Lab 11 Beers Law F23 (1)

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1 CHM 30: Studio Lab 11 Spectrophotometry and Beer’s Law *Adapted from |J. Chem. Educ. 2011, 88, 1410 – 1412 Introduction Visible light, a form of electromagnetic radiation, is comprised of the color spectrum. Each color on the spectrum has a specific wavelength(s), see color wheel below. When we see a red apple, the apple is absorbing the other color’s wavelengths, and reflecting red’s wavelengths (640 -700nm). The most absorbed wavelengths are opposite on the color wheel from the color that is observed. Therefore, the apple absorbed the green wavelengths, 480-560nm, the most. Figure 1: Color Wheel The spectrophotometer is an instrument that uses a photosensitive detector to measure the amount of visible light absorbed by matter dissolved in a solution at various wavelengths. A light source emits light that contains a range of wavelengths, infrared to ultraviolet (λ total , Figure 2). The light passes through a monochromator which contains a diffraction grating. This diffraction grating is used to select a one specific wavelength of light (λ X , nm). This wavelength of light then passes through the sample. By comparing the intensity of the light before it goes through the sample (I 0 ) with the intensity of the light after it passes through the sample (I), the spectrophotometer can determine the absorbance (A) of the sample, which is a measure of how much light was absorbed by the sample. A plot of absorbance versus wavelength is known as an absorption spectrum . Figure 5 on page 3 is an example for pure apple juice. Once you see the spectrum, you can choose a single wavelength to measure with, known as the operational wavelength (λ max ) . Typically, you want to choose the wavelength with the highest absorbance, since this will maximize your measurement signal. Figure 2. Pathway of light in a spectrophotometer.

2 Absorbance values of a solution are directly proportional to its concentration. This relationship is known as the Beer-Lambert Law , and takes the form: 𝐴 = 𝜀𝑏𝐶 (1) where 𝜀 (molar absorptivity) is a constant unique to each different substance, b is the path length of the sample through which light travels, and C is the concentration of the solution. Note that absorbance (A) is directly proportional to concentration (C) provided that the same path length of solution is used for all measurements. In order to find a quantitative relationship between A and C, you will create a series of solutions with different concentrations of 100% cranberry juice. Using the dilution equation below, you can calculate the concentrations of each solution: 𝐶 1 𝑉 1 = 𝐶 2 𝑉 2 (2) Here, the subscripts 1 and 2 refer to the solution before and after diluting, respectively. C 1 is the concentration of the stock solution . V 1 is the volume of the stock you use to dilute, and V 2 is the final, total volume of the diluted solution. Using this equation, you can calculate the concentration of your diluted samples, C 2 . Since the concentration of this series of solutions is known, these are called standard solutions . Plotting the absorbance of the standards (y-axis) versus their concentrations (x-axis) gives you a linear plot known as a calibration curve (Figure 3). Once this plot is made, a least squares fit line should be constructed in order to determine the equation of the straight line formed by the points. In the case of Figure 3, the least squares fit line has the equation: A = 1.58 C – 0.03. ( Note: while the Beer-Lambert Law does not indicate an intercept, the line may not go through zero because of random error effects during solution preparation, or other random changes to experimental conditions.) Figure 3: The direct proportional relationship of absorbance vs. concentration.

3 Given this fitted equation, if the absorbance is measured of a solution of the same substance but of unknown concentration, the unknown concentration can be calculated by substituting the measured absorbance into the equation as the value of A and solving for C , the unknown concentration. Today, you will use a spectrophotometer and Beer’s Law to determine the concentration of cranberry juice in store- bought cranberry-apple juice (a mixture of cranberry juice and apple juice). Cranberries are used to treat many diseases such as urinary tract infections and kidney diseases. There have been numerous research studies performed analyzing the effects of cranberry in the human body. The results have shown that aside from their applications as medicine, they also contain moderate levels of Vitamin C, dietary fiber, dietary minerals, manganese and micronutrients that benefit the cardiovascular and immune systems. Cranberries also contain phytochemicals, such as anthocyanins (Figure 4), which have alternating single and double bonds that give cranberries their deep red color, and which result in the high absorbance of ultraviolet radiation. Figure 4 : Anthocyanins: Phytochemicals that give cranberries their deep red color. “R” in the structure represents a sugar, and this anthocyanin (myricetin) is one of several anthocyanins present in cranberries. In this experiment, you will determine the percentage of pure cranberry juice in cranberry-apple juice. Juice manufacturers typically keep the proportion of cranberry juice and apple juice a secret. By creating a series of standard solutions of 100% cranberry juice, and plotting them as a calibration curve, you can measure the proportion of cranberry juice in a mixture to a precise degree. Figure 5: Absorption spectrum of 100% apple juice. 100% Apple Juice 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 350 400 450 500 550 600 650 700 750 Wavelength / nm Absorbance

4 Procedure Material: • Spectrophotometer (SpectroVis) • seven cuvettes • one 250-mL beaker • one 100-mL beaker • two 1000mL beakers • six more various sized beakers (>100mL) • one 10-mL graduated pipet • 100-mL volumetric flask • distilled water • cranberry juice • apple-cranberry juice • pipettor Safety and Waste : Food and drink used in laboratory experiments should never be consumed, inside or outside the laboratory. Inform your TA of any injuries, even small ones. Any waste from this experiment can go down the drain. How to use and clean a Pipet and Pipettor: 1. Hold the pipet near the upper end. Gently insert the top end of the 10mL graduated pipet into the collar of the red pipette pump. Do not bend or jam the pipet. Remember you are working with glass and you don't want it to break in your hand. 2. After the pipet is inserted, twist the white collar and the red body in opposite directions to tighten it. This should create an airtight seal. 3. Push the plunger all the way down. This will allow the maximum volume of liquid to be drawn up into the pipette. 4. To rinse the pipet: a. Set the tip into the beaker with distilled water. One hand should be holding the pipet and the other should be holding the pipettor. b. Turn thumbwheel down slowly to draw liquid up towards the top of the pipet. Stop when the liquid reaches just above the 0mL mark on the pipet. Never suck solution up into the pipettor! c. Empty this liquid into your waste beaker by pressing the white fast-release lever. d. Repeat this step 2 times with distilled water and once with 100% cranberry juice to fully rinse the pipet.

5 Part I: Preparing the Standards 1. Use labeling tape to label your beakers as shown in the table below. Beaker Size Label 100mL Cran-Apple (stock) 250mL 100% Cranberry (stock) 1000mL DI Water 1000mL Waste 2. Label 5 of the remaining beakers with S1, S2, S3, S4, and S5. These will hold your standard solutions. 3. Label the last beaker as “ cran-apple (diluted) ” . 4. Pour approximately 25 mL of the cranberry-apple juice (the unknown) into the cran-apple (stock) beaker. Put this beaker off to the side. 5. Pour approximately 80 mL of the 100% cranberry juice (the stock solution) into the corresponding beaker. This beaker is a secondary container and will be your source of stock solution for the remainder of the experiment. 6. Put approximately 500mL of distilled water into your DI water beaker from the DI water tap . The DI tap is the white faucets located on the 2 center benches in the lab. Be gentle and slowly turn the knob, water can come out fast. Do not overtighten the knob when turning off the water. You may need to refill this beaker later in the experiment. 7. Clean the volumetric flask by pouring approximately 10mL of distilled water into it (do not use your pipettor). Use a funnel if needed. Cap the flask and while holding your thumb on the cap, invert the flask 3 times. Empty the water into your waste beaker. Repeat this step 3 times. 8. Making standard 1 (S1) a. Using the same pipetting techniques as above, fill your pipet with exactly 4.00mL of 100% cranberry juice and dispense it into the 100mL volumetric flask. Make sure that all the solution is dispensed into the flask. Never put the pipet down with liquid still in it. b. Pour distilled water from the DI beaker into the volumetric flask until it reaches the bottom of the neck. c. Cap the volumetric flask. While holding the cap with your thumb, invert the flask 3 times. d. Uncap the flask, then use your DI water bottle (not the DI beaker) to carefully fill the volumetric flask with distilled water until the solution meniscus is exactly on the flask’s line. Make sure that the tip of the bottle does not touch the glass. e. Cap and invert the flask twice more, then pour this solution into the S1 beaker. 9. Repeat the previous step to make four more standard solutions. a. Each solution has an increase of 4mL of stock. See table on page 6. b. You do not need to clean the volumetric flask between samples as long as you do them in order of least concentrated to most concentrated . c. After the standard solutions are made, pour them into their corresponding labelled beakers.

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