Experiment Molecules, Light and Natural Dyes 11 Introduction Chemistry of Color The production of dyes was the basis for the creation of modern chemical industry. During the mid-19 th century all dyes were isolated from natural products before being used in other commercial industries. In 1856 William Henry Perkin first made a purple dye from a derivative of aniline. This dye, Mauveine, was much easier to produce than the naturally occurring dyes. This lead to the discovery and commercialization of many other dyes derived from aniline. As early as 1910, there was a realization that some of these new dyes were toxic if consumed in foodstuffs. While many of the aniline based dyes are safe, recent concerns about chemical safety and a desire to move toward more renewable resources has reinvigorated research into the extraction and use of natural dyes. In this lab you will develop and extraction procedure and quantify the amount of dye extracted from carrots and turmeric. Natural Dye Molecules All fruits and vegetables contain molecules that impart color. Some of these compounds can be isolated and used to as dyes for fabric or other foods. Today we will explore the dye molecules that can be extracted from carrots (beta-carotene), turmeric (curcumin) and tomatoes (lycopene). Some Common Natural Dyes Molecular Structure Name Plant H 3 C CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H 3 C H 3 C CH 3 Beta- Carotene Carrots O O O O H 3 C CH 3 Curcumin Turmeric HO OH Lycopene Tomatoes 125
Absorbance (a.u.) New Science When an atom or molecule absorbs a single photon an electron makes a transition to a higher energy orbital. Thus, the number of photons a sample of material absorbs is directly related to the number of particles in the path of the beam light. This important concept means that by measuring the change in intensity, or number of photons (I) of a light beam before (I 0 ) and after (I t ) it passes through a solution of molecules, we can measure the number of molecules in the solution. The intensity change is related to the number of molecules the beam encounters. More technically, the log of the ratio of I 0 to I t is called the absorbance (A). ( ) Red Orange Yellow Green Blue Indigo Violet Sample Red Orange Yellow Green Blue Indigo Violet 0.35 0.30 0.25 0.20 0.15 0.10 I O I t 0.05 For the red colored sample above, all of the red and some of the blue light pass through the sample, whereas the yellow, orange, and green light is absorbed. Thus, the absorbance values of yellow, orange, and green light are high and the absorbance values of red and blue are lower. 400 500 600 700 800 Absorbance specifically depends on certain properties of the sample: the wavelength of the measurement, the extinction coefficient, the path length of the sample cell, l, and the concentration of the sample c. (Beer s Law) (for mixtures) Alternatively, we can also explain this phenomenon by quantifying the transmission of a sample, or the number of photons that the sample does not absorb. Transmission, I t /I 0, is related to absorbance: - ( ) In the example above, the transmission values of red and blue would be high but those of orange, green, and yellow would be low. Scientists use both absorbance and transmission data to report the optical properties of a sample depending on what information they want to convey. During this laboratory experiment, think about how altering, l, and c would change the absorbance and transmission of the samples that you will observe. You will also be using a spectroscope and some filters to explore light and absorbance. 126
300 340 380 420 460 500 540 580 620 660 700 300 340 380 420 460 500 540 580 620 660 700 Absorbance Absorbance 300 340 380 420 460 500 540 580 620 660 700 300 340 380 420 460 500 540 580 620 660 700 Absorbance Absorbance Exp 11 Prelab Exercise, Yellow Dye Example The absorption spectrum on a unknown yellow solution is shown below. Spectra of three known solutions (Yellow #5, #6 and turmeric) are also shown. 1) Which dye is present in the unknown? 2) How can you tell? (Explain your reasoning.) 0.50 0.40 0.30 0.20 0.10 Absorption Spectra for Unknown Absorption Spectra for Yellow Dye #6 1.50 1.00 0.50 Absorption Spectra for Yellow Dye #5 1.20 1.00 0.80 0.60 0.40 0.20 Absorption Spectra for Turmeric (a natural food dye) 1.00 0.80 0.60 0.40 0.20 127
Absorbance Absorbance 1.20 Absorption Spectra for Yellow Dye #5 1.00 0.80 0.60 0.40 0.20 25 g/l 5 g/l 0.01 g/l 0.015 g/l 0.02 g/l 300 340 380 420 460 500 540 580 620 660 700 1.200 Beer's Law for Yellow #5 1.000 0.800 0.600 0.400 0.200 0 00 50 0.0100 0.0150 0.0200 0.0250 Concentration (g/l) 3) Sketch a Beer s Law Plot using the data provided. Estimate the slope of the line. 4) What is the concentration of the dye present in the unknown shown on page 1? 128
Intensity of Intensity of Absorbance of Filters Explore light, transmittance and absorbance using your own eyes as the detector. A spectroscope contains a diffraction grating that separates light into its component wavelengths. For example, when you look at fluorescent light without a spectroscope, it looks white, but when you look through the spectroscope, you can see the colors that the light is composed of. To use the spectroscope, make sure that the slit is aligned with the light source. Then look at the scale to the left without moving the spectroscope. You should see white light spread into its rainbow of colors. Schematic of aligning the spectroscope. 1. Take orange, blue, red, and purple filters from the supply shelves, their corresponding transmission spectra, and spectroscope for each individual in your group. 2. Use the spectroscope to look at the reflected sunlight through the window. What do you observe? What colors do you see? Draw the transmission spectrum in your lab notebook using the format shown below. Now look through the spectroscope with the orange filter placed over the slit. What do you observe? What colors do you see? Draw the transmission spectrum in your lab notebook. How does this spectrum compare to the outdoor or room light? 129
Intensity of Intensity of Intensity of Intensity of Repeat with the blue, red, and purple filters. 3. Does the transmission spectrum observed from the purple filter correspond to a spectrum observed when the blue and red filters are overlapped? Draw the transmission spectrum observed when the blue and red filters are overlapped. 4. You are provided with the absorbance spectra of these filters. How do these graphs correspond to your observations? 130
Investigation 1: Dye Extraction Experimental The Problem Create a procedure to extract natural dyes from carrots and turmeric. The Approach Part 1: Design your own experiment: 1) Address each of the following issues to help you design your experiment. I. Based on the structure of beta-carotene and curcumin (the dye molecules in carrots and turmeric respectively), which solvent will you use: ethanol or water? II. Besides solvent, there are three other variables for you to consider: temperature, time, and sample preparation. Design a set of three experiments that tests one of these variables. In other words, if you want to test the effect of temperature then decide on three different temperature and use the same time and sample preparation. Or you can vary the time that you run the extraction and keep the sample preparation and temperature the same. Record which variable you are going to test and what you will change. You may test a different variable for the carrots and the turmeric. III. For all of your experiments you will be using 5 g of the plant material and 50 ml of solvent. In total you will be running 6 extraction experiments, three with carrot and three with turmeric. 2) Write up a procedure and show it to your GSI who will help you locate all of your materials. 3) Follow your procedure and isolate your dye solutions. Be sure to record all of the materials and amounts of chemicals that you used and isolated during your extraction process. 131
Investigation 2: Dye Quantification The Problem Determine the concentration of a natural dye molecule from an extract. The Approach 1. Make a Beer's law plot of your peak absorption data vs. the concentration in grams per milliliter based on the data provided for both curcumin and beta-carotene. You may use the origin (0, 0) as a data point. This is equivalent to zeroing the spectrometer. Instructions for making linear graphs in Excel can be found in the appendix of this manual. 2. Determine the slope of the line using a linear regression routine on a calculator. Be careful with units. Record your value for ε (the slope); you will use this value to calculate the amount of dye in each of your solutions from Investigation 1. 3. Using your Beers law plot, determine the amount of dye in each of your solutions from Investigation 1. a. Are there any trends based on the amount variable that you tested? Did temperature, time, or sample preparation effect the amount of dye that you extracted from the carrot and curcumin)? 4. Estimate the total amount of dye that you extracted from the carrot and turmeric in your best procedure. What was the highest total amount of dye that you extracted from each starting material? Record this value on the board. 132
Experiment 11- Dyes, Molecules and Light Report Sheet Your Name Partners GSIs Name 1) Construct a Beer s Law plot (Absorbance vs. concentration) for both beta-carotene and curcumin from the data provided. Fit the data to a straight line and calculate a value for R 2. Attach the graphs to your report and report the equation for the line on the graph. 2) Calculate the extinction coefficient for your natural dye. 3) Calculate the concentration of natural dye (g/ml) that you isolated based on your Beer s Law plot for each of your extractions. 4) Calculate how many milligrams (mg) of dye you extracted from the plant material for each extraction. 133
5) Briefly describe your best extraction procedure, the one that yielded the most dye. 6) How do your best results compare to other groups? Suggest ways to make your experiment either more efficient or greener. 7) How is using a spectrometer different from using a spectroscope? Describe the benefits of using a spectrometer rather than a spectroscope for the quantitation. 134