CHM 244 B
Dr. Bauman
Isuru Wanigasooriyage Rachel Oliverio
11/1/16
Analysis of Anthocyanins and Anthocyanidins in Blackberries, Raspberries, and Blueberries
Abstract
The purpose of this experiment was to isolate anthocyanins, decompose them into their components (anthocyanidins), and to analyze the anthocyanins, and their corresponding anthocyanidins using paper chromatography, and spectrophotometric analysis procedures. Based on experimental data the blueberries contained the most amount of anthocyanins at 1.92 x 10-6 g, followed by blackberries at 1.19 x 10-6 g, finally followed by raspberries at 5.84 x 10-7 g. The paper chromatography data also supports the idea that blueberries contained the most anthocyanidins, since it contained the most streaks on the chromatography paper. The main conclusion that can be drawn from this experiment is that blueberries contain the most anthocyanins per gram compared to raspberries and blackberries.
Introduction
The purpose of this experiment was to isolate anthocyanins, decompose them into their components (anthocyanidins), and to analyze the anthocyanins, and their corresponding anthocyanidins using paper chromatography, and spectrophotometric analysis procedures. Anthocyanins, “Are responsible for most of the red, purple, and blue colors exhibited by flowers, …show more content…
fruits, and other plant tissues.”1 They are in part responsible for the antioxidant properties for many fruits. “Anthocyanidins are the basic structures of the anthocyanins.”2 Due to these features anthocyanins are widely studied. Anthocyanidins are a different but very similar chemical species to anthocyanins as seen by the structure in scheme 1 (the anthocyanin is delphinidin-3-galactoside whereas the anthocyanidin is delphinidin). The basic structure of anthocyanidins allows for fewer of them to exist, making them easier to identify and recover than anthocyanins. In order to change the anthocyanins into anthocyanidins the hydrolysis reaction of the anthocyanin can occur. An example of one of these reactions is shown in scheme 1 below. Anthocyanins and anthocyanidins can be identified as in a similar method to many other compounds via paper chromatography. Since the various anthocyanidins vary in their functional groups, they will each interact in a different manner with cellulose paper and the eluting solvent giving each anthocyanidin a distinct Rf value. The “extended conjugation of double bonds through the three rings of the anthocyanidin”3 causes “the absorption of photons in the visible region of the spectrum (wavelength of maximum absorbance between 480 and 550 nm).”3 This allows for another way to distinguish differing anthocyanidins.
A spectrophotometer can be used to analyze a solution containing anthocyanidins by locating the maximum wavelength and its corresponding absorbance value. These values can help identify which anthocyanidins are in the solution, as well as how much anthocyanidin, and by extension how much anthocyanin was present in the original
sample.
Scheme 1
This scheme shows the hydrolysis reaction of delphinidin-3-galactoside, a typical anthocyanin
Methods
Extraction and Hydrolysis: Approximately 3 raspberries were ground into a very fine powder, until .997 g of raspberries were obtained. This powder was placed into a 15 ml centrifuge tube and 10 ml of acidic methanol (MeOH/H2O/CH3CO2H, 85/15/.5). The tube was shaken vigorously until the powder was mixed thoroughly. The sample was then vortexed for 30 seconds. This process was repeated. The sample was transferred into a clean 15 ml centrifuge tube using a pipet, only transferring the liquid while leaving the solid matter in the bottom. The liquid sample was centrifuged for 10 minutes at 3200 rpm. The liquid layer was removed and placed in a test tube. 5 ml of the liquid extract was mixed with 5 ml of HCl in a 25 ml round-bottom flask. A reflux apparatus was assembled using a 250 ml beaker of water on a hot plate as the heating source. Once reflux started to occur the process was continued for 45 minutes. The mixture was cooled in an ice water bath until it reached room temperature.
Paper Chromatography: During the reflux a butanol solution (butanol/acetic acid/water 4/1/5) was used as a mobile phase for a paper chromatography. A 250 ml beaker was filled approximately 1 cm with butanol and a piece of filter paper was added lining the beaker. Tin foil was wrapped over the beaker The chromatography paper was spotted heavily from left to right with unhydrolyzed blackberry solution (from other pair), raspberry solution, and blueberry solution (from other pair) on a pencil marked line1.5 cm above the bottom. Once the filter paper in the beaker was saturated with butanol, the top of the chromatography paper was wrapped around a copper wire. The chromatography paper was placed in the butanol solution, so that the butanol was still below the pencil line. The foil was wrapped over the beaker, and the chromatography was left to run until the solvent line was close to the top of the paper. Rf values were obtained. Other Rf values were obtained from another group using a formic acid solution (formic acid/ conc HCl/water, 5/2/3) as their mobile phase. The same procedure was repeated with a new piece of chromatography paper spotted from left to right with hydrolyzed blackberry solution, raspberry solution, and blueberry solution. Rf values were obtained. Other Rf values were obtained from another group using a formic acid solution as their mobile phase.
Spectrophotometric Analysis: 1 ml of hydrolyzed raspberry solution was placed in a 10 ml graduated cylinder. The graduated cylinder was then filled with acidic methanol so that there is a 1:10 dilution factor. A cuvette filled with acidic methanol solution was used to calibrate the spectrophotometer. The 1:10 diluted raspberry solution was placed in a clean cuvette and analyzed by the spectrophotometer. An absorbance vs wavelength graph was obtained, and the maximum wavelength in the visible spectrum and its corresponding absorbance value were recorded. The same data was obtained from other groups for a 1:10 diluted blackberry solution, and a 1:40 diluted blueberry solution.
Results
Figure 1
This figure displays a graph of absorbance vs wavelength data for the 1:10 diluted raspberry solution diluted with acidic methanol.
Table 1
Type of Berry
Mass of Berry (g)
Dilution Factor max (nm)
Absorbance
Blackberry
1.010
1:10
531.0
1.797
Raspberry
.997
1:10
529.0
.873
Blueberry
1.006
1:40
542.0
.724
This table summarizes the results (mass of berries, dilution factors, maximum wavelengths, and absorbances) for each berry during the spectrophotometric analysis procedure.
Table 2
Type of Berry Rf Values in Butanol Solution Before Reflux Rf Values in Butanol Solution After Reflux
Blackberry
.405
.417
Raspberry
.473
.370
Blueberry
.405
.439
This table summarizes the results (Rf values in butanol solution before reflux and Rf values in butanol solution after reflux) for each berry during the paper chromatography procedure.
Table 3
Type of Berry Rf Values in Formic Acid Solution Before Reflux Rf Values in Formic Acid Solution After Reflux
Blackberry
.795
.741
Raspberry
.897
.862
Blueberry
.846
.810
This table summarizes the results (Rf values in formic acid solution before reflux and Rf values in formic acid solution after reflux) for each berry during the paper chromatography procedure.
For the paper chromatography all of the streaks observed were not uniform and had to measured from the middle. They were fairly faint, but were somewhat darker in the middle with the intensity of color dissipating as it radiated out from the center. The solvent line for butanol solution after reflux was slanted, and therefore each Rf value had to be calculated with a different value for how far the solvent line traveled. Also the blueberry after reflux contained more streaks than the raspberry and blackberry.
Discussion:
1. Conc of anthocyanidin UV-Vis soln = (Absorbance at λmax)/(3.0 × 104 M-1cm-1)
Raspberry: (.873)/(3.0 × 104 M-1cm-1) = 2.91 x 10-5 M
Blackberry: 5.99 x 10-5 M
Blueberry: 2.41 x 10-5 M
2. Conc of anthocyanin extract = Conc of anthocyanidin UV-Vis soln /[dilution factor × 0.5]
Raspberry: (2.91 x 10-5 M)/((1/10) x (.5)) = 5.82 x 10-4 M
Blackberry: 1.20 x 10-3 M
Blueberry: 1.93 x 10-3 M
3. Grams anthocyanin = (Conc of anthocyanin extract x .0010 L)/ grams of berry powder
Raspberry: (5.82 x 10-4 x .0010 L)/(.997 g) = 5.84 x 10-7 g
Blackberry: 1.19 x 10-6 g
Blueberry: 1.92 x 10-6 g
It appears that blueberries had the most anthocyanin at 1.92 x 10-6 g while raspberries had the least amount of anthocyanin at 5.84 x 10-7 g. This relies on the assumption that there was no loss of material between any of the steps in the experiment. For example, the entirety of the berry powder must have been transferred into the centrifuge tube. Also all of the liquid from each of the liquid transfers, must have been successfully transferred. Another assumption essential to the accuracy of the results is that the hydrolysis reaction went to completion.
4. For all of the paper chromatography runs the streaks appeared relatively the same. They were all non uniform streaks with the greatest color intensity in the middle that slowly faded as it radiated out. Since there were no identical Rf values before and after hydrolysis it can be assumed that the hydrolysis reaction went to completion. This is because the differing Rf values indicate different chemical species, which can be assumed to be the anthocyanins (before) and the anthocyanidins (after). The presence of additional streaks in the blueberry paper chromatography after reflux indicates that more anthocyanidins are present in blueberries than raspberries or blackberries.
5. For all of the berries in each solution the Rf values were different before and after hydrolysis indicating that the hydrolysis reaction successfully occurred, and that the anthocyanins and anthocyanidins were different chemical compositions for each compound. The Rf values in each solvent were still fairly close indicating that the anthocyanins and their corresponding anthocyanidins were relatively similar in structure, and functional groups. Also each berry had a different maximum wavelength in the spectrophotometric analysis portion indicating that each berry contained a different mixture of anthocyanidins, even though all of the max wavelengths appeared in the green light portion of the visible spectrum.
6. Formic acid has a carboxylic acid functional group whereas butanol has an alcohol functional group. The interactions between anthocyanins with formic acid as well as anthocyanidins with formic acid appears to be more readily interpretable. This is because there is a larger separation in Rf values for formic acid than butanol. This can be seen since the Rf values for blackberry and blueberry samples using butanol as the mobile phase before hydrolysis, is the exact same value (.405).
Conclusion
In this experiment I learned what anthocyanins and anthocyanidins are, and where they are commonly found. I also learned the theory behind paper chromatography and how anthocyanins and anthocyanidins separate in this chromatography method. Furthermore, I learned how to properly perform a spectrophotometric analysis with a solution mainly comprised of a solvent other than water. I learned about the absorption of photons by anthocyanidins in the visible spectrum. The main concept I learned in this experiment was that blueberries contain the highest amount of anthocyanins.
References
1. Takeoka, G.; Dao, L. Anthocyanins. In Methods of Analysis for Functional Foods and
Nutraceuticals; W. J. Hurst, Ed.; CRC Press: Boca Raton, FL, 2008, pp 247-276.
2. Castaneda-Ovando, M.; Pacheco-Hernandez, M.; Paez-Hernandez, M.; Rodriguez, J. A.;
Galan-Vidal, C. A. Food Chem. 2009, 113, 859.
3. Curtright, R. D.; Rynearson, J. A.; Markwell, J. J. Chem. Educ. 1994, 71, 683.