Spectrophotometric Method using Different Solvents and SPAD Chlorophyll Meter for Determination some Photosynthesis Pigments of Bean and Cowpea Plants

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1 Spectrophotometric Method using Different Solvents and SPAD Chlorophyll Meter for Determination some Photosynthesis Pigments of Bean and Cowpea Plants Ikbal M. Al-Barzinji Department of Biology Faculty of Science and Health / Koya University Erbil, Iraq Sargul A. Khudhur Department of Biology Faculty of Science and Health / Koya University Erbil, Iraq Aroul M. Anwar Department of Biology Faculty of Science and Health / Koya University Erbil, Iraq ABSTRACT In this work, some photosynthetic pigments with acetone, methanol and diethyl either were extracted spectrophotometrically, in addition to SPAD chlorophyll meter frombean (Phaseolus vulgaris L.) var. Biotack and cowpea (VignasinensisSavi) var. California black eye leaves in vegetative, flowering and podding growth stagesin Koya city, Erbil- Iraq.Results shows that concentration of chlorophyll a in bean and cowpea leaves increased significantly when acetone used in the extraction process followed by methanol and the lowest obtained when the diethyl either is used, whereas methanol was the best solvent for extracting chlorophyll b.results also show that there were nonsignificant differences in SPAD readings between different growth stages in both of bean and cowpea plants. The data shows that the SPAD reading is high in cowpea compare to bean plants.spad readings correlated high significantly with chlorophylls a and b concentrations, when acetone used as solvent in cowpea plants. Keywords: bean, extraction,,cowpea, pigments, solvents, SPAD 1. INTRODUCTION Chlorophyll pigments of the chloroplasts are responsible for the efficient capture of the solar energy in photosynthesis process. Chlorophylls a and b are the two most abundant chlorophylls. Carotenoids, the accessory pigments which assist in photosynthetic light harvest, and they prevented chlorophyll and thylakoid membrane from the damage of absorbed energy by photooxidation. The Carotenoids include majorly the red colored β-carotene and yellow colored xanthophyll. The content of foliar pigments varies depending on species. Chlorophylls and carotenoids can be isolated from green leaves in acetone, ether or other organic solvents. Chlorophyll a dissolves very well in petroleum ether while chlorophyll b in methyl alcohol. Different chlorophylls and carotenoids have a characteristic absorption spectrum, absorbing certain wavelengths of light more efficiently than the others. The absorbance properties of pigments facilitate the qualitative and quantitative analysis of them [1, 2, 3, and 4]. There is a trade-off between choosing the best solvent for efficient quantitative extraction of chlorophylls and use of a solvent best suited for spectrophotometric assay. Acetone gives very sharp chlorophyll absorption peaks, but acetone is volatile, highly inflammable,it is narcotic in high concentrations and is a skin irritant, also plastic spectrophotometer cuvettes cannot be used for acetone based chlorophyll assays. Methanol is less volatile and flammable than acetone but is notoriously toxic. It cannot be used with plastic cuvettes. Diethyl ether is a very popular solvent for chlorophylls for research purposes, particularly for preparing pure pigments. It is extremely volatile, flammable, explosive and narcotic. The explosion hazard in particular restricts its use [4, 5, 6, and 7]. Many studies were done on the effect of type of the solvent on the extracted pigments in different plants, for example[8]. Manuela et al. (2012) found that for vegetables crops 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. [9].Kumar et al. (2010) 108

2 observed that ethyl acetate showed highersignificance during the extraction process when compared to ethanol and acetone for some algal species. The Soil-Plant Analyses Development (SPAD) unit of Minolta Camera Co. has developed the SPAD-502 chlorophyll meter (Minolta Camera Co., Japan), a convenient, and nondestructive lightweight device used to calculate the amount of chlorophyll present in plant leaves [10]. The aim of this study is compares the use of three different solvents viz. acetone, methanol and, diethyl either for determining chemical extraction capabilities of chlorophyll a, chlorophyll b and total carotenoids from bean and cowpea leaves in three different growth stages, it also aims to find the relationship between the chemical method for pigments extracting and the reading of SPAD chlorophyll meter instrument. 2. Materials and Methods 2.1.Sample collection Bean (Phaseolus vulgaris L.) var. Biotack and cowpea (VignasinensisSavi)var.California black eye seeds were planted at 23 April in summer season 2015 in the Faculty of Science and Health, Koya University, Erbil- Iraq (44 o 38 E, 36 o 4N and 517 m of altitude). Fifth leaf of a three separated plants were harvested from each species in three different growth stages, vegetative, flowering and podding. The chlorophylls content were determined for each leaf separately by SPAD chlorophyll meter (SPAD-502, Japan) before the chemical extracting of pigments. 2.2.Chemical Extraction Process Fresh weighted samples were extracted with acetone (80%), methanol (99%) and diethyl either (99%). Concentration of chlorophyll a and b and total carotenoids in obtained extracts was measured spectrophotometrically ( SPECTROPHOTOMETER, China) with equations presented below (Table 1)[11], according to these measurements, each of chlorophyll a, b and total carotenoids content in fresh materials were determined. Table 1. The equations used for measure chlorophyll a, b and total carotenoids by different extractent solvents in spectrophotomete. Solvents Acetone Equations Chl a = 11.75A A645 Chl b = 18.61A A662 Car = 1000 A Chl a 81.4 Ch b /227 Methanol Diethyl either Chl a = 15.65A A653 Chl b = 27.05A A666 Car = 1000 A Chl a Ch b /245 Chl a =10.05A A644 Chl b = A A662 Car = 1000 A Chl a 56.7 Ch b /230 Where: Chla chlorophyll a [mg/l] Chlb chlorophyll b [mg/l] Car = Total Carotenoids [mg/l] A662 absorbance at wavelength 662 nm 109

3 A645 absorbance at wavelength 645 nm A470 absorbance at wavelength 470 nm A666 absorbance at wavelength 666 nm A653 absorbance at wavelength 653 nm A644 absorbance at wavelength 644 nm For converting the concentrations from mg/l to mg/100 g fresh weight, each value multiplied by (extraction volume/ (sample weight*1000)). 2.3.Statistical Analysis The treatments of all experiments replicate three times, and the comparisons between means were made by using Least Significant different (LSD) test at 5% level [12]. Correlation coefficient between SPAD reading and chemical method for pigment estimation were determined too. The statistical analysis was carried out by using SPSS program. 3. Results and Discussion In this study, chemical extraction by three different solvents and SPAD instrument were used to determine some photosynthetic pigments from fresh materials of bean and cowpea leaves on three different growth stages. Concentration of chlorophyll a, b and total carotenoids showed variations according to plant material used and to the solvent used in the chemical extraction in addition to the stage of growth. Results in table 2 and 3 shows that concentration of chlorophyll a in bean and cowpea leaves increased significantly when acetone used in the extraction process followed by methanol and the lowest obtained when the diethyl either is used. This result is disagree with [4].Nayek et al. (2014) whom found that diethyl either is the best solvent for extracting chlorophylls in some ferns,.when methanol was used as solvent, chlorophyll b concentration increased in bean and cowpea leaves non-significantly compare to acetone and significantly compare to diethyl either solvent, this result agree with [2].Verma (2008), whom state that chlorophyll b dissolves very well in methyl alcohol. This different in chlorophyll a and b solubility is due to chlorophyllb differ from chlorophylla only in one functionalgroup (i.e -CHO) bounded to the porphyrin ring, and is moresoluble than chlorophyll-a in polar solvents because of itscarbonyl group[11]. The acetone solvent enabled extracting more total carotenoids significantly compared to diethyl either solvent, this result agrees with [8]. Manuela et al. (2012) whom found that acetone extractiondetermined the best results in all analyzed vegetables: tomatoes, peppers and cucumbers carotene extracting.when performing methanol extraction, the content of total carotenoids was, difficult to detect, therefore it consider as zero. Thus the selection of the methodand the solvent to be used in the studies in connectionwith pigments according to the species will be moreuseful[13]. The concentration of chlorophyll a and b increased significantly in the flowering stage compare to vegetative and podding stages in bean and vegetative growth in cowpea, whereas there were non-significant differences in total carotenoids with changing the age of bean and cowpea plants (Table 2 and 3). Bean plants records high concentration of chlorophyll a, b and total carotenoids when acetone is used in the extraction process in the flowering stage, whereas cowpea record variation in the pigments concentration between different solvents and growth stages (Table 2 and 3). Results in table 4 shows that there were non-significant differences in SPAD meter readings between different growth stages in both of bean and cowpea plants. The data also shows that the SPAD meter reading is high in cowpea compare to bean plants.this can be attributed to inherent physiologicalcharacteristics of individual species[4]. There were positive correlations between SPAD meter readings and chlorophyll concentrations determined by chemical method with different solvents for bean and cowpea plants (Table 5), this may be due to sampling method, because SPAD meter readings were taken from same leaves used for chemical 110

4 chlorophyll extraction. SPAD meter readings correlated high significantly with chlorophylls a and b concentration, when acetone used as solvent in cowpea plants (Table 5). This may be due to effectiveness of acetone in extracting chlorophylls compare to other solvents, especially diethyl either, especially for vegetables crops [8], and attributed to inherent physiological characteristics of cowpea which may contains other types of chlorophylls which that reflects in the high value of SPAD meter reading compared to bean plant leaves (Table 4). This positive results found in previous studies of other crops like in St. Augustinegrass (Stenotaphrumsecondatum (Walt.) Kuntze, where SPAD meter readings were positively correlated with chlorophyll concentrations r 2 = 0.79 [14], and with extractable chlorophyll for eleven food crop species (r 2 >0.90) [15]. Table 2. Chlorophyll a, b and total carotenoids concentration in bean (Phaseolus vulgaris L.) extracted by different solvent in different growth stages. Treatments Chlorophyll a Chlorophyll b Total Carotenoids Solvents mg/100 g fresh weight Acetone 3.14 a 2.67 a 0.42 a Methanol 2.49 b 2.95 a 0.00 c Diethyl either 1.06 c 1.76 b 0.29 b Growth stage Vegetative 2.39 b 2.16 b 0.23 a Flowering 2.81 a 3.11 a 0.25 a Podding 2.03 c 2.14 b 0.24 a Interaction Acetone x Vegetative 2.86 b 2.78 b 0.24 c Acetone x Flowering 4.02 a 3.70 a 0.53 a Acetone x Podding 2.54 bc 1.61 c 0.50 ab Methanol x Vegetative 2.80 b 3.18 ab 0.00 d Methanol x Flowering 2.58 bc 2.91 b 0.00 d Methanol x Podding 2.10 cd 2.75 b 0.00 d Diethyl either x Vegetative 1.52 d 0.52 d 0.44 b Diethyl either x Flowering 1.83 d 2.75 b 0.23 c Diethyl either x Podding 1.60 d 2.05 c 0.21 c Mean S.E * Means followed by the same letters within column are not significantly different at p 0.05 according to the Least Significant Differences (LSD) test. 111

5 Table 3. Chlorophyll a, b and total carotenoids concentration in cowpea (VignasinensisSavi) extracted by different solvent in different growth stages. Treatments Chlorophyll a Chlorophyll b Total Carotenoids Solvents mg/100 g fresh weight Acetone 3.03 a 2.79 a 0.41 a Methanol 2.88 a 3.20 a 0.00 c Diethyl either 1.69 b 1.74 b 0.34 b Growth stage Vegetative 2.37 b 2.05 b 0.25 a Flowering 2.74 a 2.86 a 0.24 a Podding 2.49 ab 2.82 a 0.26 a Interaction Acetone x Vegetative 2.78 b 2.80 bc 0.24 b Acetone x Flowering 3.66 a 3.38 ab 0.49 a Acetone x Podding 2.65 bc 2.18 c 0.50 a Methanol x Vegetative 2.46 bcd 2.57 bc 0.00 c Methanol x Flowering 2.90 ab 2.95 abc 0.00 c Methanol x Podding 3.27 ab 4.07 a 0.00 c Diethyl either x Vegetative Diethyl either x Flowering Diethyl either x Podding 1.87 cde 0.77 d 0.52 a 1.66 de 2.24 bc 0.24 b 1.55 e 2.21 c 0.27 b Mean S.E * Means followed by the same letters within column are not significantly different at p 0.05 according to the Least Significant Differences (LSD) test. Table 4. Relationship between SPAD reading and different growth stages for bean and cowpea plants Growth stage Bean Cowpea Vegetative a a Flowering a a Podding a a Mean S.E * Means followed by the same letters within column are not significantly different at p 0.05 according to the least significant Differences (LSD) test. 112

6 Table 5. Relationship between SPAD reading and different pigments extracted by different solvents for bean and cowpea plants. SPAD reading Bean 0.39 Cowpea 0.80 ** Acetone Methanol Diethyl Either Ch. a Ch.b Car. Ch. a Ch.b Ch. a Ch.b Car ** Conclusion Results from this experiment clearly indicate that extraction of photosynthetic pigments by different solvents are depends on chemical nature of the pigments (chlorophyll a, chlorophyll b and carotenoids). Investigation reveals for each of bean and cowpea plants,acetone was best extractant solvent for chlorophyll a and total carotenoids, whereas methanol for chlorophyll b. No significant differences observed in the SPAD chlorophyll meter reading and chemically extracted pigment for three different solvents. Further experiments is needed to assess the effect solvents dilution for obtain the highest concentrations of pigments. References [1] Vechetel, B.W. and Ruppel, H.G Lipid Bodies in Eremosphaeraviridis De Bary (Chlorophyceae). Plant and Cell Phys. 31: [2] Verma, V Textbook of Plant Physiology. First Edition. Published by Ane Books. India. [3] Srivastrava, H.S Plant Physiology, Biochemistry and Biotechnology. First Edition.Rastogi publications. India. [4] Nayek, S., Choudhury, I. H., Jaishee, N. and Roy, S Spectrophotometric Analysis of Chlorophylls and Carotenoids from Commonly Grown Fern Species by Using Various Extracting Solvents. Research Journal of Chemical Sciences. 4(9): [5] Porra R.J., Thompson W.A. and KreidemannP.E Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. Biochim. Biophys. Acta. 975: [6] Scheer H Chlorophylls. CRC Press Boca Raton, Ann Arbor, Boston, Landon, [ innayek et. al 2014). [7] Ritchie R. J Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents, Photosynth. Res. 89: [8] Manuela, A.C., Gheorghe C. and Gabrila, N Studies concerning the extraction of chlorophyll and total carotenoids from vegetables. Romanian Biotechnological Letters. 17(5): [9] Kumar, P., Ramakritinan, C.M. and Kumaraguru, A.K Solvent extraction and spectrophotometric determination of pigments of some algal species from the shore of Puthumadam, Southeast coast of India. International Journal of Oceans and Oceanography. 4(1): [10] Minolta SPAD-502 owner s manual. Industrial Meter Div. Minolta Corp., Ramsey, N.J. [11] Lichtenthaler, H.K. and Wellburn, A.R Determination of Total Carotenoids and Chlorophylls A and B of Leaf in Different Solvents, Biol. Soc. Trans., 11: [12] Reza, A.H Design of Experiments for Agriculture and the Natural Sciences. New York. Chapmann& Hall. [13] Sükran, D.,Tohit G. and Rıdvan S Spectrophotometric Determination of Chlorophyll - A, B and Total Carotenoid Contents of Some Algae Species Using Different Solvents. Tr. J. of Botany. 22 : [14] Ian R. R. and Grady, L. M Using a Chlorophyll Meter to Determine the Chlorophyll Concentration, Nitrogen Concentration, and Visual Quality of St. Augustinegrass. Hortscience. 35(4): [15] Marquard, R.D. and Tipton, J.L Relationship between extractable chlorophyll and an in situ method to estimate leaf greenness. Hortscience 22: