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Rovi Gem E. Villame1
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 (α-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 25°C 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 (Gliszczyńska-Świgło 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, α-, β-, γ-, and δ for both tocopherol and tocotrioenol structures.
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a UV-Vis detector (SPD-10). The injection volume was 20-25 μL using a blunt end syringe (Hamilton). The analytical column was kept at 25°C. 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. 2000). MATERIAL S AND METHODS 2. ability to lower serum cholesterol. 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). They are present in most plant-based or plant-derived foods such as vegetable oils and nuts (Bramley et al. There are several types of HPLC systems employed in analytical procedures especially in the food industry Page 2 of 9 .1 Sample Preparation 0. The vitamin E (αtocopherols as based on the standard) present was identified by comparison of the retention times of the previously prepared standard. The mobile phase was analytical grade methanol (previously sonicated for at least 10 minutes to degas) and eluted at a flow rate of 1. 2. fiber and high concentration polyunsaturated fatty acids (PUFA) [Yoshida et al.5-g of each of the oil sample was prepared in 5. filtered in 0. The UV-Vis detector was set at 254 nm wavelength.It is said that Vitamin E are widely distributed in higher plants. carbohydrates.0 mL min-1.45μm membrane filter using a 5-mL disposable syringe. These produce are known to be inexpensive sources of important dietary nutrients and energy such as proteins. Hence. 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.6mm id). In this regard. The column was Zorbax ODS (150mm x 4. 2. 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. 2013]. 3. Resulting filtrate was collected for HPLC analysis. filtered and vortexed. RESULTS AND DISCUSSION Sample preparation The oil samples were prepared by mixing with a 5-mL methanol. the objective of the experiment is to quantify the vitamin E content of soybean and peanut oil samples employing the principles of HPLC.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).0 mL methanol. a recorder and integrator (C-R6A) and a manual injector valve with a 20μL injector loop. The total elution time was 10 minutes. Filtration was necessary to remove solid particles that may interfere during elution of components.
Polar solvents in RP-HPLC provide a competition for the adsorption sites for the analyte molecules. Going back to its increasing hydrophobicity.nowadays. 2011. Figure 2 Degree of polarity and volatility of different compound groups (Lifted from FST 202 HPLC Lecture Notes. the separation mode used was reversed-phase or RP-HPLC which employs a non-polar stationary bonded phase. adsorption. 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. Yoshida et al. it was therefore important to understand the nature including the structure and chemical components of vitamin E (as illustrated and explained in Figure 1). 2013) instead of gas chromatography which would entail derivatization of the compounds (Reuhs and Rounds 2010). Zorbax ODS and a polar mobile phase. hence increasing the degree of separation. 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. Boschin & Arnoldi. methanol. Since vitamin E was the compound of interest. (Reuhs and Rounds 2010). The principles behind these types are generally based on the physico-chemical principles in liquid chromatography such as ion exchange. 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. In this case. This could be the reason why most vitamin E analysis is mostly carried out by liquid chromatographic analyses (Gliszczyńska-Świgło et al. it can be perceived that it gears towards the region of increasing polarity or hydrophobicity and volatility. since vitamin E belongs to the fat soluble vitamin group as illustrated in Figure 2. 2007. In this particular experiment. 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. 2013. the separation of components depends on the degree of interaction with either the stationary phase or the mobile phase. size exclusion. Bele et al. etc. 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. 2014) Page 3 of 9 . Also. For instance. Also.
Also.g. Generally. it is illustrated in Figure 3 the schematic diagram and the basic parts of an HPLC system.g.g.e. One of its essential roles aside from support is the provision of suitable packing materials for effective separation of components. Figure 3 A schematic representation of an HPLC system and its basic parts (lifted from Nielsen. as well as the flow rate of the mobile phase depend on the interactions between the sample components and the stationary phase. a detector and a data integrator or computer system. it applies the concept of hydrophobic interactions in reversed-phase HPLC wherein a typical gradient elution profile may start from low (e. 95% acetonitrile over 25 minutes). The latter. For this particular example (using water:acetonitrile as gradient). precise manner.Meanwhile. 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. 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. more hydrophobic components will elute later in the chromatogram once the mobile phase gets more concentrated in acetonitrile (95% acetonitrile). 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. where mobile phase (or eluent) from the reservoir is delivered throughout the system at a specified flow rate in a controlled. 5% acetonitrile in water or aqueous buffer) to high eluting strength (e. on the other hand acts as the stationary phase and is considered as the ‘heart’ of the HPLC system. retention time). peak area. the gradient elution program is most widely used (Wang et al.0 mL min-1 for 10 minutes elution time) throughout the procedure. 4) detector. is a separation mode in which the mobile phase composition and flow rate is constant (e. The changes in the composition.g. 100% methanol at 1. starting at 90% methanol and ends at 10% methanol after 20 minutes elution). a column. 2013) and optimized because it can effectively elute all components in a sample and for optimal resolution of the chromatogram. A packing material forms the Page 4 of 9 . In most HPLC analytical procedures. 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. which was employed in this particular experiment. 3) column. 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). 2) the injector is used to place the sample into the flowing mobile phase for introduction into the column. an injector port. The HPLC system is composed of a 1) pump. which acts as both the support and the stationary phase. which translates the changes in sample concentration into electric signals and lastly. The column. it consists of a pump. 2010).
More than one type of detector may be used to provide further specificity and sensitivity for multiple types of analytes (Reuhs and Rounds 2010). However. Detection On another note. In this particular experiment. During analysis. electrochemical detectors.6mm id. etc. 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. therefore it is essential to choose an appropriate wavelength based on the type and nature of the analyte. to interpret the changes in component concentration in the effluent into electric signals. Page 5 of 9 . the UV-Vis absorption detector was used at 254 nm wavelength. UV absorbance varies with the wavelength used. If for example. HPLC systems employ various detectors such as UV-Vis absorption detectors. 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. The same is true with the column used in this experiment. refractive index or RI. the intensity of UV light observed for the mobile phase (without sample) and the eluent containing sample will differ. 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. 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). the sample passes through a flow cell. Figure 1 A Schematic diagram of the UV-Vis optical system (lifted from http://hitachi-hitec. fluorescence detectors. 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. Figure 3 illustrates the UV-Vis optical system which measures the absorbance of the sample component. Zorbax ODS with dimensions: 150mm x 4. Light from the lamp is shone onto the diffraction grating and is dispersed according to wavelengths.chromatographic bed and these should be of good chemical stability. As mentioned.. Thus. A standard UV detector can employ wavelengths between 195 to 370 nm. the measurement was set to 254 nm the angle of diffraction is adjusted so that 254 nm is projected through the flow cell. Also. sufficient resistance to withstand pressure during use and a narrow particle size distribution (Reuhs and Rounds 2010).6 or 5 mm.com).
(2013). 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 differed in the type of columns and mobile phases used.0 mL min-1. However in this study. The obtained linear correlation coefficient (r2) was 0.9918 indicating a positive linear relationship between the peak area and concentration of the prepared standards. which usually uses C18. Standard curve for Vitamin E (α-tocopherol).Analysis and quantification of Vitamin E content Varying concentrations (20-100 μg mL-1) of vitamin E (α-tocopherol) was prepared to construct a standard curve as shown in Figure 8.88) is higher than or twice as large as that of soybean oil (202. Andrés et al. (2013) as shown in Table 2. (β-+ γ-) or γ.6 mm) as column and mobile phase was acetonitrile and methanol (50:50 v/v) at a flow rate of 1. (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. 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. (2013). Page 6 of 9 . It is shown in Table 1. the other isomers of tocopherol were not distinguished due to limited availability of standards. the vitamin E content of peanut oil (437. Peak area Concentration (μg mL-1) Figure 8. used Alltima RP C-18 (250x4. Meanwhile. Having this information. In the study of Boschin & Arnoldi (2011) and Bele et al. 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. an isocratic elution system and flourimetric detector was used. Reverse-phase usually do not resolve the separation of all forms of vitamin E. lifted as reference standard curve to quantify the amount of vitamin E present in the sample obtained from HPLC. These methods were able to detect and separate α-.85) which also corresponds to their respective literature values lifted from Boschin & Arnoldi (2011) and Bele et al. However.8x – 20922. for analysis of peanut oil.tocopherols in reference to known standards.and δ. This relationship is described by the equation of the line y = 3424. Based on the results.
043 31336 6 5. This value may have corresponded to either of the other tocopherol isomers but since reverse-phase mode of separation was used.277 150118 5 7.01 19. the data in this experiment could only report the value of α-tocopherol since it is also the known standard used. This also explains the detection of a peak (Rt=7.45 Soybean Peanut Table 3.842 62555 Soybean 1 0.84). Area=48229) in peanut oil as seen in Table 3 which is perceived close to the Rt of the standard (Rt=7. Table 1.985 41 3 1.835 17383 Page 7 of 9 .15 7577 4 3. Rt (min) Area Peanut oil 1 2. these values are added together and as expressed as total tocopherols however.422 10 2 4 4.38+0.89+0.775 5747 1 0.2 γ-tocopherol (mg/100 g) 8.753 62 1 3 2.32±0.093 19 2 0. Sample Trial Peak No.12. Vitamin E (α-tocopherol) content of peanut and soybean oil samples obtained by HPLC.88 202.2 29.03 Total tocopherols (mg/100 g) 14.007 15670 5 7.12 48229 6 7.and δ-tocopherol values in soybean and peanut oil as lifted from Boschin & Arnoldi (2011) and Bele et al. HPLC data obtained from peanut and soybean oil sample employing reverse-phase mode of separation at 1.408 11 3 1. Normally.0 mL min -1 at 10 minutes elution time.22+0. (2013) α-tocopherol (mg/100 g) 0.827 292 7 7. The α-.282 40 2 0.185 9.055 107828 1 0.8 Vitamin E Peak Area 62555 17834 Vitamin E content (IU g-1 of sample) 437.85 Table 2.8 7. γ.Andrés et al.47 26 5 4. (2013) Sample Source Boschin & Arnoldi (2011) Bele et al.133 38 2 4 1.80+0. they were not readily distinguished.265 0.127 19326 oil 2 0.2 δ-tocopherol (mg/100 g) 4. Sample Peanut Soybean Vitamin E Rt (min) 7.91±0.533 73985 1 2 7. (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.
E.1016/j. 35 Niki. Dutra de Oliviera.4. Elsevier Inc. Arnoldi. P. Varelis. Handbook of food analysis instruments: high performance liquid chromatography and its application to the analysis of foods and beverages. Otero and S.and δ-tocopherol in vegetable oils in presence of hexadecyltrimethylammonium bromide/n-propanol in mobile phase. B. Desai. Lipids.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. Sci. A. 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. perhaps an attempt to further optimize the HPLC conditions such as the normal phase separation mode. and M. Tocopherol content in edible plant oils. Springer Science+BusinessMedia. Tocopherol content in vegetable oils using a rapid HPLC fluorescence detection method. Vol 1 Issue 3pp 231-238. Sikorsi. I. Reuhs. G. E. L. Khmelinskii and M. the vitamin E (α-tocopherol content) of peanut and soybean oil were 437. 157-161 Kamal-Eldin. Matea. Sikorska.. C. and Appelqvist. And in turn. J.P. 2010. Negrea. Pp. Vol 127 (2011). CONCLUSION Based on the results.88 IU g-1 and 202.016). No. Edited by: Semih Ӧtles. Not Bot Horti Agrobo. Essentials in modern HPLC separations.C.freeradbiomed.04. Salkeld. and A. Vol 41(1):93-96 Boschin. Edited by S. E. V. Rounds. R. 1199-1203. 57. A. USA. High performance liquid chromatography method for the simultaneous determination of α-. C.. 49.2010. J. Vitamin E content of crude and refined vegetable oils in Southern Brazil. Food Chem.. Food Comp. Gliszczyńska-Świgło. 1988. Legumes are valuable sources of tocopherols. London. Food Nutr. J. 4(A). The chemistry and antioxidant properties of tocopherols and tocotrienols. 2011. Assessment of antioxidant capacity in vitro and in vivo. Pp.. CRC Press Taylor & Francis Group.S. Raducu. 501-512 Page 8 of 9 . and V. 503-515 (2010) (DOI: 10. 2010. H. David. 2011. Bele. And Analysis. Pol. The Netherlands. Food Chem 126:1470-1474. Suzanne Nielsen. C. 2013. M. pp. I. Bhagavan. 5. 2013. Vera. REFERENCES: Andrés. 31. Miresan and O. J.A. S.D. Vol. 2007. Biol. γ. J. Med. Pp. Free Rad. J. Moldoveanu.. Food analysis 4th edition: high performance liquid chromatography. 671-701 (1996).
waters. Others: Vitamin E structures and chemistry.sigmaaldrich. < http://www.shu.shodex. 2013. Shuang and M.-Phase/rp_mobph.html > Accessed: February 16.com/global/science/lc/lc_basic_7. J.net/index. Waters. Undated. S. 2014.). 2014 Zorbax® HPLC Column <http://www. 2014. Tocopherol distributions and regiospecific profiles of fatty acids of Jack beans (Canavalia gladiata DC. H.chem. Sakamoto.com/waters/en_US/HPLC-SeparationModes/nav. I.hitachihitec.. 2013. Vol 1 (2). F. Pp 51-59. Kuriyama and Y. Mizushina. Pp. 7-11 Yoshida.htm> Accessed: February 17. ShodexTM. Detectors for HPLC.php?seitenid=1&applic=1485 > Accessed: February 15. 2014 HPLC Basic course: principle and featues of various detection methods. Choi.M. HPLC-UV quantitative analysis of acrylamide in baked and deep-fried Chinese foods. Yoshida..htm?cid=10049076&locale=en_US > Accessed: February 15. Y.uic. HPLC separation modes.html > Accessed: February 15. Feng. Canadian Journal of Plant Breeding. < http://www. Mobile Phases < http://hplc. Food and Composition and Analysis. H. N. 2014 Page 9 of 9 .edu/NEW/HPLC_Book/Rev. Vol 31.F. Guo.com/catalog/product/supelco/50254u?lang=en®ion=PH > Accessed: February 16. < http://www. < http://www. Y.Wang. 2014.edu/classes/phar/phar332/Clinical_Cases/vitamin%20cases/vitamin%20E/Vit amin%20E%20Chemistry.
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