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DePaul University Exp. 4 CHE 131

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Experiment 4: Introduction to Spectrophotometry Introduction Spectrophotometry is an experimental technique to measure the amount of light absorbed by a substance at a particular wavelength. In this experiment, you will use spectrophotometry to examine a series of solutions with differing concentrations of a chemical substance, cobalt(II) chloride hexahydrate. You will interpret these results using Beer’s law, which states that there is a linear relationship between the absorbance of a solution at a specific wavelength and the concentration of the absorbing species in solution. You will also determine the value of the proportionality constant between the absorbance and the concentration, known as the molar absorptivity or extinction coefficient, using a line of best fit. This line of best fit will also allow you to calculate the concentration of a solution containing an unknown amount of cobalt(II) chloride hexahydrate. Physical and Chemical Principles Many molecules and ions are colorful when isolated or in solution. Color is observed by the eye because some wavelengths of light in the visible spectrum are absorbed by a material and others are transmitted or reflected and then detected by the retina. The particular wavelengths of light that are absorbed depend on the chemical species present. Upon absorption of a photon (a particle of light with a discrete amount of energy), a molecule or ion is promoted to an excited state (a state higher in energy). As this excited state is not stable, the energy absorbed will be released at some later time when the molecule or ion relaxes to its ground, or most stable, state. Spectrophotometry is a highly useful tool in many areas of chemistry and biology. The primary reason for its utility is the fact that the amount of a particular wavelength of light absorbed is related to the concentration of the absorbing species. Therefore, spectrophotometry provides a relatively quick, straightforward, and sensitive way to determine the concentration of a species in solution that absorbs visible light. In a spectrophotometer, it is the intensity of light transmitted through a sample that is actually measured. The instrument isolates a narrow beam of light at a particular wavelength. This light, which has intensity I0, is passed through the sample. The intensity of the transmitted beam I is then measured. If a species is present that absorbs the wavelength of light passing through the sample, the intensity of the transmitted light will be decreased relative to the initial intensity. The ratio of the intensity of the transmitted light to the initial intensity, I/I0, is called the transmittance T of the sample. The transmittance T, or more often the percent transmittance %T (equation 1) is related to the absorbance of the sample according to equation 2.


% 100%IT I

  = ×   


( )log 2 log %A T T= − = − (2)

The unitless value A is the absorbance of the sample and is more commonly used over the transmittance as the measurement of light absorption in chemistry, although spectrometers can usually report both A and T. The reason that absorbance is more common (and more valuable!) in

DePaul University Exp. 4 CHE 131


chemistry is because of the relationship between the absorbance of the sample and the concentration (in units of molarity, or moles of species per liter of solution) of the absorbing species. This relationship is given by the Beer-Lambert law, more often called Beer’s law, which is expressed as:

0log IA abc I

 = =    


where a is the molar absorptivity (L∙mol−1∙cm−1), b is the pathlength (cm) that the light travels through the sample, and c is the concentration (mol/L) of the substance being analyzed. The molar absorptivity (also called the “extinction coefficient”) depends on both the substance and the wavelength. Colored species in solution generally have broad absorption peaks in the visible region of the spectrum (approx. 380–740 nm). When attempting to determine concentrations, the absorbance is usually measured at the wavelength of maximum absorbance (λmax) to obtain the most sensitive measurements. Therefore, you will first obtain the visible absorbance spectrum of cobalt(II) chloride hexahydrate to identify λmax. All subsequent absorbance measurements will be made at this wavelength. Ensuring that the spectrometer is set to the correct λmax and reporting the specific value of λmax used to collect absorbance measurements are vitally important in spectrophotometric experiments because each species has its own wavelength-dependent value for the molar absorptivity. By examining the absorbance of solutions containing varying amounts of cobalt(II) chloride hexahydrate, a plot of absorbance vs. concentration, known as a standard curve, can be constructed. If the pathlength of the cuvette used in the spectrophotometer is held constant throughout the experiment, Equation 3 indicates that such a standard curve can be used to determine the molar absorptivity of a substance at a particular wavelength. With a standard curve in hand, the concentration of an unknown solution of a substance can be determined. The generation of a standard curve is a frequently encountered task in chemistry. Because your line of best fit is dependent upon accurate and precise concentrations, careful and consistent use of volumetric glassware (volumetric pipettes and volumetric flasks) is required. The accuracy and precision of your pipetting skills will greatly affect the results of this experiment, so take care while preparing all of the necessary solutions. Experimental Table 1 lists the equipment and chemicals needed for this experiment.

DePaul University Exp. 4 CHE 131


Table 1: Equipment and Chemicals for Introduction to Spectrophotometry Equipment and Chemicals Quantity

CoCl2·6H2O No more than 3.25 g

Weigh Bottle 1

Volumetric pipettes: 1.00, 2.00, 5.00 mL 1 of each

Cuvettes (one for reference, one for sample solution) 2

Spec 20 spectrophotometer 1

Volumetric flask, 50.00 mL 1

Volumetric flasks, 10.00 mL 4

Miscellaneous beakers and flasks As needed

Wash bottle, water 1

CoCl2·6H2O solution of unknown concentration 5 mL Preparation Notebook It is necessary for you to come to lab prepared to do the exercise without the printed instructions. You should have written a list of the steps to complete the experiment. This list should not be a word-for-word re-copy of the instructions. It is better to make a “to do” list that is a reminder of both the steps and specifics of the procedure. At times, minor changes in the procedure may be announced at the start of lab. It is important to note these details as part of your notebook procedure. This requirement forces you to develop your own understanding of the concepts and procedures, and requires that you read the details of an experiment before coming to lab. The instructor and laboratory assistant will be checking your notebook to see that sufficient detail is present to complete the lab. If you cannot demonstrate that you are ready to start the experiment, you will not be allowed to enter the lab. You must come to lab with data tables already prepared in the notebook. This, too, provides you with a valuable opportunity to understand the data and the calculations that will be part of the assignment at the end of the experiment. It is not acceptable for you to paste or tape pages from the printed instructions into your notebook. Before the lab, you must calculate the mass of CoCl2·6H2O required to obtain 50.00 mL of a 0.2500 M solution of CoCl2·6H2O. Make sure this calculation and value are written in your notebook.

DePaul University Exp. 4 CHE 131


Pre-lab Assignment You must complete a pre-laboratory quiz prior to entering the laboratory, which can be accessed through the D2L website for the course. A thorough reading of these instructions should prepare you to take the quiz. A score of at least 70% is required for participation in the experiment. You may attempt the quiz as many times as you wish. The quiz must be completed (with a minimum score of 70%) by 11:59 pm the night before the start of your lab section. You will not be allowed to participate in lab without a completed pre-laboratory assignment. Procedure Be sure that the spectrophotometer is on and warmed up for 20 minutes before use. There may be different models of spectrophotometer in the lab. Each will have a quick reference card that gives specific instructions for each model. Review these instructions before acquiring data. For precise work, solutions are measured in tubes (“cuvettes”) of square or circular cross-section, made from optical-quality glass or silica plates. The bottom half of the cuvettes must be kept scrupulously clean. Any dirt specks, smudges, fingerprints, or water droplets will reduce light transmission, thus simulating higher sample absorbance. Accordingly, clean the cuvettes thoroughly, dry them with Kimwipes, handle them only at the top, and make sure they do not pick up dirt from test tube racks, beakers, etc. These cuvettes are not disposable, and should be cleaned and returned to where they came from at the end of the experiment. In all subsequent measurements, the reference cuvette should be approximately three-quarters full with an appropriate solvent, and the sample cuvette approximately three-quarters full with the sample (after rinsing it twice with approximately 1 mL of the sample solution). If your spectrophotometer does not report the absorbance directly, then you must calculate the absorbance of the sample solution from the percent transmittance (%T). Preparation of Stock Solution Before entering the lab, calculate the mass of CoCl2·6H2O required to obtain 50.00 mL of a 0.2500 M solution of CoCl2·6H2O. Make sure the calculation and value are written in your notebook. Measure out this amount of CoCl2·6H2O to a weigh bottle using the mass-by-difference technique (mass the CoCl2·6H2O vial, lightly tap some powder into the weigh bottle, then mass the CoCl2·6H2O vial again and use subtraction to determine the mass of the solid added). Your mass does not need to be exactly equal to your calculated mass–obtain a similar amount and record the exact mass obtained. Add a few milliliters of deionized water to your 50.00 mL volumetric flask and then quantitatively transfer your solid to this flask. The phrase “quantitatively transfer” means all of the solid must be transferred from the weigh bottle to the flask. You may need to use a small amount of deionized water to ensure all of the solid has been transferred from the bottle.) Gently swirl the flask to begin dissolving the solid. Keep adding small amounts of water and swirling until all of the solid is dissolved. Once the solid is completely dissolved, fill the flask to the mark with deionized water. The solid must be completely dissolved before the flask is filled to the mark with deionized water. Use a Pasteur pipette or a wash bottle to add water dropwise when nearing the mark.

DePaul University Exp. 4 CHE 131


Stopper the flask and invert it several times to sufficiently mix the solution. If you add too much water and the meniscus is above the mark, you must discard the entire solution and start again. Preparation of Diluted Solutions You will generate four diluted solutions using various quantities of the stock solution. Pour your stock solution into a clean and dry beaker. To generate these diluted solutions, first obtain four 10.00 mL volumetric flasks. Using one or more primed volumetric pipettes, add the following volumes of the stock solution, each to a different volumetric flask: 1.00, 2.00, 5.00, and 7.00 mL. Label each flask according to the volume of stock solution added to it. Fill the flasks to the mark using distilled water. Mix each solution by inverting the stoppered flask several times. If you add too much water and the meniscus is above the mark, you must discard that solution and create it again from your stock solution. Absorbance Spectrum for Determination of λmax. You will use a Nanodrop One spectrophotometer to obtain the absorbance spectrum of your stock solution. The spectrophotometer screen should say “New Experiment” at the top. Select the “Custom” tab, and then select “UV-Vis”. Select “Done”. As requested by the instrument, load 1.2 μL of deionized water using a micropipette onto the lower pedestal and lower the arm of the spectrophotometer. Once the instrument is initialized, you can touch “Blank” on the screen to acquire the blank spectrum. Raise the arm of the spectrophotometer, wipe away the water sample using a Kimwipe and load 1.2 μL of your stock solution of CoCl2·6H2O. Type a sample name using your initials and press “Measure” to acquire the spectrum. Based on your spectrum, determine the value of λmax and record it in your notebook. Absorbance of Diluted Solutions for Standard Curve Before entering the lab, prepare a table in your lab notebook with columns for volume of stock solution, concentration, and absorbance. Set the wavelength of the Spec 20 spectrophotometer to the value of λmax determined in the previous step. Be sure the Spec 20 is set to read absorbance (A), not transmittance (T). Rinse the sample cuvette twice with small (approximately 1 mL) portions of your most dilute solution. Discard the rinses in a waste beaker. Add some of the same solution to the rinsed sample cuvette and distilled water to the reference cuvette. The cuvettes should each be approximately three- quarters full. Wipe the outside walls of the cuvettes with a Kimwipe to remove any solution or fingerprints. Before measuring the absorbance of your diluted solutions, zero the spectrophotometer at λmax using the reference cuvette. Next, replace the reference cuvette with the sample cuvette and record the absorbance of this solution at λmax. Repeat the rinsing and measuring steps for the other diluted solutions and the stock solution, working in order of increasing concentration. Absorbance of Unknown Solution Following the instructions in the previous section, determine the absorbance for the CoCl2·6H2O solution of unknown concentration.

DePaul University Exp. 4 CHE 131


Waste Disposal Do not dispose of any solutions before you create your own standard curve (see Calculations section). After your plot has been reviewed by the instructor/TA, you may put all chemical waste in the container labeled Introduction to Spectrophotometry Waste. Calculations This section describes the five calculations that you will need to perform in Excel to successfully complete your assignment. Mass of CoCl2·6H2O Used Calculate the exact mass of CoCl2·6H2O used to make the stock solution from the initial mass of the weigh bottle and the final mass of the weigh bottle after adding CoCl2·6H2O. Moles of CoCl2·6H2O Used Calculate the number of moles of CoCl2·6H2O in the stock solution from the exact mass used and the molar mass of the compound. Concentration of Stock Solution Calculate the molarity of the stock solution you created, where molarity is a unit of concentration defined as the moles of solute divided by the total volume of solution in liters. Concentrations of Diluted Solutions In a dilution a small amount of a stock solution is delivered to a new volumetric flask and then filled to the line with deionized water. The number of moles of solute removed from the stock solution and present in the diluted solution are equal to one another, but the two volumes are not. Therefore, the concentration must also change. This process can be represented in a compact formula: 2211 VMVM = (4)

where M1 is the molarity of the stock solution, V1 is the volume of stock solution added, M2 is the molarity of the diluted solution, and V2 is the total volume of the diluted solution. Note how the units cancel and work out to give you the correct units on your desired value. This equation is very useful for dilutions, but should never be used for stoichiometry calculations or other applications. Slope and Intercept of Standard Curve Use the appropriate Excel formulas to calculate the slope (=SLOPE) and intercept (=INTERCEPT) for the line of best fit for your plot of A vs. c corresponding to the data from the stock solution and the four diluted solutions. Make sure your concentrations are all in units of molarity. The slope of a plot of A vs. c is equal to ab according to Beer’s law (A = abc). The molar absorptivity a of your aqueous CoCl2·6H2O solution can be calculated from this slope and the pathlength b, which is 1.00 cm in this experiment.

DePaul University Exp. 4 CHE 131


Concentration of Unknown Sample Calculate the molarity of the unknown solution from the absorbance of the unknown solution and your line of best fit from the standard curve. Standard Curve Generate a plot of the five absorbance measurements (A of the stock and diluted solutions at λmax) vs. concentration in molarity. Fit these data to a line and include it on the plot. The plot and line must be created using Excel or a similar program. Note: Everyone should construct a standard curve of their own. Assignment You must hand in a partial report and your Excel file for this experiment at the beginning of your next laboratory meeting. In addition, you must submit both files in the appropriate submissions folder on the D2L website for your section. You should review the Lab Report Guidelines, Sample General Chemistry Lab Report, and Appendix E on D2L while writing to ensure that your drafts are correctly formatted. The rubric for the lab report can be found on the D2L site for your lab section. The partial report must be neatly typewritten and contain the following elements: Procedure It is sufficient to cite the procedure provided above (remember to provide a suitable reference or references if you used additional sources). The format for the citation can be found in the Lab Report Guidelines posted on the D2L site. Note any changes to the procedure in a bulleted list. If there were no changes made to the experimental procedure, please say so. Results The results section must contain a description of the main data, as well as any tables and figures. A table or figure can never stand alone. There must be narrative text that accompanies each one, describing and explaining the results in full sentences. Each table and figure must be introduced by number before appearing in the report. The data should be presented succinctly in a clear, organized fashion. Do not present all of the text, then all of the tables, then all of the figures. Everything should be interspersed within the narrative in a logical manner. Refer to the Lab Report Guidelines posted on D2L for more information. In your results section, be sure to include the following elements specific to this experiment. You should present sample calculations in the report by including only the equation in algebraic symbols with clearly defined variables (similar to how it was done in this lab manual). The actual numerical calculations, on the other hand, must be performed in Excel. The order of presentation should be dictated by the logical flow of your narrative, not necessarily by the order below.

DePaul University Exp. 4 CHE 131


Sample calculations For each calculation performed for the experiment, you must include the equation used in symbolic form along with definitions for each variable in the appropriate juncture in the results section. Equations should be logically interspersed within the results section. Equations should not include numerical values. The numerical values will be reported in tables and figures in the results section and in an Excel spreadsheet, as described below. All necessary calculations must be performed in an Excel spreadsheet and submitted electronically on D2L with your lab report electronic submission. The Excel spreadsheet calculations must be organized and clearly labeled so that they can be followed by your instructor. You must use Excel formulas (i.e., don’t use a calculator and then manually type in all of your calculated numbers!) to earn full credit for the calculations. If the instructor cannot follow the sample calculations or if the formulas are not present in the submitted Excel spreadsheet, no credit will be awarded. Concentration of your stock solution The concentration of the stock solution you prepared should be presented at a logical point in the Results section with the correct significant figures and units Identification of λmax for your stock soluion The wavelength of maximum absorbance (λmax) for your stock solution should be presented at a logical point in the Results section with the correct significant figures and units Concentration table Create a table containing the volume of the stock solution used and the concentration of each of the dilutions along with their corresponding absorbance measurements. The table should be neat and organized with appropriate labels for each row and column that include correct units. All information should be reported with the correct number of significant figures. The table must also have a descriptive title. Standard curve Make a figure for the standard curve. The figure caption should contain the equation of best fit for the plot with appropriate units and the molar absorptivity of the analyte. Once again, see the Lab Report Guidelines for examples of properly formatted figures with captions. Identification of unknown concentration The identity (e.g., “Unknown A”) and concentration of the unknown solution should be presented at a logical point in the Results section with the correct significant figures and units. Discussion Question Discuss the quality of your standard curve, specifically the accuracy of the slope and intercept of the line of best fit and the precision of the data overall. The known value of the molar absorptivity

DePaul University Exp. 4 CHE 131


for an aqueous solution of CoCl2·6H2O is 4.8 M−1·cm−1 at 512 nm. (Hint: If your value of λmax was not 512 nm, would you expect your molar absorptivity to be larger or smaller than the value at 512 nm?)

  • Experiment 4: Introduction to Spectrophotometry
    • Introduction
    • Physical and Chemical Principles
    • Experimental
    • Preparation
      • Notebook
      • Pre-lab Assignment
    • Procedure
      • Absorbance of Unknown Solution
    • Waste Disposal
    • Calculations
    • Assignment
      • Procedure
      • Results
        • Sample calculations
        • Concentration of your stock solution
        • The concentration of the stock solution you prepared should be presented at a logical point in the Results section with the correct significant figures and units
        • Identification of λmax for your stock soluion
        • The wavelength of maximum absorbance (λmax) for your stock solution should be presented at a logical point in the Results section with the correct significant figures and units
        • Concentration table
        • Standard curve
        • Identification of unknown concentration
      • Discussion Question

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