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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.
A pH indicator is just a weak acid with differently colored acid and conjugate base forms. and cranberry juice appear red and blueberries. The powder is about 10% carminic acid. strawberries. In an 0. Several synthetic modifications of alizarin are also used as acid/base indicators. CURCUMIN OR TURMERIC YELLOW Is a natural dye found in curry powder. Here are few examples of natural pH indicators: ALIZARINI Is an orange dye present in the root of the madder plant. You'll have to grind about 70. found in Mexico and Central America. [875-884] INTRODUCTION All pH indicators change color depending on whether they donate or accept proton (acids are proton donors and bases are proton acceptors). Selective natural indicators (such as litmus) to indicate specific pH levels have been developed based on their color changes. EJEAFChe . Esculin changes from colorless at pH 1. blackberries and red cabbage appear purple due to the presence of cyanidin based colorant molecules. Cochineal solutions are not used much as acid/base indicators these days. Persia. Many flowers. (2001) as a natural pH indicator which is known to contain anthocynins.5 to fluorescent blue at pH 2. The blue and red pigments of flowers were isolated and extensively studied by Willstatter in 1913. alizarin is yellow at pH 5.5% alcohol solution. The substances in the plant products such as tea. fruits and vegetables contain chemical substances that change color in solutions of different pH.8. which is yellow in acidic solution and deep violet in alkaline solution. roses.5 and red at pH 6. cornflowers.4 to red at pH 8. it was used to dye wool in ancient Egypt. 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. 2010. Apples.000 insects to make one pound of dry indicator.6.Padma S Vankar et al. These plant pigments can act as acid-base indicators. Red cabbage juice has been used by I. and India. COCHINEAL Is an acid-base indicator made from the bodies of dried female cochineal insects. Many indicators have been extracted from plants since then. 876 . Therefore pH indicators are themselves acids or bases. It turns from yellow at pH 7. Jansons.(5). which has pigments that react in a different way to acids and bases. and violets appear blue while grapes. 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. autumn leaves. 9.
They have utilized plant flowers of Celosia cristate Linn. Litmus is red at pH 4. rosa-sinensis) or at 525 nm (I.(1978) utilized the extracts of anthocyanin pigments from red cabbage The solution was mixed with various strengths of acids and bases. The pH-changing anthocyanin pigments of the red cabbage act as indicator. LOGWOOD Is a dye obtained from the heartwood of a tree that grows in Central America and the West Indies. Litmus paper from red cabbage was patented by Hueseyin ( 2006). Between pH 5 to 7 wavelength maxima changed from 421 nm (yellow) to 552 nm (green) and reverted back when pH 12.an exercise for the junior high school Forster.(5). [875-884] LITMUS Is a blue dye extracted from various species of lichens. and blue colors. some is used as a coloring for beverages. which may be prepd. 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. Plant pigments as acid-base indicators . The acetone extracts of the pigments turn red in presence of acid and show absorption max. B( 1983). showed higher stability than in vivo. violet. which turn bright red in alkaline solution. have been shown by using common flowers like Hibiscus rosa-sinensis. The method consists of the production of blue litmus paper by soaking up the blue juice of red cabbage to copy paper. 877 . while the red litmus paper is produced by soaking up the pink juice of red cabbage.3. The in vitro pigment ext.(2000).Padma S Vankar et al. and the color and pH range were monitored. The extracts when treated with alkali turn green and show a greater absorbance in the range 575-625 nm depending upon the flowers used. turnetea).0 from 611 nm (olive green) was changed to (yellow) at 421 nm.1 to 13. ANTHOCYANIN Is probably the most readily available acid/base indicator. and poppies red. EJEAFChe . cornflowers blue. it is the plant pigment that makes red cabbage purple. resp. M.5 and blue around pH 8. fistulosa and C. therefore they are used as pH indicators. It changes color from red in acid solution to purplish to green in mildly alkaline solution to yellow in very alkaline solution. at 500 nm (H. 9.. by dropping the citric acid or other acids to the red cabbage juice. Ipomoea fistulosa and Clitoria turnetea contain various anthocyanin pigments in the form of purple-red. Usefulness of some plant flowers as natural acid-base indicators has also been demonstrated by Soltan et al ( 2002). While most litmus is used to make litmus papers. 2010. Although these lichens grow in many parts of the world. The extract contains hematoxylin and hematein. These flowers contain anthocyanin pigments whose color depends on pH. almost all litmus is extracted and packaged in Holland. Bombax ceiba L. Poinciana regia and Hibiscus subdariffa. A potential chemical indicator from pheasant's eye (Adonis aestivalis L. 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.) plant has been reported by Hammouri et al.
PLANT MATERIAL Rosa rosa flowers were collected from the I. 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 λmax. Significant changes were observed when stannous salt was added to the rose anthocyanin extract. [875-884] 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. Campus. 2010. tartaric acid (S D fine) Distilled water (DW. The colorant present in the rose flower is mainly consisting of cyanidin.T he anthocyanin was used as natural indicator in acid – base titration. 9. citric acid and tartaric acid.I.T. Figure-1 Colorants of Rose flowers MATERIALS AND METHODS CHEMICALS Hydrochloric acid. citric acid. pelargonin or peonidin or to mixtures of these pigments as shown in figure-1. 878 . The intrinsic pH of the extract was 2.88. The extract showed wavelength maxima change from 517 nm (dark pink) to 592 nm (green) between the pH range 2 to 9. Kanpur and they were kept in cold (20°C) and dark storage until processed.Padma S Vankar et al.(5).1% HCl. Millipore) were used All chemicals used were of analytical grade. EJEAFChe .
The extract showed wavelength maxima change from 517 nm (dark pink) to 592 nm (green) between the pH range 2 to 9. Abs 520nm 550.0% citric acid in methanol until a clear solution was obtained. [875-884] ANTHOCYANIN EXTRACTION Anthocyanin extraction was carried out by three methods by 1. 2. is 560 and 582 nm respectively as shown in table-1. 0. HCl.57 517.1% HCl (v/v) in methanol for 2-3 h at room temperature.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 combined filtrates were dried using a rotary evaporator at 30°C. 2. 3.82 Change in pH withSnCl2 1.88. EJEAFChe . The mixture was filtered on a Buchner funnel and the remaining solids were washed with 4.85 592. With the addition of Stannous salt the pH falls from 2. 2010. 0.99 578. The combined filtrates were dried using a rotary evaporator at 30°C.0% citric acid in methanol until a clear solution was obtained. The mixture was filtered on a Buchner funnel and the remaining solids were washed with 0.36 9 879 582. 0.Padma S Vankar et al.72 with change in λmax to 520 nm.0 % Citric acid (w/v) in methanol for 23 h at room temperature. in darkness. 0. The mixture was filtered on a Buchner funnel and the remaining solids were washed with 4.45 535. Similar trend was observed at pH 6 and 9 where the change in λmax. 2006). Anthocyanins were extracted from flowers with 4. Significant changes were observed when stannous salt was added to the rose anthocyanin extract.(5). The concentrate was dissolved in 0. Anthocyanins were extracted from flowers with 0. 9. The concentrate was dissolved in 0.29 6 560. Citric acid and 3. 1.72 3 λmax. Tartaric acid 1.05 . in darkness. Abs 517nm. 0. Anthocyanins were extracted from flowers with 4.34 540. The concentrate was dissolved in 0. 0.4 % citric acid (w/v) in DW and the solution obtained was used as indicator. 0. The intrinsic pH was 2. 0.46 552.52 527. The combined filtrates were dried using a rotary evaporator at 30°C. 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 λmax. 0.0 % tartaric acid (w/v) in methanol for 2-3 h at room temperature.88 to 1. in darkness (Longo and Vasapollo.1% HCl in methanol until a clear solution was obtained.88 3 4 5 6 7 8 9 Λmax. Table-1 Shift in λmax in rose anthocyanin with change in pH: pH Intrinsic 2. 0.01% HCl (v/v) in DW and the solution obtained was used as indicator.
Bb-) which is blue. The substances in plant products react with acids(or bases). 1977). 2010. pH indicators are themselves acids or bases. EJEAFChe . there are three forms of cyanidin: the flavylium cation AH+. the carbinol B.(5). is yellow in acid and blue in base and intermediate in between at pH 7. has also been reported (Brouillard. a prototropic tautomer of the carbinol B. Hydration of the flavylium cation involves the formation of a C-O bond and a proton transfer. Equilibrium between the two neutral forms occurs exclusively by way of the flavylium cation. [875-884] Figure-2 Anthocyanin extract at intrinsic and different pH In acidic aqueous media (pH 2-9). (base-form. 9. and the quinonoidal base A. change colors depending upon whether they donate or accept protons.ions at higher pH changes to anion. HBb) when donates a proton to OH. The choice of indicators are made based on for acid strength and specific pH levels.Padma S Vankar et al. HBb. (acid-form. et al. Therefore. resulting in changes at the molecular level which causes their colors to be different at different pH levels. For example. The existence of very small amounts of a third neutral form. All pH indicators. named Bromthymol Blue. 880 . (acids areproton donors and bases are proton acceptors). a commonly used synthetic acid-base indicator. The point at which the color changes from intermediate to blue gives information on the acid strength of the acid-form. the chalcone C. Yellow form of Bromthymol Blue. such as litmus paper. The selection natural dyes to indicate specific pH levels based on their color changes.
40 0.0 4.53 MeOH/Tartaric Acid Ext. [875-884] Table 2 CIELab values pH 2.0 18.93 2.50 60.08 33.29 45.0 39.46 44.96 4. 2.02 38.N.55 pH 8.39 23. S.46 47.03 4.03 5 2.88 51.3 5.72 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.68 24.96 4. 7.89 55. 10. 1.43 5. 2. 5.15 20.14 23.23 5.43 4.03 5.5 5.23 5.16 881 .13 5.0 15.73 36.0 28. 3.6 5.03 5.73 5.96 5.09 0.53 15.36 4.27 25.67 pH 10.0 25. 3.41 0.40 pH 6.62 24. 10.2 5.87 44.26 4. 7. 4.93 5. 2.06 4.31 41. 4.4 5.13 5.36 4.03 4.2 5.72 0.86 4.14 1.46 4.2 5. Normality (HCl) 0. Normality (NaOH) 0.53 48.23 5.46 5.01 31.1 5.93 5.06 32.80 pH 4.03 5.66 MeOH/Tartaric Acid Ext.03 5.50 pH 9.16 16.0 12. 9.74 18. 6.51 0.76 18.74 36.1N 1N 2N 3N 4N 5N 6N 7N 8N MeOH/HCl Ext.0 23.39 pH 7. 5.58 24. 9.43 MeOH/Citric Acid Ext.0 16.96 4.20 29.96 21.01 61.16 5. 5. S.79 28.03 4.Padma S Vankar et al.01N 0.03 5. 8. 6.74 59.03 5.1 5. 5.73 0.80 pH 3. 1.1N 1N 2N 3N 4N 5N 6N 7N 8N MeOH/HCl Ext.78 52.9 4.03 4.03 5. 2010. 8.39 14.23 4.70 27. EJEAFChe .33 5. 9. 3.29 42.N.3 4.46 pH 5.96 Table 3a: Titer values in Acid Base titration by differently extracted rose anthocyanin as indicator at different Normality of NaOH.13 4.26 5.46 MeOH/Citric Acid Ext.23 4.46 4.75 33. 5.01N 0.41 0. 4.51 30.(5).
[875-884] Figure-3 Rose anthocyanin extracted by HCl method as acid. 2010. 9.base indicator 882 .(5).base indicator Figure-4 Rose anthocyanin extracted by citric acid method as acid. EJEAFChe .Padma S Vankar et al.
In this work. 9. light absorption. To determine the highest absorption wavelength and demonstrate the shape changing of spectra in function of the acidity. EJEAFChe . Among various pigments in rose petals. The specie used was Rosa rosa whose flowers crude extract was used to obtain molecular absorption spectra.Padma S Vankar et al. effects of changes in conjugated double bounds. usually flavones and flavonols.0%. pKa. cause a bathochromic shift of anthocyanins when they stack with anthocyanins (Goto and Kondo 1991). and redder in acidic pH. Addition of Stannous salt to the extract further enhanced the changes in the pH and the absorption wavelength.base titrations. The color changed from dark pink to mehndi green between pH 2 and 9. i. The 883 . to verify the Lambert-Beer law and as an indicator in acid. Anthocyanin pigments of rose flowers whose color changes depending on the pH. [875-884] Figure-5 Rose anthocyanin extracted by tartaric acid method as acid. CONCLUSION All pH indicators changes colors depending upon whether they donate or accept protons. anthocyanins. The selective of rose anthocyanin as natural dyes to indicate specific pH levels was based on its chemical changes. the flower extract of rose are suggested as pH indicator in the form of either solutions. the absorption spectra in UV and visible regions were obtained in different pH values. the number of hydroxy groups on the B-ring and/or aromatic acyl moieties modifying anthocyanins increase. To be useful in illustrating chemical principles such as acid –base equilibria. pH indicators are themselves acids or bases. etc. The goal of this work was to propose a simple and cheap indicator. the chemical structures are primarily determined by their color. 2010. Anthocyanins change their color depending on the pH of the vacuole in which anthocyanins localize.e.base indicator. causing a bathochromic shift (Honda and Saito 2002). Co-pigments. their color is bluer in weakly acidic or neutral pH.1% and 1. The end point results obtained with the acidified methanolic extracts when compared to the conventional ones showed relative errors between 0. Therefore.(5). a class of flavonoids.
Ed. Engl. TR 2004001355 A2 20060123. Honda T. Chem.E. Brouillard. Phanstiel . J-E Dubois. K. Acidified methanolic extract of Rosa rosa were prepared and used as new indicators in quantitative analysis of standardization of NaOH and HCl solutions. Journal of the American Chemical Society.K. 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. L. M. International Journal of Chemistry 10(2) (2000) 151.I. 3. 13. (2006). and G. Natural pH Indicators: What are they and how do they work?. 55(2) (1978) 107. Summerlin.Padma S Vankar et al.frostburg. Journal of Chemical Education. 11. Chem. Effect of using such natural extracts as indicators gave quantitative results as compared with conventional indicators with good agreement.shtml. Turk. Shibli. 6. 9. 49 (1) (2002) 63. Food Chemistry. 7. S. 2. Ereifej.edu/chem/senese/101/features/water2wine. Everst. Justus Liebigs Ann.A. REFERENCES 1. L. Appl. Silver Burdett. Longo. 62 (1985) 322. Heterocycles 56 (2002) 633–692. 99(5) (1977) 1359. Journal of Chemical Education. National Academy Science Letters.N. Vasapollo. 14. Angew. Nelson Science (2001) 10. M. Pat. http://antoine. R. 884 . B. New York. 5. Bose. O. Jansons. 30 (1991) 17–33. R. Ajlouni. Saito N. 401 (1913) 189 I. Hammouri. M..M. 94(2) (2006) 226. 9. EJEAFChe .M. Chemistry of Common Substances. E. Willstätter.R. E. G. M. Goto T. Kondo T. 4. Sirry. 12.. Journal of the Chinese Chemical Society. 1986. Hueseyin. Al-Karaki. 2010. A. 8.(5). 6(8) (9183) 249. R. Forster. Int. 10. [875-884] pH indicator solutions were prepared from the acidified methanolic extracts of the rose flower. Chapter 8: Acids and Bases. M. Soltan.