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Romanian Biotechnological Letters Vol. 17, No.5, 2012Copyright © 2012 University of BucharestPrinted in Romania. All rights reservedORIGINAL PAPERStudies concerning the extraction of chlorophyll and total carotenoids fromvegetablesReceived for publication, June 13, 2012Accepted, September 12, 2012AbstractMANUELA ADRIANA COSTACHE 1) , GHEORGHE CAMPEANU 2) ,GABRIELA NEATA 3)1)Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania;E-mail: manuela_bth@yahoo.com2 ) Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania;3 ) Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania;Corresponding author: E-mail: manuela_bth@yahoo.comThe main pigments found in vegetables are chlorophyll a, b and c. Chlorophyll a converts lightenergy into chemical energy through photosynthesis process. The content of chlorophyll pigmentsvaries by species. The aim of the research was to determine simultaneously chlorophyll and carotenecontent from different vegetables by spectrophotometric method, testing three organic solvents to obtainthe best extraction solution. 17 samples of cherry tomatoes, peppers and cucumbers, collected fromdifferent greenhouses located in southern part of Romania were analyzed. The carotenoids andchlorophyll pigments extracted in methanol, diethyl ether and acetone, were determined based on theabsorbance of three wavelengths: 470 nm, 645 nm-653 nm and 662 nm-666 nm. The results werecalculated according to the three "trichromatic equations" of Lichtentaler and Wellburn. The bestextraction solvents for simultaneously determination of chlorophyll a and b and carotene weremethanol and acetone. For cherry tomatoes and tomatoes the best extraction solvent was methanol forchlorophyll a and acetone for carotene. For peppers the best extraction solvent was acetone and forcucumbers it was methanol. Variation coefficients were high, both between vegetable species andbetween tested varieties or hybrids.Keywords: a and b chlorophylls, total carotenoids, extraction, vegetablesIntroductionThe presence of pigments in plant tissues gives color to vegetables and fruit, which isdifferent depending on variety and species. Pigments are substances with very differentchemical structure; they are present in the form of porphyrin pigments, carotenoids,anthocyanins and flavones. The main porphyrin pigments found in vegetables are chlorophyll a,b and c. Chlorophyll a, the main pigment in plants, converts light energy into chemical energythrough photosynthesis process. The content of chlorophyll pigments varies by species.Several studies concerning chlorophyll pigments determination using phytoplankton(ARAR [1, 2], BARLOW et al. [3], DON SANTOS et al. [7], FLANDER et al. [9], andHALLENGRAEFF [10]) and some species of algae (DERE et al. [8]) are reported.The change of chlorophyll pigment content from fruit and vegetables have a greatimportance for their color variation during the maturation phase. CIOFU [5] states that thechlorophyll content of tomatoes during ripening period varied from 0.95 mg/100 g freshproduct to 0.12 mg/100 g fresh product after 7 day of storage (CȂMPEANU et al. [4],COSTACHE et al. [6]).Carotenoid pigments can be located in chromoplasts, contributing to the color ofvegetables and fruits, or in chloroplasts, where, together with chlorophylls, are involved in the7702 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

Studies concerning the extraction of chlorophyll and total carotenoids from vegetablestwo phytosystems. Carotenoids group and their derivatives, include about 70 compounds thatare present in most vegetables and fruits. Among carotenoid pigments with 40 carbon atomsidentified in vegetables and fruits, β-carotene is the most popular and widespread. Inhorticultural products lycopene has been extensively identified. It is found mostly in tomatoand other 70 plant species. Total carotenoid content of tomato pigments varies between 6.55mg/100 g and 12.0 mg/100 g fresh product. In pepper, the reported range is between 8.0 and25.0 mg/100 g fresh product (CIOFU [5]).The aim of the research was to determine the chlorophyll and carotene pigmentssimultaneously from different vegetables, using three organic solvents, in order to obtain thebest extraction solution. Absorptive properties of the pigments were followed up inquantitative analysis using a spectrophotometric method. The literature presents two methodsto determine these pigments - the trichromatic method and monochromatic method, the twomethods differing by the used wavelengths. It is known that chlorophyll pigments have broadabsorption band from blue to red. Also the coextracted carotenoids have maximum absorptionin blue band so that the chosen determination method is based on measuring the absorbance atthree wavelengths for each type of matrix and then calculate chlorophyll and carotene usingthe three "trichromatic equations" of LICHTENTALER and WELLBURN [11, 12, 13].Materials and methodsThe research was conducted on 17 samples of several vegetable species collected fromdifferent greenhouses located in southern part of Romania. Investigated species were cherrytomatoes, common tomatoes, peppers and cucumbers, in several varieties and hybrids existingin culture.Extraction of pigments was carried out in stoppered tubes. Vegetables samples wereprepared with a laboratory homogenizer using about 1 g fresh material. Three extractionsolutions were used for each sample: 95% aqueous diethyl ether solution (petroleum ether),90% aqueous methanol solution and 100% acetone. The extraction ratio was 1:50.Homogenized mixture is separated by centrifugation at 3000 rpm, for 10 minutes.The analytical determination was performed with Helios α spectrophotometer at thefollowing wavelengths: 645, 653, 662 and 664 nm, for chlorophyll a and b (according to eachextraction solvent) and 470 nm for carotene.Equations used for calculation are presented below.Diethyl etherChlorophyll a = 10.05 A662 -0.766 A644Chlorophyll b = 16.37 A664 – 3.140 A662Carotene = 1000 A470 – 1.280 Chl a – 56.7 Chl b/230MethanolChlorophyll a = 15.65 A666 – 7.340 A653Chlorophyll b = 27.05 A653 – 11.21 A666Carotene = 1000 A470 – 2.860 Chl a – 129.2 Chl b/245AcetoneChlorophyll a = 11.75 A662 – 2.350 A645Chlorophyll b = 18.61 A645 – 3.960 A662Carotene = 1000 A470 – 2.270 Chl a – 81.4 Chl b/227Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7703

Studies concerning the extraction of chlorophyll and total carotenoids from vegetablesMeasurements were performed in triplicates.Statistical interpretation of results was done using mean, standard deviation and coefficient of variation values (ANOVA programme).Results and discussionsAnalyses performed on several vegetables showed a large variety of chlorophyll a and b and also of carotene content.When performing diethyl ether extraction, the content of chlorophyll a was small, difficult to detect, except the values obtained atcucumber determinations that had ranged from 7.21 mg/100 g fresh product to 33.76 mg/100 g fresh product (Table 1). The averaged was 23.14mg/100 g, the standard deviation was 10.31 and the coefficient of variation was 44.55% (Table 2).In the case of methanol extraction, the contents were higher, varying from 10.49 mg/100 g fresh product to 626.15 mg/100 g freshproduct. In the case of cherry tomatoes, chlorophyll a content varied between 10.49 mg/100 g to 28 mg/100 g fresh product and in commontomatoes, range values were between 10.49 mg/100 and 45.89 mg/100 g fresh product.In pepper, chlorophyll a results obtained by methanol extraction ranged from 45.89 mg/100 g and 127.52 mg/100 g fresh product and incucumber the values ranged from 354.06 mg/100 g to 626.15 mg/100 g fresh product.The acetone extraction of chlorophyll a, detected in tomatoes was low, ranging from 3.98 mg/100 g to 5.54 mg/100 g fresh product and inpepper the quantities of pigment had values between 181.06 mg/100 g and 416.07 mg/100 g fresh product. The highest values of chlorophyllextracted in acetone ranged between 259.89 mg/100 g and 466.27 mg/100 g fresh product.Table 1. Chlorophyll a and b and total carotenoids content, mg/100 g fresh productSpeciesCherrytomatoesCultivarDiethyl ether 1:50 Mehtanol 1:50 Acetone 1:50Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll bCaroteneBelle F1 7.21 4.50 16.93 28.19 3.51 14.34 4.50 7.21 52.79Yellow pears 7.41 4.65 1.39 10.49 4.50 3.39 4.12 6.23 6.37Gold nugget 7.11 4.20 1.20 28.19 3.51 5.98 4.79 6.89 2.99Muscato F1 6.85 4.45 5.78 10.49 4.50 11.95 5.54 6.58 54.78Diamont F1 7.32 4.58 21.91 28.19 3.51 20.52 4.50 7.11 48.80Tomatoes Carabella F1 7.21 4.12 15.54 10.49 4.50 10.76 4.50 7.21 32.677704 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

MANUELA ADRIANA COSTACHE, GHEORGHE CAMPEANU, GABRIELA NEATAPeppersKaterina F1 16.06 4.01 3.98 19.34 4.01 10.76 4.65 6.53 34.26Ursula F1 7.11 4.50 11.95 19.34 4.01 7.77 3.98 6.89 37.65Winona F1 7.31 4.62 4.98 45.89 2.52 12.55 4.56 7.48 26.89Kaptur F1 7.11 4.15 1.59 45.89 2.52 4.78 48.02 105.86 11.95Fat papperBianca F1 7.21 4.50 1.20 47.87 63.45 2.59 181.06 357.56 35.46MaradonnaF1 7.02 4.32 8.96 127.52 59.00 42.83 416.07 714.78 74.70Bettina F1 7.21 4.50 2.39 540.93 412.31 28.09 390.80 201.82 27.49Asterix F1 16.06 4.01 1.00 478.64 359.84 25.10 440.80 272.28 21.31Cucumbers Alibi F1 33.76 3.02 4.98 626.15 397.38 33.86 466.27 255.23 25.10Fabrio 33.76 3.02 2.39 400.94 287.88 20.52 277.84 117.28 16.53Mirabell F1 24.91 3.51 2.19 354.06 254.90 18.13 259.89 175.3 13.94Table 2. Statistically interpretation of pigments content using different extraction solvents, mg/100 g fresh productDiethyl ether Methanol AcetoneSpeciesCherrytomatoesCultivars Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b CaroteneAverage 7.18 4.4761 9.44 21.11 3.9071 11.23 4.69 6.804 33.15Max 7.41 4.65 21.91 28.19 4.5 20.52 5.54 7.21 54.78Min 6.85 4.2 1.2 10.49 3.51 3.39 4.12 6.23 2.99Standarddeviation0.16 0.26 8.46 8.67 0.538 6.1 0.46 0.424 23.34Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7705

Studies concerning the extraction of chlorophyll and total carotenoids from vegetablesCv% 2.22 5.81 2.75 41.07 13.79 54.31 9.8 6.23 70.4TomatoesAverage 9.422 4.311 9.11 23.765 3.758 10.46 4.42 7.02 32.87Max 16.06 4.62 15.54 45.89 4.5 12.55 4.65 7.48 37.65Min 7.11 4.006 3.98 10.49 2.52 7.77 3.98 6.53 26.89Standarddeviation3.83 0.27 4.82 13.3 0.734 1.7 0.28 0.479 3.87Cv% 40.65 6.26 52.9 55.97 19.57 16.25 6.33 6.82 11.77PeppersAverage 7.113 4.323 3.92 73.76 41.658 16.73 215.05 392.73 40.7Max 7.21 4.5 8.96 127.52 59 42.83 416.74 714.78 74.7Min 7.02 4.15 1.2 45.89 2.52 2.59 48.02 105.86 11.95Standarddeviation0.218 0.21 3.57 38.01 27.73 18.46 4.81 7.93 25.88Cv% 3.06 4.86 91.07 51.53 66.57 90.27 22.39 20.23 91.27CucumbersAverage 23.14 3.61 2.59 480.144 342.461 25.14 367.11 204.38 20.88Max 23.76 4.5 4.98 540.93 412.31 33.86 466.26 272.28 27.49Min 7.21 3.07 1 354.89 254.89 18.13 259.89 117.28 13.94Standarddeviation10.31 0.574 1.3 97.85 61.39 4.16 84.2 57.3 5.05Cv% 44.55 15.9 50.19 52.15 47.95 16.55 46.3 48.56 24.187706 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

MANUELA ADRIANA COSTACHE, GHEORGHE CAMPEANU, GABRIELA NEATAComparing the values of the three extraction variants of chlorophyll a, best resultswere obtained with methanol and acetone.Chlorophyll b extraction with diethyl ether registered low results obtained in all thetested vegetables, being close to the limit of detection of the spectrophotometer.Extraction with methanol resulted in high values of chlorophyll b in peppers andcucumbers; the peppers values ranged between 2.52 mg/100 g and 63.45 mg/100 g freshproduct and the cucumbers values ranged from 254.90 mg/100 g to 412.31 g/100 g freshproduct.When using acetone, the extraction of chlorophyll b is decreased in tomatoes, near thelimit of detection of the device and high in peppers and cucumbers.Results of the extraction of chlorophyll b showed that acetone solvent determined thebest results followed by extraction with methanol.Statistical interpretation of the results for chlorophyll a determination showed that thebest results were obtained from extraction with methanol where the coefficient of variationwas 41.07% in cherry tomatoes, 55.97% in common tomatoes, 51.53% in peppers and52.15% in cucumbers (Table 2).In the case of chlorophyll b extraction, the best coefficients of variation were obtainedusing extraction with methanol respectively 13.79% in cherry tomatoes 19.57% in commontomatoes, 66.57% in peppers and 47.95% in cucumbers (Table 2).Diethyl ether solvent determined a different extraction of carotene, so at tomatoes thevalues ranged between 1.20 mg/100 g and 21.91 mg/100 g in fresh product, while at peppersand cucumbers low extraction values were obtained (Table 1).Methanol extraction determined higher levels of carotene pigment in tomatoes,varying between 3.39 mg/100 g and 20.52 mg/100 g in fresh product, in pepper theconcentrations of carotene were between 2.59 mg/100 g and 42.83 mg/100 g in fresh product,and in cucumbers the concentrations obtained ranged from 18.13 mg/100 g and 33.86 mg/100g fresh product.In the third extracting solution – acetone, the obtained carotene values were higher,thus in tomatoes were between 2.99 mg/100 g and 54.78 mg/100 g fresh product, in peppersbetween 11.95 mg/100 g and 74.70 mg/100 g fresh product, and in cucumbers theconcentrations ranged from 13.94 mg/100 g fresh product and 27.49 mg/100 g fresh product(Table 1).Comparing the results of the three extractions of carotene, acetone extractiondetermined the best results in all analyzed vegetables: tomatoes, peppers and cucumbers.Statistical interpretation of results of carotene showed that acetone extraction resultedin the best variation coefficient respectively 70.4% at cherry tomatoes, 11.77% at commontomatoes, 91.27% at peppers and 24.18% at cucumbers. Methanol extraction also showedcoefficient of variation of the results slightly lower than for acetone extraction, respectively54.31% for cherry tomatoes, 16.25% for common tomatoes, 90.27% for peppers and 16.55%for cucumbers (Table 2).ConclusionsThe best extraction solvents for simultaneously determination of chlorophyll a and band carotene were methanol and acetone. For cherry tomatoes and tomatoes the bestextraction solvent was methanol for chlorophyll a and acetone for carotene. For peppers thebest extraction solvent was acetone and for cucumbers is methanol;Variation coefficients were high, both between vegetable species and between testedvarieties or hybrids.Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7707

Studies concerning the extraction of chlorophyll and total carotenoids from vegetablesDetermination of the three pigments from different vegetables can be provided withspectrophotometric method using methanol as extractant. Beacause the methanol is toxic,extraction with acetone is preferred, providing reliable results that must be corrected with asuitable coefficient according to the vegetable species.AknowledgementThese researches were supported by the POSDRU/88/1.5/S/52614 Programme.References1. E.J. ARAR, Determination of Chlorophyll a, b, c1 and c2, and Pheophytin a in marine and freshwaterphytoplankton by Spectrophotometry, EPA, (1997).2. E.J. ARAR, Determination of Chlorophyll a,b, c1 and c2, and Pheophytin a in marine and freshwaterphytoplankton by High Performance Liquid Cromatography, EPA, Cincinnati (1997).3. R.G. BARLOW, R.F.C. MANTOURA, D.G.CUMMINGS, Phytoplankton pigments distribution andassociated fluxes in the Bellinghauser Sea during the austral spring 1992, J. Mar. Sys. 17:97-113 (1998)4. GH. CÂMPEANU, , M.A. COSTACHE, , G. NEAŢĂ , Research on the methodology of chlorophyll andcarotene content extraction in cucumbers and peppers grown in the south of Romania area, Annals’ OfThe University Of Craiova, XVI (LII), pp. 74-82 (2011).5. R. CIOFU, N. STAN, V. POPESCU, P. CHILOM, S. APAHIDEAN, A.HORGOŞ, V.BERAR, K.F.LAUER, N. ATANASIU, Tratat de legumicultură, Ed. Ceres, București, (2003).6. M. A. COSTACHE, GH. CÂMPEANU, G. NEAŢĂ, Research on the methodology of extraction ofchlorophyll and carotene content of tomatoes grown in the south of Romania area, Scientifical PapersU.S.A.M.V. Bucharest, seria B, LV, pp. 069-073 (2011).7. DON SANTOS, A.C.A., CALIJURI, M.C., MORAES, E.M., ADORNO, M.A.T., FLACO, P.B.,CARVALHO, D.P., DEBERDT, G.L.B. & BENASSI, S.F., Comparison of three methods for Chlorophylldetermination: Spectrophotometry and Fluorimetry in samples containing pigment mixtures andspectrophometry in samples with separate pigments through High Performance Liquid Chromatography,Acta Limnol. Bras., 15(3):7-18 (2003).8. Ş.DERE, T.GUNES,R. SIVACI, , Spectrophotometric Determination of Chlorophyll - A, B and TotalCarotenoid Contents of Some Algae Species Using Different Solvents, Tr. J. of Botany, 22, pp. 13-17(1998).9. P.V. FLANDER, T.SENKA, M. ALENKA, A. MARIJAN, Effect of mucilage event on the distribution ofsummer phytoplankton as reflected by phytoplankton pigments, Per. Biol.,102:169-177 (2000).10. G.M. HALLENGRAEFF, Seasonal study of pkytoplankton pigments and species at a coastal station offSidney: Importance of diatoms and the nanoplankton, Mar. Biol., 61:107-118 (1981).11. H.K. LICHTENTHALER, A.R. WELLBURN, Determination of Total Carotenoids and Chlorophylls Aand B of Leaf in Different Solvents, Biol. Soc. Trans., 11, pp. 591-592 (1985).12. D.P. SATORY, Extraction of Chlorophyll A from Freshwater Phytoplankton for SpectrophotometricAnalysis, M. Sc. Hesis. Univ. Orange free state, Bloemfontein, Republic of S. Africa (1982).13. A.R. WELLBURN, The Spectral Determination of Chlorophylls A and B, as well as Total Carotenoids,Using Various Solvents with Spectrophotometers of Different Resolution, J. Plant Phys. 144, 307-313(1994).7708 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012