CHM 114: Bio-Organic Molecules Week 5 Laboratory Separation of Wine Phenolics 1
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1 CHM 114: Bio-rganic Molecules Week 5 Laboratory Separation of Wine Phenolics 1 Background: The reds and purples that make beautiful fall colors in the leaves of trees result from pigment compounds known as anthocyanins. Flowers have their distinctive colors because of their unique combinations of anthocyanins and another class of compounds called carotenoids. The colors of fruits and vegetables such as apples, plums, rasberries, beets, radishes and grapes also depend on anthocyanins. The actual color of a specific anthocyanin depends upon the acidity of their environment. In fact, anthocyanins can actually be used as acid-base indicators. The earliest definition of acids was given by Robert Boyle, who stated that acids turned plant juices red. In his book Experiments upon Colours, published in 1663, Boyle described a means of making indicator paper from the juice of violets, cornflowers, roses, snowdrops, brazilwood, primroses, cochineal, and litmus. Use of such natural acid-base indicators predates the use of synthetic indicators such as phenophthalein by over 200 years. H H Caffeic acid + 3 CH 3 C 2 H H Chalcones H Cyanidin λ max = 535 nm violet-blue color Anthocyanins H (Quercitin) Flavonols Figure 1. Biosynthesis of Anthocyanins H R R' 3 position Aglycone R R cyanidin H peonidin CH 3 H delphinidin petunidin CH 3 malvidin CH 3 CH 3 pelargonidin H H Table 1. Structures of Different Anthocyanins There are three main classes of natural pigments: chlorophyll (green), carotenoids (yellow and orange) and flavonoids (red, yellow, orange, blue, and violet). The anthocyanins belong to the flavonoid class of pigments. 1 This is a modification of an experiment described in Brenneman, C. A.; Ebeler, S. E. J. Chem. Educ. 1999, 76,
2 Anthocyanins are biosynthesized from caffeic acid and three acetate units through such intermediary compounds as chalcones, flavanones, flavones, and flavonols (Figure 1.). All of these compounds belong to the general class of compounds called flavonoids. There are hundreds of different anthocyanins which all have the same aglycone unit (core molecular structure) but vary in their exact substitution patterns and groups (Table 1.). The exact color of a particular anthocyanin depends upon its structure. Looking at Table 1 on the previous page, the compound cyanidin has a violet blue color while malvidin-3-glucoside has a red color. The 3-glucoside portion of the name indicates that a glucose molecule is connected to hydroxy group at position 3 in the molecule (see structure in Table 1). The flavylium ion has a red color. Anthocyanins, extracted from grape skins during fermentation, are largely responsible for red wine color. The anthocyanins exist in several different forms depending on ph (Figure 2): at low ph (<2.5), the red flavylium ion is the predominant form in equilibrium with the colorless pseudobase (and it s corresponding tautomer, the chalcone), while at ph s > 4.25 the blue quinone form dominates. Normally, wine has a ph in the range of , and approximately 10% -30% of the total anthocyanins are in the red, flavylium form 2. At the beginning of the experiment, the wine ph is adjusted to 7.0, and a distinct change in color should be observed due to quinone formation. H Red Flavylium ion ph<2 add lose H Colorless pseudobase H Blue Quinone Form ph>4.5 Figure 2. ph Dependence of Anthocyanin Structure In this experiment, the principles of chromatographic separation are visually demonstrated using solid phase extraction (SPE) columns. The columns are packed with a nonpolar stationary phase (e.g., octadecyl silyl or C18), and following sample application, individual components of a mixture are sequentially eluted by changing the composition of the mobile phase (solvent). SPE columns are also commonly used in environmental, food, and biomedical laboratories for recovery and isolation of organic compounds from water, air, food, and biological samples. The theory which explains why the individual compounds can be separated on this column is similar to that seen previously with the gas chromatographic (GC) analysis of cough syrup extracts and the dextromethorphan standard. However, in this experiment the stationary phase will be nonpolar instead of polar like the GC column. Wine is a complex beverage containing a diverse array of natural products. Among these constituents, polyphenols have been extensively studied for their biological effects as antioxidants, antimutagens, and cancer preventive agents 3. Polyphenols are also present in a wide range of other fruits and beverages, including blueberries, green tea, and chocolate, and they contribute to the color and to the bitter taste and astringent mouthfeel properties of these foods 7. Structurally, the term polyphenol refers to molecules which have several phenolic groups. A phenolic is an bonded to a six-membered ring with alternating single and double bonds (Figure 3). The falvonoids present in red wine can all be classified as polyphenols. 2 Ribéreau-Gayon, P. In Anthocyanins as Food Colors, Markakis, P., Ed.; Academic Press, Inc. New York, 1982, pp Bailey, G. S.; Williams, D. E. Food Tech. 1993, 47, Clifford, A. J.; Ebeler, S. E.; Ebeler, J. D.; Bills, N. D.; Hinrichs, S. H.; Teissedre, P.-L.; Waterhouse, A. L. Am. J. Clin. Nutr. 1996, 64, Watkins, T. R. Wine. Nutritional and Therapeutic Benefits, American Chemical Society: Washington, DC, 1997, pp Middleton, E.; Kandaswami, C. In The Flavonoids. Advances in Research Since 1986, Harborne, J. B., Ed.; Chapman & Hall: New York, 1994, pp Ebeler, S. E. In Functionality of Food Phytochemicals, Johns, T.; Romeo, J. T., Eds.; Plenum Press: New York, 1997, pp
3 Reverse Phase Chromatography Normal phase chromatography utilizes a polar stationary material or column. A mixture of compounds then travel through the column at different rates depending upon their interactions with the polar stationary phase (Note: this is intermolecular forces again). Polar compounds will interact more strongly with the polar column and move at a slower rate. Nonpolar compounds will have relatively weak attractions to the polar stationary phase and move through the column at a faster rate. In reverse phase chromatography, we use a non-polar stationary phase and use solvents with different polarities to move compounds through the column. For example, if one of the compounds in a mixture can be put into an ionic form while the rest of the compounds remain nonpolar, a polar solvent will move the ionic compound through the non-polar, reverse phase column while the nonpolar compounds remain stuck to the nonpolar column. We will utilize this concept to separate the mixture of flavonoids present in a sample of red wine. H Phenol A Polyphenol Figure 3. Phenolic Groups and Polyphenols Typical polyphenols and their concentration in wine are shown in Table 2 and Figure 4. These polyphenols can be separated into 3 fractions, based on their polarity: 1) phenolic acids, 2) catechins and anthocyanins, and 3) flavonols. By adjusting ph and solvent strength (polarity) you will isolate these fractions from a wine sample using a nonpolar (C18) reverse phase SPE column. Initially, the wine is adjusted to ph 7.0, resulting in ionization of the phenolic acids. Thus, the phenolic acids are not absorbed to the hydrophobic C18 stationary phase and are collected in the eluant. The other polyphenols are more hydrophobic and are absorbed onto the column. Following addition of HCl to the column to ionize the anthocyanins (Figure 4), the catechins/anthocyanin and flavonol fractions are then eluted by decreasing the polarity of the mobile phase: eluting first with 16% aqueous acetonitrile followed by ethyl acetate. Class Fraction Example Compound Concentration White Wine (mg/l) Concentration Red Wine (mg/l) UV/Vis Absorbance λ max (nm) Phenolic Acids 1 Caffeic acid , 320 Catechins 2 Catechin Anthocyanidins 2 Malvidin , 520 glucoside Flavonols 3 Quercetin Trace , 365 Polymeric Flavonoids Retained on column , 520 Table 2. Concentrations of Polyphenols in Wine and their Separation Using Reverse Phase Chromatography Separation of Polyphenols At ph 7, the polyphenols listed in Table 2 will all be neutral; except for the phenolic acids. The phenolic acids will be ionized, appearing brown in color. They will not absorb or bind to the neutral nonpolar C18 column and will move with the water solvent used to elute fraction 1 from the column. The other polyphenols will be able to interact with the nonpolar C18 stationary phase and not able to be solvated by the water moving through the column. As a result, they will not move through the column at this time. There are other compounds besides the phenolic acids which can move through the column with water at ph 7 such as residual sugars but these compounds do not interfere with the UV/Vis absorbance spectrum of the phenolic acids. After the phenolic acids have been separated (eluted from the column) the column is reconditioned to acidic conditions using 0.01 M HCl. This will convert the malvidin-3-glucoside into its red flavylium ion form (polar) which will no longer be able to strongly interact with the nonpolar C18 stationary phase. You should notice that the material bound to the column becomes red during the column reconditioning step. The solvent mixture (16% CH 3 CN/H 2 ) is polar but not quite as polar as 100% H 2 and will be able to solvate both malvidin-3-glucoside and
4 catechin. These compounds will move through the column with the acetonitrile/water solvent. You will collect a red fraction with this solvent. However, acetonitrile/water solvent will not be able to solvate the less polar flavonols. Compounds such as quercetin will remain bound to the nonpolar C18 stationary phase during this elution step. H H H Caffeic acid H Ionized form predominates at ph = 7and will move with water as the solvent H Catechin Catechin does not have an ionized form which exists at ph< 9. It will move with aqueous acetonitrile CH 3 CH 3 H Malvidin-3-glucoside form at ph 7 (blue) CH 3 H Ionized form at ph 2 will move with aqueous acetonitrile CH 3 H Quercitin is similar to catechin but is less polar. It will move with the least polar solvent used (ethyl acetate) Figure 4. Structures of Polyphenols at Different ph Values After fraction 2 has been collected, the solvent will be changed to ethyl acetate which is the least polar of the solvents you will use. Quercetin will be able to be solvated by the relatively nonpolar ethyl acetate and finally move through the column. These separations of catechins/anthocyanins and flavonols are largely due to decreasing the polarity of the eluting solvents and altering the partitioning behavior of the polyphenols between the nonpolar stationary phase and the increasingly nonpolar mobile phase. The flavonols are colorless. Carefully observe fraction 3. You may notice a red/pink bottom layer in the test tube with a clear, colorless top layer. The ethyl acetate will not mix with the polar acetonitrile/water solvent system. The pink bottom layer should be considered part of fraction 2 and not used to analyze fraction 3. Polymeric flavonoids will remain on the column throughout this separation process. These polymeric materials have a reddish/brown color and consist of combinations of ~4 or more monomeric catechins and other flavonoids (e.g., anthocyanins) joined together in various chemical linkages 8. Formation of these polymers are important in wine aging reactions and affect the color, taste, and mouthfeel properties of the wine. 8 Sun, B.; Leandro, C.; Ricardo da Silva, J. M.; Spranger, I. J. Agric. Food Chem. 1998, 46,
5 Spectroscopic Analysis Although differences in color of each fraction are observed, the identity of the major phenolic groups in each fraction can be more accurately determined with a scanning spectrophotometer. Each phenolic class absorbs maximally at a specific wavelength depending on the degree of conjugation present in the molecule: the phenolic acids at 320 nm, flavonols at 365 nm, anthocyanins at 520 nm. Each of the collected fractions will be analyzed by UV/Vis spectroscopy to identify the type of compounds present. Table 2 gives the exact λ max for each of the different types of compounds which we will have isolated from red wine. This is the wavelength of light which the molecule absorbs to the greatest extent and will appear as the highest point on the absorption curve. Shown below are the typical absorption curves for the different types of flavonoids all superimposed on the same graph. Your spectra should look similar. If the flow rates were not carefully monitored during the separation, then there may be mixtures of the different compounds in the fractions. The spectrum of a mixture would show the λ max of all of the different components present. Experimental Procedure: Separation of Wine Fractions 1. Adjust the alcohol-free wine to ph 7.0 ± 0.1 (use 1 M Na or 0.01 M Na as appropriate) noticing the change in color. Using Figures 2 and 4, determine the predominant form of malvidin-3-glucoside (the predominant anthocyanin in red wines) at wine ph (ph ) and at ph Clamp a Sep-Pak C18 SPE column to a ring stand with a 10 ml graduate cylinder below to collect fractions. Note: For column conditioning and all subsequent elution steps, mobile phase flow through the columns should be approximately 1 ml/min. Sufficient time must be given to allow for sorption/desorption of the sample/analyte between the stationary and liquid phase. Elution rates that are excessive will lead to column breakthrough and incomplete recoveries of the compounds of interest. Use the pipet bulb carefully to keep flow rates at 1 ml/min. Water will need more help being pushed through the column than the organic solvents such as methanol and ethyl acetate. 3. Activate the column with 2.0 ml of methanol followed by conditioning with 2.0 ml neutral ph water. Discard the eluant. 4. Load ~1.5 ml of the dealcoholized, ph 7.0 wine onto the column (actual amount of sample loaded is dependent on the phenolic content of the wine; an overloaded column is easily observed by breakthrough of the red color during the sample loading step). Note the color of the column and the eluant throughout each step of the fractionation. 5. Elute 6 ml (one column volume) of neutral water through the column. This may be done in two steps. The eluant collected in this step is Fraction 1. Note the color of the eluant. This fraction should contain the phenolic acids. 6. Recondition the column by eluting 2.0 ml of 0.01 N HCl through the column. The eluant is discarded. Notice how the color of the column changes. What is happening to the anthocyanins? 7. Pour 6 ml of 16% aqueous acetonitrile (ph 2) through the column. Allow the 16% acetonitrile solution to run all the way through the column. The collected eluant is Fraction 2. Note the color of the fraction. 8. Pour 6 ml of ethyl acetate through the column, collecting the eluant. The collected eluant is Fraction 3. Note the color of the eluant. The eluant will have 2 noticeable layers present. The lower, more polar liquid is 16% acetonitrile that was not completely removed from the column in the previous step. Spectrophotometric Analysis of Fractions 1. Using a quartz cuvette, scan a solvent blank (e.g., for Fraction 1 the blank is neutral water) from 250 nm to 600 nm. Store the spectrum in the computer. 2. btain the spectrum of Fraction 1 over the wavelengths nm. Dilution of the fraction may be necessary depending on the concentration of polyphenols present in the fractions. 3. Record the experimental λ max for the fraction. 4. Repeat steps 1-3 for each fraction, using the appropriate solvent blank (e. g., 16% aqueous acetonitrile for Fraction 2; ethyl acetate for Fraction 3) to correct for background absorbance.
6 5. From the graph and the information provided in Table 2, identify the major phenolic group in each fraction. Name a representative compound that may be contained in each fraction. Questions: 1. Why was the ph of the wine adjusted to 7 at the begining of the experiment? 2. What accounts for the colors of the different fractions? 3. Explain why red wine could be used as a ph indicator. ver what ph range would it be an indicator? 4. Explain why polar or ionic compounds will move through a nonpolar C18 column with a polar solvent. 5. What keeps the nonpolar compounds on the column when a polar solvent moves through the column?
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