eosin

Pakistan Journal of Agriculture Sciences, 2009 Fatima et al.,

SYNTHESIS AND APPLICATION OF EOSIN

Kabeer Fatima1, Sofia Nosheen,2 Humera and Munazza Azhar3

Department of Chemistry and Biochemistry, Faculty of Sciences, University of Agriculture, Faisalabad, Pakistan

Dyes are colored substances having affinity for one or more textile fibers on which they can be applied. Nature does provide with dyes, but with the advent of civilization, chemists are trying to synthesis dyes by new approaches. The present project involved the synthesis and application of eosin by new protocols involving effect of catalyst and temperature. Different Phenolic components (Resorcinol, Caticol, β-napthanol, Anisole, o-Cresol), Catalyst (zinc chloride, ferric chloride, calcium chloride) and temperature (150, 200 and 250oC) were used for synthesis of fluorescein. Only resorcinol gave the product at different temperatures with different catalysts. Higher temperature 250 oC and zinc chloride were found to be best because they gave higher yield of product. For synthesis of eosin different bromine concentrations (5, 10 and 50%) were used and higher bromine concentration (50%) was found to be best as this gave higher yield of product. Finally the obtained product was applied as ink and as dye on silk and wool. Eosin ink was also compared with the commercial red ink. It was observed that as the temperature increases the quantity as well as quality of dye also increases. Thus samples prepared at higher temperature gives good and dark tones of colors. Key words; Eosin, application of eosin, synthesis of eosin, fluorescent dyes, eosin ink

1. INTRODUCTION

Dyes are colored substances having affinity for one or more

textile fibers on which they can be applied by suitable means light, dry and wet rubbing. www.DyesOnline.com

All the colored compounds are not necessarily dyes, only

those colored compounds are called dyes which can fix to a cloth. (Tyagi and Yadav, 1990). They are retained in these substances by physical adsorption, mechanical retention or by the formation of covalent bonds. (Shah, 1994).

Many plants yield dyestuffs that will dye wool or silk, but

there are few of these that will dye cotton as well. In addition, the natural dyes, with a few exceptions, do not cover a wide range of colors, nor do they yield “brilliant” colors. Also, synthetic dyes that will dye the popular synthetic fibers can now be manufactured. Thus, today we have available an almost infinite variety of colors, as well as dyes to dye any type of fabric. (Benjamin et al., 2004). Ecological problems that have arisen in the last years from the application of different organic products, including dyes and pigments, have necessitated investigations directed to the synthesis of more tolerant materials (Konstantinova and Bonjinov, 1998).

Fluorescent labeling techniques have become increasingly

popular in many areas. Thus the extensive use of fluorescent compound explores new ways to synthesized new and stable fluorescent dyes. (Deerinck et al., 1994).

Fluorescein an important fluorescent compound, is a

fluorophore commonly used in microscopy, in a type

of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein was first synthesized by Adolf von Baeyer in 1871. It can be prepared from phthalic anhydride and resorcinol in the presence of zinc chloride via the Friedel-Crafts reaction. (Sun et al., 1997). The general reaction for the synthesis of fluorescein is shown in Fig.1

Fig. 1. Synthesis of fluorescein

Eosin yellowish (2', 4', 5', 7'-tetrabromofluorescein,

EYH2) is a well-known organic dye, characterized by a large conjugated ח system, allowing л-л* transitions at low energy, i.e., in the visible part of the spectrum. (Hazebroucq et al., 2008). Eosin can be prepared by Bromination of fluorescein, (which exists in two Tautomeric forms), using bromine in glacial acetic acid or alcohol medium yields eosin. (Vasudevan and Anantharaman, 1993). The general reaction for the synthesis of eosin is shown in Fig.2

Fig. 2. Synthesis of eosin

1 2007-ag-404 Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan Email; [email protected] 2 Corresponding Author Lecturer, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan. Email; [email protected] 3 2007-ag-403 Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan Email; [email protected]


This acidic molecule has an affinity for basic proteins, it gives different colors to the cells or to the tissues, according to the pH of the medium. For such peculiar characteristics EYH2 has been widely used as a dye in many biological and medical fields, such as cytology, histology, and hematology. In chemistry, it has been studied as a photocatalyst or as the main component in the fabrication of holographic optical elements, this latter application being related to its fluorescent properties. (Hazebroucq et al., 2008). The familiar haematoxylin and eosin (H&E) preparation employs eosin as a counter stain. Eosin stains those structures that are positively charged and have no affinity for a metal mordant. Basic groups (primarily amino) are positively charged so that at a pH below their isoelectric point they will bind anionic dyes such as eosin. Such dyes are therefore called acid dyes and the substance stained is termed acidophilic (acid-loving), or eosinophilic in the case of cell components that stain with eosin. (Awgustafson, 2006).

In Pakistan domestic production of dyes is very insufficient in comparison to their use in textile and non- textile purposes thus we have to import and consume huge amount of foreign exchange. Keeping in view, the consumption of foreign exchange on dyes, it is imperative to synthesis dyes in our own country with improved qualitative and quantitative aspects. Major objectives of this project include optimization of reaction conditions for the better quality of eosin, improvement in percentage yield and application of eosin dye.

2. MATERIALS AND METHODS 2.1. Materials Following chemicals were used for synthesis and application of eosin; Phthalic anhydride, Resorcinol, Catechol, β-napthanol, Anisole, o-Cresol, Anhydrous-Zinc chloride, Ferric chloride, Calcium chloride, Conc. Hydro chloric acid, Sodium hydroxide, Bromine, Ethanol and Ammonium chloride. All the chemicals were Merk greded. 2.2. Synthesis of fluorescein

The preparation of fluorescein was done by the condensation of various phenolic components with phthalic anhydride catalyzed with different catalyst at various temperatures as follows; (Waheed and Gupta, 1996)

2.2.1. By using resorcinol

Nine experiments were performed with resorcinol using three catalysts (zinc chloride, ferric chloride and calcium chloride) at three different temperatures (150, 200 and 250oC). 5g phthalic anhydride and 7.4g resorcinol was grinded in a mortar. Then transferred the mixture to a

250ml Erlenmeyer flask, a thermometer was immerse and the flask was heated slowly to desired temperature (150 200 or 250oC) on a sand-bath. In the meantime 2.3g of desired catalyst (zinc chloride, ferric chloride or calcium chloride) added to the reaction mixture in small lots by Stoppard bottle, stirring with the thermometer after each addition. Heating was continued till the mixture became dark red and highly viscous. Flask was cooled to about 90oC and to this 70ml water and 3.5 ml conc. HCl was added. Heated again till the salts of zinc, calcium and ferric have dissolved. The colored salt was filtered on Buchner funnel, washed with water, drained well and dried. The propose mechanism is given here in Fig.3

Fig. 3. General mechanism for synthesis of fluorescein 2.2.2. By using β-napthanol

Nine experiments were performed with β-napthanol

using three catalysts (zinc chloride, ferric chloride and calcium chloride) at three different temperatures (150, 200 and 250oC). 5g phthalic anhydride and 7.4g β-napthanol was grinded in a mortar. Then transferred the mixture to a 250ml Erlenmeyer flask, a thermometer was immerse and the flask was heated slowly to desired temperature (150, 200 or 250oC) on a sand-bath. In the meantime 2.3g of desired catalyst (zinc chloride, ferric chloride or calcium chloride) added to the reaction mixture in small lots by Stoppard bottle, stirring with the thermometer after each addition. Heating was continued till the mixture became dark red and highly viscous. Flask was cooled to about 90oC and to this 70ml water and 3.5 ml conc. HCl was added. Heated again till the salts of zinc, calcium and ferric have dissolved. The colored salt was filtered on Buchner funnel, washed with water, drained well and dried. The propose mechanism is given herein fig 4.

Fig.4. Mechanism for synthesis of fluorescein by

β-napthanol

2


2.2.3. By using o-cresol One experiment was performed with o-cresol using

catalyst zinc chloride at temperature 200oC. 5g phthalic anhydride and 7.4g o-cresol was grinded in a mortar. Then transferred the mixture to a 250ml Erlenmeyer flask, a thermometer was immerse and the flask was heated slowly to 200oC on a sand-bath. In the meantime 2.3g of catalyst zinc chloride added to the reaction mixture in small lots by Stoppard bottle, stirring with the thermometer after each addition. Heating was continued till the mixture became dark red and highly viscous. Flask was cooled to about 90oC and to this 70ml water and 3.5 ml conc. HCl was added. Heated again till the zinc salts have dissolved. The colored salt was filtered on Buchner funnel, washed with water, drained well and dried. The propose mechanism is given here in fig.5.


o-cresol

2.2.4. By using anisole One experiment was performed with anisole using catalyst zinc chloride at temperature 200oC. 5g phthalic anhydride and 7.4g anisole was grinded in a mortar. Then transferred the mixture to a 250ml Erlenmeyer flask, a thermometer was immerse and the flask was heated slowly to 200oC on a sand-bath. In the meantime 2.3g of catalyst zinc chloride was added to the reaction mixture in small lots by Stoppard bottle, stirring with the thermometer after each addition. Heating was continued till the mixture became dark red and highly viscous. Flask was cooled to about 90oC and to this 70ml water and 3.5 ml conc. HCl was added. Heated again till the zinc salts have dissolved. The colored salt was filtered on Buchner funnel, washed with water, drained well and dried. The propose mechanism is given herein fig. 6


Anisole

2.2.5. By Using Catechol One experiment was performed with Catechol using

catalyst zinc chloride at temperature 200oC. 5g phthalic anhydride and 7.4g Catechol was grinded in a mortar. Then transferred the mixture to a 250ml Erlenmeyer flask, a thermometer was immerse and the flask was heated slowly to 200oC on a sand-bath. In the meantime 2.3g of catalyst zinc chloride added to the reaction mixture in small lots by Stoppard bottle, stirring with the thermometer after each addition. Heating was continued till the mixture became dark red and highly viscous. Flask was cooled to about 90oC and to this 70ml water and 3.5 ml conc. HCl was added. Heated again till the zinc salts have dissolved. The colored salt was filtered on Buchner funnel, washed with water, drained well and dried. The propose mechanism is given herein fig. 7


Catechol

2.3. Characterization of fluorescein For characterization of product the λ max is

determined by scanning the solution between the range 475-575nm.

2.4. Synthesis of eosin

Only those samples were brominenated which were confirmed to be fluorescein by observing the λ max of sample. Bromination was done with three different concentrations of bromine solution (5, 10 and 50%) by the following procedure. 5g of the dried fluorescein was taken in a 250 ml Erlenmeyer flask and 25ml of ethanol was added. solution of bromine water was prepared and transferred to a burette. Bromine was added slowly to fluorescein solution with constant stirring. When about half of bromine was added a solid disappears. Tetrabromofluorescein was appeared as the addition continued. The mixture was allowed to stand for 1 hour. The solid was filtered, washed twice with ethanol and dried in oven at 100OC. The propose equation is given here in fig. 8

Fig. 8. Mechanism for synthesis of eosin

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2.6.4. Application of eosin on cellulose fiber 2.5. Characterization of eosin

2g of eosin salt was dissolved in 150ml of water. 3cm×6cm cotton piece was taken. The cotton was heated in eosin salt solution for 30 min. after 30min piece was taken out washed with water dried and pressed. (Norris, 1915).

For characterization of dye the λ max is determined by scanning the solution between the range 475-575nm for eosin. 2.5.1. Solution preparation

3. RESULTS AND DISCUSSION About 2mg of salt was taken and dissolved in 10ml distilled water. The absorbance of all samples was noted from 475-575nm for fluorescein and from 475-575nm for eosin. (Spectrophotometer = T60U)

3.1. Products by changing Phenolic components

3.1.1. By using resorcinol 2.6. Application of eosin No product was obtained in case of β–napthanol, o-cresol,

anisole and catechol. Fluorescein was obtained only by resorcinol at different temperatures by changing catalysts. Sun et al; (1997) also prepared the fluorescein from resorcinol. Table No. 1. showes the % age yield and Characteristics of Fluorescein at different temperatures and catalysts using Resorcinol

2.6.1. Preparation of ammonium salt

About 25cc. of conc. Ammonium chloride solution was prepared and NaOH was added in it, to evolve ammonia. This solution was placed in the desiccator. 1g of eosin salt was also placed in the desiccator. The desiccator was covered. After some time the solubility of salt in water was checked when salt was completely soluble the reaction was stopped and salt became ready for use. (Norris, 1915)

In case of β–napthanol, o-cresol anisole and catechol

there is no active hydrogen at position next to the hydroxyl group which helps in the elimination of water molecule. While in case to resorcinol active hydrogen is present at the position next to the hydroxyl group which helps in the elimination of water molecule. This hydrogen is active due to the presence of hydroxyl (OH) group on its opposite position. While in case of β–napthanol there is no substituent is present which can make active to the hydrogen at the position next to the hydroxyl group.

2.6.2. Application of eosin as ink

About 0.2g of eosin ammonium salt was dissolved in 10ml of water. This solution was filled in a pen and used for witting on paper and compared with commercial ink. (Norris, 1915)

2.6.3. Application of eosin on silk In case of anisole there is another problem that instead of

hydroxyl group an ester group is present due to which no water can eliminated. The structures of all compounds are shown in the fig.9

2g of eosin salt was dissolved in 150ml of water. 3cm×6cm silk piece was taken. The silk was heated in eosin salt solution for 30 min. after 30min piece was taken out washed with water dried and pressed. (Norris, 1915)

Fig. 9. Structures of resorcinol, β–napthanol, o-cresol, anisole and catechol

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Table No. 1: % age yield and Characteristics of Fluorescein at different temperatures and catalysts using Resorcinol Sample no Temperature Catalyst % age yield Time required for salt formation Color of salt λ max (oC)

A 150oC Anhyd-Zncl2 37.9 readily on cooling Red color 494nmB 200oC Anhyd-Zncl2 48.9 readily on cooling Dark Red color 494nm C 250oC Anhyd-Zncl2 95.46 readily on cooling Brownish Red color 494nm D 150oC CaCl2 - no product was obtained ---- - E 200oC CaCl2 43.37 after long time Dark Red color 494nm F 250oC CaCl2 73.4 after long time Brown color 494nm

G 150oC FeCl3 58.4 after keeping over night Blackish Brown color 494nm H 200oC FeCl3 45.9 after keeping over night Blackish Brown color 494nm

I 250oC FeCl3 97.55 after keeping over night Dark Brown color 494nm 3.2. Effect of temperature on% age yield At higher temperature 250oC the yield was higher and at lower temperature 150oC the yield was low. Thus Sample C, F and I gave the higher yield then the other samples, and sample A, D and G gave lower yield then the other samples. However sample B, E and H gave medium yield at 200oC. The results obtained are similar to the results of Sethi (2003) who also observed the prepared the fluorescein from resorcinol and the obtained yield was more than 90% at 200 oC. This shows that at higher temperature reactants can fused well with each other thus large number of molecules can react with each other so more product can obtained at higher temperature. Following graph shows that at higher temperature yield is higher. The trend obtained is shown in the following fig.10.

Effect of temperature on %age yield

0

20

40

60

80

100

120

A B C D E F G H I

SAMPLES

% a

ge y

ield

Fig. 10. Effect of temperature on % age yield 3.3. Effect of catalyst on the yield of product

Ferric chloride and zinc chloride both gave better yield then calcium chloride. Thus Ferric chloride and zinc chloride are the better catalysts for the synthesis of fluorescein. The trend obtained is shown in the following fig.11.

Effect of catalyst on %age yield

0

20

40

60

80

100

120

A B C D E F G H I

SAMPLES%

age

yie

ld

Fig. 11. Effect of catalyst on % age yield

3.4. Effect of bromine concentration on yield Only 50% bromine solution gave the eosin product 5% and 10% bromine solution did not show any change in fluorescein. From the above results it is clear the high concentration of bromine is required for better yield of eosin. The data of obtained yield is given in the table No.2 Table No 2: The % age and λ max yield of Eosin by 50% bromine solution Sample no. Theoretical yield % age yield λ max A 4.69 g 68.75 514nm B 6.06 g 77.39 514nm C 11.82g 82.2 514nm E 5.37 g 18.2 514nm F 9.10 g 85.18 514nm G 7.24 g 3.72 514nm H 5.68 g 33.93 514nm I 12.08 g 87.7 514nm The trend obtained of % age yield is shown in the following fig. 12.

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% AGE YIELD OF EOSIN

0102030405060708090

100

A B C E F G H I

Samples

% a

ge y

ield

Fig. 12. % age yield of eosin. 3.5. Characterization of eosin

The absorbance of all samples was taken from 475-575nm. The observed λ max of the dye is 514nm which confirms the eosin dye. Kyzylel et al., (2004) also observed the λ max of eosin dye. All the sample gives the same λ max 514 nm which indicates that all samples are of eosin only yield is different at different conditions. Table No.2 shows the λ max of all samples. Absorbance of all samples at different wavelengths are shown in fig. 13

Fig. 13. Absorbance of all eosin samples 3.6. Application of eosin 3.6.1. Application of eosin on silk As the temperature increases the quantity as well as quality of dye also increases. Thus sample C. F and I give good tone of color on silk. Norris (1915) also observed the dyeing application of eosin on silk. The entire dyed samples give color near to red and pink tone. Matthews (2007) also noted that eosin dye gives delicate red and pink shades. 3.6.2. Application of eosin on cellulose fiber

As the temperature increases the quantity as well as quality of dye also increases. Thus sample C. F and I give

g

osin dye gives

nk

e quantity as well as uality of dye also increases. Thus sample C. F and I give

g

roject involves qualitative and quantitative provements in production of eosin dye by changing the

d

for fluorescein

edge with deep sense of gratitude for ncere and able guidance of my supervisor Mrs. Sofia

06. Methods of Study. HistoNotes, 9: 1-10. enjamin, W., Gung and T. T. Richard, 2004. A

Deesien, D. L. Spetor, S. Huang and M. H.

Hazof

ood tone of color on cellulose. Norris (1915) also examines the dying application of eosin. The entire dyed samples give color near to red and pink tone. Matthews (2007) also noted that edelicate red and pink shades. 3.6.1. Application of eosin as i

As the temperature increases thq

ood results as ink. Norris (1915) applied the eosin as ink.This work makes this clear that eosin can be used as red ink. Thirumurthy (2002) also used eosin as ink. 3.7. Summary

The present pimifferent reactants catalyst and conditions.

From the results it is quite clear that 250oC, resorcinol and zinc chloride were found to be best synthesis. 50% bromine solution was found to be best for bromination of Fluorescein. Finally the obtained product was applied as ink and as dye on silk and wool. Eosin ink was also compared with the commercial red ink. It was observed that as the temperature increases the quantity as well as quality of dye also increases. Thus sample C. F and I give good tone of color on cellulose and silk and give good results as ink and dye. Samples prepared at higher temperatures gives darker tone of color. Acknowledgment

Absorbance of eosin samples

0

0.4

0.8

1.2

1.6

2

475nm 495nm 515nm 535nm 555nm 575nm

Wave lenght (nm)

Abs

orba

nce

I wish to acknowlsiNosheen and to my parents for their full cooperation and ever strengthening prayers which made me able to pursue my goals and made them true. 3.8. References Awgustafson. 20B

Combinatorial Experiment Suitable for Undergraduate Laboratories. Journal of Chemical Education, 81(11): 1-8. rinck, T. J., M. E. Martone, V. Lev-rem, D. P. L. Green, R. Y. TEllisman, 1994. Fluorescence photooxidation with eosin. The Journal of Cell Biology, 126(4):901-910. ebroucq, S., F. Labat, D. Lincot, and C. Adamo. 2008. Theoretical insights on the electronic properties eosin Y, an organic dye for photovoltaic applications. J. Phys. Chem. A., 112(31): 7264–7270.

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Konstantinova, T. N. and V. B. Bonjinov, 1998. Synthesis of some unsaturated 9-phenyle xanthene dyes. Dyes and Pigments, 39(2):69-75

Kýzýlel, S., V. H. Perez-Luna and F. Teymour. 2004. Photopolymerization of Poly(Ethylene Glycol) Diacrylate on Eosin-Functionalized Surfaces. Langmuir, 20 (20): 8652-8658.

Matthews, J. M. 2007. Laboratory Manual of Dyeing and Textile Chemistry. Application of acid dyes, 87

Norris, F. J. 1915. Experimental Organic Chemistry. Dyes and Dyeing, 26: 24-25.

Sethi, A. 2003. Systematic Lab Experiments in Organic Chemistry. New age international publishers. 772-774.

Shah, K. M. 1994. Classification of dyes, Hand book of synthetic dyes and pigments. Multi. Tech. pulishing co. 2nd edition 1:1-10.

Sun, W. C., K. R. Gee, D. H. Klaubert and R. P. Haugland, 1997. Synthesis of fluorinated fluoresceins. J. Org. Chem., 62 (19): 6469-6475.

Tyagi. O. D. and M. Yadav. 1990. A text book of synthetic dyes. Anmol Publications, New Dehli, 1st Ed. 1(10): 66-67.

Thirumurthy, M. and C. Srinivas. 2002. Demonstration of fungus by using parker's India ink and eosin- a simple technique. Indian J Dermatol Venereol Leprol, 68(6):376

Vasudevan, D. and P. N. Anantharaman, 1993. Electrochemical synthesis of eosin from fluorescein. Journal of Applied Electrochemistry, 23: 808-812.

Waheed, A. A and P.D. Gupta. 1996. Application of an eosin B dye method for estimating a wide range of proteins. J. Biochem. Biophys. Methods. 33: 187-196.

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