2025-12-30

Lab 5: Beer’s Law Investigation

In this lab, you will use volumetric glassware and the dilution equation to create solutions of specific concentrations. You will measure the absorbance of these solutions to investigate how concentration affects the amount of light absorbed by a solution. Using these measurements, you will create a calibration curve and use that to determine the concentration of an unknown solution using its measured light absorption.

Background Reading

Molarity: Chap 6.3

Beer’s Law

The absorbance of light is directly proportional to the amount of concentration of pigment in a solution. This is described by Beer’s Law, also known as the Beer-Lambert Law (Equation 1). In this equation, the absorbance of light (A, unitless) is directly proportional to the path length (l, measured in cm), the concentration of the solution (c, measured in M), and the molar absorptivity of the solution (, measured in M-1 cm-1). The path length is generally the width of the cuvette, which holds the solution in the spectrophotometer.

A= ϵ*l*c Equation 1

In this experiment, you will construct a calibration curve for Allura Red (Red 40), relating the absorbance of the sample (A) to the solution concentration (c). You will then use this calibration curve and the measured absorbance of a Kool Aid sample with an unknown concentration of Allura Red to determine the concentration of the sample.

Prelab Assignment

Before lab, neatly complete the following on a separate sheet of paper and submit the assignment to Moodle. Include your name and the experiment title at the top of the page. This must be completed prior to completing the experiment.

Write one experimental, one mathematical, and one theoretical purpose for this experiment.
Write out all relevant equations for the mathematical data analysis (in this case, Beer’s Law).
Practice problems: show all of your work and follow all sigfig rules.
1. Some sample with a concentration of 0.05999 M has a measured absorbance of 0.998. The molar absorptivity of the sample is 11.1 L cm-1 mol-1. What is the molar concentration of this sample?
2. Calculate the mass of Allura Red needed to make 250.0 mL of a 1.90 x 10-4 M Allura Red stock solution.
Summarize the experimental procedure. You can use bullet points, write a list, draw out diagrams, or draw a flowchart. This summary does not need to include all specific quantities or equipment details. Remember – this is a summary, so it should not exceed one page. If you choose to write it in words, you must not copy (plagiarize) the procedure from the packet.
Summarize the hazards of the chemicals used in this experiment and the safety precautions taken to mitigate these hazards (for this lab, Allura Red). Also summarize the waste disposal procedures for each part of the experiment.

Experiment Procedure

Preparation and Materials

Obtain a LabQuest and SpectroVis unit, two cuvettes, two disposable pipettes, 250 mL volumetric flask, 5 mL graduated pipet, red pipet filler, 100 mL beaker, unknown Kool Aid sample

Procedure

Measure out the mass of Allura Red you calculated in the prelab to make 250.0 mL of a 1.90 x 10-4 M stock solution onto weigh paper and record the exact mass.
Transfer all of the Allura Red to the 250 mL volumetric flask, rinsing it in with a wash bottle to ensure that all of the powder gets added. Continue adding deionized water until the flask is about 1/3 full. Swirl the flask until all of the Allura Red is dissolved. Continue to fill the flask with water. As you get close to the mark, use a pipet to add water dropwise. Swirl the solution to ensure that the final volume is 250.0 mL. If you go over the mark, you will have to start over.
Obtain four 100 mL volumetric flasks and label them #1, #2, #3, and #4. Rinse them out with tap water, then with deionized water. Pour about 50 mL of stock solution into the clean, dry 100 mL beaker. Using the graduated pipet, dispense 2.50 mL of stock into Flask #1, 5.00 mL into #2, 7.50 mL into #3, and 10.00 mL into #4. Then fill the flasks to the mark using deionized water, swirling to ensure the solution is homogeneous. Have each team member prepare one flask. These are the standard solutions.
Connect the SpectroVis unit to the LabQuest. Set the mode to “Full Spectrum.” Click on the “USB: Abs” block and select “Calibrate.” Wipe the cuvette with a KimWipe (not a paper towel). Hold the cuvette only by the ridged sides. Allow the lamp to warm up (90 seconds).
Place the calibration cuvette in the SpectroVis unit with a smooth side facing the triangle on the unit (and the other smooth side facing the white lamp symbol). Then click “Finish Calibration.” Leave this cuvette in the SpectroVis unit while you proceed. If you notice the reading drift away above “0.010” while preparing your other solutions, you should calibrate the unit again using the procedure above. Keep this cuvette on hand for doing calibration checks as you go through the rest of the procedure.
Measure the absorbance of the stock solution as follows:
- Obtain an empty cuvette and rinse it three times with a small amount of the stock solution, then fill it about 2/3 full with stock solution (use a disposable pipette). Wipe it down with a KimWipe and handle ONLY by the ridged sides.
- Place the cuvette in the compartment, align as before, and collect the full absorbance spectrum (press the button with the green arrow on the bottom left of the screen). Let this run for 30 seconds before stopping the collection.
- Once done, select Analyze, then from the drop-down menu select Statistics, then the box next to Abs. Record the wavelength of maximum absorbance as λmax and check this value with your instructor.
Now that the wavelength of maximum absorbance is known, you will measure the absorbance of the standard solutions and the unknown sample at this wavelength. On the LabQuest, e the mode to “Time Based.” Click on the “USB: Abs” block and select “Change Wavelength.” Set the wavelength to λmax.
Empty the stock solution cuvette and rinse it three times with a small amount of the solution in Flask #1 (use a new pipet this time), then fill it and place in the SpectroVis as you did before. Record the absorbance value in the data table.
- Remove the cuvette and re-insert the calibration cuvette. If the absorbance reads more than 0.010 or less than –0.010, recalibrate the SpectroVis unit.
- Empty the cuvette containing the solution from Flask #1 and rinse it three times with a small amount of the solution from Flask #2 (using the same pipet), then repeat as above, doing a calibration check before each new solution. Continue until absorbance values are recorded for all five solutions. Be sure and measure the solutions in order from 1-4.
Obtain an unknown sample from your instructor. Measure the absorbance of this sample following the same procedure as for the standard solutions. 11.
Dispose of all solutions in the appropriate waste container, wash all glassware, and return the LabQuest and SpectroVis.

After experiment

Complete the Questions.
Check in with your instructor before leaving.

Submission: submit the pages containing Data, Processing and Questions, and the rubric (pages 5-8), as well as your completed prelab to your instructor at the start of the next lab period. Submit your calibration curve (copied into a Word document, with a figure caption) to Moodle by the start of the next lab period.

Lab 5: Beer’s Law Investigation

Data

Sample wavelength of maximum absorbance: ___________

Standard XXX

Solution	Concentration (M)	Absorbance
Stock	1.9 x 10-4
1
2
3
4
Unknown sample

Questions

Now that you have identified all your solutions, write an equation (or a “No Reaction” symbol) for each mixture made (reference the Data section; there should be 3-8 equations). Each equation should be balanced and include phases for all species. For the amphoteric hydrides, only write an equation for their original precipitation reactions (not for redissolution). Use the examples on pages 1 and 2 to help.

Grading Rubric

Score	Prelab	Flowchart
5 pts	Both purposes accurate; flowchart completed correctly; chemical equations complete and correct; chemical safety and waste disposal complete and accurate; submission well-organized and easy to read	Each step in the identification included; test for each step; observations and results for each step; solutions identified and those unknown at each step
3.75 pts	Criteria mostly met	Criteria mostly met
2.5 pts	Criteria somewhat met	Criteria somewhat met
1.25 pts	Criteria mostly not met	Criteria mostly not met
0 pts	Section largely incomplete, missing, or copied from packet	Nothing included

Score	Data collection	Questions
10 pts	All relevant data are collected (result, interpretation, solution information for each step); example precisely followed	All chemical equations are written; product and reactant formulas correct; equations balanced; phase states included and correct for all reactants and products
7.5 pts	Criteria mostly met	Criteria mostly met
5 pts	Criteria somewhat met	Criteria somewhat met
2.5 pts	Criteria mostly not met	Criteria mostly not met
0 pts	Section largely incomplete or carelessly completed	Section largely incomplete or carelessly completed

Total: /30

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