Absorption Spectroscopy and Solution Stoichiometry


Denice Gamper

Bard High School Early College, Manhattan

Summer Research Program for Science Teachers

August 2012




Course:  Chemistry


Grade Level:  10 and 11 


Purpose:  Students will make use of the spectrophotometer to determine the protein concentration in a variety of sports drinks, food samples and different types of milk.



Students will be able to (SWBAT):

·    develop an understanding of how the spectrophotometer works.

·    explain the basic principles of spectrophotometry.

·    understand the relationship between the amount of light a solution transmits (or absorbs), and its concentration as given by Beer's Law.

·    prepare a series of standard solutions by making serial dilutions from a stock solution.

·    generate a calibration curve that relates absorbance to concentration.

·    use the calibration curve to determine the protein concentration of an unknown solution.

·    use a simple protein assay and a spectrophotometer to design their own experimental protocol to determine the protein content in sports drinks and/or milk.

·    collect and analyze data as well as draw valid conclusions from data.

·    present their experimental findings to the class.


Prior Knowledge:

            Students will have an understanding of the wave and particle nature of electromagnetic energy.  In addition, students will have had experience with graphing data and drawing lines of best fit.  Students will also know how to calculate the molarity of a solution and determine concentrations of solutions prepared during a serial dilution using the dilution formula C1V1 = C2V2.  Prior to this activity, students will have completed an introductory laboratory activity on how to use the spectrophotometer.


Time Required: 

1.      10 fifty minute class periods

a)      Part 1

·         1 class period to explore the principles of spectrophotometry.

·         1 class period to complete a worksheet that explores the effect of cell path length, molar absorptivity, and concentration on absorbance (or transmittance) of a solution using the Beer’s Law Lab PhET simulation.

·         2 class periods to conduct an introductory laboratory activity to learn how to use the spectrophotometer.

b)      Part 2

·         1 class period to complete a pre-lab simulation activity on the Biuret Protein Assay.

·         2 class periods to design an experimental procedure to determine the protein concentration in sports drinks and milk. 

·         2 class periods to conduct the lab activity.

·         1 class period to present data and results to the class. 


Essential Questions:

1.      Describe the basic principles of the spectrophotometry.

2.      What is the difference between transmittance and absorbance?

3.      Explain Beer’s Law (Beer-Lambert Law) and its significance.

4.      Explain how the absorbance of a solution varies with molar absorptivity, cell path length and concentration

5.      What is a serial dilution?

6.      What is a standard or calibration curve and what is it used for?

7.      How can the Beer-Lambert Law be used to determine the concentration of compounds in solution?


Preparation and Procedure:

Part 1: - WebQuest, Simulation Activity and Spectrophotometry Introductory Laboratory Activity (class periods)

1.      Students will work in groups of two or three to complete a WebQuest and worksheets that explore

a)      principles behind spectrophotometry.

b)      the relationship between absorbance, molar absorptivity, path length, and concentration in Beer’s Law.

c)      Biuret Protein Assay

2.      Use the Essentials Questions to guide students during the WebQuest.

3.      Teacher Tip: 

a)      Print out hard copies of each of the websites used for the WebQuest in the event that students are unable to connect to the Internet.  For a class size of twenty-four students working in groups of 3, print out eight copies of each website to be used in during the WebQuest.

b)      The Beer’s Law Lab PhET simulation can be downloaded to the computer or run directly from the PhET website.

c)      Limit the number of websites that address the principles of spectrophotometry to only two.  

4.      Students will conduct a preliminary laboratory activity to learn how to use the spectrophotometer, express the relationship of concentration vs. absorbance graphically and use the graph to determine the unknown concentration of a food dye sample.

5.      Students will work in groups of two or three to devise an experimental procedure to determine the protein concentration in sports drinks, food products or milk.  Students will submit a draft copy of their procedure to the instructor for review before conducting the laboratory activity.


Website Resources:


1.      Animations/Simulations

a)      Bio 121:  Biuret Protein Assay

b)      Beer’s Law Lab

c)      Virtual Chemistry Experiments:  Spectrophotometry

2.      Tutorials

a)      The Beer-Lambert Law Tutorial

b)      Spectrophotometry

c)      Experiment: Spectrophotometric Analysis Of Food Dyes

d)      Section 17:  Spectrophotometry

e)      Introduction to Spectrophotometry

f)       Detecting and Quantifying Biological Molecules Using Spectrophotometry


3.      WebQuest Resources:

a)      WebQuest

·         Website that describes how to develop and implement a WebQuest in the classroom.


Part 2:  Guided Inquiry Based Lab Activity (5 class periods)

Background Information: 

      Many substances have color and the intensity of that color relates to the amount of substance present.  For example, the more drops of red food coloring that are added to water, the more intense the red color.  Quantitative and qualitative measurements based upon the property of a substance to absorb visible light are used extensively in chemical laboratories.  The basis for what the chemist calls colorimetric analysis is the variation in the intensity of the color of a solution with changes in concentration.  By comparing the intensity of the color of a solution of unknown concentration with the intensities of solutions of known concentrations, the concentration of an unknown solution may be determined.

1.      Students will work in groups of two or three to:

a)      Devise an experimental procedure to measure protein content in sports drinks and/or milk.

·         Homework:  Students will complete their procedures at home.  Students may conduct research using the Internet in order to design their procedure.

b)      Students will review each other’s procedures in class before deciding on a final procedure to submit to the instructor for approval. 

2.      Once the instructor has approved the procedure students will carry out their experiment during their assigned laboratory period.

a)      Teacher Tips for Laboratory Preparation: 

·         When reviewing student experimental procedures be sure that:

(1)   Students use bovine serum albumin (BSA) to prepare the standard solutions needed for the calibration curves.

(2)   The concentration for the standard BSA stock solution needed for the serial dilution should be 5mg/mL.   Prior to the laboratory activity the instructor should make a stock solution containing 2.5 grams of BSA dissolved in 500 mL of distilled/deionized water. 

·         Safety: 

(1)   Students will be expected to include a list of safety precautions as part of their experimental design.

(a)   Teacher Tip:  Be sure students include safety procedures/precautions for the following:

(i)            Electrical safety

1.      Check all electrical equipment for worn cords or loose plugs before use.

2.      Keep work area free of water.

(ii)          checking glassware for chips, cracks and stars

(iii)        cleanup and disposal of broken glassware

(iv)        cleanup and disposal of spilled liquids

(v)          disposal of unused liquids

(vi)        use of personal protective equipment:  goggles, apron or lab coat, latex or nitrile gloves (some students may have an allergic reaction to latex so it is advisable to have a non-allergenic substitute)

The students will be given time during the class period following the laboratory activity to summarize their data and present their results to the class using newsprint or an overhead projector and transparencies or an LCD projector.


3.      Teacher Preparation for Part 2:

a)      Possible experimental procedures for this laboratory activity can be found by reviewing the following:

·         Biuret Protein Assay

·         Got Protein? Kit

·         Protein Standard Curve and Protein Assay of Milk and Protein Drinks

·         Lab 3 – Protein Determination            

·         Spectrophotometric Determination of Total Protein-Biuret Method      

·         Absorption Spectrophotometry and Protein Measurement



Part 1: 

1.      WebQuest

a)      WebQuest Worksheet for the principles of spectrophotometry.

b)      Print copies of websites used for the WebQuest.

c)      Laptop computers with Internet access

d)      LCD projector

2.      Computer Simulation Activity

a)      Worksheets for the completion of the Beer’s Law Lab PhET simulation and Biuret Protein Assay


Part 2 – Guided Inquiry Based Lab




Spec 20 Spectrophotometer



test tube holder


test tubes

Newsprint for presentation of results


Sharpie marker

graph paper

10 mL graduated cylinder

30 cm ruler

Bovine Serum Albumin (BSA) stock solution (5mg/mL)


15 mL capped tubes

100–1,000 μl adjustable-volume micropipette


5mL disposable graduated pipettes


100–1,000 μl pipet tips

pipette pump

wash bottle with distilled/deionized water

1X Phosphate Buffered Saline (PBS) pH 7.4

disposable transfer pipettes


Milk samples (suggestions: low fat, fat free, soy, baby formula)



Protein enriched drink s (suggestions:  Gatorade G Series Pro 03 Recover, Gatorade G Series Fit 03 Recover, Carnation Instant Breakfast)   


Biuret Reagent

Commercial Beef, Chicken or Pork Bouillon


Part 2:  Presentation of Results

Newsprint, an overhead projector and transparencies or an LCD projector for each group to organize and present their laboratory findings.


Suggestions for Assessment:

·    Evaluate the completion of the WebQuest worksheet and worksheets for the computer simulations.

·    Evaluate the group presentation of data and results.

·    Evaluate a formal laboratory report submitted at the completion of the laboratory exercise.


New York State Standards:

1.      Standard 1:  Students will use mathematical analysis, scientific inquiry, and engineering design, as appropriate, to pose questions, seek answers, and develop solutions.

a)      Key Idea 1 - The central purpose of scientific inquiry is to develop explanations of natural phenomena in a continuing and creative process.

·         Performance indicators 1.1, 1.2, 1.3

b)      Key Idea 2 - Beyond the use of reasoning and consensus, scientific inquiry involves the testing of proposed explanations involving the use of conventional techniques and procedures and usually requiring considerable ingenuity.

·         Performance indicators 2.1, 2.2, 2.3, 2.4

c)      Key Idea 3 - The observations made while testing proposed explanations, when analyzed using conventional and invented methods, provide new insights into natural phenomena.

·         Performance indicators 3.1, 3.2, 3.3, 3.4, 3.5

2.      Standard 4:  Students will understand and apply scientific concepts, principles, and theories pertaining to the physical setting and living environment and recognize the historical development of ideas in science.

a)      Key Idea 3 - Matter is made up of particles whose properties determine the observable characteristics of matter and its reactivity.

·         Performance Indicator 3.1 - Explain the properties of materials in terms of the arrangement and properties of the atoms that compose them.

1)      Major Understandings 3.1oo, 3.1pp

b)      Key Idea 4 - Energy exists in many forms, and when these forms change energy is conserved.

·         Performance Indicator 4.1 - Observe and describe transmission of various forms of energy.

1)      Major Understandings 4.1a