of hydrogenated oil. Fractions containingcis- andtrans-18:1 isomers were readily separated. The positional fatty acid isomers were separated by rechromatographing these fractions. The elution order and percent compositions were compared with results obtained by gas chromatography. Of the Δ8 to Δ14trans-18:1 isomers, only the Δ8 and Δ9 pair could not be separated. The Δ8 and Δ9cis-18:1 pair also could not be separated, and the Δ10 isomer was poorly separated from this pair. Area percents were comparable to results obtained by gas chromatography.     

Assessment of olive oil adulteration by reversed-phase high-performance liquid chromatography/amperometric detection of tocopherols and tocotrienols

A method involving reversed-phase high-performance liquid chromatography with amperometric detection has been developed for the analysis of tocopherols and tocotrienols in vegetable oils. The sample preparation avoids saponification. Recoveries of α-tocotrienol and γ-tocotrienol in extra virgin olive oil were 97.0 and 102.0%, respectively. No tocotrienols were detected in olive, hazelnut, sunflower, and soybean oils, whether virgin or refined. However, relatively high levels of tocotrienols were found in palm and grapeseed oils. This method could detect small quantities (1–2%) of palm and grapeseed oils in olive oil or in any tocotrienol-free vegetable oil and might, therefore, help assess authenticity of vegetable oils.     

Development of high-performance liquid chromatography criteria for determination of grades of commercial olive oils. Part I. The normal ranges for the triacylglycerols

Criteria for authentic olive oils were developed from isocratic high-performance liquid chromatography analyses of 99 olive oils from the major Mediterranean producers in the 1983–1986 crop years. Authentic olive oils include extra virgin, virgin and pure or refined oils, but exclude all reesterified and adulterated oils. The extra virgin through pure grades will have a combined area for the LOO (C_18:2C_18:1C_18:1), LOP (C_18:2C_18:1C_16:0), OOO (C_18:1C_18:1C_18:1), POO (C_16:0C_18:1C_18:1), POP (C_16:0C_18:1C_18:1), and SOO (C_18:0C_18:1C_18:1) peaks between 82.0 and 92.6% of the total area (L, linoleic; O, oleic; P, palmitic; S, stearic). Authentic oils will have ratios of LOO/LOP and OOO/POO that coincide with a line defined by OOO/POO=0.7844(LOO/LOP)+0.0968; correlation coefficient is 0.885. Authentic oils will not have a trilinolein (LLL) peak over 0.5% in area. Neither triolein (OOO) nor any other single peak suffices to characterize an olive oil sample as one of the authentic grades.     

Organogelation of plant oils and hydrocarbons by long-chain saturated FA,fatty alcohols,wax esters,and dicarboxylic acids

Conversion of oils into gels generally involves altering the chemical characteristics of the liquid. We describe here the gelling of vegetable oils, essential oils, and hydrocarbons at ambient temperature, without changing the chemical characteristics of the liquids, using saturated FA having carbon chain lengths of 10 to 31. The gelling ability of the added FA increased linearly with their chain lengths. Structure-function studies demonstrated that the carboxyl group, position of an additional hydroxyl group, and acyl chain length played an important role in gelation. Long-chain saturated fatty alcohols, wax esters, and dicarboxylic acids also had the ability to gel plant oils and hydrocarbons.     

Solvent extraction of ethanol from aqueous solutions using biobased oils, alcohols, and esters

Distribution coefficients and separation factors were determined for the partitioning of ethanol and water from aqueous mixtures into several vegetable oils and their fatty alcohol and fatty ester derivatives. Castor oil, ricinoleyl alcohol, and methyl ricinoleate all show higher ethanol distribution coefficients, and similar or reduced separation factors, relative to other oils and derivatives studied here or reported by others. Of particular interest, ricinoleyl alcohol has an ethanol distribution coefficient 50% higher than that of oleyl alcohol, a commonly studied solvent for ethanol extraction from fermentation broths.     

Fatty acid distribution of fats,oils and soaps by high-performance liquid chromatography without derivatization

A high-performance liquid chromatography (HPLC) procedure without derivatization was developed for quantitating fatty acid components of various soap-related fats and oils, as well as for the direct quantitation of fatty acids from soap. The fatty acids are detected by refractive index after isocratic reverse-phase chromatography. The method has been developed with radial compression and stainless-steel column technology. The triglycerides are saponified and acid-hydrolyzed into fatty acids, and they are dissolved in a solvent and injected. The soaps are dissolved in methanol and injected into the HPLC, where they are acid-hydrolyzed directly on the column by an acid-modified mobile phase. The total run time after injection is approximately 20 min, with quantitation performed on an NEC Powermate^® computer driven by PE/Nelson Analytical Software. The typical carbon chains analyzed are from C6 to C20.     

High-performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, and pomace and 1D- and 2D-nuclear magnetic resonance characterization

Phenolic compounds are the most important antioxidants of virgin olive oil. This paper reports on the application of solid phase extraction (SPE) in the separation of phenolic compounds from olive fruit, olive oil, and by-products of the mechanical extraction of the oil and the complete spectroscopic characterization by nuclear magnetic resonance of demethyloleuropein and verbascoside extracted from olive fruit. SPE led to a higher recovery of phenolic compounds from olives than did liquid/liquid extraction. SPE also was used to separate phenolic compounds from pomaces and vegetation waters. Phenylacid and phenyl-alcohol concentrations in extracts obtained from SPE and liquid/liquid extraction were not significantly different (P<0.05). The recovery of the dialdehydic form of elenolic acid linked to 3,4-(dihydroxyphenyl)ethanol and an isomer of oleuropein aglycon, however, was low.     

Separation and identification of eugenol in ethanol extract of cloves by reversed-phase high-performance liquid chromatography

A method has been developed for the separation and identification of eugenol in the alcohol extract of cloves directly without having to previously separate other components. Extraction of eugenol with alcohol is followed by analysis with high-performance liquid chromatography. Separation by reversed-phase chromatography on low-polarity μBondapak C₁₈ was achieved with isocratic elution. Tentative identification is based on chromatographic appearance of a peak with retention time 5.54 min for pure standard reference eugenol. The identification of eugenol was confirmed by gas chromatography/mass spectrometry analysis.     

Separation of perillyl alcohol from the peel of citrus unshiu by supercritical CO2 and preparative high-performance liquid chromatography

To separate perillyl alcohol (POH), a potential anti-cancer agent, the peel of citrus unshiu was extracted by supercritical CO₂ extraction (SCE) system at 50 ‡C, 200 bar and 6 kg CO₂/hr/kg sample. The extracts were partitioned by acetonitrile/hexane (90/10, vol%). POH was eluted in the acetonitrile phase. An open-tubular chromatography with silica gel (40–63 Μm) was used to purify POH from the acetonitrile phase. Mobile phase was hexane/ethyl acetate (90/10, vol%). To obtain POH in a pure form, finally preparative high-performance liquid chromatography was applied. The collection of POH from citrus unshiu peel was achieved on a laboratory-prepared Chromatographic column (300x3.9 mm) packed with 15 Μm C₁₈ preparative packings. The composition of mobile phase was water/acetonitrile (50/50, vol%). The flow rate of the mobile phase was 1 ml/min and UV wavelength was fixed at 205 nm. It was found that the total yield of POH was 1.6xl0^-3 (wt%) as the dry powder of citrus unshiu peel.     

Pharmacokinetics and tissue distribution study of praeruptorin d from radix peucedani in rats by high-performance liquid chromatography (HPLC)

Praeruptorin D (PD), a major pyranocoumarin isolated from Radix Peucedani, exhibited antitumor and anti-inflammatory activities. The aim of this study was to investigate the pharmacokinetics and tissue distribution of PD in rats following intravenous (i.v.) administration. The levels of PD in plasma and tissues were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The biosamples were treated by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) and osthole was used as the internal standard (IS). The chromatographic separation was accomplished on a reversed-phase C(18) column using methanol-water (75:25, v/v) as mobile phase at a flow rate of 0.8 mL/min and ultraviolet detection wave length was set at 323 nm. The results demonstrate that this method has excellent specificity, linearity, precision, accuracy and recovery. The pharmacokinetic study found that PD fitted well into a two-compartment model with a fast distribution phase and a relative slow elimination phase. Tissue distribution showed that the highest concentration was observed in the lung, followed by heart, liver and kidney. Furthermore, PD can also be detected in the brain, which indicated that PD could cross the blood-brain barrier after i.v. administration.