Visible Spectrophotometry Analysis of a Food Dye in a Commercial Beverage: Skill Building Lab  2011, Sharmaine S. Cady East Stroudsburg University Skills to build:      Using a volumetric flask Using a micropipettor Using a Vernier Spectrometer Solving dilution problems Preparing calibration curves Visible Light and Spectrophotometry When white light passes through a prism, it forms a continuous spectrum of colors found in the visible region of the electromagnetic spectrum (Figure 1). chemical species absorbs photons of visible light when the photon energies match energy required to move the valence electrons to higher energy levels. The color of compound is a blend of the wavelengths of visible light that are transmitted and absorbed by the sample. Figure 1. Continuous spectrum of visible light the A the the not Food Dye in Beverages There are two ways by which color arises from absorption of visible light. A color wheel will aid in the explanation (Figure 2). First, a species may transmit one color and absorb all the rest. For example, if a chemical species absorbs all wavelengths of visible light except red, the observed color of the solution is red. Second, the compound may absorb several wavelengths of light and transmit the rest. The color perceived will be a blend of the transmitted wavelengths. For example, if a chemical species absorbs blue light, then the remaining red, orange, yellow, green, indigo, and violet will recombine to give an orange color since orange lies opposite of blue on the color wheel. This color is known as the complementary color. Copper(II)        Violet: 400 - 424 nm Indigo: 424 - 430 nm Blue: 430 - 491 nm Green: 491 - 575 nm Yellow: 575 - 585 nm Orange: 585 - 647 nm Red: 647 - 700 nm Figure 2. Color wheel and wavelengths of visible light complexed with ammonia, [Cu(NH3)4]2+appears blue in solution (Figure 3) because it absorbs red and orange light and transmits the remaining wavelengths that produce the complementary blue-green color. Figure 3. Visible absorption of [Cu(NH3)4]2+ in solution (Source: http://www.chem.purdue.edu/gchelp/cchem/color.html) Usually a species has a maximum absorption of light at a particular wavelength. This maximum wavelength of absorption allows for measurement of the concentration of 2 Food Dye in Beverages a colored species in dilute solutions. Table 1 gives the observed complementary color for the range in which a maximum wavelength falls. For example, -carotene is a carotenoid found in plant pigments. It absorbs light at 453 and 483 nm. From the table, the expected complementary color is yellow-orange. Table 1. Maximum Wavelength and Their Complementary Colors Beer-Lambert Law To determine the concentration of a colored species in solution, the BeerLambert Law is applied to the spectral data obtained. The Beer-Lambert Law states that the concentration of a colored species is directly proportional to its absorbance at a given wavelength. This can be seen from the mathematical representation of the law given below, where A is the absorbance, is a constant known as the molar absorptivity or extinction coefficient, b is the cell path length in cm, and c is the concentration of the colored species. This equation gives a straight-line graphical plot (y = mx + b) at concentrations below 0.10 M, A   bc where y = A, the slope m = b, and x = c. In a Beer-Lambert plot, the y-intercept b = 0. Figure 3 shows a typical Beer-Lambert plot with the equation for the straight line shown. 3 Food Dye in Beverages Absorbance Absorbance vs. Fe(II) Concentration 0.900 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 0.00E+00 y = 11006x 2.00E-05 4.00E-05 6.00E-05 8.00E-05 Concentration (M) Figure 3. Graphical representation of Beer-Lambert Law The above plot represents a calibration curve used to determine an unknown concentration. A calibration curve is prepared by plotting the absorbance of standard solutions of known concentration vs. concentration as shown in Figure 3. The equation for the straight line is shown. The absorbance value for an unknown is substituted for y in the equation, and the value of x is determined. For example, an unknown solution with an absorbance of 0.680 has a concentration of 6.18 x 10-5 M. Food Dyes The Food and Drug Administration (FDA) began its regulatory functions in 1906 with the passage of the Federal Food and Drugs Act. The FDA is responsible for the regulation of all color additives used in the United States since the passage of the Federal Food, Drug, & Cosmetic (FD&C) Act of 1938, which made food color certification mandatory. There are nine certified food color additives that have received FDA approval at the present time, seven of which have no restrictions as to use (Table 1). Color additives are dyes, pigments, or other substances that impart color when added or applied to food, drugs, cosmetics, or the human body. Color additives may be classified as a dye or lake. Dyes are water-soluble and are manufactured as powders, granules, liquids, or other special purpose forms. Dyes are used in beverages, dairy products, pet foods, and other various consumer products. Lakes are water-insoluble and more stable than dyes. They are often used to color products that have high fat or oil content or lack sufficient moisture to dissolve dyes, such as coated drug tablets, cake and donut mixes, hard candies, and chewing gum. 4 Food Dye in Beverages Table 1. Certified Food Color Additives Name Color Common Food Use FD&C Blue No. 1 Brilliant Blue FCF Bright blue FD&C Blue No. 2 Indigotine Royal blue FD&C Green No. 3 Fast Green FCF Sea green FD&C Red No. 40 Allura Red AC Orange-red FD&C Red No. 3 Erythrosine Cherry-red FD&C Yellow No. 5 Tartrazine Lemon yellow FD&C Yellow No. 6 Sunset Yellow Orange Beverages, icings, jellies, condiments, extracts, confections Ice cream, snack foods, confections, cereals, baked goods Beverages, pudding, ice cream, confections, baked goods Beverages, gelatins, pudding, confections, condiments Fruit cocktail cherries, baked goods, snack foods, confections Beverages, ice cream, custard, cereals, confection, preserves Beverages, snack foods, cereals, ice cream, baked goods, confections Note the number of conjugated double bonds in the molecular structures (FD&C Blue No. 1 (1), FD&C Blue No. 2 (2), FD&C Green No. 3 (3), FD&C Red No. 40 (4), FD&C Red No. 3 (5), FD&CYellow No. 5 (6), and FD&C Yellow No. 6 (7)) that can establish a set of bonding and anti-bonding molecular orbitals that allow electronic transitions from the ground to the excited state through the absorption of visible light. _ SO3 Na + O + H N _ Na SO3 N N H _ SO3 Na + O 2 _ SO3 Na _ SO3 + + N +_ Na O3S N HO + _ Na SO3 1 + N 3 _ SO3 5 Food Dye in Beverages I OCH3 I +_ O Na O O HO +_ Na O3S N I N I _ + COO Na H3C _ + SO3 Na 4 5 +_ HO Na OOC +_ Na O3S +_ N Na O3S N N N N N H3C HO _ + SO3 Na 6 _ + SO3 Na 7 Determination of Food Dye Concentration in a Beverage To determine the dye concentration, standard solutions of known dye concentration are prepared to obtain data to plot a calibration curve. A stock solution of dye is used to prepare five standard solutions whose concentrations are determined using dilution calculations. (See supplement under the experiment listing at the web site.) Then the absorbances of the five standard solutions are determined, and a Beer-Lambert plot is obtained as a calibration curve. This calibration curve is used to determine the concentration of the dye in the beverage. In this experiment, you will determine the concentration of FD&C Blue No. 1 or FD&C Red No. 40 in a beverage. FD&C Blue No. 1 and FD&C Red No. 40 have maximum absorbances at 629 nm and 503 nm, respectively. The measured absorbance for the beverage will be used to determine the dye concentration. 6 Food Dye in Beverages Experimental Methods and Materials Safety considerations Wear suitable protective clothing, gloves, and eye/face protection! You should read the online MSDS for: FD&C Blue No.1 Preparation of Standard Solutions With a micropipettor, place 125 L of the stock FD&C Blue No. 1 solution or FD&C Red No. 40 solution into a 100.00-mL volumetric flask. Record the concentration of the dye in the stock solution. Add distilled water to the mark on the flask. Invert 10 times to insure thorough mixing. In a similar fashion, prepare four more standard solutions with the values given in the table below. Determine the concentration of dye in each solution. 7 Food Dye in Beverages Table 2. Data for Standard Dye Solutions Standard Solution Volume Stock Solution, L 1 120.00 2 250.00 3 500.00 4 750.00 5 1000.00 Concentration, M Calibrating the Vernier Spectrometer Click on Logger Pro 3.5 from the Programs list on the computer. Click on Experiment  Calibrate  Spectrometer on the menu bar. Allow the spectrometer to warm up for 3 minutes. Place a cuvette ¾ full with distilled water in the cuvette holder. Click Finish Calibration. Once the OK button is ready, click it Collecting Data for the Calibration Curve Fill a cuvette ¾ full with Solution 5. Click on the Configure Spectrometer Data Collection icon on the right-hand side of the menu bar. Click Abs. vs. Concentration under Set Collection Mode. Click the OK button to close the dialog box.Click Experiment Start Collection and then Keep. Enter the concentration of the solution into the dialog box. Check that the wavelength for absorbance readings is correct for the color dye you are sampling. If so, repeat Start Collection and then Keep with Solutions 4, 3, 2, and 1. If not, repeat the calibrationuntil the correct wavelength is showing. 8 Food Dye in Beverages Beer-Lambert Plot Click the Linear Fit icon on the menu bar. Is the correlation factor acceptable (close to one)? If not, prepare a new set of standard solutions. Record the wavelength for which the data set was collected. Determination of Dye Concentration in Beverage Record the name of the beverage and the dye present based on the label information. Fill a cuvette ¾ full with the beverage. Click on AnalyzeInterpolation Calculator on the menu bar. Click the OK button. Record the absorbance and concentration. Print the screen. Laboratory Report Answer the following questions as part of your conclusions. 1. Did the standard concentrations and absorbances provide an acceptable linear calibration curve? Discuss the correlation value for your curve. The closer the correlation value is to 1, the more reliable the experimental data and the more the line representsan accurate relationship between absorbance and the concentration. When the value = 1, all experimental data points are on the line. See Figure 3. 2. What is the class average concentration for the beverage dye? Do any values seem unreasonable? 3. Calculate the m/v% dye in the solution based on the class average. Is the concentration of the dye within the OSHA standard (see MSDS) for a nonhazardous solution? 9 Food Dye in Beverages References Reusch, W. Visible and Ultraviolet Spectroscopy. http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm (accessed September 2005) U. S. Food and Drug Administration. Food Color Facts.http://www.cfsan.fda.gov/~Ird/colorfac.html (accessed January 24, 2007) Sigmann, S. B.; Wheeler, D. E. The Quantitative Determination of Food Dyes in Powdered Drink Mixes.J. Chem. Ed.2004, 81, 1475-1478. 10