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

The absorption spectra of olive oilSpain, extra virgin olive oilSpain, extra virgin olive oilItaly , olive oil composed of refined olive oil and virgin olive oilItaly, soya bean oilturke

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

I, Tewodros Taye Assefa, declare that this thesis is my own work and all sources or materialsused for this thesis have been duly acknowledged This thesis is submitted to the department ofphysics 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

1 Introduction 1

1.1 Background 1

1.1.1 Chlorophyll Benefits 3

1.1.2 Carotenoids Properties and Functions 4

1.1.3 -Carotene 5

1.1.4 Beta-carotene Benefits 6

1.2 Objective of the study 7

1 2 1 General objective 7

1 2 2 Specific objectives 7

1.3 Literature review 7

1.3.1 Optical properties of chlorophyll and beta-Carotenoids of edible oils 7

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

2 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

4 Conclusion 45

5 References 47

Above all I thanks God; nothing can be done without his will Behind the realization of thisthesis 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 generaland Federal Police Crime Investigation Bureau in particular for giving me this opportunity

I want to express my gratitude to my advisorA.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 greatcontribution and dedication, Yet again many thanks for inspiring me to do throughout this thesiswork

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 beta- carotene 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

List of figures

Figure 1.1 molecular structure of chlorophyll a 2

Figure 1.2 molecular structure of chlorophyll b 2

Figure1 6 Structure of beta- carotenoid 5

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

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

Caventou and Pierre JosephPelletier in1817 (Speer, Brian R 1997).

Molecules that are good absorbers of light in the visible range are called pigments Organismshave evolved a variety of different pigments, but there are only two general types used in greenplant photosynthesis: Carotenoids and chlorophylls There are two kinds of chlorophyll in plants,chlorophyll a and chlorophyll b, which preferentially absorb blue and red light Chlorophyllsabsorb photons within narrow energy ranges Neither of these pigments absorbs photons withwavelengths between 500 - 600 nm, and light of these wavelengths is, therefore, reflected byplants When these photons are subsequently absorbed by the pigment in our eyes, we perceivethem as green Chlorophyll a is the main photosynthetic pigment and is the only pigment that canact directly convert light energy to chemical energy However, chlorophyll b, acting as anaccessory or secondary light-absorbing pigment, complements and adds to the light absorption ofchlorophyll a Chlorophyll b has an absorption spectrum shifted toward the green wavelengths.Therefore, chlorophyll b can absorb photons that chlorophyll a cannot Chlorophyll b thereforegreatly increases the proportion of the photons in sunlight that plants can harvest An importantgroup of accessory pigments, the Carotenoids, assists in photosynthesis by capturing energy fromlight 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 theirground state to an excited state Light absorption takes place at the reaction centers of photosystems 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 amethyl group (-CH3) as shown the figure 1.1 & 1.2 below This aldehyde group is also thereason that chlorophyll b has a greater molecular weight than chlorophyll a Along withchlorophylls, 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 893.48g/mol and Chlorophyll b has a structural formula of C55H70O6N4Mg and a molecular weight of907.46 g/mol (Paech K and M.V Tracey 1955) The differences in these structures cause thered 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 thevisible spectrum, which accounts for their green color

Figure 1.2 molecular structure of chlorophyll b Figure 1.1 molecular structure of chlorophyll a

During storage, chlorophyll encounters many elements, which cause degradation.Chlorophyll a is formed about five times faster than chlorophyll b Chlorophyll is present in theseed being part of the chloroplasts present in the cotyledons While the amount of chlorophyll inseeds decreases during maturation, significant amounts are often left, especially in areas wherethe seed is maturing and being harvested into a cool fall or winter season The problem is mostprominent in B napus L summer sown seed types but may occur in other species and typesoccasionally (James K Daun 2012).

Carotenoids are organic pigments that are produced by plants and algae, as well as severalbacteria and fungi Carotenoids can be produced from fats and other basic organic metabolicbuilding blocks by all these organisms Carotenoids from the diet are stored in the fatty tissues ofanimals, and exclusively carnivorous animals obtain the compounds from animal fat (BoranAltincicek 2011) There are over 700 known Carotenoids; they are splits into two classes,xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, andcontain no oxygen) All are derivatives of tetraterpenes, meaning that they are produced from

8 isoprene molecules and contain 40 carbon atoms In general, Carotenoids absorb wavelengthsranging from 400-520 nanometers (violet to green light) in the visible spectrum This causes the

compounds to be deeply colored yellow, orange, or red (Armstrong GA, Hearst JE 1996).

Carotenoids serve two key roles in plants and algae: they absorb light energy for use inphotosynthesis, and they protect chlorophyll from photo damage Carotenoids that containunsubstitude beta-ionone rings (including beta-carotene,carotene, beta-cryptoxanthin and gamma-carotene) have vitamin A activity (meaning that they can be converted to retinol), and these andother Carotenoids can also act as antioxidants In the eye, certain other Carotenoids(lutein, astaxanthin, and zeaxanthin) apparently act directly to absorb damaging blue and near-ultraviolet light, in order to protect the macula of the retina, the part of the eye with the sharpest

vision.( Armstrong GA, Hearst JE 1996).

1.1.1 Chlorophyll Benefits

Several research results have demonstrated that plant pigments play important roles in health Ithas been reported that chlorophyll concentrations encountered in chlorophyll-rich green

vegetables can provide substantial cancer chemo protection, and suggested that they do so byreducing carcinogen bioavailability (Chipmunka, 2008).

 Chlorophyll benefits include helping fight cancer, improving liver detoxification, speeding up

wound healing, improving digestion and weight control, and protecting skin health (Sudakin DL

2003)

 Chlorophyll is a super food because of its strong antioxidant and anticancer effects

 Chlorophyll benefits the immune system because it’s able to form tight molecular bonds with

certain chemicals that contribute to oxidative damage and diseases like cancer or liver disease(Kidd, Parris 2011)

 The very best sources of chlorophyll are found on the planet are green vegetables and algae

Here are some of the top food sources to incorporate into your diet to experience all of thechlorophyll benefits This includes dark green leafy veggies like kale, spinach and Swiss chard.Cooking these foods decreases the nutrient content and lowers the chlorophyll benefits, so eatthem raw or lightly cooked to preserve the nutrients (Kidd, Parris 2011, Boran Altincicek 2011,

Sudakin DL 2003)

1.1.2 Carotenoids Properties and Functions

Due to the coloring properties of Carotenoids, they are often used in food, pharmaceutical andcosmetics In addition to their extensive use as colorants, they are also used in food fortificationbecause of their possible activity as provitamin A and their biological functions to health benefit,such as strengthening the immune system, reducing the risk of degenerative diseases, antioxidantproperties and anti obesity/hypolipidemic activities (N Mezzomo, L Tenfen and M S Farias2015)

From the non enzymatic compounds, on antioxidant defense, some minerals (copper,manganese, zinc, selenium and iron), vitamins (ascorbic acid, vitamin E, and vitamin A),carotenoids ( -carotene, lycopene, and lutein), bioflavonoids (genistein, quercetin), and tannins(catechins) can be detached

1.1.3 -Carotene

Figure1 6 Structure of beta- carotenoid

-Carotene is an organic, strongly colored red-orange pigment abundant in plants andfruits It is a member of the carotenes, which are terpenoids (isoprenoids), synthesizedbiochemically from eight isoprene units and thus having 40 carbons Among the carotenes, β-carotene is distinguished by having beta-rings at both ends of the molecule, β-Carotene is the

most common form of carotene in plants When used as a food coloring (Susan D Van Arnum 1998).

Among the Carotenoids, the -carotene is the most abundant in foods that has the highestactive pro vitamin A It is found in some varieties of pumpkin, carrot, nuts, carrot noodles rosehip fruits and palm oil, extra virgin olive oil, olive oil, which is an excellent source due to beingfree from any barrier of vegetal matrix and thus has increased bioavailability of this pigment

“beta-carotene” (G Britton S, volume 5, 2009)

In the uv visible, spectroscopy for β-Carotene shows the absorption for this compound to

be strongest from 400-500 nm, which lies in the green/blue part of the electromagnetic spectrum.Hence, precisely due to the absorbance at the green/blue range, the red/yellow colors of visiblelight are reflected back and carrots appear orange As the position of blue-green, the colorabsorbed by these pigments, is exactly complementary to red-orange, the color that is reflected(H., Keiler M 2011)

1.1.4 Beta-carotene Benefits

 Beta-carotene is the main safe dietary source of vitamin A, essential for normal growth and

development, immune system function, and vision (G Britton S.2009)

 Beta-carotene and other Carotenoids can facilitate communication between neighboring cells by

stimulating the synthesis of proteins that form pores in cell membranes, allowing communicationthrough the exchange of small molecules This effect appears unrelated to the vitamin A orantioxidant activities of various Carotenoids

 Beta-carotene is thought to possess many positive health benefits and in particular helps prevent

night blindness and other eye problems

 It also effective in skin disorders, enhances immunity, protects against toxins and cancer

formations, colds, flu, and infections It is an antioxidant and protector of the cells while slowingthe aging process

 It is considered that natural Beta-Carotene aids in cancer prevention It is important in the

formation of bones and teeth No vitamin overdose can occur with natural Beta-Carotene (VanPoppel G 1993, Hughes DA 1997, Bertram JS 1999)

In view of above the beneficial effects of chlorophyll and beta-Carotenoids it’s of interest to study these compounds in edible oils which are used for daily consumption.

1.2 Objective of the Study

1 2 1 General Objective

The overall objective of the study is to investigate Chlorophyll a, Chlorophyll b and

beta-Carotene found in edibleoil by using absorption and fluorescence spectroscopy through

quantitative determination

1 2 2 Specific objectives

The study enfolds the following specific

objectives:- To determine 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 spectroscopy in the edible oils

 To compare the performance of absorbance and fluorescence spectroscopy in differentiating

the composition of Chlorophyll a found in edible oils

1.3 Literature review

1.3.1 Optical properties of chlorophyll and beta-Carotenoids of edible oils

Fats and oils constitute one of the major categories of food products, as they containmany nutrients The great interest in studying the chemical composition of oils since suchinformation is valuable for the assessment of oil quality Apart from the major components, tri-fatty acid esters of glycerol, vegetable oils contain about 2–5% of minor compounds in a widerange of chemical classes These compounds have a marked influence on the oil quality Forinstance, tocopherols and carotenoids affect the oxidative stability of oils, whereas chlorophyllsare responsible for oil photo oxidation (De Man, J M 1999)

Edible oils (olive oil, seed oil) have interesting optical properties (absorption andfluorescence) due to the content of optically active compounds, such as chlorophyll, beta-carotene and others These properties can be used to recognize and characterize the various types

of oil In the spectrum of “Extra Virgin” olive oil, obtained by cold pressing you notice the

absence of the products of per oxidation of fatty acids, which give fluorescence at about 470nm.This happens both because the oil is cold worked and because the high content of natural anti-

oxidants (carotenes and poly phenols) prevents oil from oxidative degradation Seed oils show allcomponents to varying degrees, a clear fluorescence at about 470nm, a sign of the content inperoxides resulting from oxidative degradation of fatty acids, both because they are supposedlyhot worked and because of the lower content of molecules anti-oxidants Olive oil absorptionspectrum has absorption maxima in the red band at around 660-670nm and in the blue band, lessthan 500nm, Olive oil absorption is due to Carotenoids and chlorophylls (physicsopenlab.org ›English Posts 2015).

Sunflower oil fluorescence spectrum excited by uv-visible emission at 405nm.Thefluorescence band with peaks at 464nm and 476nm are presumably due to the products of peroxidation (degradation ) of polyunsaturated fatty acids in the oil (oleic and linoleic)(

physicsopenlab.org › English Posts 2015)

The weak fluorescence peak around 666 nm for the sunflower oil is caused by the

presence of chlorophyll-a No such peaks were discovered in soybean oil since they do not contain or contain very little chlorophyll-a (Krastena Nikolova 2012).

Carotenoids, present in olive oil with a relatively high concentration, are characterized by

a less intense emission In fact, carotenoids strongly compete for incident light with otherchromophores present in olive oil due to their concentration and high extinction coefficient.Carotenoids signals can be observed in the 430–480 nm regions, but their quantification remainsvery difficult by fluorescence techniques (Maurizio Zandomeneghi 2005)

Soybean oil fluorescence spectrum excited by UV-vis emission at 405nm,Thefluorescence band with the peak at 477nm is presumably due to the products of per oxidation(degradation ) of polyunsaturated fatty acids present in the oil (oleic and linoleic) The sameapplies to the band with a peak at 533nm, to assess the contribution of vitamin E on theemissions of the green fluorescence (physicsopenlab.org › English Posts 2015)

For some oils, namely linseed (seed oils) and olive oil, a long-wavelength band is observedwith excitation at about 350 nm–420 nm and emission at about 660 nm–700 nm This band wasattributed to pigments of chlorophyll group, based on its excitation and emission characteristics.This group includes chlorophylls a and b, and pheophytins a and b, derived from chlorophylls byloss of magnesium (B Schoefs 2003)

Visible absorption spectra of extra-virgin olive oils have characteristic features a threepeak band in the range 390–520 nm and a sharper band around 660–675 nm This last absorption

band is due to the electronic transition of chlorophylls and their derivatives, while the first band

is more complex, since it is due to the overlap among Carotenoids and chlorophylls absorptionsignals In the visible absorption spectrum of extra-virgin olive oils is very different from theabsorption spectra of other seeds and fruits oils This evidence is at the basis of several worksdevoted to the authentication of extra virgin olive oils and the identification of specific frauds,such as the mixing between olive oils and oils obtained from other seeds Visible (vis) light

absorption of extra virgin olive oils is associated with the pigments’ content, and this specificity

is at the origin of several research works aiming to substitute the chromatographic methods withmuch faster and direct spectrophotometry methods (Cayuela J.A., ousf K., Martinez M.C 20014,Torrecilla J.S., Rojo E 2010)

The colour of olive oil related to its pigment content varies from a light gold to a richgreen Green olives produce green oil because of the high chlorophyll content, while ripe olivesyield yellow oil because of the carotenoids (yellow red) The exact combination and proportions

of pigments determine the final colour of the oil Their presence in olive oil depends on olivefruits (Olea europa, L.), but also on genetic factors (olive cultivar), the stage of fruit ripeness,environmental conditions, the extraction processing and storage conditions The role ofchlorophylls as natural pigments accounting for greenish colours and in photosynthesis is wellknown There are also some reports about the benefits of dietary chlorophylls for human health(Gandul‐Rojas B.2000, Giuliani A 2011)

The structure of chlorophyll pigments, consisting of one tetrapyrrole macro cycle,coordinated to Mg2+ion to form a planar complex, is responsible for the absorption in the visibleregion of the spectrum of olive oils Here, both the bluish‐green chlorophyll‐a and the

yellowish‐green chlorophyll‐b can be found Chlorophylls in olive oils are mostly converted to

pheophytins, due to the exchange of the central Mg2+ ion with acid protons Pheophytin‐a is

100% Products from Olive Tree predominant with respect to Pheophytin‐b In the case of bad

storage conditions, Pheophytin are further degraded to pyropheophytins The main carotenoids

present in olive oils are lutein and β‐carotene (Cristina Lazzerini, 2016) Fluorescence

spectroscopy can be more diagnostically helpful due to the selective excitation of pigments, such

as chlorophylls, but useless in case of carotenoids, since their fluorescence is very weak (Sayago,A.2004)

A useful technique to study chlorophylls and Carotenoids is fluorescence spectroscopywhich is a photoluminescence process Fluorescence spectroscopy can be performed by rightangle with several possibilities of investigation Different types of signals can be indeedacquired: emission spectra, excitation spectra Several works have been published based onfluorescence spectroscopy applied to extra virgin olive oils since several chemical constituents,including pigments, give fluorescence in specific conditions and they can be identified Thequantification of fluorophores is less straightforward than in absorption spectroscopies Inparticular, this work shows that the right angle method gives several artifacts, while front facetechnique is more appropriate for quantification of fluorophores In emission spectra of oliveoils, the band centered at 670–680 nm is clearly due to chlorophyll like chromophores(Zandomeneghi M 2005) The fluorescence excitation-emission (λex=300-390 nm and

λem=415-600 nm) were used in studies of the Spanish extra virgin, virgin, pure, and olive

pomace oils, to investigate the relationship between oil fluorescence and the conventional qualityparameters, including peroxide value (Guimet F.2005c)

Total synchronous fluorescence spectra measured emission peaks between 500-700 nm,depending on for both classes of oils Classification of virgin olive oils based on theirsynchronous fluorescence spectra was performed by hierarchical cluster analysis and partial leastsquares using the 429–545 nm spectral range The authors conclude that the fluorescence in the429–545 nm range, which they used for data analysis, originates from oleic acid (Poulli K.2005)

The total fluorescence spectrum of diluted extra virgin olive oils, measured with the use

of right angle geometry with excitation at about 330–440 nm and emission at about 660–700 nm

An additional band appears in spectra of refined olive oil, located in the intermediate range, withexcitation at 280-330 nm and emission at 372-480 nm The long-wavelength band has a lowerintensity in refined as compared to virgin olive oil (Ewa Sikorska, 2004, 2005)

Direct Fluorescence of Oils: Right Angle Emission, Presents the fluorescence spectra of oliveoil The excitation wavelengths are in the region 280-450 nm, where the absorbance of the oil ishigh, and in the visible region, 500-650 nm, where the absorbance is much lower Focusing onthe 300-600 nm region, the emission spectra of an olive oil with λexc= 320 and 350 nm(Kyriakidis & Skarkalis (2000)), with λexc = 365 nm, at least for the position of the minima andmaxima For example, the spectrum measured with λexc =320 nm for wavelengths up to 500 nm,

shows peak maxima at 385, 439, and 470 nm and minima at 415, 453, and 480 nm Theseminima correspond almost exactly to the absorption peaks centered at 417, 455, and 482 nm ofthe oil absorption spectrum This correspondence indicates the presence of fluorescence self

absorption, whose magnitude, however, cannot be quantified unless the “true” fluorescence ismeasured The “true” fluorescence can be defined as the fluorescence measured in a sufficiently

dilute solution in an ideal solvent, which does not affect the fluorescence properties of oil Sincethe absorbance of the oil in the excitation and emission regions is quite high, we applied thecorrection for inner filter effects due to the absorption both of the excitation and the fluorescentradiation We note, a wavelength-dependent intensification of the spectrum, such that the minima

in the uncorrected spectrum become maxima in the corrected one and vice versa This behavior

is due to the high value of the absorbance correction factor and its rapid variations in the 375-525

nm range where the correction factor acts on the very low values of the emission intensity Incontrast, in the 525-640 nm regions, the correction factor is almost constant and about 2 orders ofmagnitude lower than at shorter wavelengths (Maurizio Zandomeneghi 2005)

Spectra give a complete description of the fluorescent components of the mixture andexhibit different features for various oils Assignment of emission bands to the specific chemicalcomponents was based on an analysis of the respective excitation and emission fluorescencespectra A relatively intense band, with excitation in the region of about 270–310 nm andemission in the region of about 300–350 nm was ascribed to tocopherols and tocotrienols Along-wavelength band, at 350–420 nm in excitation and 660–700 nm in emission, present inolive oil and linseed oil, is characteristic for fluorescence of the chlorophyll group pigments.This group includes chlorophylls a and b and pheophytins a and b The spectra of oils reveal anadditional emission band in the intermediate region, at about 400–450 nm The shape andintensity of this intermediate emission vary for different oils (Ewa Sikorska 2003)

By performing linear discriminate analysis, 100% correct classification was achieved Byused excitation wavelength of 360 nm to differentiate between common vegetable oils, includingolive oil, olive residual oil, refined olive oil, soybean oil, sunflower oil, and cotton oil All theoils studied showed a strong fluorescence band at 430–450 nm, except for virgin olive oil, whichexhibited a low intensity at 440 and 455 nm, a medium band around 681 nm and a strong one at

525 nm The latter two bands have been ascribed to chlorophyll and vitamin E compounds,

respectively The very low intensity of the peaks at 445 and 475 nm is due to the high content ofphenolic antioxidants, which provide more stability against oxidation All refined oils showedonly one intense peak at 445 nm, which is due to fatty acid oxidation products formed as a result

of the large percentage of polyunsaturated fatty acids present in these oils (Kyriakidis andSkarkalis 2000)

The intensity of the tocopherols and chlorophyll bands decreased in all of the storedsamples, to the extent dependent on the storage conditions The chlorophyll bands disappearedcompletely in oil stored in clear glass bottles under light The oil stored under light in greenbottles also exhibited a reduced intensity of the chlorophyll pigments after 10 months of theexperiment, with a very small reduction in the oil stored in darkness Interestingly, a newfluorescence band appeared in the oil stored under light both in green and clear glass bottles,with a maximum at 300 nm in excitation and 400 nm in emission A similar band had been noted

by us previously in a variety of edible oils, which revealed an emission band at 400–450 nm Theshape and intensity of such emissions varied for different oils (Ewa Sikorska 2004, 2005)

Olive oil is obtained from the fruit of the olive tree (Olea europaea L.) There are different

grades of olive oils [e.g., extra virgin, virgin, pure (or simply olive oil) and olive pomace oil].Each of these grades must fulfill some specifications Due to its nutritional and economicimportance, olive oil authentication is an issue of great interest in the manufacturing countries.Authenticity covers many aspects, including adulteration, mislabeling, characterization, andmisleading origin Olive oil authentication is usually based on chemical parameters [acidity,major fatty acids composition, peroxide value (PV), ultraviolet absorbance, trinolein content, andsterol composition (Zandomeneghi M 2005)

Olive pomace is one of the main by-products of oil fruit processing It contains fragments

of skin, pulp, pieces of kernels and some oil The oil present in the olive pomace undergoes rapiddeterioration due to the moisture content that speeds up triacylglycerol hydrolysis Refinedolive–pomace oil is obtained from olive pomace after an extraction with authorized solvents and

a refining process, which includes neutralization, deodorization and decolorization This oil isimproved with virgin olive oil to obtain the oil known as olive pomace oil Owing to the lowprice of this oil, it is sometimes used for adulterating extra virgin olive oil For this reason, arapid method to detect such a practice is important for quality control and labeling purposes(Francesca Guimet 2005)

Fluorescence spectroscopy has been used in the past for determining olive oilauthenticity The advantages of this technique are its speed of analysis, lack of solvents andreagents, and requirement of only small amounts of sample In addition, it is a noninvasivetechnique (Kyriakidis and Skarkalis 2000) showed that useful information can be extractedfrom the fluorescence spectra of vegetable oils They showed that the fluorescence spectra ofvirgin olive oils between 400 and 700 nm measured at an excitation wavelength (λex) of 365 nmhave clear differences compared to the spectra of other vegetable oils Virgin olive oils presenttwo low peaks at 445 and 475 nm, one intense peak at 525 nm, and another peak at 681 nm.Kyriakidis and Skarkalis (2000) suggested that the peaks at 445 and 475 nm were related to fattyacid oxidation products and that the one at 525 nm was derived from vitamin E However, theyalso showed that addition of vitamin E acetate to virgin olive oil increased fluorescence intensitynot only at 525 nm but also at 445 and 475 nm They stated that this was due to oxidized vitamin

E, which emits fluorescence approximately in this region Finally, the peak at 681 nm wasrelated to the chlorophylls The very low intensity of the peaks at 445 and 475 nm of virgin oliveoils is due to their large content of monounsaturated fatty acids and phenolic antioxidants, whichprovide them more stability against oxidation All refined oils show only one intense and widepeak at around 400-560 nm, which is due to a larger oxidation state of these oils as a result oftheir large content of polyunsaturated fatty acids (Sayago, A 2004, Ramon Aparicio 2000)

Niger (Guizotia abyssinica (L f.) Cass, Compositae) its cultivation originated in theEthiopian highlands, and has spread to other parts of Ethiopia Common names include:noog/nug is an oilseed crop cultivated in Ethiopia and India It constitutes about 50% ofEthiopian and 3% of Indian oilseed production In Ethiopia, it is cultivated on waterlogged soilswhere most crops and all other oilseeds fail to grow and contributes a great deal to soilconservation and land rehabilitation The genus Guizotia consists of six species, of which five,including Niger, are native to the Ethiopian highlands It is a dicotyledonous herb, moderately towell branched and grows up to 2 m tall The seed contains about 40% oil with fatty acidcomposition of 75-80% linoleic acid, 7-8% palmitic and stearic acids, and 5-8% oleic acid(Getinet and Teklewold 1995)

1.4 Theoretical Background of the instruments

1.4.1 Absorption Spectroscopy

Absorption is a transfer of energy from the electromagnetic wave to the atoms or molecules ofthe medium Energy transferred to an atom can excite electrons to higher energy states Energytransferred to a molecule can excite vibrations or rotations The wavelengths of light that canexcite these energy states depend on the energy-level structures and therefore on the types ofatoms and molecules contained in the medium The spectrum of the light after passing through amedium appears to have certain wavelengths removed because they have been absorbed This iscalled an absorption spectrum

UV-visible radiation comprise only a small part of the electromagnetic spectrum, Whenradiation interacts with matter, a number of processes can occur, including reflection, scattering,absorbance, fluorescence (absorption and reemission), and photochemical reaction (absorbanceand bond breaking) In general, when measuring UV-visible spectra, we want only absorbance tooccur Because light is a form of energy, absorption of light by matter causes the energy content

of the molecules (or atoms) to increase The total potential energy of a molecule, in somemolecules and atoms, photons of UV and visible light have enough energy to cause transitionsbetween the different electronic energy levels The wavelength of light absorbed is that havingthe energy required to move an electron from a lower energy level to a higher energy level (Tony

Owen 2000).

The energy associated with electromagnetic radiation is defined by the following equation:

Where E is energy (in joules), h is Planck’s constant (6.62 × 10-34 Js), ν is frequency (in

seconds) and c is the speed of light (3 × 108ms-1), and λ is wavelength (in meters) In UV-visible

spectroscopy, wavelength usually is expressed in nanometers (1 nm = 10-9m) In UV-visiblespectroscopy, the low-wavelength UV-vis light has the highest energy

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 particular frequencies at which light is absorbed are affected by thestructure and environment of the chromophore (light absorbing species).

1.4.1.1 Absorbance and the Beer – Lambert Law

When electromagnetic radiation passes through a material medium the intensity of light decreases exponentially when increase of path length in the medium and

concentration of absorbing molecules.

Figure 1.7 absorbance and beer Lambert law

The absorbance, A is defined as

dI is the intensity absorbed in the slab, and Izis the intensity of light leaving the sample

Then, the fraction of photons absorbed will be σ · N · dz 1.4

σ- Absorption of Cross sectional area

If the concentration of the absorbing molecules = N / 3

The fraction of the area occupied by the molecules in the slab = × N× / rea

The negative sign represents a decrease in intensity

Integrating equation 1.5 from z = 0 to z = b

Now, the molar concentration of the molecules, c can be given by

C (moles/liter) = N (molecules/cm³) · (1 mole/6.023·1023molecules) · 1000 cm³ / liter

Substituting for N in equation 6 and converting natural logarithm, ln into log10 gives

- 2.303 × log I/I O = ×c × (6.022 × 1020/2.303) × b 1.9

1/log (e) = 2.303 and ε = α /2.303 1.10

Then - log (I / Io) = σ · (6.023x1020/ 2.303) · c · b

-log I/I Ois defined as the absorbance and × (6.022 × 1020/2.303) is defined as the molar

absorption coefficient, denoted by the Greek alphabet, ε

Where ε = σ · (6.023x1020/ 2.303) = σ · 2.61x1020Therefore, equation 6 can be written as:

b- Length of light path through the sample cuvette is “b”

c- Sample concentration

ԑ- Molar absorptive coefficient

This equality showing linear relationship between absorbance and the concentration of the

absorbing molecule (or chromophore, to be precise) is known as the Beer Lambert law or Beer’slaw

Transmittance is another way of describing the absorption of light Transmittance (T) is simply

the ratio of the intensity of the radiation transmitted through the sample to that of the incident

radiation Transmittance is generally represented as percentage transmittance (%T)

As is clear from the definition of absorbance and transmittance, both are dimensionless

quantities Absorbance and transmittance are therefore represented in

arbitrary units (AU)

1.4.2 Fluorescence Spectroscopy

Fluorescence is the emission of light subsequent to absorption of ultraviolet or visible light of afluorescent molecule or substructure, called a fluorophore Thus, the fluorophore absorbs energy

in the form of light at a specific wavelength and liberate energy in the form of emission of light

at a higher wavelength (Papa Georgiou 2004, Romdhane Karoui & Christophe Blecker 2011)

Figure 1.8 Jablonski diagram

Absorbance: S0 state with 0th vibrational level is the state of lowest energy and therefore,the highest populated state Absorption of a photon of resonant frequency usually results in the

population of S1 or S2 electronic states; but usually a higher vibrational state Transition of

electrons from low energy molecular orbital to a high energy molecular orbital through

absorption of light is a femtosecond (10-15s) phenomenon The electronic transition, therefore, istoo quick to allow any significant displacement of the nuclei during transition.

Internal conversion: Apart from few exceptions, the excited fluorophores rapidly relax to

the lowest vibrational state of S1 through non-radiative processes Non-radiative electronic

transition from higher energy singlet states to S1 is termed as internal conversion while relaxation of a fluorophore from a higher vibrational level of S1 to the lowest vibration state is termed as vibrational relaxation The terms ‘internal conversion’ and ‘vibrational relaxation’,

however, are often interchangeably used The timescale of internal conversion/vibrationalrelaxation is of the order of 10-12seconds

Fluorescence: Fluorescence lifetimes are of the order of 10-8 seconds, implying that the

internal conversion is mostly complete before fluorescence is observed Therefore, fluorescence

emission is the outcome of fluorophore returning back to the S0 state through S1 → S0 transition

emitting a photon This also explains why emission spectra are usually independent of theexcitation wavelength, also known as Kasha’s rule (However, there are exceptions wherein

fluorescence is observed from S2 → S1 transition) The S1 → S0 transition, like S0 → S1

transition, typically results in the population of higher energy vibrational states The moleculesthen return back to the lowest vibrational state through vibrational relaxation

Quantum yield: As has been mentioned earlier, an excited molecule can come back to

the ground state through non-radiative pathways

Fluorescence quantum,

Ԛ=

Fluorescence quenching: A decrease in fluorescence intensity is referred to asquenching A molecule that quenches the fluorescence of a fluorophore is called aquencher A quencher can be either a collisional quencher or a static quencher Acollisional quencher brings about decrease in fluorescence intensity by de-exciting theexcited fluorophore through collisions Addition of another non-radiative process tothe system leads to lower quantum yield A static quencher forms a non-fluorescentcomplex with the fluorophore It effectively leads to a decrease in the concentration ofthe fluorophore thereby decreasing the fluorescence emission intensity

1.4.3 Excitation and Emission Spectra

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)

1.4.3.1 Excitation Spectrum

Figure.1.9 shows the excitation spectrum defined as the relative efficiency of differentwavelengths of exciting radiation in causing fluorescence The shape of the excitation spectrumshould be identical to that of the absorption spectrum of the molecule and independent of thewavelengths at which fluorescence is measured However, this is seldom the case because thesensitivity and the bandwidth of the spectrophotometer (absorbance spectrum) and thespectrofluorimeter (excitation spectrum) are different In addition, for many food samples,scattering properties and energy transfer between neighboring molecules could contribute to thisdifference A general rule of thumb is that the strongest (generally the longest) wavelength peak

in the excitation spectrum is chosen for excitation of the sample This minimizes possibledecomposition caused by the shorter wavelength, higher energy radiation (Romdhane Karoui

2010)

1.4.3.2 Emission Spectrum

Figure.1.9 shows the emission spectrum of a compound which results from the radiationabsorbed by the molecule The emission spectrum is the relative intensity of radiation emitted atvarious wavelengths In theory, the quantum efficiency and the shape of the emission spectrumare independent of the wavelength of the excitation radiation In practice, this is not the case Ithas been shown that fluorescence of chlorophyll from a green leaf has a lower short wavelength

emission maximum when excited with green light than when excited with blue light (Buschmann2007) Green light penetrates more deeply into the leaf since it is less absorbed than blue lightand the green light excited fluorescence from more inside the leaf is more readily re-absorbed bythe chlorophylls on its way to the sample surface The re-absorption of fluorescence isparticularly high in the short wavelength fluorescence where it overlaps with the absorptionspectrum of chlorophyll If the exciting radiation is at wavelength that differs from thewavelength of the absorption peak, less radiant energy will be absorbed and hence less will beemitted.

1.4.4 Stokes Shift

According to the Jablonski diagram (Fig 1.8), the energy of emission is lower than that ofexcitation This implies that the fluorescence emission occurs at higher wavelengths than theabsorption (excitation) The energy of emission is typically less than that of absorption.Fluorescence typically occurs at lower energies or longer wavelength The difference betweenthe excitation and emission wavelengths is known as Stokes shift (Romdhane Karoui &Christophe Blecker 2011), as indicated with the arrow in Fig 1.10, below marking the differencebetween the excitation and emission spectrum

Figure 1.10 Stokes shift difference between the excitation and emission spectrum.

Stokes shift (cm -1 ) = ( - )

where λexand λemare the maximum wavelengths (nanometer) for excitation and emission,respectively (Romdhane Karoui2010)

2 Material and methods

2.1 Materials

2.1.1 Oils

Many oilseed crops are cultivated for extraction and production of edible oils Edible oilsare most often plant-based oils Edible oils may be solid or liquid at room temperature Edible orcooking oil is fat of plant, animal or microbial origin, which is liquid at room temperature and issuitable for food use

Edible oils can be broken into three large groups: saturated, monounsaturated, andpolyunsaturated oils It's easy to distinguish between these groups:

Saturated oils are solid at room temperature; this describes shortenings, margarines and a fewother commonly eaten oils

Monounsaturated oils are liquid at room temperature but begin to solidify in the refrigerator.These include olive oil

Polyunsaturated oils remain liquid when at room temperature or when chilled These includeNiger seed, sunflower, and soybean oils etc

Saturated oils are considered to be relatively unhealthy because they can contribute to highercholesterol levels and heart disease Thus, monounsaturated and polyunsaturated oils are usually

a better choice for overall health

Edible oils are natural esters formed from glycerol and triglycerides Some of them havetwo or three different fatty acids Fatty acids are saturated if the carbon atoms in the molecule areconnected with single bonds Sometimes, fatty acids are converted from saturated tomonounsaturated, with the loss of one hydrogen atom, and the single bond between carbon atomsbecomes double If there is more than one double bond, the fatty acid becomes polyunsaturated.Every vegetable oil has its own composition with different ratios of unsaturated with saturatedfatty acids Oils with a higher content of unsaturated acids are liquid at room temperature Thenature of the oil depends on its constituent fatty acids Their variation can change the oil’sphysical and chemical properties, which enables them to be a good replacement for mineral oil