Essential Question
To make stock solutions and determine the best wavelength for measuring total antioxidant capacity using the CUPRAC assay by collecting a UV-VIS absorbance spectrum before and after reacting Trolox. All of these solutions, except sodium carbonate, are light sensitive and should be stored in amber vials or clear vials wrapped in foil. The teacher may have made some of these solutions for the students.
Background Information
Spectrophotometric measurement for determination of the wavelength of maximum absorbance (?max) for the Copper (II) complex product, versus the Copper (II) complex reagent Always allow the spectrophotometer to warm up for at least 30 minutes. Students should have obtained instruction on how to operate the spectrophotometer. If they have not, the teacher should demonstrate this for them. Make sure students use distilled water and set the “blank” for the spectrophotometer every time they change wavelengths. Students should add the following to two test tubes, labeled “ethanol” and “trolox” 1 ml of the copper chloride solution 1 ml of the neocuproine 1 ml of acetate buffer 0.75 ml of water To the tube labeled “ethanol,” add 1 ml of ethanol, and to the tube labeled “trolox,” add 1 ml of 100 µM trolox. Mix the solutions by covering with parafilm and inverting. Transfer each solution to a cuvet and tap the side to release any air bubbles. Starting at 400 nm, measure the absorbance of your control and your sample at intervals of 25 nm from 400 to 700 nm. Remember that students should blank the spectrophotometer using distilled water every time they change wavelengths. Students should record their data in their notebook, and then repeat the measurement for the second test tube.
Student Work
Copper chloride dihydrate– CuCl2•2H2 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, TE) epicatechin quercetin dihydrate ethanol sodium carbonate – Na2CO3 amber storage vials in 20 ml and 250 ml sizes aluminum foil if amber vials are not available volumetric glassware for 10 ml, 25 ml and 100 ml sizes volumetric glassware for 100 µL-1000 µL (0.1mL – 1mL)Spectrophotometer Glass tubes Antioxidants Student Manual (PDF)
Procedure
Preparing SolutionsPreparation of CUPRAC Method Reagents: Copper Chloride and Neocuproine Make 250 ml of a 0.010 M copper chloride solution in deionized water. (Students should have calculated the amount of copper chloride to weigh out as part of their pre-laboratory assignment. If they have not, they should complete this before they begin.) If they do not weight the exact amount of copper chloride needed, recalculate to determine the concentration of the solution. Transfer the final solution to an clear vial. Cover with foil to block light. Label. Put parafilm around the cap of the vial to avoid solvent evaporation. Make 25 ml of 0.0075 M neocuproine in ethanol. Transfer the solution to a clear vial covered with foil to block light. Put parafilm around the cap of the vial for weekly storage to avoid solvent evaporation. Preparation of Standard Antioxidants: Trolox, Epicatechin and Quercetin Make 10 mL of 0.010 M Trolox solution in ethanol. Make a dilution of this solution to 100µM. Make 10 mL of 0.010 M epicatechin solution in ethanol. Make 10 mL of 0.005 M solution of quercetin in ethanol. In another laboratory period they will dilute these stock solutions to lower concentration. For now, be sure to label and store the solutions.
Duration
Starting Your Notebook Students should read the laboratory activity below and make a pre-laboratory entry before they come to lab. If they need help making a pre-laboratory entry, use the notebook guidelines provided. They will need to include an objective, materials, procedure, and results sections for this laboratory To make solutions in this lab they will need to calculate the amount of solute to use and the volume of solvent in which to dissolve the solute. Concentration is commonly expressed as molarity, molality, percent by mass and percent by volume. Below are different ways to express concentration that they will need to calculate amounts to make their solutions. Students should read them and complete the following pre-laboratory assignment. Pre-Laboratory Assignment (PDF)
Reflections and Feedback
Laboratory 2: Learning the CUPRAC Method using Epicatechin, Quercetin, and TroloxLaboratory 3: Determination of Ascorbate (Vitamin C) Concentration in Common JuicesLaboratory 4: Independent Research Project
Learning Context/ Introduction
In this first lab (of three) students will make solutions that they will later use for measuring antioxidants in their samples. Some of these solutions will consist of very polar solutes and solvents whereas others will utilize less polar solutes and solvents. Close attention should be paid to both the solute and solvent instructions as these are based on polarity. Students will also determine the wavelengths of light absorbed for measuring total antioxidant activity using the CUPRAC method.
Assessment
Students should plot the absorbance versus wavelength (wavelength on the x-axis and absorbance on the y-axis.), making sure to label the axes appropriately. For which wavelength did they measure the highest absorbance for the sample? For which wavelength did they measure the highest difference between the sample and the control? Students should answer these questions in their lab notebook: Based on your results, what is the best wavelength to detect copper (I) neocuproine? Explain your answer using your absorbance data. Which test tube was a control? What is a control?
Content Provider
The original module was created and formatted by: Dr. J. Burgess, in collaboration with The Center for Authentic Science Practice in Education, Purdue University. This resource was modified for Secondary Chemistry by: Dr. V. Frerichs, P. Kresic and S. Small with support from the Dreyfus Foundation Special Grant Program in the Chemical Sciences
