First Order Reaction:

Methylene Blue and Ascorbic Acid

OBJECTIVE

In this experiment, you will determine the rate constant, initial reaction rate, and rate law
of the reaction between methylene blue, ascorbic acid using absorption spectroscopy.

INTRODUCTION

One of the most common methods for determining the rate of a reaction is to follow the
change in the absorption spectrum of one or more of the reactants as the reaction
progresses. From this, we can follow the concentration as a function of time, if Beer’s Law is
followed. In this experiment we will study the reduction of methylene blue by ascorbic acid
(Vitamin C). This laboratory was adapted from the article by Mowry and Ogren, listed in
the references. Vitamin C is a well-known antioxidant, in that it provides a source of
electrons to react with species generated by metabolism that are toxic due to their free
radical chemistry. The reaction is between methylene blue:

and ascorbic acid:

Methylene blue is, as you might have suspected, blue in aqueous solution. Vitamin C is
colorless. We will use the SpectraVis to follow the loss of the blue color as the ascorbic acid
reacts with the methylene blue. From the absorbance vs. time data, we will determine the
initial rate. We will further analyze the data via first and second order integrated rate
equations via Excel. These analyses will allow us to determine the rate law for the reaction
and the rate constant.

PRE-LABORATORY QUESTIONS

1. What are the integrated rate laws for first and second order reactions?

2. Define what the term “initial rate” means.

3. State Beer’s law, and how it will facilitate our kinetic study.

4. State the conditions necessary for Beer’s law to be obeyed.

MATERIALS
2.0 x 10-4 M methylene blue SpectroVis Plus
0.10 M ascorbic acid plastic cuvettes
1.0 M hydrochloric acid LoggerPro and Excel with Analysis
small beakers Toolpak software

PROCEDURE
1. Open the “Logger Pro” program on your computer
2. Plug the cable into the USB port of the computer, and the other end into the
SpectroVis
3. Calibrate the SpectroVis:
a. Click “Experiment”
b. Scroll down and click “Calibrate” and select “Spectrometer: 1”
c. Allow the lamp to warm up, then place the blank cuvette in the device
d. Click “Finish Calibration” and wait until you are prompted to click “OK”
4. Generate a spectrum of methylene blue
a. Fill a cuvette about 3/4 full with 3.9 x 10-6 M methylene blue sample
b. Place the sample in the spectrophotometer and click "Collect"
c. Wait approximately 5 seconds and then click "Stop" to end the data collection
d. Hit Ctrl + J to autoscale the spectra to fit your screen
5. Set up the spectrophotometer for kinetics experiments
a. Click the "Configure Spectrophotometer Data Collection button,
b. Select Absorbance vs. Time as the data-collection mode. The wavelength of
maximum absorbance should be selected. Click "OK" to continue or click
 "Clear" and select the maximum wavelength from the graph or in the list of
wavelengths. Make sure to store this data for the remaining experiments.
c. NOTE: The default settings are 1 sample per second for 200 seconds. To
change the data-collection parameters for your experiment, choose Data
Collection from the Experiment menu and make the necessary changes. Click
"Done". You may also type Ctrl + D and the window will pop open.
6. From the stock solutions of methylene blue, ascorbic acid, and hydrochloric acid,
obtain approximately 10 mL of each substance.
7. Accurately measure 1.0 mL of methylene blue, 1.0 mL of hydrochloric acid, and 2.0
mL of de-ionized water, in that order.
8. Thoroughly rinse the graduated cylinder with de-ionized water and measure 1.0 mL
of ascorbic acid.
9. Add the ascorbic acid to the test tube, mix the solutions and quickly add the solution
to a cuvette and begin absorbance vs. time data collection.
10. Because the reaction is first order, you can determine the rate constant, k, by
plotting a graph of ln Absorbance vs. time.
a. Choose New Calculated Column from the Data menu.
b. Enter “ln Absorbance” as the Name, and leave the unit blank.
c. Enter the correct formula for the column into the Equation edit box by
choosing “ln” from the Function list, and selecting “Absorbance” from the
Variables list. Click .
d. Click on the y-axis label. Choose ln Absorbance. A graph of ln absorbance vs.
time should now be displayed. Change the scale of the graph, if necessary.
e. Click the Linear Regression button, . Write down the slope value in your
data table as the rate constant, k.
f. Close the Linear Regression box by clicking the X in the corner of the box.
HINT: you can toggle between the absorbance vs. time graph and the ln
absorbance vs. time graph by clicking on the y-axis and selecting whichever
graph you desire.
11. To prepare for the next trial, choose Store Latest Run from the Experiment menu.
12. Repeat the experiment according to the table below.

Volume Volume
Volume Volume
Mixture Methylene Ascorbic
HCl Water
Blue Acid
I 1.0 1.0 1.0 2.0
II 2.0 1.0 1.0 1.0
III 1.0 2.0 1.0 1.0
IV 1.0 1.0 2.0 1.0
PROCESSING THE DATA
1. From your plot of ln A (absorbance) versus time determine the rate constant k for
each mixture and the average rate constant. HINT: rate constant can be determined
from the slope of the regression line.

2. Determine the initial rate for each reaction mixture and the average rate. HINT: the
initial rate is the negative slope of the absorbance vs. time graph when t (time) is
closest to zero
a. Make sure the absorbance vs. time graph is showing.
b. Click the tangent button. Move the cursor to the desired location.
Record the negative slope of the line for the four mixtures.

3. Determine the exponent variables for each compound in the rate law.

4. Determine the overall rate law equation

POST-LABORATORY QUESTIONS
1. Write the rate law for this reaction as you determined it from your experiments.

2. Based on these analyses, is the order with respect to methylene blue first or second?

3. What are the orders with respect to ascorbic acid and hydrochloric acid?

4. What is the overall rate constant for the reaction?

5. Using ChemDraw, draw the structures of the products of the reaction between
methylene blue and ascorbic acid. Include these figures in your laboratory report.

REFERENCES
Mowry, S.; Ogren, P. J. J. Chem. Ed. 76, 1999, 970-73
DATA AND CALCULATION TABLE

Mixture [Methylene Blue] [Ascorbic Acid] [HCl] k Initial Rate

II

III

IV