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Analysis of Vitamin E content of soybean (Glycine max) and peanut (Arachis hypogea) oil by High Performance Liquid Chromatography

(HPLC)
Rovi Gem E. Villame1
1Food

Science Program, Graduate School, University of the Philippines Los Banos ABSTRACT

A reversed phase high performance liquid chromatography with UV-Vis detector for measuring vitamin E (-tocopherol) in soybean and peanut oil samples was conducted. Oil samples were diluted in methanol and after being vortexed and filtered, an aliquot of the overlay was injected directly into a Zorbax ODS column. Analytical grade methanol was used as a mobile phase with a flow rate of 1 mL min-1. Quantification of vitamin E was performed by UV-Vis detector at 254 nm wavelength. Vitamin E was detected and eluted at 25C in less than 10 min after injection. The resulting vitamin E content of soybean oil and peanut oil was 202.85 IU and 437.88 per gram of sample respectively. Keywords: vitamin E, tocopherol, soybean, peanut, HPLC 1. INTRODUCTION Vitamin E is an antioxidant in which its biological role is to prevent or retard lipid oxidation by scavenging free radicals (lipid peroxyl radicals) by donating hydrogen atoms and react with reactive oxygen and nitrogen species (Nikki, 2010). It is the collective term used to describe groups of compounds that occur naturally in plant materials all are derived from a 6chromanol with a 2-phytyl substituent (Gliszczyska-wigo et al. 2007). There are two types of vitamin E, the tocopherols which are vitamin E compounds with saturated (single bonds) phytyl chain while tocotrienols have three (3) bonds at the positions 3, 7 and 11 of the alkyl side chain.

Figure 1 Chemical structures of two types of Vitamin E (A) tocopherol and (B) tocotrienols (lifted from http://lipidlibrary.aocs.org/). Both structures are similar but tocopherols have saturated phytyl side chains while tocotrienols have isoprenyl side chains with three (3) double bonds in its chromanol ring. The different positioning of the methyl groups on the aromatic ring describes its vitamers, -, -, -, and for both tocopherol and tocotrioenol structures.

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It is said that Vitamin E are widely distributed in higher plants. They are present in most plant-based or plant-derived foods such as vegetable oils and nuts (Bramley et al. 2000). In this regard, examining the vitamin E content of some locally available legumes and nuts (soybean and peanuts) could be important in contributing to readily available baseline information for locally produced crops as sources of vitamin E. These produce are known to be inexpensive sources of important dietary nutrients and energy such as proteins, carbohydrates, ability to lower serum cholesterol, fiber and high concentration polyunsaturated fatty acids (PUFA) [Yoshida et al. 2013]. Hence, the objective of the experiment is to quantify the vitamin E content of soybean and peanut oil samples employing the principles of HPLC. 2. MATERIAL S AND METHODS 2.1 Sample Preparation 0.5-g of each of the oil sample was prepared in 5.0 mL methanol, filtered in 0.45m membrane filter using a 5-mL disposable syringe. Resulting filtrate was collected for HPLC analysis. 2.2 Analytical working conditions and reagents Separation by HPLC was carried out using a Shimadzu liquid chromatograph system equipped with an isocratic delivery pump (LC-9A), a UV-Vis detector (SPD-10), a recorder and integrator (C-R6A) and a manual injector valve with a 20L injector loop. The column was Zorbax ODS (150mm x 4.6mm id). The mobile phase was analytical grade methanol (previously sonicated for at least 10 minutes to degas) and eluted at a flow rate of 1.0 mL min-1. The analytical column was kept at 25C. The UV-Vis detector was set at 254 nm wavelength. The total elution time was 10 minutes. The injection volume was 20-25 L using a blunt end syringe (Hamilton). The vitamin E (tocopherols as based on the standard) present was identified by comparison of the retention times of the previously prepared standard. The concentration of the total vitamin E expressed in IU g-1 present in the samples was calculated using the linear equation obtained from the standard curve (calculated employing linear regression). 3. RESULTS AND DISCUSSION Sample preparation The oil samples were prepared by mixing with a 5-mL methanol, filtered and vortexed. Filtration was necessary to remove solid particles that may interfere during elution of components. It was also necessary to sonicate or degas the methanol solvent system before using in order to exhaust air bubbles which may in turn affect the results and since the HPLC system used is not equipped with a degasser. HPLC Principle High performance liquid chromatography or HPLC employs the principle of liquid chromatography with the application of high operating pressures (relatively high compared to gravity-mediated liquid chromatography) generated by the columns used. There are several types of HPLC systems employed in analytical procedures especially in the food industry

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nowadays. The principles behind these types are generally based on the physico-chemical principles in liquid chromatography such as ion exchange, size exclusion, adsorption, etc. (Reuhs and Rounds 2010). In this particular experiment, the separation mode used was reversed-phase or RP-HPLC which employs a non-polar stationary bonded phase, Zorbax ODS and a polar mobile phase, methanol. Polar solvents in RP-HPLC provide a competition for the adsorption sites for the analyte molecules, hence increasing the degree of separation. Also, the separation of components depends on the degree of interaction with either the stationary phase or the mobile phase. In this case, the choice and the type of the mobile and stationary phases greatly affect the retention and separation as well as the resolution of the chromatogram. For instance, the mobile phase can either promote or suppress the ionization of the analyte molecules or the stationary phase can affect the degree of separation of sample components. This is also highly influenced by the nature of the compounds to be analyzed and the number of compounds present in the sample to be analyzed. It is therefore essential to know and understand the chemical structure and polarity of the compound/(s) to be analyzed in choosing the right mobile and stationary phases as well as the optimization methods needed to be employed in the HPLC analysis. Since vitamin E was the compound of interest, it was therefore important to understand the nature including the structure and chemical components of vitamin E (as illustrated and explained in Figure 1). Also, since vitamin E belongs to the fat soluble vitamin group as illustrated in Figure 2, it can be perceived that it gears towards the region of increasing polarity or hydrophobicity and volatility. This could be the reason why most vitamin E analysis is mostly carried out by liquid chromatographic analyses (Gliszczyska-wigo et al. 2007, Boschin & Arnoldi, 2011, Bele et al. 2013, Yoshida et al. 2013) instead of gas chromatography which would entail derivatization of the compounds (Reuhs and Rounds 2010). Going back to its increasing hydrophobicity, it can be generally deduced that it will have more affinity towards the mobile phase (which is polar) and will therefore elute later in the chromatogram.

Figure 2 Degree of polarity and volatility of different compound groups (Lifted from FST 202 HPLC Lecture Notes, 2014)

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Meanwhile, it is illustrated in Figure 3 the schematic diagram and the basic parts of an HPLC system. Generally, it consists of a pump, an injector port, a column, a detector and a data integrator or computer system. The pump which delivers the mobile phase through the system can either be applied in two ways: 1) gradient elution program and the 2) isocratic elution program. The latter, which was employed in this particular experiment, is a separation mode in which the mobile phase composition and flow rate is constant (e.g. 100% methanol at 1.0 mL min-1 for 10 minutes elution time) throughout the procedure. While the other is a separation mode which varies the concentration and even the flow rate of the mobile phase by combining mobile phases from two or more reservoirs (e.g. starting at 90% methanol and ends at 10% methanol after 20 minutes elution). In most HPLC analytical procedures, the gradient elution program is most widely used (Wang et al. 2013) and optimized because it can effectively elute all components in a sample and for optimal resolution of the chromatogram. Also, it applies the concept of hydrophobic interactions in reversed-phase HPLC wherein a typical gradient elution profile may start from low (e.g. 5% acetonitrile in water or aqueous buffer) to high eluting strength (e.g. 95% acetonitrile over 25 minutes). The changes in the composition, as well as the flow rate of the mobile phase depend on the interactions between the sample components and the stationary phase. The separation would take place in a two-step process depending on the affinity of the components to either the stationary phase or the mobile phase. For this particular example (using water:acetonitrile as gradient), more hydrophobic components will elute later in the chromatogram once the mobile phase gets more concentrated in acetonitrile (95% acetonitrile).

Figure 3 A schematic representation of an HPLC system and its basic parts (lifted from Nielsen, 2010). The HPLC system is composed of a 1) pump, where mobile phase (or eluent) from the reservoir is delivered throughout the system at a specified flow rate in a controlled, precise manner; 2) the injector is used to place the sample into the flowing mobile phase for introduction into the column; 3) column, which acts as both the support and the stationary phase, usually constructed with stainless steel tubing with terminators that connects it between the injector and the detector (some HPLC systems employ guard columns or precolumns that are usually shorter and is placed in between the injector and the column to protect the column from strongly adsorbed components); 4) detector, which translates the changes in sample concentration into electric signals and lastly, the 5)data system or integrator which displays the chromatogram and provide information on the electronic signal related to the composition of the HPLC column effluent (i.e. peak area, retention time).

The column, on the other hand acts as the stationary phase and is considered as the heart of the HPLC system. One of its essential roles aside from support is the provision of suitable packing materials for effective separation of components. A packing material forms the

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chromatographic bed and these should be of good chemical stability, sufficient resistance to withstand pressure during use and a narrow particle size distribution (Reuhs and Rounds 2010). As mentioned, the choice of the appropriate column to be used for analytical procedures highly depends on the nature and polarity of the compound to be analyzed and the goals of the separation method. According to Reuhs and Rounds (2010) HPLC columns are usually built with stainless steel metal casing to provide protection from the pressure build-up inside during analysis and usually are 10, 15 or 25-cm long with an internal diameter (id) of 4.6 or 5 mm. The same is true with the column used in this experiment, Zorbax ODS with dimensions: 150mm x 4.6mm id. It is believed that using columns with smaller diameter with a decrease in the consumption of the mobile phase could result to increase resolution and peak area. Detection On another note, HPLC systems employ various detectors such as UV-Vis absorption detectors, fluorescence detectors, refractive index or RI, electrochemical detectors, etc., to interpret the changes in component concentration in the effluent into electric signals. More than one type of detector may be used to provide further specificity and sensitivity for multiple types of analytes (Reuhs and Rounds 2010). In this particular experiment, the UV-Vis absorption detector was used at 254 nm wavelength. Figure 3 illustrates the UV-Vis optical system which measures the absorbance of the sample component. The difference in the intensity of light scattered between the mobile phase and the sample is measured hence the amount of sample can be determined. Also, UV absorbance varies with the wavelength used, therefore it is essential to choose an appropriate wavelength based on the type and nature of the analyte. A standard UV detector can employ wavelengths between 195 to 370 nm. However, one of the most commonly used wavelengths is 254 nm (as used in this case) because this wavelength corresponds to the maximum emission or a mercury lamp that been applied as a UV source in simpler spectrophotometric analyses (Moldoveanu and David 2013).

Figure 1 A Schematic diagram of the UV-Vis optical system (lifted from http://hitachi-hitec.com). During analysis, the sample passes through a flow cell. Light from the lamp is shone onto the diffraction grating and is dispersed according to wavelengths. If for example, the measurement was set to 254 nm the angle of diffraction is adjusted so that 254 nm is projected through the flow cell. Thus, the intensity of UV light observed for the mobile phase (without sample) and the eluent containing sample will differ.

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Analysis and quantification of Vitamin E content Varying concentrations (20-100 g mL-1) of vitamin E (-tocopherol) was prepared to construct a standard curve as shown in Figure 8. The obtained linear correlation coefficient (r2) was 0.9918 indicating a positive linear relationship between the peak area and concentration of the prepared standards. This relationship is described by the equation of the line y = 3424.8x 20922.

Peak area

Concentration (g mL-1)

Figure 8. Standard curve for Vitamin E (-tocopherol), lifted as reference standard curve to quantify the amount of vitamin E present in the sample obtained from HPLC.

It is shown in Table 1, the computed values (kindly refer to Appendix I for sample computations) of Vitamin E content expressed in IU g-1 of sample of peanut and soybean oil employing HPLC with analytical working conditions described earlier. Based on the results, the vitamin E content of peanut oil (437.88) is higher than or twice as large as that of soybean oil (202.85) which also corresponds to their respective literature values lifted from Boschin & Arnoldi (2011) and Bele et al. (2013) as shown in Table 2. In the study of Boschin & Arnoldi (2011) and Bele et al. (2013), an isocratic elution system and flourimetric detector was used; however differed in the type of columns and mobile phases used. Boschin & Arnoldi for analysis soybean oil sample employed a Lichosorb Si60 column with a precoloumn Si60 and mobile phase n-hexane:isopropanol (98:2 v/v) with a flow rate of 1.0 mL min-1. While Bele et al. (2013), for analysis of peanut oil, used Alltima RP C-18 (250x4.6 mm) as column and mobile phase was acetonitrile and methanol (50:50 v/v) at a flow rate of 1.0 mL min-1. These methods were able to detect and separate -, (-+ -) or - and - tocopherols in reference to known standards. Having this information, it can be readily deduced that Boschin & Arnoldi (2011) employed a reversedphase mode of separation and would have been at least more comparable to this experiment for its soybean sample. However in this study, the other isomers of tocopherol were not distinguished due to limited availability of standards. Meanwhile, Andrs et al. (2011) explains that normal phase chromatography (opposite that of reverse-phase which uses a non-polar stationary phase and a polar mobile phase) could provide complete separation of all tocopherols than reverse-phase columns, which usually uses C18. Reverse-phase usually do not resolve the separation of all forms of vitamin E. However,

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Andrs et al. (2011) also argued that RP-HPLC is still more preferred over NP-HPLC of the reproducibility of the retention times and mechanical strength of the columns. This also explains the detection of a peak (Rt=7.12, Area=48229) in peanut oil as seen in Table 3 which is perceived close to the Rt of the standard (Rt=7.84). This value may have corresponded to either of the other tocopherol isomers but since reverse-phase mode of separation was used, they were not readily distinguished. Normally, these values are added together and as expressed as total tocopherols however, the data in this experiment could only report the value of -tocopherol since it is also the known standard used. Table 1. Vitamin E (-tocopherol) content of peanut and soybean oil samples obtained by HPLC.
Sample Peanut Soybean Vitamin E Rt (min) 7.8 7.8 Vitamin E Peak Area 62555 17834 Vitamin E content (IU g-1 of sample) 437.88 202.85

Table 2. The -, - and -tocopherol values in soybean and peanut oil as lifted from Boschin & Arnoldi (2011) and Bele et al. (2013)
Sample Source Boschin & Arnoldi (2011) Bele et al. (2013) -tocopherol (mg/100 g) 0.80+0.01 19.22+0.2 -tocopherol (mg/100 g) 8.89+0.185 9.320.2 -tocopherol (mg/100 g) 4.38+0.265 0.910.03 Total tocopherols (mg/100 g) 14.2 29.45

Soybean Peanut

Table 3. HPLC data obtained from peanut and soybean oil sample employing reverse-phase mode of separation at 1.0 mL min -1 at 10 minutes elution time. Sample Trial Peak No. Rt (min) Area Peanut oil 1 2.533 73985 1 2 7.055 107828 1 0.093 19 2 0.408 11 3 1.422 10 2 4 4.277 150118 5 7.12 48229 6 7.842 62555 Soybean 1 0.127 19326 oil 2 0.753 62 1 3 2.15 7577 4 3.007 15670 5 7.775 5747 1 0.282 40 2 0.985 41 3 1.133 38 2 4 1.47 26 5 4.043 31336 6 5.827 292 7 7.835 17383

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4. CONCLUSION Based on the results, the vitamin E (-tocopherol content) of peanut and soybean oil were 437.88 IU g-1 and 202.85 and IU g-1 respectively and that the vitamin E content of peanut oil is perceived to be almost twice as large as that of soybean oil. 5. RECOMMENDATION It is recommended to also quantify the concentration of other tocopherol isomers to produce a better data presentation on vitamin E content hence employment of the tocopherol isomer standards. And in turn, perhaps an attempt to further optimize the HPLC conditions such as the normal phase separation mode.

REFERENCES: Andrs, M.P.S., J. Otero and S. Vera. 2011. High performance liquid chromatography method for the simultaneous determination of -, - and -tocopherol in vegetable oils in presence of hexadecyltrimethylammonium bromide/n-propanol in mobile phase. J. Food Chem 126:1470-1474.

Bele, C., C. Matea, C. Raducu, V. Miresan and O. Negrea. 2013. Tocopherol content in vegetable oils using a rapid HPLC fluorescence detection method. Not Bot Horti Agrobo. Vol 41(1):93-96
Boschin, G. and A. Arnoldi. 2011. Legumes are valuable sources of tocopherols. J. Food Chem. Vol 127 (2011). Pp. 1199-1203. Desai, I.D., H. Bhagavan, R. Salkeld, J.E. Dutra de Oliviera. 1988. Vitamin E content of crude and refined vegetable oils in Southern Brazil. J. Food Comp. And Analysis. Vol 1 Issue 3pp 231-238. Gliszczyska-wigo, A., E. Sikorska, I. Khmelinskii and M. Sikorsi. 2007. Tocopherol content in edible plant oils. Pol. J. Food Nutr. Sci. Vol. 57, No. 4(A), pp. 157-161 Kamal-Eldin, A. and Appelqvist, L. The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids, 31, 671-701 (1996). Moldoveanu, S.C. and V. David. 2013. Essentials in modern HPLC separations. Elsevier Inc. The Netherlands. Pp. 35 Niki, E. Assessment of antioxidant capacity in vitro and in vivo. Free Rad. Biol. Med., 49, 503-515 (2010) (DOI: 10.1016/j.freeradbiomed.2010.04.016). Reuhs, B. and M.A. Rounds. 2010. Food analysis 4th edition: high performance liquid chromatography. Edited by S. Suzanne Nielsen. Springer Science+BusinessMedia. USA. Varelis, P. 2010. Handbook of food analysis instruments: high performance liquid chromatography and its application to the analysis of foods and beverages. Edited by: Semih tles. CRC Press Taylor & Francis Group. London. Pp. 501-512

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Wang, H., F. Feng, Y. Guo, S. Shuang and M.M.F. Choi. 2013. HPLC-UV quantitative analysis of acrylamide in baked and deep-fried Chinese foods. J. Food and Composition and Analysis. Vol 31. Pp. 7-11 Yoshida, H., N. Yoshida, Y. Sakamoto, I. Kuriyama and Y. Mizushina. 2013. Tocopherol distributions and regiospecific profiles of fatty acids of Jack beans (Canavalia gladiata DC.). Canadian Journal of Plant Breeding. Vol 1 (2). Pp 51-59. Others: Vitamin E structures and chemistry. Undated. < http://www.uic.edu/classes/phar/phar332/Clinical_Cases/vitamin%20cases/vitamin%20E/Vit amin%20E%20Chemistry.htm> Accessed: February 17, 2014 Zorbax HPLC Column <http://www.sigmaaldrich.com/catalog/product/supelco/50254u?lang=en&region=PH > Accessed: February 16, 2014. Mobile Phases < http://hplc.chem.shu.edu/NEW/HPLC_Book/Rev.-Phase/rp_mobph.html > Accessed: February 16, 2014 HPLC Basic course: principle and featues of various detection methods. < http://www.hitachihitec.com/global/science/lc/lc_basic_7.html > Accessed: February 15, 2014. Detectors for HPLC. ShodexTM. < http://www.shodex.net/index.php?seitenid=1&applic=1485 > Accessed: February 15, 2014. HPLC separation modes. Waters. < http://www.waters.com/waters/en_US/HPLC-SeparationModes/nav.htm?cid=10049076&locale=en_US > Accessed: February 15, 2014

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