Investigation of chlorophyll and beta carotenoids in edible oils by absorption and fluorescence spectroscopy

INVESTIGATION OF CHLOROPHYLL AND BETA CAROTENOIDS IN EDIBLE OILS BY ABSORPTION AND FLUORESCENCE SPECTROSCOPY By Tewodros Taye Assefa A THESIS SUBMITTED TO THE DEPARTMENT OF PHYSICS PRESENTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHYSICS ADDIS ABABA UNIVERSITY ADDIS ABABA, ETHIOPIA JUNE 2017 © Copy right by Tewodros Taye Aassefa, 2017 Declaration I, Tewodros Taye Assefa, declare that this thesis is my own work and all sources or materials used for this thesis have been duly acknowledged This thesis is submitted to the department of physics in Partial Fulfillment of the Requirements for the awarded Degree of Master of Science Signed by the Examination committee: Advisor: Name: Signature: Date: _ Examiner: Name: Signature: _Date: _ Examiner: Name: Signature: _Date: _ June, 2017 Addis Ababa – Ethiopia Table of content……………………………………………………………… page Acknowledgement i Abstract ii List of figures iii List of Table……………………………………………………………………………………… 1 Introduction 1.1 Background 1.1.1 Chlorophyll Benefits 1.1.2 Carotenoids Properties and Functions 1.1.3 -Carotene 1.1.4 Beta-carotene Benefits 1.2 Objective of the study General objective 2 Specific objectives 1.3 Literature review 1.3.1 Optical properties of chlorophyll and beta-Carotenoids of edible oils 1.4 Theoretical Background of the instruments 14 1.4.1 Absorption Spectroscopy 14 1.4.1.1 Absorbance and the Beer – Lambert Law 15 1.4.2 Fluorescence Spectroscopy 17 1.4.3 Excitation and Emission Spectra 19 1.4.3.1 Excitation Spectrum 19 1.4.3.2 Emission Spectrum 19 1.4.4 Stokes Shift 20 Material and methods 21 2.1 Materials 21 2.1.1 Oils… ………………………………………………………………………………….21 2.2 Instruments 22 2.2.1 Absorption spectra measurements 22 2.2.2 Fluorescence spectra measurements 22 2.3 Sample preparation 22 2.4 Data Collection and Spectral Data Analysis 23 2.5 Experimental set up of the instruments 24 3.Results and discussion 25 3.1 Absorption and fluorescence Spectra 25 3.1.1 Chlorophyll and beta-Carotenoid from extra virgin olive oil (Spain) 25 3.1.2 Chlorophyll and beta carotenoid from extra virgin olive oil (Italy)…………………… 28 3.1.3 Chlorophyll and beta-carotenoid from Niger seed oil (Ethiopia) 30 3.1.4 Chlorophyll and beta-carotenoid from olive oil (Spain) 32 3.1.5 Chlorophyll and Beta-carotenoid from olive pomace oil (Italy) 34 3.1.6 Chlorophyll and beta-carotenoid from composition of refined olive oil and virgin olive oil (Italy) 35 3.1.7 Chlorophyll and beta-carotenoid from sunflower oil (Ethiopia) 37 3.1.8 Chlorophyll and beta-carotenoid from soya bean oil (turkey) 38 3.9 Wavelengths of edible oils corresponding to their Absorbance maximum 39 3.10 Fluorescence Spectra of chlorophyll a and fatty acid composition in edible Oils 42 Conclusion 45 References 47 Acknowledgement Above all I thanks God; nothing can be done without his will Behind the realization of this thesis there are some wonderful people who must be mentioned and acknowledged, without them it would have been very challenging I want to forward a special recognition to my organization Federal Police Commission in general and Federal Police Crime Investigation Bureau in particular for giving me this opportunity I want to express my gratitude to my advisor A.V Gholap (Professor) for his support encouragements and guidance throughout this thesis work I express heartfelt appreciation to Mr.Tesfaye Mamo (chief technical assistance) for his great contribution and dedication, Yet again many thanks for inspiring me to throughout this thesis work i Abstract The absorption spectra of olive oil(Spain), extra virgin olive oil(Spain), extra virgin olive oil(Italy) , olive oil composed of refined olive oil and virgin olive oil(Italy), soya bean oil(turkey) , sunflower oil(Ethiopia), Niger seed oils (Ethiopia), were studied along with the relative peaks of chlorophyll a, chlorophyll b and beta-Carotenoids in each of the eight types of edible oils The oil was diluted by n-hexane (1% v/v) in a 10 mm quartz cuvette and the absorption spectrum of chlorophyll a, chlorophyll b and beta-Carotenoids was determined over a range of 400-750 nm The relative peaks in the blue region 410nm-414nm of chlorophyll a and in the red region chlorophyll a peaks appear from 660nm-671nm, chlorophyll b peaks appear in the blue region from 440nm-455nm and beta-Carotenoids peaks from 470nm-483nm were determined by using the given sample of edible oils based on the absorbance data The absorbance spectrum of most of the edible oils in the visible light range of chlorophyll and betacarotene gives interesting results The fluorescence spectrum of chlorophyll a and fatty acid composition of eight edible oil samples were obtained the emission spectra in the range from 420 to 750 nm, at excitation wavelengths from 350 to 420 nm, with a wavelength difference(steps) of 10nm, 15nm and 20nm There are two spectral regions in which Fluorescence spectra of edible oils are observed: The first one is a base width in the interval 640-750 nm and a top width 650–675nm with a maximum at 671 nm occurs for chlorophyll a The second one is a broad peak with a base width of in the interval 420-630 nm and a top width of 450–470 nm With a maximum at 466 nm, is due to the products of per oxidation (degradation) of polyunsaturated fatty acids in the oil Key words: Edible oils, absorbance spectroscopy, fluorescence spectroscopy, chlorophyll and beta- carotenoids ii List of figures Figure 1.1 molecular structure of chlorophyll a Figure 1.2 molecular structure of chlorophyll b Figure1 Structure of beta- carotenoid Figure 1.7 absorbance and beer Lambert law 15 Figure 1.8 Jablonski diagram……………………………………….…………………………………………… 17 Figure.1.9: Excitation and emission spectra of a fluorophore The emission spectra are plotted for excitation at three different wavelengths (EX1, EX2, and EX3) 19 Figure 1.10 Stokes shift difference between the excitation and emission spectrum 20 Figure 2.1 Experimental set up of absorption spectroscopy 24 Figure 2.2 Experimental set up of fluorescence spectroscopy 24 Figure 3.1 absorption spectrum of chlorophyll and beta-carotene from extra virgin olive oil (Spain) 26 Figure.3.2 fluorescence Spectrum of Chlorophyll a from extra virgin olive oil (Spain) 27 Figure 3.3 absorbance of chlorophyll and beta–carotenoid of extra virgin olive oil (Italy) 28 Figure 3.4 chlorophyll a fluorescence spectra of extra virgin olive oil (Italy) 29 Figure 3.5 Absorption Spectrum of Chlorophyll and beta-carotenoid Niger seed oil 30 Figure 3.6 fluorescence spectrum of chlorophyll a of Niger seed oil 31 Figure 3.7 Absorption Spectrum of Chlorophyll and beta-carotenoid from olive oil 32 Figure 3.8 chlorophyll a fluorescence of olive oil (Italy) 33 Figure 3.9 Absorption Spectrum of Chlorophyll and beta-carotenoid from olive pomace oil 34 Figure 3.10 chlorophyll a fluorescence spectra of olive pomace oil 35 Figure 3.11 Absorption Spectrum of Chlorophyll and beta carotenoid from composition of Refined olive oil and virgin olive oil……………………………………… 35 Figure 3.12 chlorophyll a fluorescence spectra of composition of refined olive oil and virgin Olive oil……………………………………………………………………… 36 Figure 3.13 Absorption Spectrum of Chlorophyll and beta- carotenoid from sunflower oil 37 Figure 3.14 fluorescence of chlorophyll a of sunflower oil………………………………… 37 Figure 3.15 Absorption Spectrum of Chlorophyll and beta carotene from soya bean oil 38 Figure 3.16 shows that the fluorescence of soya bean oil 39 Figure 3.17 shows wave length maxima from extra virgin olive oil and Niger seed oil… 40 Figure 3.18 wavelength maxima of extra virgin olive oil (Italy), olive oil (Spain), composition Of refined and virgin olive oil (Italy) and olive pomace oil………………… 41 Figure 3.19 wavelength maxima of sunflower (Ethiopia) and soybean oil (turkey) 41 iii Figure 3.20 Fluorescence of the eight edible oils 43 List of table Table 3.1 Wavelengths of edible oils corresponding to their Absorbance maxima 42 iv Introduction 1.1 Background Chlorophyll is any of several closely related green pigments found in cyanobacteria and the chloroplasts of algae and plants Chlorophyll is essential in photosynthesis, allowing plants to absorb energy from light Chlorophyll absorbs light most strongly in the blue portion of the electromagnetic spectrum, followed by the red portion Conversely, it is a poor absorber of green and near-green portions of the spectrum, which it reflects, producing the green color of chlorophyll-containing tissues Chlorophyll was first isolated and named by Joseph Bienaimé Caventou and Pierre JosephPelletier in1817 (Speer, Brian R 1997) Molecules that are good absorbers of light in the visible range are called pigments Organisms have evolved a variety of different pigments, but there are only two general types used in green plant photosynthesis: Carotenoids and chlorophylls There are two kinds of chlorophyll in plants, chlorophyll a and chlorophyll b, which preferentially absorb blue and red light Chlorophylls absorb photons within narrow energy ranges Neither of these pigments absorbs photons with wavelengths between 500 - 600 nm, and light of these wavelengths is, therefore, reflected by plants When these photons are subsequently absorbed by the pigment in our eyes, we perceive them as green Chlorophyll a is the main photosynthetic pigment and is the only pigment that can act directly convert light energy to chemical energy However, chlorophyll b, acting as an accessory or secondary light-absorbing pigment, complements and adds to the light absorption of chlorophyll a Chlorophyll b has an absorption spectrum shifted toward the green wavelengths Therefore, chlorophyll b can absorb photons that chlorophyll a cannot Chlorophyll b therefore greatly increases the proportion of the photons in sunlight that plants can harvest An important group of accessory pigments, the Carotenoids, assists in photosynthesis by capturing energy from light of wavelengths that are not efficiently absorbed by either chlorophyll (C B van Niel, 1930) The absorption of light by different pigments causes excitation of electrons from their ground state to an excited state Light absorption takes place at the reaction centers of photo systems that contain accessory and primary pigments (Gross, Jeana, 1991) All green plants contain chlorophyll a and chlorophyll b in their chloroplasts Chlorophyll b differs from chlorophyll a by having an aldehyde (-CHO) group in place of a methyl group (-CH3) as shown the figure 1.1 & 1.2 below This aldehyde group is also the reason that chlorophyll b has a greater molecular weight than chlorophyll a Along with chlorophylls, the chloroplast also contains a family of accessory pigments called Carotenoids (Campbell, N.A 1996) In higher plants, chlorophyll a is the major pigment and chlorophyll b is an accessory pigment The structural formula of Chlorophyll a is C55H72O5N4Mg with a molecular weight of 893.48 g/mol and Chlorophyll b has a structural formula of C55H70O6N4Mg and a molecular weight of 907.46 g/mol (Paech K and M.V Tracey 1955) The differences in these structures cause the red absorption maximum of chlorophyll b to increase and lower its absorption coefficient (Goodwin, T.W 1965) Chlorophyll pigments strongly absorb in the red and blue regions of the visible spectrum, which accounts for their green color Figure 1.1 molecular structure of chlorophyll a Figure 1.2 molecular structure of chlorophyll b Figure 3.11 shows the absorption spectrum of Chlorophyll from composition of refined olive oil and virgin olive oil In the blue region chlorophyll a absorbs in the absorbance range of 0.4830.906 at a wavelength 412.4nm, with absorbance maximum 0.906, while in the red region it absorbs in the range of 0.158-0.333 at a wavelength 670.8 nm, with absorbance maximum 0.333.In addition, there is no visible absorbance maxima of chlorophyll b in the blue region and beta-carotenoid absorbs in the absorbance ranges from 0.210-0.393 the peak at 480.8nm with absorbance maximum 0.393 Figure 3.12 chlorophyll a fluorescence spectra of composition of refined olive oil and virgin olive oil The figure.3.12 shows that the fluorescence spectrum of chlorophyll a in composition of refined and virgin olive oil at λex=412nm and emission spectra in the range 420 to 750 nm There are two emission peaks located between 420 to 750 nm The peak located at 671 nm is pigments of the chlorophyll a and another peak at 466nm it is composition of fatty acids groups The emission spectra were measured in the range from 420 to 750 nm Stokes shift = ( =( =107( ) - =9345.1 (cm-1) 36 ) ) 3.1.7 Chlorophyll and beta-carotenoid from sunflower oil (Ethiopia) Figure 3.13 Absorption Spectrum of Chlorophyll and beta- carotenoid from sunflower oil Figure3.13 shows that the chlorophyll and beta carotenoid in sunflower oil has absorbance maxima in both the blue and red regions of the visible spectrum In the blue region there is no chlorophyll and beta-carotenoid absorbs maximally, while in the red region chlorophyll a absorbs in the absorbance range from 0.132-0.143 at a wavelength 664 nm with absorbance maximum 0.143 Figure 3.14 fluorescence of chlorophyll a of sunflower oil 37 Figure 3.14 shows fluorescence spectra of Sunflower oil at λex=410nm and emission spectra in the range 420 to 750 nm, the fluorescence peaks at 467 nm and 669 nm are observed The peak at 669nm is the pigment of chlorophyll a where the peak at 467 is due to the high products of per oxidation (degradation) of polyunsaturated fatty acids in the oil (oleic and linoleic) 3.1.8 Chlorophyll and beta-carotenoid from soya bean oil (turkey) Figure 3.15 Absorption Spectrum of Chlorophyll and beta carotene from soya bean oil Figure 3.15 shows that the excitation spectra in the range from 350 to 750 nm Chlorophyll and beta-carotenoid of soya bean oil has absorbance maxima in both the blue and red regions of the visible spectrum there is no chlorophyll and beta- carotenoid peaks absorbs 38 Figure 3.16 shows that the fluorescence of soya bean oil Figure 3.16 shows that the Fluorescence spectra chlorophyll a of soybean oil at λex=411nm and emission spectra from 420-750nm is a weak, long-wavelength peak with a maximum at 660 nm is observed for soya bean oil is the pigments of the chlorophyll a and an additional peak of a large base width a maximum at 466 nm is fatty acid composition The presence of chlorophylls and beta-Carotenoids group of in this oil is absent in the uv-visible absorbance maxima, soya bean oil is different from other edible oils it is less chlorophyll groups and less florescence peaks comparing with other edible oils 3.9 Wavelengths of edible oils corresponding to their Absorbance maximum Visible spectra were recorded in the range of wave length 375–750 nm In figure below (3.173.19) of obtained spectral curves regions of maximum absorption can be seen: 410 to 415 nm, 445 to 455nm, 470 to 483 nm, 530 to 540 nm, 560 to 565 nm, 610 to 615 nm, and 660 to 673 nm For clarity, the absorbance maxima were divided into three groups according to wavelength (Tab 1) 39 Visible spectra absorption (375 - 750 nm) of edible oils Figure 3.17 wavelength maxima of extra virgin olive oil and Niger seed oil 40 Figure 3.18 wavelength maxima of extra virgin olive oil (Italy), olive oil (Spain), composition of refined and virgin olive oil (Italy) and olive pomace oil Figure 3.19 wavelength maxima of sunflower (Ethiopia) and soybean oil (turkey) 41 Table 3.1 Wavelengths of edible oils corresponding to their Absorbance maxima Edible oils Maximum wavelength (nm) violet- Blue Green- Yellow Orange-Red Extra virgin olive oil(Spain) 412, 444, 474 532*,561*, 611* 669.0 Niger seed oil(Ethiopia) 414, 446, 473 532*, 561*,611* 669.0 Extra virgin olive oil(Italy) 412.8, 448.8, 474 535.2*,560.8*, 610* 669.6 Olive oil(Spain) 411.2,452.8,482.4 535.2*, 604.8* 670.0 Olive pomace oil(Italy) 537.2*,608.4* 670.4 Composition of refined and 412.4, 480.8 536.4*,560.4*,610.8* 670.8 virgin olive oil(Italy) Sunflower oil(Ethiopia) - - 664.0 Soybean oil(turkey) - - - The results from the above table obtained allow us to formulate the following conclusions The visible spectra of edible oils in the visible range provide a basis for grouping of edible oils in three basic groups depending on the fact, which of the absorbance peak dominates 3.10 Fluorescence Spectra of chlorophyll a and fatty acid composition in edible Oils Chlorophyll a is a specific form It absorbs most energy from wavelengths of violet-blue and orange-red light It also reflects green/yellow light, and as such contributes to the observed green color of most plants Fluorescence spectra chlorophyll a of extra virgin olive oil(Spain),extra virgin olive oil((Italy), composition of refined olive oil and virgin olive oil(Italy), olive oil(Spain), sunflower oil(Ethiopia), Niger seed oil(Ethiopia), olive pomace oil(Italy) and soybean oil(turkey) were examined 42 The representative fluorescence spectrums of all examined edible oils are presented in Figure 3.20 below The excitation spectra measured in the range from 350 to 415 nm and the emission spectra in the range from 420 to 750 nm with a step difference of 10,15and 20 There are two spectral regions are observed Figure 3.20 Fluorescence of the eight edible oils  The first one is a base width of in the interval 640-750 nm and a top width of 650–675nm Peak of almost all olive oils and Niger seed oil samples examined in this series of experiments occurred at 671 nm, indicating that the chlorophyll a has almost the same concentration in all these samples However, the fluorescence spectra of soya bean oil samples were different and had low overall chlorophyll a compared with olive oils and Niger seed oil Moreover, the peak of fluorescence spectra for soya bean samples occurred at 660nm 43  The second is a broad peak with a base width of in the interval 420-630 nm and a top width of 450–470 nm With a maximum at 466 nm for soya bean oil; emission spectra in the range 450– 630 nm, is good separation between each edible oils The shape and intensity of these emissions varies from one oils to another The analysis of these spectra shows that chemical compounds responsible for this emission in different oils may belong to the same class, although their chemical structure may vary slightly to cause the variations observed, mainly shows peaks due to oxidation products 44 Conclusion Chlorophylls and beta-Carotenoids are pigments that give color to vegetables and several fruits, where they play key roles in photosynthesis Chlorophyll pigments are responsible for the greenish hues of edible oils The existence of chlorophyll in edible oil give rise to this study to know their composition level in the eight types of edible oils selected for this study The composition of chlorophyll is studied by using Absorption and fluorescence spectroscopy Moreover the fatty acid compositions in the eight types of edible oils were also examined through fluorescence spectroscopy Absorbance spectroscopy measures how much of a particular wavelength of light gets absorbed by a sample It’s usually used to measure the concentration of a compound in a sample so, the more light that is absorbed, the higher the concentration of the compound in the sample Absorption of energy causes transitions between electronic energy levels from the ground state to various excited states The absorption spectra of chlorophyll and beta- Carotenoids pigment from the samples of each edible oil absorption is with maxima in the visible spectrum over the range of 375 to 750 nm, olive oil group and Niger seed oils have much chlorophyll and beta-Carotenoids while soya bean oils have lower chlorophyll and beta-carotene to compared to others oils Looking at the composition of chlorophyll and beta-Carotenoids found in Niger seed oil pigments are similar in content with those imported high quality edible oils like extra virgin olive oil Due to their molecular structure, chlorophylls are good fluorescent probes Chlorophyll content is also an important quality control parameter in the edible oils This study has found out that, a long-wavelength band, at 350–420 nm in excitation and 660–750 nm in emission is presented in olive oil group and seed oil (Niger seed, sunflower and soya bean) oils, are characteristic for fluorescence of the chlorophyll a pigments This group includes chlorophylls a and b The spectra of oils reveal an additional emission band in the intermediate region, at about 420–600 nm The shape and intensity of this intermediate emission vary for different oils Looking at the fatty acid composition in the edible oils, extra virgin olive oil (Spain and Italy) have low fatty acid composition with high chlorophyll While soya bean oil has high fatty acid composition with low chlorophyll 45 The existence of chlorophyll a in the edible oils chosen for this study has been analyzed in both Absorption and fluoresce spectroscopy The findings in these two instruments were the same in the level of composition those edible oil 46 References  Armstrong GA, Hearst JE (1996) Carotenoids 2: Genetics and molecular biology of carotenoid pigment biosynthesis  B Schoefs (2003) Chlorophyll and carotenoid analysis in food products Properties of the pigments and methods of analysis  B Schoefs(2004), Advanced Food Sci Nutrition  B.Schoefs(2004) Determination of pigments in vegetables  Benjamin/Cummings(1996) Campbell, N.A Biology 4th Edition, Menlo Park, CA  Bertram JS (1999) Carotenoids and gene regulation Nutrition Stahl W, Nicolai S, Briviba K, et al Biological activities of natural and synthetic carotenoids  Buschmann, C (2007) Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves  C B van Niel(1930) The Role of Light  Carotenoids Functionality, Sources, and Processing by Supercritical Technology: A Review Natália Mezzomo and Sandra R S Ferreira EQA-CTC/UFSC, Chemical and Food Engineering Department, Federal University of Santa Catarina, CP 476, 88040-900 Florianopolis, SC, Brazil  Carotenoids Volume 5: Nutrition and Health Edited by G Britton S Liaaen-Jensen H Pfander Birkhäuser Verlag Basel · Boston · Berlin  Cayuela J.A., ousf K., Martinez M.C., Garcia J.M (2014) Rapid determination of olive oil chlorophylls and carotenoids by using UV‐Vis spectroscopy  Cristina Lazzerini, Mario Cifelli and Valentina Domenici(2016), Pigments in Extra‐Virgin Olive Oil: Authenticity and Quality  deMan, J M (1999) Principles of food chemistry New York: Kluwer Academic/Plenum Publishers  Diffraction Grating Spectrometer Design and Collected Spectra Theremino System – Spettrometro_ENG Spectroscopy of Edible Oils  Dorothy H Forster(2008) Nutrition and the Imprisoned SplendourThe Green and White Diet 47  E Marasco and C Schmidt-Dannert(2003) Towards the biotechnological production of aroma and flavor compounds in engineered microorganisms Applied Biotechnology Food Science and Policy, vol  Ewa Sikorska (2003) characterization of edible oils using synchronous scanning fluorescence spectroscopy  Ewa Sikorska (2004,2005), Classification of edible oils using synchronous scanning fluorescence spectroscopy  Francesca Guimet(2005) Rapid detection of olive–pomace oil adulteration in extra virgin olive oils from the protected denomination of origin Siurana using excitation–emission fluorescence spectroscopy and three-way methods of analysis  Gandul‐Rojas B., Roca‐L (2000) Cepero M., Mínguez‐Mosquera M.I Use of chlorophyll and carotenoid pigment composition to determine authenticity of virgin olive oil  Getinet, A and A Teklewold( 1995) An agronomic and seed-quality evaluation of Niger(Guizotia abyssinica Cass.) germ plasm grown in Ethiopia  Giuliani A (2011) Chlorophylls in olive and in olive oil: chemistry and occurrences Critical Reviews in Food Science and Nutrition  Goodwin (1965) Chemistry and Biochemistry of Plant Pigments  Guimet F., Ferre J., Boque R.; Vidal M & Garcia J (2005) Excitation-Emission Fluorescence Spectroscopy Combined with Three-Way Methods of Analysis as a Complementary Technique for Olive Oil Characterization  H., Keiler M (2006) A UV-Vis Spectroscopic Study." University of Wisconsin-La Crosse  Hughes DA, Wright AJ, Finglas PM, (1997) The effect of beta-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers  J Toxicol Clin Toxicol (2003) Sudakin DL Dietary aflatoxin exposure and chemoprevention of cancer  James K Daun Spectrophotometric analysis of chlorophyll pigments in canola and rapeseed oils  Jose´ A.(2014) Rapid Determination of Olive Oil Chlorophylls and Carotenoids by Using Visible Spectroscopy  Kidd, Parris (2011) Astaxanthin, Cell Membrane Nutrient with Diverse Clinical Benefits and Anti-Aging Potential 48  Krastena Nikolova(2012), Quick Fluorescence Method for the Distinguishing of Vegetable Oils  Kyriakidis, N and B, Skarkalis(2000), P Fluorescence spectra measurement of olive oil and other vegetable oils  Lichtenthaler ,H.K., & Wellburn, A.R Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents: Biochemical Society Transactions  M Rahmani and A Saari Csallany(1998) Role of Minor Constituents in the Photooxidation of Virgin Olive Oil  Matthews CK, van Holde Ke (1996) Biochemistry 2nd ed  Maurizio Zandomeneghi (2005), Fluorescence of Vegetable Oils:  Olive Oils  N Mezzomo, L Tenfen, M S Farias, M T(2015).“Evidence of antiobesity and mixed hypolipidemic effects of extracts from pink shrimp (Penaeus brasiliensis and Penaeus paulensis) processing residue  Nicole M Gerardo ( 2011).Horizontally transferred fungal carotenoid genes in the twospotted spider mite Tetranychus urticae Boran Altincicek,  Nicole M Gerardo (2011) Horizontally transferred fungal carotenoid genes in the twospotted spider mite Tetranychus urticae  Paech K and M.V Tracey( 1955) Modern Methods of Plant Analysis  Papageorgiou, G C Govindjee (2004) Chlorophyll a fluorescence: a signature of photosynthesis  Poulli K I.; Mousdis G A & Georgiou C A (2005) Classification of Edible and Lampante Virgin Olive Oil Based on Synchronous Fluorescence and Total Luminescence Spectroscopy  Primer Tony Owen (2000)Fundamentals of modern UV-visible spectroscopy  Ramon Aparicio , Ramon Aparicio-Ruız (2000)Authentication of vegetable oils by chromatographic techniques  Roldan B & Cáceres M I R (2003) Simultaneous Fluorometric Determination of Chlorophylls A and B and Pheophytins A and B in Olive Oil by Partial Least-Squares Calibration 49  Romdhane Karoui & Christophe Blecker (2010)Fluorescence Spectroscopy Measurement for Quality Assessment of Food Systems  Sayago, A.; Morales, M T.; Aparicio, R (2004) Detection of hazelnut oil in virgin olive oil by a spectrofluorimetric method  Scott SM (2003) Total luminescence spectroscopy with pattern recognition for classification of edible oils  Speer, Brian R (1997) "Photosynthetic Pigments"  Susan D Van Arnum (1998), "Vitamin A", Kirk-Othmer Encyclopedia of Chemical Technology  Teresa Galeano Diaaz (2003) Simultaneous Fluorometric Determination of Chlorophylls a and b and Pheophytins a and b in Olive Oil by Partial Least-Squares Calibration  Torrecilla J.S A (2010) novel method to quantify the adulteration of extra‐virgin olive oil  with low grade olive oils by UV‐Vis  Van Poppel G, Spanhaak S, Ockhuizen T (1993) Effect of beta-carotene on Van Nostrand Reinhold (1991) Pigments in Vegetables: Chlorophylls and Carotenoids immunological indexes in healthy male smokers  W A Schroeder and E A Johnson(1995) Singlet oxygen and peroxyl radicals regulate carotenoid biosynthesis in Phafa rhodozyma  Zandomeneghi M., Carbonaro L., Caffarata C (2005) Fluorescence of vegetable oils: oliveoils 50 ... most of the edible oils in the visible light range of chlorophyll and betacarotene gives interesting results The fluorescence spectrum of chlorophyll a and fatty acid composition of eight edible. .. properties of chlorophyll and beta- Carotenoids of edible oils Fats and oils constitute one of the major categories of food products, as they contain many nutrients The great interest in studying the... an absorption spectrum of Chlorophyll a , Chlorophyll b and beta- carotene in the visible range of wavelengths of light absorbed by the molecules  To determine chlorophyll a and fatty acid by fluorescence