The synthesis and characterization of 7-hydroxy-4-methylcoumarin and theinvestigation of the fluorescence properties of its 7-hydroxy-4-methylcoumarin-chitosan filmsDeana Wahyuningrum, Muhammad Zulqarnaen, and Veinardi Suendo

Citation: AIP Conference Proceedings 1589, 169 (2014); doi: 10.1063/1.4868775 View online: http://dx.doi.org/10.1063/1.4868775 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1589?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Synthesis and properties of nanosized silver catalyst supported on chitosan-silica nanocomposites AIP Conf. Proc. 1502, 255 (2012); 10.1063/1.4769149 Synthesis of Schiff Base Bearing Phenolic Hydroxy Group and Its Anion Recognition Chin. J. Chem. Phys. 24, 330 (2011); 10.1088/1674-0068/24/03/330-334 Synthesis And Characterization Of Silver Sulfide Nanoparticles Of Various Morphologies Using Chitosan AsStabilizer AIP Conf. Proc. 1276, 62 (2010); 10.1063/1.3504343 Synthesis Of Graphene/Chitosan Nanocomposite Thin Films AIP Conf. Proc. 1276, 158 (2010); 10.1063/1.3504291 The photophysical behavior of 3-chloro-7-methoxy-4-methylcoumarin related to the energy separation of the twolowest-lying singlet excited states J. Chem. Phys. 107, 6062 (1997); 10.1063/1.474274

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The Synthesis and Characterization of 7-Hydroxy-4-Methylcoumarin and the Investigation of the FluorescenceProperties of Its 7-Hydroxy-4-Methylcoumarin-Chitosan

Films

Deana Wahyuningrum 1*, Muhammad Zulqarnaen 2*, Veinardi Suendo 3

1Organic Chemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung,Jl. Ganesha 10, Bandung 40132, Indonesia

2Chemistry Study Program, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha10, Bandung 40132, Indonesia

2Inorganic and Physical Chemistry Research Division, Faculty of Mathematics and Natural Sciences, InstitutTeknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia

* Email: deana@chem.itb.ac.id

Abstract. Chitosan fluorescent films containing 7-hydroxy-4-methylcoumarin (7H4MC) have been successfullyprepared. Used chitosan was obtained from chitin isolated from skin of tiger prawns (Penaeus monodon) through thedeproteination, demineralization, and deacetylation process. The yields of chitin and chitosan are 10.66% and 23.83%,respectively. The chitosan has 55.00% degree of deacetylation based on FTIR spectroscopy. Average molecular mass ofchitosan which was determined by Ostwald viscometry method is 8.55 x 106 g/mol. The 7H4MC was synthesized fromresorcinol and ethyl acetoacetate using amberlyst-15 as catalyst based on Pechmann reaction with chemical yields of90.01% and the melting point of 189-190˚C. The FTIR, 1H-NMR, and 13C-NMR spectroscopies confirmed the structurewhich corresponds to the structure of 7H4MC. The films of chitosan containing 7H4MC were prepared by solventevaporation method in 2% (v/v) acetic acid. The 7H4MC content in each film was 0% (blank), 0.2%, 0.4%, 0.6%, and0.8% (w/w). The UV-Vis spectrum of 7H4MC in methanol showed λmax at 235 and 337 nm. The observed fluorescenceis the fluorescence color of cyan. The excitation wavelengths are 200, 235, 275, 337, and 365 nm. The highest intensityof cyan color fluorescence of chitosan containing 7H4MC films was obtained at the concentration of 0.2% of 7-hydroxy-4-methylcoumarin at the excitation wavelength of 275 nm.

Keywords: chitosan, coumarin, fluorescence, Pechmann.PACS: 81

INTRODUCTION

Recently, fluorescence materials are beingdeveloped to be applied in display technology, laserand solar cells [1]. Coumarin derivative compounds insolution have been widely used as dye laser, brighteneragents in detergent and as fluorescence markers [2].However, so far coumarin derivatives in solid matricesare scarcely used or have not been applied yet.

Chitosan derived from isolated chitin of Penaeusmonodon shrimp shells does not have goodfluorescence at visible light wavelength range; on theother hand, coumarin derivatives could have excellentfluorescence at the same wavelength region.However, chitosan could be formed as strong fiber ormembranes, while coumarin derivatives are smallmolecules that lack of those properties [3,4]. Bycombining the advantages of the good properties ofchitosan and coumarin derivatives, therefore coumarin

derivatives-chitosan film can be developed to fulfillthe properties needed as potent fluorescence materials.

EXPERIMENTS

GeneralOrganic solvents are analytical grade and purified

further prior to use. All starting materials are analyticalgrade and used without further purification. Thesource of chitin is Penaeus monodon shrimp shellswaste. The characterizations of compounds wascarried out using Fisher-Johns Melting PointApparatus and spectrophotometry methods: FTIR(Alpha Bruker FTIR at Physical Chemistry andMaterials Research Laboratory, ITB); 1H-NMR (500MHz) and 13C-NMR (125 MHz) (AgilentTechnologies NMR at Basic Science Center A, ITB);UV-Vis and Fluorescence Spectrophotometer

4th International Conference on Mathematics and Natural Sciences (ICMNS 2012)AIP Conf. Proc. 1589, 169-173 (2014); doi: 10.1063/1.4868775

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(JENWAY 6305 UV/Vis Spectrophotometer atPhysical Chemistry and Materials ResearchLaboratory, ITB); and LC-MS (LC-MS MarinerBiospectrometry Work Station with C-18 Column atLIPI Serpong).

Experimental Details

Isolation of Chitin from Penaeus monodon ShrimpShell and Its Transformation to Chitosan

The isolation of chitin was carried out according tothe modified procedures of previous work [5] viadeproteination and demineralization of Penaeusmonodon shrimp shells, and deacetylation of chitin in50% (w/v) NaOH solution. Dried shrimp shell wasgrinded into powder and dissolved in 3.5% (w/v)NaOH solution in ratio of shrimp shell to NaOHsolution is 1:10 (w/v). The mixtures were heated to 65oC for 2 hours. The residue was collected, washed anddried in oven at 60°C for 2 hours. The residue wasdissolved in 1 M HCl solution in ratio of shrimp shellsto HCl solution is 1:15 (w/v) and stirred for 1 hour atroom temperature. The residue was collected, washed,and dried in oven at 60°C. The isolated chitin wasdissolved in 50% (w/v) NaOH solution in ratio ofchitin to NaOH solution is 1:15 (w/v). The mixtureswere refluxed for 4 hours. The residue was collected,washed and dried in oven at 60° C to obtain chitosan.Dried chitosan was further characterized using FTIRand its average viscosity molecular weight ( ) wasdetermined using Ostwald viscometer method [6].

The Synthesis of 7-Hydroxy-4-Methylcoumarin(7H4MC)

The synthesis of 7H4MC was carried out usingmodification of Pechmann reaction procedure (Figure1) [7,8].

FIGURE 1 The reaction scheme of synthesizing 7-hydroxy-4-methylcoumarin (7H4MC) from the reaction betweenresorcinol and ethyl acetoacetate

Resorcinol (0.6655 g), ethyl acetoacetate (0.76mL), toluene (10 mL) and Amberlyst-15 (0.6565 g)were placed in 50 mL round flask. The mixtures wererefluxed at 150 oC for 45 minutes to form solidprecipitation. The reaction was monitored using TLCin silica gel 60 GF254 using n-hexane:ethyl acetate(3:2) as eluents resulting the Rf value of the product =0.46. Warm methanol (20 mL) was added to dissolvethe solid product and the amberlyst-15 was removedby filtration. The filtrate was evaporated in vacuum toremove the solvents and the product was recrystallizedfrom methanol/water [8].

7-Hydroxy-4-methylcoumarin: 90.01% yields;white crystalline solid; m.p. 189-190 oC; FTIR (KBr,ν, cm-1): 3500 (O–H), 3113 (=C–H), 1671 (conjugatedC=O), 1607 (C=C) and 1394 (symmetry deformationof –CH3); 1H NMR (500 MHz, DMSO-d6, , ppm):2.34 (s, 3H), 6.10 (s, 1H), 6.69 (d, J = 2.34 Hz, 1H),6.78 (dd, Ja = 2.36 Hz, Jb = 8.67 Hz, 1 H), 7.56 (d, J =8.69 Hz, 1H), 10.51 ppm (s, broad, 1H). 13C-NMR(125 MHz, DMSO-d6, , ppm): 161.17 (lactonecarbonyl), 160.31; 154.85; 153.52; 126.59; 112.87;112.03; 110.27; 102.19 (-aromatics and alkenes);18.12 (-CH3). LC-MS: retention time 2.8 minutes;[M+H]+ with m/z = 177.23 (base peak) and [M+Na]with m/z = 199.17.

The Preparation of 7-Hydroxy-4-Methylcoumarin-Chitosan Film

The preparation of chitosan film and 7HMC-Chitosan film was carried out using solventevaporation method. The composition of eachprepared film was presented in Table 1. The solventused in each preparation was 40 mL of 2% (v/v) aceticacid. The template for film casting was 9 cm Petridishes. Dried casted films were removed from Petridishes by soaking in water. The films were furthercharacterized to analyze its fluorescence properties.

TABLE 1 The composition of 7H4MC-Chitosan filmsNo. Chitosan (mg) 7H4MC (mg)1 513.6 -2 513.6 1.03 513.6 2.14 513.6 3.15 513.6 4.2

RESULTS AND DISCUSSIONThe chitin was isolated from shrimp shells in yields

of 10.66% (5.33 g chitin from 50 g shrimp shells).The chitin has been transformed to chitosan in yieldsof 23.83% (1.27 g chitosan from 5.33 g chitin) and hasthe degree of deacetylation of 55.00% according toFTIR spectra analysis. The average viscositymolecular weight ( ) of chitosan is 8.55 x 106 g/mol

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based on the results of Ostwald viscometer methoddetermination.

The coumarin derivative, 7-hydroxy-4-methylcoumarin (7H4MC), has been successfullysynthesized with the chemical yields of 90.01%according to the modified Pechmann reaction. TheLC-MS measurements showed one peak with retentiontime of 2.8 minutes on LC chromatogram as well asthe peak of [M+H]+ with m/z 177.23 as base peak andthe peak of [M+Na] with m/z 199.17 on MS spectrum.The FTIR spectrum of 7H4MC showed the vibrationsof O–H at ν = 3500 cm-1, =C–H at ν = 3113 cm-1,conjugated C=O at ν = 1671 cm-1, C=C at ν = 1607cm-1, and the vibration of symmetrical deformation ofmethyl group at ν = 1394 cm-1. The spectrum of 1H-NMR showed six different type of hygrogen signals atchemical shifts of 2.34 ppm which represents methylgroup; the chemical shifts of 6.10, 6.69, 6.78, and 7.56ppm which represent aromatics hydrogens; and thechemical shifts of 10.51 ppm which represents thehydrogen of hydroxyl (-OH) group of coumarin. Thespectrum of 13C-NMR showed one lactone carbonyl(161.17 ppm), eight carbons of aromatic rings (160.31;154.85; 153.52; 126.59; 112.87; 112.03; 110.27;102.19 ppm), and one methyl carbon (-CH3) (18.12ppm). All of spectroscopy characterizations confirmedthe structure of 7H4MC. The chemical shifts data ofthe synthesized 7H4MC shown in NMR spectra waspresented on Table 2.

TABLE 2 The chemical shifts data of the synthesized7H4MC shown in 1H-NMR and 13C-NMR spectra

HO

CH3

O O1

2

345

6

7

89

10

11

12Numbering of

matching nuclei incorrespondingstructure above

H, ppm (multiplicity,J (coupling constant)

in Hz, numbers ofhydrogens)

C, ppm(numbers

of carbons)

1 - -2 - 161.173 6.10 (s, 1H) 112.034 - 153.525 7.56 (d, J = 8.69 Hz,

1H)126.59

6 6.69 (d, J = 2.34 Hz,1H)

112.87

7 - 160.318 6.78 (dd, Ja = 2.36

Hz, Jb = 8.67 Hz, 1 H)102.19

9 - 154.8510 - 110.2711 2.34 (s, 3H) 18.1212 10.51 (s-broad, 1H of

hydroxyl group)-

The UV-Vis absorption spectrum of 7H4MC inmethanol shows two peaks at wavelength of 235 and337 nm. The maximum wavelength of UV-Visabsorption spectrum occurred because of the S1←S0transition, which is the absorption peak at 337 nm.The absorption peak at shorter wavelength occurredbecause of the S2←S0 transition. The S1←S0 hashigher probability to occur compare to S2←S0transition; therefore the height of absorption peak at337 nm is larger than the peak at 235 nm [9].

The 7-hydroxy-4-methylcoumarin-Chitosan filmshave been prepared and its fluorescence propertieswere analyzed at the maximum absorption of 7H4MC(337 nm) and at other wavelengths beside of themaximum absorption (at 200, 235, 275, 337, and 365nm). The 7-hydroxy-4-methylcoumarin-Chitosan filmsgave cyan fluorescence with low intensity under UVlightning at shortwave mode (254 nm). The 7H4MCitself still has UV absorption at 254 nm which is thetail part of the peak at 235 nm, therefore it still has thecapability to fluoresce with low intensity because ofthe absorption near 235 nm is less favorable. All 7-hydroxy-4-methylcoumarin-Chitosan films gave cyanfluorescence with relatively high intensity under UVlightning at long-wave mode (365 nm). The 7H4MCitself still has UV absorption at 365 nm; therefore itstill has the capability to fluoresce with higherintensity than at shorter wavelengths. On the otherhand, the fluorescence of 7-hydroxy-4-methylcoumarin-Chitosan films at visible light regionis also cyan at the wavelength of 480-490 nm.Therefore, the analysis of fluorescence spectra wasfocused at the wavelength emission of 480-490 nm.

The fluorescence spectra at wavelength of 200,235, 275, 337, and 365 nm for each 7-hydroxy-4-methylcoumarin-Chitosan film at the same conditionwere presented on Figure 2. All films show the cyanintensity fluorescence at various excitationwavelengths with the sequence of excitationwavelength from the lowest to the highest intensity asfollows: 200, 337, 235, 365, and 275 nm. At theexcitation wavelength of 200 nm, the intensity of cyanis very low (below 1000) which is caused by the lackof electronic energy transition at the excitationwavelength of 200 nm that give rise to the cyanfluorescence. The cyan fluorescence at its excitationwavelengths (235 and 337 nm) has lower intensitythan at the other wavelengths. This phenomenonhappened because of the excitation wavelengths favorthe fluorescence at the same wavelengths as theresonance fluorescence excitation wavelengths. Theresonance fluorescence at 337 nm is more favorablebecause at this wavelength the probability of S1←S0transition is high and almost the same as its reversetransition process, which is S0←S1 transition. Theprobability of S2←S0 transition at excitation

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wavelength of 235 nm is also high and also almostsimilar to the reverse transition process, which isS0←S2, therefore the resonance fluorescence is alsofavorable at 235 nm. The probability of S1←S0transition is higher than the S2←S0 transition;therefore the cyan fluorescence at excitationwavelength of 235 nm has higher intensity than atexcitation wavelength of 337 nm. At the excitationwavelengths which are not the peaks on UV-Visabsorption spectra (except at 200 nm), the cyanfluorescence gave higher intensities because theexcitation at wavelength of 275 and 365 nm is lessfavorable, therefore once the electron was excited, theelectron has more difficulty to directly go back to theprevious state and would rise to the non-radiationenergy transfer which has higher probability. Thiswould lead to the more favorable Stokes shifts inproducing the higher intensities of cyan fluorescence[10].

The fluorescence spectra at excitation wavelengthof 200 nm did not give significantly the cyan color. Atthe excitation wavelengths of 235, 275, and 365 nmthe cyan fluorescence of 7-hydroxy-4-methylcoumarin-Chitosan films gave similar intensity.

The highest intensity, qualitatively (Figure 2), wasgiven by the 7-hydroxy-4-methylcoumarin-Chitosanfilm with the composition of 0.2% of 7-hydroxy-4-methylcoumarin in chitosan film. This phenomenonwas caused by the secondary absorption, which is theabsorption of radiation emission of the same type ofmolecules that increase with the increasing ofconcentration after reaching its saturated point offluorescence [10]. The intensity of cyan fluorescenceof 0.8% of 7-hydroxy-4-methylcoumarin-chitosan filmwas higher than 0.6% of 7-hydroxy-4-methylcoumarin-chitosan film, which showed that theincrease in the amount of 7-hydroxy-4-methylcoumarin within chitosan film would affect thefluorescence intensity. In the same point of view, thehighest cyan fluorescence intensity at the excitationwavelength of 337 nm occurred at the concentration of0.4% of 7-hydroxy-4-methylcoumarin-chitosan filmbecause at this wavelength the saturation offluorescence have not been accomplished at the lowerconcentration, therefore its fluorescence intensity stillincreased after the addition of 0.4% of 7-hydroxy-4-methylcoumarin.

FIGURE 2 The fluorescence spectra of 7-hydroxy-4-methylcoumarin-Chitosan films at various excitation wavelenghts: (A). 0.2%of 7H4MC-Chitosan film; (B) 0.4% of 7H4MC-Chitosan film; (C) 0.6% of 7H4MC-Chitosan film; (D) 0.8% of 7H4MC-Chitosan film

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CONCLUSIONSThe synthesis of 7-hydroxy-4-methylcoumarin

(7H4MC) have been accomplished according tomodified Pechmann reaction. The chitosan fluorescentfilms containing 7H4MC have also been successfullyprepared with various compositionss of 7-hydroxy-4-methylcoumarin (0.2%, 0.4%, 0.6%, and 0.8% (w/w)).The observed fluorescence in each film is thefluorescence color of cyan. The highest intensity ofcyan color fluorescence of chitosan containing7H4MC films was obtained at the concentration of0.2% of 7-hydroxy-4-methylcoumarin at the excitationwavelength of 275 nm. These 7-hydroxy-4-methylcoumarin-Chitosan films have the potent to beapplied as fluorescent materials.

ACKNOWLEDGMENTSWe would like to express our gratitude to Prof.

Yana Maolana Syah of ITB for the kind assistance inNMR measurements; Mrs. Teni Ernawati, M.Sc. ofLIPI Serpong for kind contribution in LC-MSmeasurements; and Dr. Rer.nat. Didin Mujahidin forproviding Amberlyst-15 for the synthesis of 7H4MC.

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