Absorbance Introduction to Spectroscopy: Analysis of Copper Ore Introduction The goal of this lab is to determine the unknown concentration of two different copper solution samples, taken from fictitious mining sites (sites A and B), by generating a standard curve, called a calibration curve. A calibration curve or standard curve is a mathematical tool used by chemists to determine the concentration of a substance based off a set of known reference points. First the reference point solutions (referred to as standard solutions here on out) need to be made and analyzed. Your group will make a series of diluted solutions of copper(ii) sulfate pentahydrate (CuSO4 5H2O). These diluted solutions will be your standard solutions. The relationship between concentration and absorption of these standard solutions will be analyzed using a spectrophotometer (Genesys Spec-20). Spectrophotometric analysis is one method used to determine the concentration of a colored substance in solution. Concentration is determined by calculating the amount of light that passes through a sample and hits a detector. The more light that gets through, the less concentrated the solution is (fewer molecules of the colored substance that absorb light). Concentration may be expressed in several ways. In this experiment, it is expressed as the milligrams of copper per milliliter of solution (mg/ml). To produce accurate results the appropriate wavelength at which to measure the absorbance of the standard and unknown solutions must be determined. To select the best wavelength for measurements, we must find the wavelength of maximum absorption. To do this, we plot wavelength (x-axis) versus absorbance (y-axis) for one of the solutions and simply find the wavelength that gives the maximum absorbance. The example below shows a peak absorbance occurring at about 610 nm. The concentrations of solutions A and B will then be determined using graphical analysis of the standard solutions data. A regression line representing the best straight-line fit of the absorbance data (y-axis) versus standard solutions concentrations (x-axis) will produce an equation of the form y = mx + b. This is considered a Beer s Law Plot (see below). The regression line equation will then be used to mathematically determine the concentration of the solutions taken from site A and B. 60 50 40 30 20 10 0 Beer's Law Plot y = mx + b R² = 1 0 0.2 0.4 0.6 Concentration (mg/ml) GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 1 of 11
For more information: Chemistry: Atom s First by OpenStax section 6.3 Molarity - Dilution of Solutions Equations to use for the calculations: Volume delivered: VTotal = VFinal VInitial Dilution Formula: M1V1=M2V2 y=mx + b Materials: 5 50-mL volumetric flasks with lids 25-mL buret CuSO4 5H2O stock solution buret stand small plastic pipets Genesys Spec-20 Spectrometer 2 plastic cuvettes Kim wipes access to Microsoft Excel or Google Charts plastic funnel DI water bottle calculator Procedure Part I: Preparing the Standard Solutions 1. Turn on the Genesys Spec-20 so it will have plenty of time (about 15 minutes) to warm up. 2. Record the concentration of the stock copper(ii) sulfate pentahydrate solution in the data section of your lab report. 3. Condition a clean 50.00 ml buret with the stock copper(ii) sulfate pentahydrate solution and prepare it to deliver solutions into the 4 50-mL volumetric flasks. See technique Preparing Solutions. 4. Use the buret of stock copper solution to deliver volumes as close to whole numbers as you can measure. See technique Using a buret to deliver a solution. Record the initial volume from the buret and deliver as close to 1 ml as you can to the first volumetric flask. Record the final volume from the buret. Add deionized (DI) water from your wash bottle to the solution in the volumetric flask. When the meniscus gets close to the line, you may use the plastic pipet to add water drop-wise. Be sure the bottom of the meniscus does not go above the line. If it does, you will have to remake that solution! Stopper the flask and invert it several times to thoroughly mix the solution. 5. Repeat this process to make the next 4 solutions. Label your flasks with pencil or tape. Deliver approximately 1 ml, 2 ml, 4 ml, 6 ml, and 8 ml of stock solution, and fill each volumetric flask to the line with deionized water. Remember to record the exact volume of the buret before and after delivering the stock solution. GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 2 of 11
6. Stopper all your labeled flasks, invert them several times to thoroughly mix the solutions, and set them aside for part III. Part II: Finding the Maximum Absorbance for Copper Solutions 1. Press the A / T / C button until an A appears in the upper right corner of the display. 2. Use a disposable pipet to fill one cuvette 2/3 full with DI water and a second cuvette 2/3 full with your last (8.00 ml stock) solution. Check each cuvette for air bubbles. If you see air bubbles clinging to the walls of a cuvette, tap the cuvette gently to get the bubbles to rise to the top of the solution and escape. Use a Kim-wipe to wipe off the outside of the cuvettes. 3. Follow steps a e below to determine the absorbance for each wavelength. a) Place the water cuvette in the holder with the clear sides aligned with the arrow. (This arrow shows the direction in which light is aimed.) b) Select the first wavelength to test (400 nm) using the up/down nm buttons. (They scroll quickly if you hold the button down.) c) Press the 0 Abs/100 % T button to calibrate the instrument for that wavelength. d) Remove the cuvette with water and replace it with the cuvette of solution. DO NOT press any buttons!!! e) Read the absorbance on the display for your solution. If it reads a small negative number, record a value of 0 for that reading. 4. Repeat steps a e for water and the first solution cuvettes at a wavelength of 500 nm. Record the absorbance values in the table under Part II on the Report sheet. Also repeat steps a-e to obtain absorbance readings at wavelengths of 600, 700, 800, and 900 nm. 5. Examine your absorbance readings to find the maximum value for the 100-nm intervals. Take readings 10, 20, and 30nm above and below the wavelength with the maximum absorbance. For example, if you found 600 nm to be the highest value, repeat steps a e for wavelengths of 610, 620, 630, 590, 580, and 570 nm. 6. Plot Part II - Plot and connect the 12 data points on the graph provided in the lab report sheet. The wavelength that gives the maximum absorbance, λ max (lambda max), is the wavelength you want to use for Part III of this experiment. Note: If the appropriate wavelength is not selected, the sample will not absorb enough light to make an accurate measurement. 7. Have your instructor sign off on your chosen wavelength before you continue to Part III. Part III: Measuring Absorbance Values for the Known and Unknown Solutions 1. Use a cuvette filled 2/3 full with deionized water to calibrate the Spec-20. Select the λ max wavelength found in part II that your instructor signed off on. Wipe the cuvette off with a clean Kim-Wipe, and place the water cuvette in the sample holder. Close the lid, and press the 0 Abs/100% T button to set the absorbance of the water sample to zero. Do NOT press this button again during Part III. 2. Empty the cuvette, and rinse it twice with small amounts of the 1 ml solution made in part I. Fill the cuvette 2/3 full with that solution. Wipe the cuvette off with a Kim-Wipe before GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 3 of 11
placing it in the sample holder. After closing the lid, read and record the absorbance of that solution. 3. Repeat step 2 for all of the standard solutions you made in Part I (1 ml, 2 ml, 4 ml, 6 ml, and 8 ml). 4. Save the solutions until all data collection and graphing is complete. 5. There are two unknown solutions, A and B. The unknowns contain the same colored metal (copper) solutions as the standards you prepared, but at different concentrations. You will analyze your two undiluted unknowns in the same manner as the standards. The unknowns are in dropper bottles. Add enough unknown solution from the Site A bottle to rinse the cuvette with small amounts and then fill the cuvette 2/3 full. Wipe the cuvette off with a Kim-Wipe before placing it in the sample holder. 6. Without pressing any buttons on the spectrophotometer, place your cuvette with the unknown solution from Site A in the spectrophotometer and record its absorbance reading. 7. Repeat steps 5 and 6 with your unknown solution from Site B. 8. Plot Part III Creating a Beer s Law Plot (see page 1 of this lab report for an example of the correct Beer s Law Plot). Plot your data and results in either Microsoft Excel or Google Charts. Enter exact concentrations of the standard solutions only as the x-axis data and absorbance values as the y-axis data. Be sure to include the origin (0, 0) as a data point (from the calibration of water) for a total of 6 data points. Add a regression line to your data points to find the best fit of a linear equation to your data. See technique Graphing and trendlines. Please refer to the Laboratory Techniques Document on the CHM151LL Course Website for more detailed techniques and images of lab equipment. Clean-Up: Discard all waste in the specified containers in the hood. Rinse everything well with tap water followed by a quick DI water rinse. Clean your benchtop. Put all equipment back exactly where you found it. GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 4 of 11
Name: Introduction to Spectroscopy Pre-lab assignment Date: Purpose: Summary of procedure: Drawing of apparatus used: Instructor signature: Pre-lab points: GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 5 of 11
Page Intentionally Left Blank for Double-Sided Printing GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 6 of 11
Name: Data: Introduction to Spectroscopy Lab Report Data Table 1: Standard Solution Buret Readings Data Table Solution Initial Buret Reading Final Buret Reading 1 ml 2 ml 4 ml 6 ml 8 ml Partners: Concentration of stock solution: Data Table 2: Finding Maximum Absorbance Using 8 ml Solution Data Table Wavelength Absorbance Wavelength Absorbance 400 nm Record the max value here: Instructor Signature: Data Table 3: Absorbance Values for Standard and Unknown Solutions Data Table Solution Absorbance 1 ml 2 ml 4 ml 6 ml 8 ml Site A Site B GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 7 of 11
Observations: Graph: Plot Part II - Finding the Maximum Absorbance for Copper Solutions (5 points) Calculations for Results Table 1: (volume delivered, dilution concentration) GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 8 of 11
Graph: Plot Part III - Attach your Graph of the data from part III as either an Excel plot or a Google Charts plot (this is different than the part II data and the part II plot). (7 points) See pg 4, #8 for instructions. Calculations for Results Table 2: (Unknown solutions Site A and Site B concentrations from linear fit equation. The graph from part III data will give you the linear fit equation in the form y=mx + b. Recall y is absorbance and x is concentration) Results: Results Table 1: Standard Solution Concentrations Solution 1 ml 2 ml 4 ml 6 ml 8 ml Volume Delivered Concentration Results Table 2: Site A and Site B Concentrations Solution Site A Concentration Site B GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 9 of 11
Discussion Questions: 1. Just like the Cu 2+ ion used in today s lab, some other metal ions have a distinctive color when in an aqueous solution. Some examples of ions that give colors in aqueous solution are Cr 3+ and Ni 2+. However, other metallic ions such as Mg 2+ and Ti 4+ are relatively colorless. a. (4 pts) Write the short-hand electron configurations for the following ions: Ni 2+ Cr 3+ Mg 2+ Ti 4+ b. (2 pts) Based on the electron configurations, what is the main difference between the metal ions that are colored and the metal ions that are colorless? 2. (5 pts) You measure a sample of the filtered waste in the Spec-20 at a wavelength of 810 nm and get an absorbance reading of 0.873. Based on your regression line equation of the standard solutions, calculate the copper concentration of the waste jar. 3. (5 pts) How many milliliters of a stock solution of 60.0 mg/ml NaCl would you have to use to prepare 0.500 L of a 12.0 mg/ml solution? 4. (2 pts) Discuss two sources of error and how they can be corrected in the future. 1. 2. GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 10 of 11
5. (2 pts) Now that you have completed the experiment please write a revised Purpose statement that more accurately reflects the function of this lab. Conclusion: (5 pts) Summarize the results for determining the concentration of two unknown solutions, Site A and Site B, from a standard curve. Use data to support. Remember to attach your Part III graph from Microsoft Excel or Google Charts. GCC CHM 151LL: Intro to Spectroscopy GCC, 2018 page 11 of 11