ISSN:

1579-4377

ROSE ANTHOCYANINS AS ACID BASE INDICATORS

Padma S Vankar* and Dhara Bajpai Facility for Ecological and Analytical Testing, (FEAT) Indian Institute of Technology, Kanpur 208 016, India psv@ iitk.ac.in

ABSTRACT A convenient method of extraction of anthocyanin from Rose flowers (Rosa rosa) has been developed using methanolic solution of 3% citric acid which gave better yield of anthocyanins than the methanolic solution of 0.1% hydrochloric acid. It has been shown that by changing the pH of the extract from 2-9, the hue color changes from dark pink to mehdi green. The extract showed wavelength maxima change from 517 nm (dark pink) to 592 nm (green) between the pH range 2 to 9. In the present study we have made use of anthocyanin extracted from Rose flowers as natural indicator for acid base titration. The intrinsic pH of the extract was 2.88. Significant changes were observed when stannous salt was added to the rose anthocyanin extract. The spectroscopic data of the extracted anthocyanin pigments indicate a shift in wavelength maxima of the colors by addition of stannous salt and the complex with a change in pH of the aqueous medium. Usefulness of rose flowers as natural acid-base indicator has been exemplified.

KEYWORDS Anthocyanin, Citric acid extraction, Rosa rosa, natural acid-base indicator.

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INTRODUCTION All pH indicators change color depending on whether they donate or accept proton (acids are proton donors and bases are proton acceptors). A pH indicator is just a weak acid with differently colored acid and conjugate base forms. Therefore pH indicators are themselves acids or bases. Many flowers, fruits and vegetables contain chemical substances that change color in solutions of different pH. These plant pigments can act as acid-base indicators. The blue and red pigments of flowers were isolated and extensively studied by Willstatter in 1913. Many indicators have been extracted from plants since then. Selective natural indicators (such as litmus) to indicate specific pH levels have been developed based on their color changes. The substances in the plant products such as tea, red cabbage or grapes react with acids or bases resulting in changes at the molecular level which causes their color to be different at different pH. Red cabbage juice has been used by I. Jansons, (2001) as a natural pH indicator which is known to contain anthocynins, which has pigments that react in a different way to acids and bases. Apples, autumn leaves, roses, strawberries, and cranberry juice appear red and blueberries, cornflowers, and violets appear blue while grapes, blackberries and red cabbage appear purple due to the presence of cyanidin based colorant molecules. Here are few examples of natural pH indicators: ALIZARINI Is an orange dye present in the root of the madder plant; it was used to dye wool in ancient Egypt, Persia, and India. In an 0.5% alcohol solution, alizarin is yellow at pH 5.5 and red at pH 6.8. Several synthetic modifications of alizarin are also used as acid/base indicators. COCHINEAL Is an acid-base indicator made from the bodies of dried female cochineal insects, found in Mexico and Central America. You'll have to grind about 70,000 insects to make one pound of dry indicator. The powder is about 10% carminic acid, which is yellow in acidic solution and deep violet in alkaline solution. Cochineal solutions are not used much as acid/base indicators these days. CURCUMIN OR TURMERIC YELLOW Is a natural dye found in curry powder. It turns from yellow at pH 7.4 to red at pH 8.6. ESCULIN Is a fluorescent dye that can be extracted from the leaves and bark of the horse chestnut tree. You'll need to shine a black (ultraviolet) light on the indicator to get the full effect. Esculin changes from colorless at pH 1.5 to fluorescent blue at pH 2.
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LITMUS Is a blue dye extracted from various species of lichens. Although these lichens grow in many parts of the world, almost all litmus is extracted and packaged in Holland. Litmus is red at pH 4.5 and blue around pH 8.3. While most litmus is used to make litmus papers, some is used as a coloring for beverages. LOGWOOD Is a dye obtained from the heartwood of a tree that grows in Central America and the West Indies. The extract contains hematoxylin and hematein, which turn bright red in alkaline solution. ANTHOCYANIN Is probably the most readily available acid/base indicator; it is the plant pigment that makes red cabbage purple, cornflowers blue, and poppies red. It changes color from red in acid solution to purplish to green in mildly alkaline solution to yellow in very alkaline solution. Researchers who have used anthocyanin as acid base indicators are listed below: Economic use of the pigments of some common flowers as acid/alkali indicators Bose, B( 1983), have been shown by using common flowers like Hibiscus rosa-sinensis, Ipomoea fistulosa and Clitoria turnetea contain various anthocyanin pigments in the form of purple-red, violet, and blue colors, resp. The acetone extracts of the pigments turn red in presence of acid and show absorption max. at 500 nm (H. rosa-sinensis) or at 525 nm (I. fistulosa and C. turnetea). The extracts when treated with alkali turn green and show a greater absorbance in the range 575-625 nm depending upon the flowers used. A potential chemical indicator from pheasant's eye (Adonis aestivalis L.) plant has been reported by Hammouri et al,(2000). The spectroscopic data of the purified pigments indicate a shift in wavelength maxima of the colors developed by changing the environmental pH of the aqueous medium. Between pH 5 to 7 wavelength maxima changed from 421 nm (yellow) to 552 nm (green) and reverted back when pH 12.1 to 13.0 from 611 nm (olive green) was changed to (yellow) at 421 nm. The in vitro pigment ext. showed higher stability than in vivo. Plant pigments as acid-base indicators - an exercise for the junior high school Forster, M.(1978) utilized the extracts of anthocyanin pigments from red cabbage The solution was mixed with various strengths of acids and bases, and the color and pH range were monitored. Litmus paper from red cabbage was patented by Hueseyin ( 2006). The method consists of the production of blue litmus paper by soaking up the blue juice of red cabbage to copy paper, while the red litmus paper is produced by soaking up the pink juice of red cabbage, which may be prepd. by dropping the citric acid or other acids to the red cabbage juice. The pH-changing anthocyanin pigments of the red cabbage act as indicator. Usefulness of some plant flowers as natural acid-base indicators has also been demonstrated by Soltan et al ( 2002). They have utilized plant flowers of Celosia cristate Linn, Bombax ceiba L., Poinciana regia and Hibiscus subdariffa. These flowers contain anthocyanin pigments whose color depends on pH; therefore they are used as pH indicators.
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The acid-base equilibrium of these pigments were investigated using spectrophotometric and potentiometric methods. In the present study we have extracted the anthocyanin from rose flowers by 3 methods using methanolic solutions of 0.1% HCl, citric acid and tartaric acid.T he anthocyanin was used as natural indicator in acid base titration. The intrinsic pH of the extract was 2.88. The spectroscopic data of the extracted anthocyanin pigments indicate a shift in wavelength maxima of the colors by addition of stannous salt and the complex not only showed change in pH of the aqueous medium but it also showed change in max. The extract showed wavelength maxima change from 517 nm (dark pink) to 592 nm (green) between the pH range 2 to 9. Significant changes were observed when stannous salt was added to the rose anthocyanin extract. The colorant present in the rose flower is mainly consisting of cyanidin, pelargonin or peonidin or to mixtures of these pigments as shown in figure-1.

Figure-1 Colorants of Rose flowers

MATERIALS AND METHODS CHEMICALS Hydrochloric acid, citric acid, tartaric acid (S D fine) Distilled water (DW, Millipore) were used All chemicals used were of analytical grade. PLANT MATERIAL Rosa rosa flowers were collected from the I.I.T. Campus, Kanpur and they were kept in cold (20C) and dark storage until processed.
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ANTHOCYANIN EXTRACTION Anthocyanin extraction was carried out by three methods by 1. HCl, 2. Citric acid and 3. Tartaric acid 1. Anthocyanins were extracted from flowers with 0.1% HCl (v/v) in methanol for 2-3 h at room temperature, in darkness (Longo and Vasapollo, 2006). The mixture was filtered on a Buchner funnel and the remaining solids were washed with 0.1% HCl in methanol until a clear solution was obtained. The combined filtrates were dried using a rotary evaporator at 30C. The concentrate was dissolved in 0.01% HCl (v/v) in DW and the solution obtained was used as indicator. 2. Anthocyanins were extracted from flowers with 4.0 % Citric acid (w/v) in methanol for 23 h at room temperature, in darkness. The mixture was filtered on a Buchner funnel and the remaining solids were washed with 4.0% citric acid in methanol until a clear solution was obtained. The combined filtrates were dried using a rotary evaporator at 30C. The concentrate was dissolved in 0.4 % citric acid (w/v) in DW and the solution obtained was used as indicator. 3. Anthocyanins were extracted from flowers with 4.0 % tartaric acid (w/v) in methanol for 2-3 h at room temperature, in darkness. The mixture was filtered on a Buchner funnel and the remaining solids were washed with 4.0% citric acid in methanol until a clear solution was obtained. The combined filtrates were dried using a rotary evaporator at 30C. The concentrate was dissolved in 0.4 % citric acid (w/v) in DW and the solution obtained was used as indicator. RESULTS AND DISCUSSION In the present study we have made use of anthocyanin extracted from Rose flowers and used for acid base titration. The intrinsic pH was 2.88. The spectroscopic data of the extracted anthocyanin pigments indicate a shift in wavelength maxima of the colors by addition of stannous salt and the complex not only showed change in pH of the aqueous medium but it also showed change in max. The extract showed wavelength maxima change from 517 nm (dark pink) to 592 nm (green) between the pH range 2 to 9. Significant changes were observed when stannous salt was added to the rose anthocyanin extract. With the addition of Stannous salt the pH falls from 2.88 to 1.72 with change in max to 520 nm. Similar trend was observed at pH 6 and 9 where the change in max. is 560 and 582 nm respectively as shown in table-1.
Table-1 Shift in max in rose anthocyanin with change in pH: pH Intrinsic 2.88 3 4 5 6 7 8 9 max, Abs 517nm, 0.57 517, 0.52 527, 0.45 535, 0.34 540, 0.46 552, 0.99 578, 0.85 592, 0.82 Change in pH withSnCl2 1.72 3 max, Abs 520nm 550, 0.29

560, 0.36

9 879

582, 1.05

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Figure-2 Anthocyanin extract at intrinsic and different pH

In acidic aqueous media (pH 2-9), there are three forms of cyanidin: the flavylium cation AH+, the carbinol B, and the quinonoidal base A. Equilibrium between the two neutral forms occurs exclusively by way of the flavylium cation. Hydration of the flavylium cation involves the formation of a C-O bond and a proton transfer. The existence of very small amounts of a third neutral form, the chalcone C, a prototropic tautomer of the carbinol B, has also been reported (Brouillard, et al, 1977). All pH indicators, such as litmus paper, change colors depending upon whether they donate or accept protons, (acids areproton donors and bases are proton acceptors). Therefore, pH indicators are themselves acids or bases. The substances in plant products react with acids(or bases), resulting in changes at the molecular level which causes their colors to be different at different pH levels. The selection natural dyes to indicate specific pH levels based on their color changes. For example, a commonly used synthetic acid-base indicator, named Bromthymol Blue, is yellow in acid and blue in base and intermediate in between at pH 7. Yellow form of Bromthymol Blue, (acid-form, HBb) when donates a proton to OH- ions at higher pH changes to anion, (base-form, Bb-) which is blue. The point at which the color changes from intermediate to blue gives information on the acid strength of the acid-form, HBb. The choice of indicators are made based on for acid strength and specific pH levels.

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Table 2 CIELab values pH 2.88 51.74 59.53 15.01 61.39 14.14 1.80 pH 3.0 39.46 47.96 21.78 52.68 24.41 0.80 pH 4.0 28.73 36.62 24.46 44.08 33.72 0.46 pH 5.0 25.06 32.27 25.31 41.02 38.09 0.40 pH 6.0 23.20 29.51 30.29 42.29 45.72 0.39 pH 7.0 16.58 24.14 23.75 33.87 44.51 0.55 pH 8.0 15.16 16.39 23.79 28.89 55.41 0.50 pH 9.0 12.76 18.15 20.70 27.53 48.73 0.67 pH 10.0 18.74 18.01 31.74 36.50 60.40 0.65

L a b C H Abs

Table 3: Titer values in Acid Base titration by differently extracted rose anthocyanin as indicator at different Normality of HCl. S.N. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Normality (HCl) 0.01N 0.1N 1N 2N 3N 4N 5N 6N 7N 8N MeOH/HCl Ext. 5.2 5.13 5.43 5.03 5.2 5.23 5.03 5.03 4.36 4.43 MeOH/Citric Acid Ext. 3.5 5.23 5.23 4.86 4.96 4.93 5.03 4.96 4.36 4.66 MeOH/Tartaric Acid Ext. 2.3 5.26 5.03 5.03 5.1 5.03 5 2.46 4.43 4.96

Table 3a: Titer values in Acid Base titration by differently extracted rose anthocyanin as indicator at different Normality of NaOH. S.N. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Normality (NaOH) 0.01N 0.1N 1N 2N 3N 4N 5N 6N 7N 8N MeOH/HCl Ext. 4.1 5.03 5.46 5.23 5.2 5.23 4.96 5.03 4.3 4.46 MeOH/Citric Acid Ext. 5.4 5.03 5.13 5.0 4.93 5.06 4.9 4.96 4.26 4.53 MeOH/Tartaric Acid Ext. 5.6 5.03 5.16 5.13 4.73 5.03 4.93 2.46 4.33 5.16

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Figure-3 Rose anthocyanin extracted by HCl method as acid- base indicator

Figure-4 Rose anthocyanin extracted by citric acid method as acid- base indicator

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Figure-5 Rose anthocyanin extracted by tartaric acid method as acid- base indicator.

Anthocyanin pigments of rose flowers whose color changes depending on the pH. The goal of this work was to propose a simple and cheap indicator. Among various pigments in rose petals, anthocyanins, a class of flavonoids, the chemical structures are primarily determined by their color, i.e. the number of hydroxy groups on the B-ring and/or aromatic acyl moieties modifying anthocyanins increase, causing a bathochromic shift (Honda and Saito 2002). Anthocyanins change their color depending on the pH of the vacuole in which anthocyanins localize; their color is bluer in weakly acidic or neutral pH, and redder in acidic pH. Co-pigments, usually flavones and flavonols, cause a bathochromic shift of anthocyanins when they stack with anthocyanins (Goto and Kondo 1991). To be useful in illustrating chemical principles such as acid base equilibria, pKa, light absorption, effects of changes in conjugated double bounds, etc. The specie used was Rosa rosa whose flowers crude extract was used to obtain molecular absorption spectra, to verify the Lambert-Beer law and as an indicator in acid- base titrations. The end point results obtained with the acidified methanolic extracts when compared to the conventional ones showed relative errors between 0.1% and 1.0%. The color changed from dark pink to mehndi green between pH 2 and 9. To determine the highest absorption wavelength and demonstrate the shape changing of spectra in function of the acidity, the absorption spectra in UV and visible regions were obtained in different pH values. Addition of Stannous salt to the extract further enhanced the changes in the pH and the absorption wavelength. CONCLUSION All pH indicators changes colors depending upon whether they donate or accept protons. Therefore, pH indicators are themselves acids or bases. The selective of rose anthocyanin as natural dyes to indicate specific pH levels was based on its chemical changes. In this work, the flower extract of rose are suggested as pH indicator in the form of either solutions. The
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pH indicator solutions were prepared from the acidified methanolic extracts of the rose flower. The different pH leads to color changes in the indicator solutions and it can be used for teaching elementary chemistry concepts of acid-base titrations. Acidified methanolic extract of Rosa rosa were prepared and used as new indicators in quantitative analysis of standardization of NaOH and HCl solutions. Effect of using such natural extracts as indicators gave quantitative results as compared with conventional indicators with good agreement. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. R. Willsttter, A. E. Everst, Justus Liebigs Ann. Chem., 401 (1913) 189 I. Jansons, Natural pH Indicators: What are they and how do they work?, Chapter 8: Acids and Bases, Nelson Science (2001) 10. O. Phanstiel , Journal of Chemical Education, 62 (1985) 322. L.R. Summerlin, Chemistry of Common Substances, Silver Burdett, New York, 1986. http://antoine.frostburg.edu/chem/senese/101/features/water2wine.shtml. B. Bose, National Academy Science Letters, 6(8) (9183) 249. M.K. Hammouri, K.I. Ereifej, M.M. Ajlouni, R.A. Shibli, G.N. Al-Karaki,. International Journal of Chemistry 10(2) (2000) 151. M. Forster, Journal of Chemical Education, 55(2) (1978) 107. E. M. Hueseyin, Turk. Pat. Appl. (2006), TR 2004001355 A2 20060123. M.E. Soltan, S.M. Sirry, Journal of the Chinese Chemical Society, 49 (1) (2002) 63. L. Longo, and G. Vasapollo, Food Chemistry, 94(2) (2006) 226. R. Brouillard, J-E Dubois, Journal of the American Chemical Society, 99(5) (1977) 1359. Goto T, Kondo T. Angew. Chem. Int. Ed. Engl. 30 (1991) 1733. Honda T, Saito N. Heterocycles 56 (2002) 633692.

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