Research Article Improved method for the determination of wax esters in vegetable oils Amalia A. Carelli, Erica B. Ba ¨umler and Liliana N. Ceci Planta Piloto de Ingenierı ´a Quı ´mica (PLAPIQUI, UNS-CONICET), Bahı ´a Blanca, Buenos Aires, Argentina In this work, a modified International Olive Council (IOC) method for wax determination involving a double-adsorbent layer of silica gel and silver nitrate-impregnated silica gel is presented (SN method). Column chromatography by the SN method did not show retention of wax esters standards with an even number of carbon atoms (C34–C44), observing recovery percentages higher than 90% even for unsaturated wax esters. All wax fractions were lower by the SN method than by the IOC method, resulting in a percentage decrease in the total wax content (olive oils: 20–50%, crude sunflower oil: 38%, crude soybean oil: 58% and crude grape seed oil: 13%). Olive oils analysed by the SN method showed increases of up to 27% in C40 relative percentage with respect to the IOC method. Additionally, decreases were observed by the SN method in the relative percentages for odd-carbon atom waxes for the seed oils in comparison to the IOC method (crude sunflower oil: 27%, crude soybean oil: 28% and crude grape seed oil: 13%). The main advantages of the proposed modification consist in its easy implementation and a better determination of wax esters (C34–C60) by controlling their complete recovery and removing interfering substances. The method is suitable for quality control and for authentication of olive oil and seed oils as well as in processing monitoring. Practical applications: The proposed method is useful in the quality, authentication and processing control of fruit and seed oils. Moreover, it can be an important tool for vegetable oil industries to control the efficiency of the wax separation process to prevent turbidity in the refined oil. Keywords: Argentation chromatography / GC / Olive oil / Seed oils / Wax esters Received: March 7, 2012 / Revised: June 4, 2012 / Accepted: June 26, 2012 DOI: 10.1002/ejlt.201200098 1 Introduction Vegetable oils contain a number of minor compounds that are removed along with the oil in the extraction process. Among them there is a group of esters of fatty acids and long-chain aliphatic alcohols known as wax esters. Wax esters in the range of 36–60 carbon atoms were reported for several vegetable oils [1–6]. Wax esters with a high melting point and low solubility in the oils contribute to the development of cloudiness when stored at low temperatures. Waxes with chain lengths lower than 40 carbon atoms (C40) are called soluble waxes, those with lengths between C40 and C43 are called partially soluble waxes and those with 44 or more carbon atoms are called crystallisable waxes. The last fraction is reduced by winter- ization or dewaxing in order to improve the cold stability of the oil. Thus refined oils retain mostly soluble waxes [1, 2]. The content and profile of the waxes depend on the oil origin, seed or fruit variety, pretreatment of the raw material and the temperature and technology used in the extraction process. Oils obtained by solvent extraction have a higher wax content than those obtained by mechanical extraction [6–8]. Therefore, the distribution and amount of waxes in a veg- etable oil is a good indicator of its quality and authentication. For example, the sum of C40, C42, C44 and C46 waxes is a parameter used to detect olive-pomace oil in virgin olive oil. This sum has been established as 250 mg/kg for edible virgin olive oils [9, 10]. Wax esters are very abundant in olive-pomace oil, whereas low concentrations are found in virgin oils [7, 8]. Extra virgin olive oils of premium quality Correspondence: Dr. Amalia A. Carelli, Planta Piloto de Ingenierı ´a Quı ´mica (PLAPIQUI, UNS-CONICET), Camino Carrindanga Km. 7, 8000 Bahı ´a Blanca, Buenos Aires, Argentina E-mail: acarelli@plapiqui.edu.ar Abbreviations: CC, column chromatography; CCGC, capillary column gas chromatography; CGC, capillary gas chromatography; CV, coefficients of variation; FID, flame ionization detector; GC, gas chromatography; GC-MS, gas chromatography-mass spectroscopy; IOC, International Olive Council; SCS, second centrifugation sludge Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000 1 ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com