Page 137: undigested).

In addition, the class will investigate the effect of temperature, volume of enzyme mix added and reaction time on the determination of glucose in Gatorade. You will work in groups of three, or four if there are an odd number of students, for todays experiment. Each group will construct a standard calibration plot from the absorbance measurements obtained from the analysis of the prepared standards (Table 3). The Gatorade, digested sugar and undigested sugar solutions will be prepared at the same time as the standard solutions. In addition, the class will be broken up by bench (Table 2) to further investigate the effect changing temperature, amount of glucose oxidase added, and reaction time on the determination of glucose in the Gatorade.
Bench A&B C&D E&F Parameter to Investigate Room Temperature vs. 40 C 20 min vs. 45 min Reaction Time in Water Bath 0.10 mL vs. 0.50 mL *Enzyme Mix

Table 2: Bench assignments for the investigation of various experimental parameters. *The enzyme mix contains both glucose oxidase and horseradish peroxidase. Each group member will prepare one of the three Gatorade solutions (Solutions 8, 9, and 10) based on Table 4 and each group member will prepare one Gatorade solution (11, 12, and 13) and carry out the change in the assigned experiment parameter (Table 2). Each group, not group member, will prepare a digested and undigested sugar solution and run them under normal conditions, as described below. At the end of the lab period, the entire class will pool all the Gatorade data so that every student has data acquired under all the different experimental parameters.

Solution Preparation
Table 3 summarizes the procedure that you will use to prepare the glucose standards. Solutions 1 through 7 will serve as your glucose standards, the concentrations of these solutions range from 0 mM to 0.60 mM. You should note that after preparing all the solutions, the total volume in each test tube should be 5 mL. 1. Obtain the following solutions and bring them back to your lab bench.

Glucose Solution (1 mM): 15 mL Enzyme Mix: 1 Vial Ferrocyanide Solution (0.0625 M): 15 mL Phosphate Buffer (0.05 M; pH 6.00): 40 mL Gatorade (Diluted 50x): 1 mL Acid-Digested Sugar Solution (Diluted 50x): 1 mL Undigested Sugar Solution (Diluted 50x): 1 mL

Page 139: Solution # Volume of Ferrocyanide Solution (mL) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Volume of Phosphate Buffer (mL) 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Volume of Sports Drink or Soft Drink (mL) 0.25 mL of Gatorade solution (diluted 50x) 0.25 mL of Gatorade solution (diluted 50x) 0.25 mL of Gatorade solution (diluted 50x) 0.25 mL of Gatorade solution (diluted 50x) 0.25 mL of Gatorade solution (diluted 50x) 0.25 mL of Gatorade solution (diluted 50x) 0.5 mL of undigested sugar solution (diluted 50x) 0.25 mL of digested sugar solution (diluted 50x) Volume of Enzyme Mix (mL) 0.50 0.50 0.50 **0.50 **0.50 **0.50 0.50

8 (Gatorade) 9 (Gatorade) 10 (Gatorade) *11 (Gatorade): Altered Condition *12 (Gatorade): Altered Condition *13 (Gatorade): Altered Condition 14 (Undigested Sugar Solution) 15 (Digested Sugar Solution)

1.00

3.25

0.50

Table 4: Summary of procedure for preparing the Gatorade, digested and undigested sugar solutions to be analyzed. *These 3 solutions will either be prepared differently (Table 5) or will be run under different conditions depending on bench assignment. **This volume may be different if you are one of the groups running the investigation to look at a different amount of added enzyme. 2. Clean 15 test tubes. Use a grease pencil to number the test tubes. Do not use labels because they will come off in the water bath. 3. Prepare the 15 solutions as indicated in Tables 3 and 4.

If you are one of the groups that are assigned to look at varying the amount of enzyme mix added you will prepare 3 glucose solutions as described in Table 4 and you will prepare 3 additional glucose solutions as summarized in Table 5. You will note that the total volume is still 5 mL.

Page 140: Volume of Ferrocyanide Solution (mL) 1.00 Volume of Phosphate Buffer (mL) 3.65 Volume of Sports Drink or Soft Drink (mL) Volume of Enzyme Mix (mL)

Solution #

0.25 mL of Gatorade 0.10 solution (diluted 50x) 0.25 mL of Gatorade 1.00 3.65 0.10 12 (Gatorade) solution (diluted 50x) 0.25 mL of Gatorade 1.00 3.65 0.10 13 (Gatorade) solution (diluted 50x) Table 5: Summary of procedure for preparing Gatorade solutions based on altering the volume of enzyme mix. 11 (Gatorade)

You will use an autopipette to deliver the volumes of liquid indicated in Tables 3, 4 and 5. The autopipette delivers microliter (L) volumes so remember to convert mL to L before pipetting. Add the solutions to the test tubes in the order that they are listed in Tables 3, 4, and 5, with the enzyme mix added last. The volumes you need to dispense are already provided in the table, you do not need to calculate the volumes of the solutions.

4. Cover the top of each test tube with a piece of Parafilm. Invert each test tube 1-2 times, remove the Parafilm and place the test tube rack in the water bath for 45 min.

The temperature of the water bath should be at 40 C ( 1 C). Use your thermometer to measure the temperature of the water bath and record the actual temperature in your lab notebook. If you are one of the groups that are assigned to look at the varying temperature conditions, all your solutions will go in the 40 C water bath except for 3 of your 6 Gatorade solutions, which will be left on the bench top for the 45 min reaction time.

5. Remove test tubes from the water bath and allow them to cool before making any measurements.

If you are one of the groups that are assigned to look at the varying reaction time, all your solutions will remain in the water bath for 45 min except for 3 of your 6 Gatorade solutions, which will be removed after 20 min, and the absorbance measured after the solutions have cooled.

Page 146: from the linear region. Perform a linear regression analysis and record the equation for the line. This plot will serve as the calibration plot.

WASTE DISPOSAL
All your waste should be emptied in the liquid waste bottle located in the lab hood. Do not dump any liquid containing ferrocyanide down the drain.

FORMAL LABORATORY REPORT

The report for this experiment is a formal laboratory report. The specific requirements for writing a formal lab report are presented in the Introduction to Chemistry 241L chapter of the Chemistry 241 Lab Manual and must be followed. In addition to the required components, your report should include the following 1. All data and results properly presented. Remember that you are pooling all the class data for the glucose solutions that were run under the 3 different conditions (reaction time, volume of glucose oxidase and temperature).

Table of absorbance data taken for the standard solutions. Table of experimentally determined absorbance data for all the sample solutions. Table of concentration data from digested and undigested sugar solution measurements. Table of concentration data (average and standard deviation) from glucose measurements comparing different reaction times. Table of concentration data (average and standard deviation) from glucose measurements comparing different volumes of Enzyme Mix. Table of concentration data (average and standard deviation) from glucose measurements comparing different temperatures. Calibration plot using the data from all the standards and the calibration plot showing the linear region only.

2. Calculate the concentration of glucose in each of the original, undiluted samples (Gatorade, undigested sugar solution and acid- digested sugar solution). This means you have to account for all the dilutions performed, including the dilution that was carried out by the prep room to produce the 50x-diluted solutions. 3. Discuss the results you obtained for the acid-digested and undigested sugar solutions. Were the concentrations of glucose determined for these two solutions similar or different? Why? 4. Discuss the effect of changing the various experimental parameters and conditions had on the glucose concentration experimentally determined.