Solutions and Spectroscopy - Food Dyes

Solutions and Spectroscopy – Food Dyes Prior Knowledge: • Basic algebra skills. • How to use a spreadsheet program. Learning Objectives Students will be able to: Content: • Give a proper description of the term solution and identify the parts thereof. • State the relation between the wavelength of light absorbed by the solution and the observed color of the solution. Process: • Predict changes that will be observed in absorbance spectra upon changing color and concentration of a solution. Background Question: How would you describe a solution? A solution is a homogeneous mixture of two or more substances. A homogeneous mixture is defined as a mixture that has the same chemical composition throughout and is stable (none of the substances will settle out of the mixture over time). Solutions can be composed of the same phase of matter (i.e., air, alloy, rubbing alcohol, etc.) or different phases of matter (i.e., tea, Styrofoam, whip cream, saltwater, etc.) A solution is composed of a solute and a solvent. The solute is the substance that is being dissolved into the solvent to make a solution. In general, the solute is the substance that is present in the smaller amount and the solvent is the substance that is present in the greater amount. Looking at the three beakers below, the amount of blue dye present in the solution is indicated by the intensity of the solution’s color. Predict the order of the beakers when arranged from most to least amount of solute (i.e., the blue dye) would be C > A > B. A B C Speaking only in terms of color, they are similar for they are blue solutions; they differ in the intensity of blue color. 1. Affirm or refute these statements: a. The amount of solute present in the solutions changes the intensity of the color. b. The amount of solute present in the solutions changes the identity of the color. Question: What is concentration? The concentration of a solution is defined as 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑉𝑜𝑙𝑢𝑚𝑒 The concentration of the original solution can be changed by adding more solvent; the solution is now diluted. When a solution is diluted, the volume of the solution increases, but the amount of solute remains the same as illustrated below: 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛) = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒(𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛) Beaker A Concentration: 10 red balls/10 mL or 1 red ball/1mL Beaker B Concentration: 5 red balls/5 mL or 1 red ball/1 mL Beaker B with additional 5 mL of water. Beaker C Concentration: 5 red balls/10 mL or 1 red ball/ 2 mL Beakers A & B have different numbers of red balls yet have the same concentration: 1 red ball/1 mL. Beaker C represents what happens if 5 mL of water is added to Beaker B. The concentration of Beaker C is 1 read ball/2 mL, but the number of red balls in Beaker C is the same as Beaker B; each have 5 red balls. Chemists refer to the process shown in the figures above as a dilution. To calculate dilutions the following mathematical relationship is used 𝐶1 𝑉1 = 𝐶2 𝑉2 In this equation, C1 is the solution original concentration before the dilution, V1 is the amount of the original solution used to make the new solution, C2 is the diluted solution concentration and V2 is the new volume of the dilute solution prepared. Example: How would you prepare 100.0 mL of 0.100 M NaOH. You have 1.00 M stock solution on hand. • • • Identify what variables (i.e., C1, V1, C2 or V2) you know. That will tell you which variable to compute to answer the problem. C1 is always associated with the more concentrated (i.e., higher) value so that means that C1 is 1.00 M and C2 is always associated with the less concentrated value. o 0.100 M NaOH is less than the 1.00 M NaOH given in the problem which means C2 = 0.100 M. o Since 0.100 M NaOH is associated with the volume 100.0 mL then 100.0 mL is V2. We are solving for V1 in this problem; the amount of the concentrated solution that will be diluted to make the new solution. 𝐶1 𝑉1 = 𝐶2 𝑉2 rearrange to 𝑉1 = 𝐶2 𝑉2 𝐶1 Plug in the values we were given in the problem. 𝑉1 = 0.100 𝑀 𝑥 100.0 𝑚𝐿 1.00 𝑀 𝑉1 = 10.0 𝑚𝐿 2. What is the concentration of a solution if you dilute 10.0 mL of 2.3 g/L NaOH to 100.0 mL with deionized water? 3. If you want to make 200.0 mL of a solution that is 1.75 ppm and you have a 12.00 ppm stock solution, how much of the stock solution should you measure out and dilute with water? Question: How can light be used to determine physical properties of a solution? Electromagnetic radiation can interact with an object either by being reflected or absorbed, which determines the color the object appears. When light passes through a solution without being absorbed, we say that it has been transmitted. If the solution is colored, when a beam of white light passes through a solution, some of the wavelengths are absorbed by the solution and the rest are transmitted. The wavelengths of light between 400 and 700 nm that are transmitted by the solution are observed by your eyes as color. White light containing all wavelengths Transmitted light Figure 1: The observed (transmitted) color of a solution is the light (i.e., wavelength) that was not absorbed by the water (i.e., media). Spectrophotometry is a technique based on the interaction of matter with light. A spectrophotometer is an instrument that measures the absorption of light. Light from the lamp (light source) falls on the sample; light that reaches the sample, but is not absorbed, passes through, or is transmitted by, the sample. The transmitted light is focused on a prism where it is dispersed into separate wavelengths. The diode array (detector) measures the intensity of this light electronically and converts the energy of this light into electrical current. The amount of current is directly proportional to the amount of light transmitted by the sample. Figure 2 below is a schematic of a simple spectrophotometer. Figure 2: Schematic of a Simple Spectrophotometer Figure 3 shows the amount of light absorbed by a dye at various wavelengths. Such a data plot is referred to as an absorbance spectrum. Absorbance spectral plots can illustrate the relationship between Absorbance and wavelength. Lambda max (max) is the wavelength at which the highest absorbance value is observed (i.e., peak of the curve). The absorbance at the max in a spectrum plot is the color that is being absorbed. Normally, a well-planned experiment results in the measurement of one physical or chemical property and its dependence on another. A graph represents the relationship between a pair of variables. The independent variable is plotted on the horizontal, or x-axis, while plotted on the vertical, or y-axis. Figure 3: Absorbance Spectrum of Blue Dye Information Statement • Experiments have quantities that can be changed and measured, called variables. There are three different kinds of variables: independent, dependent, and controlled. o Independent variable is the property which is changed in a controlled manner by the experimenter. There should only be one of these in an experiment. o Dependent variable is the variable which responds to the change. respond to the change made in the independent variable. There may be more than one of these in an experiment. o Controlled variables are quantities that the experimenter holds constant. There are usually more than one of these, and not controlling these quantities can result in your experiment not being a fair test. 4. In Figure 3, is wavelength the dependent or independent variable? 5. In Figure 3, is absorbance the dependent or independent variable? Each unique type of matter, including molecules and atoms, has a characteristic absorbance spectrum which is obtained by plotting absorbance as a function of wavelength. We can take advantage of this fact to identify compounds in solutions. This is a qualitative application. The wavelength at which the absorbing specie (solute) absorbs maximum radiation (i.e., light) is referred to as lambda max (max). See Figure 4. Absorbance Spectra for Various Colored Dyes 1.200 Absorbance 1.000 0.800 0.600 0.400 0.200 0.000 400 425 450 475 500 525 550 575 600 625 650 675 Wavelength (nm) Red, λmax 500 nm Yellow, λmax 425 nm Blue, λmax 625 nm Figure 4. Each unique dye has a unique pattern of light absorbance and a unique max value. 700 Absorbance spectroscopy also has a quantitative application. If radiation of one wavelength is passed through a solution, then the quantity of light absorbed will be proportional to the concentration of the solute in that solution. As the concentration increases, the amount of light absorbed increases. The relationship between concentration (c) and absorbance (A) is linear and is known as Beer's Law. Beer’s Law: The absorbance and concentration relationship The Beer’s Law equation is: A = a l c • The symbol, “a”, represents the absorptivity coefficient or constant for the solution. • The symbol, “l”, represents the path length or the length the light travels through the solution. • The symbol, “c”, represents concentration of the solute. Absorbance does not have units. When “l” has units of centimeters and “c” is in molarity (moles per liter), “a” has the units M-1cm-1. The value of the absorptivity constant is unique for each absorbing species. Its value can be derived from a Beer's Law calibration curve. A calibration curve shows the relationship between the absorbance and the concentration. A calibration curve is a method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. To construct a Beer’s Law calibration plot, a series of standard solutions are prepared and then analyzed. The absorbance of each standard solution is measured at max. Figure 5 is an example of a Beer’s Law Calibration plot. The calibration plot yields a trendline. Calibration plots and their associated trendline are specific for that set of prepared standards. In Figure 5, the trendline is y = 1.1929x + 0.286. The trendline (y = mx+b) allow us to determine the concentration of the solute in unknown solutions from their absorbance values. Informational statement: • A standard solution has a known concentration of solute. • Standards are carefully prepared solutions; their concentrations need to fall within a range known as the linear range. o Linear range: the range of concentrations that give absorbance values that fit a linear function when measured at a specific wavelength, which is usually max. ▪ Example: The linear range of the calibration curve for copper by AA analysis is 0.15 – 10 ppm using a wavelength of 324.7 nm. ▪ Solutions that are “too dark” (i.e., too concentrated) give absorbance values that do not fall within the linear range. Beer’s Law is not linear over all concentrations. Absorbance Beer's Law Calibration Curve 1.300 1.200 y = 1.1929x + 0.0286 1.100 1.000 0.900 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 0.0000.1000.2000.3000.4000.5000.6000.7000.8000.9001.0001.100 Copper(II) Concentration (M) Figure 5: The amount of light absorbed by a solute in a solution is directly proportional to the concentration of that solute. 6. In a sentence describe the relationship between concentration and absorbance that is illustrated in Figure 5. 7. When analyzing a sample using visible spectroscopy, the instrument’s wavelength setting is often set at max. Speculate why this wavelength is chosen. 8. Which components of Beer’s Law (A = alc) relate to the trendline equation 𝑦 = 𝑚𝑥 + 𝑏? a. b. c. d. y: m: x b: 9. How would you describe the relationship (i.e., direct or inverse) between absorbance and concentration? 10. Speculate why only standard solutions and their associated absorbance values are required to construct a calibration plot and not absorbance values of the unknowns? 11. What is the advantage for obtaining unknown absorbance reading within the linear range? 12. If an unknown solution’s absorbance reading falls outside the linear range, what can you do with solutions that fall outside the linear range to make them usable in your analysis? Determination of an unknown concentration from Beer’s Law Plot trendline. In the Figure 4, the trendline, y = 1.11929x + 0.0286, is given. The y-intercept should, theoretically, be zero (0), but under experimental conditions, it is usually a small value, close to zero. In this case, it is 0.0286. The trendline, generated by the calibration curve allows us to determine concentration of solute in an unknown. Example: The absorbance of a copper (II) solution of unknown concentration was measured and determined to be 0.581. Calculate the concentration of this solution. Using the equation, y = 1.11929x + 0.0286, we solve for x (since it represents concentration). 𝑥= 𝑥= 𝑦−0.0286 1.11929 0.581−0.0286 1.11929 plugging in the y absorbance value of 0.581 = 0.494 𝑀 𝑐𝑜𝑝𝑝𝑒𝑟(𝐼𝐼) 13. The absorbance value of an unknown copper (II) ion solution was 0.145. Determine the unknown solution’s concentration (M). 14. The concentration (M) for an unknown copper (II) ion solution was determined to be 0.285 M. Determine the absorbance reading of this solution. 15. The absorbance value for a 1:10 dilution of an unknown copper (II) ion solution was determined to be 0.312. Determine the concentration (M) of the unknown copper (II) ion solution. Experimental Blank Informational statement • A blank is the solvent that was used to prepare the standards and unknown sample solutions. Prior to any analysis using the technique of spectroscopy, the instrument must be set-up according to the parameters of the experiment. Experimental parameters are determined by the substance to be analyzed, the wavelength used (i.e., max) and the linear range specific for the chosen wavelength. Once the instruments parameters have be set, one more step must be taken prior to analysis of the standards and unknown sample solutions. The instrument needs to be blanked. Blanking the instrument, electronically removes any absorbance that would be associated with the solvent. Water, the most common solvent absorbs light. The analysis is about the solute; not the solvent or solvent + solute. Experimental Procedure Part A. Setup the spectrophotometer. It needs to warm up prior to use. 1. Assemble, Reboot and Set Parameters. a. Plug in the LabQuest. Use the USB cable to attach the SpectroVis to the LabQuest. Wait for the LabQuest to turn on. b. Using the stylus, tap the “Home” icon at the bottom. i. Select the “System” folder, and “Reboot”. ii. After rebooting is complete, you should see a red banner that shows “USB: Abs”. c. Using the stylus, select “Sensors” at the top of the screen. d. From the dropdown menu, select “Data Collection”. i. The “Mode” should be “Full Spectrum”, if not, select it from the dropdown menu. e. Set the “Wavelength Range” to 400 to 700 nm. Select “OK”. f. Tap the red banner with “USB: Abs”, select “Calibrate”. i. A 90 second countdown labeled “Waiting for lamp to warmup” will begin. While that is running move on to the next step. 2. Run a blank. a. Obtain 1 clean cuvette with two rough sides and two smooth sides (the windows). b. While holding the cuvette with your fingers on the rough sides - rinse and fill the cuvette ¾ full of deionized water. i. Gently tap the cuvette to remove any bubbles and use a Kimwipe tissue to polish the smooth sides. c. Insert the cuvette into the SpectroVis with the clear sides (windows) between the arrow and sun symbol. At this point, the warmup should be complete. d. Select “Finish Calibration”. e. Select “OK”. You should see the red banner that shows “USB: Abs”. Part B: Collect Absorbance Spectrum Data for Three Dyes (colors) 1. Remove the cuvette from the SpectoVis. 2. Empty the cuvette, rinse it 2-3 times with the unknown color solution of the same color that is found in your assigned drawer. a. Ensure that the curvette is filled ¾ full (~0.5cm from the top of the curvette. b. Gently tap the curvette to remove any bubbles. 3. Use a Kimwipe tissue to polish the smooth sides of the curvette and insert into the Spectro Vis with clear sides between the arrow and sun symbol. 4. Select the green “Play” icon at the bottom left. a. You may see a warning screen at this point. If not, move on to step 5. b. If you so, select “Store” and then move on to step 5. 5. Wait a few seconds for the graph to appear. 6. Select the red “Stop” icon at the bottom left. a. Record the “run number.” 7. Using the stylus, tough the graph at the top of the highest peak on the curve. Record the wavelength (on the x-axis of your graph) and the absorbance intensity (on the y-axis) values. a. THIS WAVELENGTH IS THE max for this particle dye color. 8. Repeat step 1-7 with the other two unknown dyes. These dyes are located on the top bench shelf. a. Rinse out the curvette with deionized water and shake out the remaining droplets. b. You will use the Beral pipet to add the dye directly to the pre rinse curvette. i. Fill ¾ of the curvette with the unknown dye. c. Take filled curvette and run spectra of this dye as directed in steps 3-7. d. When spectra run is complete, pour out dye, rinse curvette. 9. Repeat for the second unknown dye solution as directed above. Part C: Prepare Standard Solutions 1. In your assigned drawer you will find your unknown dye. Note and record the dye color and its concentration given on the label. 2. Obtain a 100 mL volumetric flask, five clean, dry 150 and/or 250 mL beakers, a 10.0 mL graduated cylinder and a Beral pipet. a. Label the 5 beakers as 1, 3, 5, 8 & 10 mLs. 3. Rinse the 100 mL volumetric flask with deionized water. 4. Measure out 1 mL of the dye solution as follows: a. Using a Beral pipet, draw up the dye and carefully transfer 1 mL of dye into a 10.0 mL graduated cylinder. b. Carefully transfer the 1.0 mL to the 100 mL volumetric flask. Rinse out the graduated cylinder and pour the rinse into the volumetric flask. 5. Add deionized water to the volumetric flask to its mark. The bottom of the meniscus should rest on the mark. 6. Cover the opening of the volumetric flask with Paraffin. Your instructor will demonstrate how to use the parafilm. 7. Thoroughly mix the solution 8. Transfer to diluted dye appropriately labeled beaker. 9. Repeat steps 3-8, measuring out 3,5,8 & 10 mL solution of dyes instead of the 1 mL amount of dye. Part D: Collect Beer’s Law and Unknown Data - Collect Absorbance Spectrum Data 1. Remove the cuvette from the SpectoVis. 2. Empty the cuvette, rinse it 2-3 times with a small amount of the solution in the “1 mL” beaker, and then fill it with this solution. a. Gently tap the cuvette to remove any bubbles. Use a Kimwipe tissue to polish the smooth sides. b. Insert the cuvette into the SpectroVis with the clear sides (windows) between the arrow and sun symbol. 3. Select the green “Play” icon at the bottom left. a. Note: You may see a warning screen at this point. If not, move on through the remaining steps. If you do see a warning, select “Store” and then move to 3b. b. Wait a few seconds for the graph to appear. Select the red “Stop” icon at the bottom left. c. Record the Run number along with its associated beaker label. d. Using the stylus, touch the graph at λmax for this dye and record the absorbance at this wavelength. 4. Repeat step 1-3 for the solutions in the beakers labeled 3, 5, 8 and 10 mL. 5. NOTE: for your unknown you will use the data collected in Part B Step #7. Part E: Export Data to USB Drive and Email Data to Partners 1. 2. 3. 4. 5. 6. 7. 8. Remove the USB cable from the LabQuest. Plug your USB drive into the LabQuest. At the top of the screen, select “File”. From the dropdown menu select “Export”. a. Ensure that the USB drive is selected. (Look at the top left of the screen. b. In the “Name” field, you should see files you recognize since this is your personal USB drive.) c. Swipe over “untitled” at the top of the screen and give this file an appropriate name, for example, “DyeSpecData”. d. Select “Done”. Select “OK” next to “Export to Text” at the bottom of the screen. Remove the USB drive from the LabQuest. Plug your USB into a device with Excel and internet access. Open Excel. a. From within Excel, select “Open”. b. Change the dropdown menu that says, “All Excel Files” to “All Files”. c. Search for and open your USB drive. d. Search for and open the text file you just exported from the LabQuest. Select “File”, “Save As”, change the file type to “Excel Workbook”, and then select “Save”. Email the Excel file to all your partners and to the lab instructor. Part F: Clean-up 1. All solutions can be discarded done the LARGE sink with water. 2. Rinse all glassware thoroughly prior to putting away in assigned drawer. 3. Turn of Vernier; disassemble and put away instruments. 4. Wash hands prior to leaving the laboratory.