A Kinetics Experiment of the Reaction of Coumarin-102 with Base, Using

Fluorescence

Prelab Questions

Coumarin-102 Coumaric Acid

Reaction of Coumarin-102 with NaOH

1. Adding NaOH to Coumarin-102 will generate a new compound with:

a) same fluorescence properties
b) different fluorescence properties
Please justify your answer.

2. Does the concentration of Coumarin-102 increase or decrease over time in

reaction with base? What do you expect to happen with the light being emitted by
Coumarin-102?

3. What is the integrated rate law?

4. How do you determine the order of a reaction from a graph? (hint: plots of zero-,
first- and second-order reaction)

5. Explain in few words what fluorescence is.

1
A Kinetics Experiment of the Reaction of Coumarin-102 with Base, Using
Fluorescence

Important Notes
 This experiment presents lab work with a learning- and technology-centered approach. The learning-
centered approach structures the work to include chemistry content (kinetics) and the demonstration of
a competency (mathematical analysis). The technology-centered approach revolves around the use of
smartphone cameras that offer advantages in terms of student involvement in data management along
with the speed of the measurements, low cost, ease-of-operation, data management, and minimal
equipment.
 This is a two-week procedure, with Parts I and II in the first week and then Part III in the second
week with your own experiment design.
 You will carry out some of the data analysis during the lab. For this reason, you will need a computer
that can run Java® available during the lab. Ideally, each member of a pair of students will have a
computer.

Introduction
The overall purpose of this experiment is learning the content of kinetics and developing a competency
in analyzing fluorescence images mathematically to determine the kinetics of a reaction using data from a
cell-phone camera processed using the software ImageJ from the National Institutes of Health. You will
examine the loss of fluorescence of coumarin-102 in reaction with a base using a simplified and direct way
to detect light with pictures taken with a smartphone camera. Then you will get information on the change
in the concentration and determine the kinetics (rate law and rate constant) of the reaction.

Performance expectations
 Students will know important content, at the level specified in the Anchoring Concept Content Map
of the ACS Exams Institute: VII.B.a. Kinetics: “Rate laws are always determined experimentally, and
the methods of initial rates or graphical depiction of rates provide the key method for determining rate
laws”.
 Students will gain the ability to perform specified MCAT2015 competencies from the AAMC-HHMI
(Association of American Medical Colleges and Howard Hughes Medical Institute) and new
MCAT2015 (Medical College Admission Test). There are 15 Core Competencies and at least half of
these relate to chemistry. The competency for this lab is Competency E1: “Apply quantitative
reasoning and appropriate mathematics to describe or explain phenomena in the natural world”. In
particular, we want to support and assess student learning with Learning Objective 5: “Make
inferences about natural phenomena using mathematical models”.

Taken together, the goal of content and competency are expressed in the form of an evidence of learning
goal: a claim that, if students can provide the evidence, they both understand the content and have
demonstrated the competency.

Evidence of learning goal (Claim)

Interpret mathematical models of data in graph form to determine the order and rate constant of a
reaction using fluorescence data from cellphone images
Evidence of learning specification
Collect kinetic data, generate graphs and then interpret them with respect to integrated rate laws.

2
 In Part I of this lab you will examine how the amount of fluorescence given off by a solution of coumarin-
102 is proportional to the concentration of the coumarin-102 using data we will give to you. In Part II, you
will prepare a solution of coumarin-102 and treat it with a solution of strong base. As the coumarin-102
reacts with the base, it becomes a molecule that does not fluoresce. You will collect fluorescent data with your cell
phone camera. By examining the loss of fluorescence, you can get information on the change in the
concentration of coumarin-102 and determine the kinetics of the reaction. You can determine the order of
the reaction with respect to coumarin-102 from the graphs of the fluorescence. You will have time during
the first week to prepare some of these graphs. In Part III, in the second week, you will do the reaction
with different concentrations of base. This will allow you to determine the order of the reaction with respect
to the base. Once you have the order of the reaction, you can then determine the overall rate constant.

Fluorescence and Chemical Substances

This lab is built around the phenomenon of fluorescence, where a molecule absorbs light and then emits
the light at a different wavelength. For your data collection you have to analyze fluorescence images and
extract the information you need (while aware of the limitations in image quality). No background in image
analysis or computer programming is assumed, and the requirement of mathematics knowledge is that
discussed earlier in the semester in the Linear Regression Lab.
Fluorescence is now an incredibly important phenomenon in bioanalytical chemistry, since the ability
for instruments to sense even a few fluorescent photons against a dark background makes it very easy to
detect when a process induces fluorescence. Fluorescence was even the subject of a Nobel Prize in 2008,
awarded for the discovery and development of green fluorescent protein for use in biochemistry.
In this experiment we are studying a particular fluorescent molecule. The molecule has the name
coumarin-102 (Figure 1) and is commonly used as organic fluorophore in super-resolution microscopy.
Fluorophores are chemical compounds that contain aromatic groups, or planar/cyclic molecules with π
bonds. Coumarin-102 absorbs long wave ultraviolet light and can do one of two things: it can simply release
the energy into its surroundings as heat or it can release the energy as light (light that we can see as blue).

Coumarin-102 (Fluorescent) Coumaric Acid (Non-Fluorescent)

Figure 1. Reaction of Coumarin-102 with NaOH

The chemistry of the reaction of coumarin-102 with NaOH involves some organic chemistry, which
you may study later in your coursework. For a chemical reaction to occur some of the bonds are broken and
new bonds are formed. The molecules have to collide with each other in order to react (the collision model).
The rate of collision and the particular steps of the reaction (the mechanism) determine how fast the reaction proceeds.
Hence, the rate law for the reaction, describing the overall change of the concentration of the reactants and the products
with time, can depend mathematically on the concentrations of different substances. Determining this mathematical
dependence is known as the rate law of the reaction. In the case of this experiment, there are two reactants. The general
rate law will be:
Rate = k[coumarin-102]a [NaOH]b

3
 At the start of the experiment, we do not know either a or b. Because of the relative amounts of the
substances, during the course of a given reaction [coumarin-102] will change dramatically. But the
concentration of NaOH will hardly change at all. Therefore, we use two different methods to get the values
of a and b. In Part II, you use the method of the integrated rate laws to follow the change in [coumarin-102]
over time to get a. Then, in Part III, you will vary the amount of [NaOH] at the beginning of the experiment
to determine b.
To determine the rate law, we need to follow the changes in concentration of reactants, products, or both. In this
experiment, we take advantage of the fact that one of the reactants (coumarin 102) is fluorescent. Fluorescence occurs
when the molecule emits light and returns to the ground state. The energy of the fluorescent photon is less
than the energy of the excitation photon. To illustrate fluorescence activity, an energy diagram (Jablonski
diagram) is used (Figure 2).

Figure 2. Jablonski diagram for fluorescence

Figure 3. Optical properties of coumarin-
102: absorption (____) and emission spectra (- -
-). The wavelengths of excitation (purple)
and STED (green) lasers were also indicated

The actual reaction is very simple: the reaction converts a fluorescent molecule (coumarin-102) into a
different molecule that is not fluorescent. The particular reaction of coumarin-102 and NaOH is a hydrolysis
reaction, which causes ring opening of β-unsaturated lactone to form coumaric acid with isomerization of
the double bond (trans to cis). Coumarin-102 has very little apparent color, because it absorbs visible light
weakly. But it does absorb long wave ultraviolet light (just outside of our sight range) very well. When it
absorbs the light, it can do one of two things: it can simply release the energy into its surroundings as heat
or it can release the energy as light - it gives off light that we see as blue/green! (Figure 3). The intensity of
the light emitted by the coumarin-102 is directly proportional to its concentration. If we illuminate the
sample with light that we cannot see—ultraviolet light—we can get a blue/green glow from the sample.
This blue glow is fluorescence. Because of this, coumarin-102 is used in super-resolution microscopy
settings. You will not be using spectrometer in this experiment, but instead you will introduce the light via
UV-lamp. The light intensities will be measured by analyzing the data via ImageJ program.

Analyzing fluorescence using technology

The kinetics of this reaction is analyzed by fluorescence by measuring the light intensity over a period
of 10 minutes and recording the observations. This experiment does not require the use of a spectrometer
for concentration analysis. Instead, you will analyze the data via the ImageJ software

4
(https://imagej.nih.gov/ij/download.html). You will take photographs (using a camera on your cell phone)
as the reaction proceeds, analyze them for fluorescence intensity, and get data that allows you to infer
changes in concentration vs. time. From those changes, you can then study kinetics. You will get data such
as that in Figure 4, from which we can determine intensity vs time.

Figure 4: Schematic of analysis of a digital photograph using ImageJ

To use ImageJ, transfer the raw photo to a folder on your computer. Open ImageJ, then open the file
you want to analyze. Then, select “color-split channels”. These images all look black and white but the
program recognizes them in different colors: red, green, and blue. Each picture is titled differently, so you
have to look closely for labels and find the picture entitled “blue” from which you will deduce the intensity
data (Figure 5).

Figure 5: Obtaining coumarin-102 intensity data with ImageJ. The picture name (top frame) will
contain the color label (red, green or blue). Keep the BLUE one only

To obtain the light intensities, you have to select each sample separately and analyze the data for that
specific set (Figure 6a, 6b). Your selection should be consistent in size and shape (rectangular) to the best

5
of your ability. Work to ensure you are just measuring the light in the sample—not getting any part of the
top of the solution or the sides of the cuvette, which can distort the results.

A new window with

students have to copy
into their Excel sprea
the correct cuvette.

Intensity Data
a) b) c)
Figure 6: a), b) Selection should be consistent in size and shape (rectangular). c) A new window
with the intensity data will open and the students have to copy the value for MEAN (light intensity)
into their Excel spreadsheet. Each data has to correspond to correct cuvette.

The data is obtained by use of the “area” tool in ImageJ and highlighting the region of the image we
are interested in. Once this is done, we can measure (“Ctrl-M” or select “analyze – measure”) that value
and the mean (which represent the intensity I is shown in a measurement window (Figure 6c).
In this experiment we will study the kinetics using this technology. We will take photographs as the
reaction proceeds, analyze them for fluorescence intensity, and get data such as that in Figure 7 for intensity
vs time. ImageJ will allow you to determine intensity data from the images which you will then directly
plot in Excel, determine the trendline and R2 value and analyze the order of the reaction. As shown in Figure
7, we have taken data for a sample and used ImageJ to create a table of the data.

Figure 7: Example of Excel spreadsheet and graph of I / Io vs time data

Safety Considerations

In other parts of the experiment, you will be using a “black light,” designed to give very long wavelength
UV light (about 390 nm). This light is not visible to the eye but is not damaging in the way shorter
wavelengths are. Still, take care not to stare directly into the black light.
You are also working with very concentrated solutions of strong base (6M NaOH). Proper eye
protection is necessary at all points. Even a drop of this can damage the eye very quickly. Wear protective
gloves and wash your hands thoroughly after handling solutions.

6
Procedure

Gather Materials and Supplies

You will be provided with 1.0 mL Mohr pipettes and 10 mL graduate cylinders. You will also be provided
with stock solutions with the following approximate concentrations. Make note of the exact concentration
on the solution bottle.
 Coumarin-102, 1.0 × 10–4 M or 100.0 micromolar.
 NaOH, 6.0 M (Note: this is a very concentrated solution. Handle cautiously!)
Using clean, dry glassware, you should obtain approximately 20 mL of the coumarin-102 stock solution
and 20 mL of the 6.0 M NaOH.

Part I: Interpret graphical models of concentration and fluorescence

You are asked to construct a mathematical model in graph form that will help you to solve the actual
problem using your prior knowledge. In chemical kinetics, the chemical reactions are studied as natural
phenomena. You are asked to determine the dependence of fluorescence on intensity using a mathematical
model. This model is a graphical model that compares the intensity of light vs concentration.

Generating and Interpreting a Mathematical Model from Data

In Table 1 you have random data of fluorescence intensity (I) and coumarin-102 concentration
([C102]). Using the same method that you used in the regression analysis experiment from earlier in the
semester, generate a graph of I vs [C102].

Sample I [C102] µM
1 132.262 2.5
2 182.456 5
3 192.730 7.5
4 246.135 10
Table 1. Intensity vs coumarin-102 concentration

Mathematical analysis of data

During the lab period, analyze the data to determine:
 The best fit of trendline.
 The slope and its numerical value.
 The value of R2 (how close the data are to the fitted regression line).
The graph you have created is a model of the data in mathematical form, both graphically and in terms
of the numbers you have generated in the analysis.

In your Lab Report:

Interpret the model. To do this, answer the following questions:
 What is the general trend within the data?
 Why might the data have this trend?
 What is the chemical interpretation you can provide? This should specifically relate the model
to a molecular-level interpretation.
 Attach the graph that you generate on your computer.

7
Demonstrate your model by performing the experiment

Obtaining fluorescence measurements with a digital camera

As you know, the amount of light detected is related to the amount of the fluorescent molecule
coumarin-102. In this experiment, you will track the change of the light to indicate the change of the amount
of coumarin-102 in the reaction vessel.
Prepare five samples of coumarin-102 with different concentrations, from 0.0 M up to 4.0 x 10-5 M (40
micromolar). You should do this using stock solution and water so that the total volume is 10 mL. You
then use 1.0 mL of each solution in a different cuvette for the fluorescence measurements (make sure you
are using a different pipette for different solutions). Measure the fluorescence for all five samples. Position
the five cuvettes under the UV light and fix the position of the camera to make sure that in the picture the
cuvettes are sitting in the middle and well-focused. Turn on the UV light and turn down the room light.
Take at least three digital photographs using your cell phone camera, which you will analyze with ImageJ
software. Compare the results and characterize the concentration dependence of fluorescence on
concentration.

In your Lab Report:

For this part of the experiment, you should write out the analysis that you conducted in your notebook.
Plot the intensity (I) vs concentration of coumarin-102 ([C102]) to demonstrate the concentration
dependence of fluorescence. Attach the graph that you generate on your computer. Compare your
experimental data with the model and analyze the results.

Part II: Kinetics of Reaction of Coumarin-102 with NaOH

This part of the experiment is based on the reaction between coumarin-102 and NaOH. Here, you need
to find the order of the reaction with respect to coumarin-102, using the integrated rate laws. To set up the
reaction, refer to Table 2:

Reference cuvette Reaction cuvette

Vol. of 100 µM C102 stock (µL) 100 100
Vol. of water (µL) 900 600
Vol. of 6.0 M NaOH (µL) - 300
Table 2. Reaction setup

Use a pipet to measure out the solutions and make sure you’re using a different pipette for different
solutions. Fill the reference cuvette first and then fill the reaction cuvette. Mix carefully and well. Once the
cuvettes are filled, place the experimental cuvette about 1 cm from the reference cuvette. Turn on the UV
light and turn down the room light. Start taking pictures immediately and every 30 seconds. 12 to 15 pictures
are enough to analyze.

Repeat the experiment two times!

Determination of Concentration Dependence of Coumarin-102 Fluorescence

During the experiment, you obtained images of the reaction over about 5-8 minutes. Transfer the images
to your computer and use ImageJ to determine the fluorescence intensity in an appropriate region of each
of the images. Because the camera can make many different adjustments to the exposure, it is essential that
the light be compared to the standard source in each of the images. So, for each image, you obtain two
intensities: the intensity of the standard cuvette (Io) and the intensity of the experimental solution (I). Select

8
an area of the reference cuvette and record the light intensity (noted as Io), then select the same area at the
same position of the reaction cuvette and record the light intensity (noted as I).
Generating and Interpreting a Mathematical Model from Data (to be done during lab for at least one
trial)
A mathematical model will allow you to analyze kinetics of the reaction of coumarin and NaOH
(determine the order of the reaction and the rate constant).
You need to determine the reaction order for two components: NaOH and coumarin-102. In Part II, you
will look at the way the fluorescence changes during the reaction to determine the reaction order with respect
to coumarin-102. Next week, (in Part III), you will compare the rate constants for different NaOH solutions
to obtain the dependence of the reaction on NaOH. You carry out the determination of the coumarin-102
order by plotting the intensity of the fluorescence. As noted, you actually use the ratio of the intensity relative
to a standard (I/Io) so that camera effects are minimized. You will compare the mathematical models of
graphs of the data with those expected for the integrated law for the reaction. [MORE INFORMATION
ON THE USE OF INTEGRATED RATE LAWS IS AVAILABLE IN THE SPRING 2019
CHEMISTRY 122 TEXTBOOK, CHAPTER 16]. Table 3 is a quick overview of the same material:

Zero order reaction (a = 0): First order reaction (a = 1): Second order reaction (a = 2):
rate = k rate = k[C102] rate = k[C102]2
[C102] = - kt + [C102]0 ln[C102] = - kt + ln[C102]0 1/[C102] = kt + 1/[C102]0
Table 3. Integrated rate laws

When you plot the fluorescent intensity (or a function of the fluorescent intensity), you can investigate
different orders of the reaction. The overall rate law (ignoring the NaOH for this week) is that Rate =
k[C102]a, where a is the order of the reaction with respect to coumarin-102. By using calculus, you can
determine different conditions.
For a zero order reaction (a=0), rate = k (k = slope of line of graph of I/I0 vs. time)
The integrated rate law for this zero order reaction is: I/I0 = kt
A straight line is obtained if you plot [C102] vs. t, and the rate constant is determined by the slope.
If the reaction is zero-order, a plot of I/I0 vs. t will be a straight line, and the slope will be - k.
For a first order reaction (a=1), rate = k[C102], (k = - slope of line of graph of ln(I/I0) vs. time)
The integrated rate law for this first order reaction is: ln(I/I0) = kt
A straight line is obtained if you plot ln([C102]/[C102]0) vs. t, and the rate constant is determined by the
slope. In the logarithmic form, if you plot ln[C102] vs. t a straight line is supposed to be obtained and the
slope indicates the rate constant - k.
If the reaction is first-order, a plot of ln(I/I0) vs. t will be a straight line, and the slope will be - k.
For a second order reaction (a=2), rate = k[C102]2, (k = slope of line of graph of 1/(I/I0) vs time)

Mathematical analysis of data

During the lab period, take at least one of your data sets and generate a table of I/I0, ln (I/I0), and 1/(I/I0)
vs time. Examine them to see which is the best line. For your work to prepare for next week, graph the data
2
using Excel. In Excel, plot I/I0 vs. time, add the trend line and record the R value. Plot ln (I/I0) vs. time,
2 2
add the trend line and record the R value. And finally, plot 1/(I/I0) vs time record the R value. Compare
2
the R values. The higher one indicates a better linear fit and can determine the order of reaction.

In your Lab Report:

Compare the mathematical models of graphs of the data with those expected for the integrated law for
the reaction. Model the change of [C102] as a function of change of light. Interpret model to get reaction
order. The rate constant k for the reaction can also be determined from the data. Obtain this for each of
the trials. The one that fits best is correct. Attach the graphs that you generate on your computer.

9
Part III: Reaction Order with Respect to NaOH

Part III is done in the second week.

In week 1, you determined the order of the reaction with respect to coumarin-102. You also determined
k for the reaction for a given concentration of NaOH. To determine the order of the reaction with respect to
NaOH, you will repeat the experiment from Part II, with different values of [NaOH]. You will obtain
k at different [NaOH] and, by seeing how k varies with [NaOH] you can determine the reaction order with
respect to NaOH.
Carry out the reaction with at least three different values of [NaOH]. Use the 6.0 M NaOH stock
solution to prepare 4.5 M, 3.0 M, and 1.5 M NaOH solutions by dilution. Calculate the effective NaOH
concentrations ([NaOH]eff - these are the concentrations of NaOH in each reaction cuvette; see Table 2:
reaction setup) and [NaOH]eff2. You already know (from Part II) the reaction order with respect to [C102],
therefore you can graph only the appropriate data (intensity vs time) in Excel. Collect the slope values from
all of the reactions and record in Table 4 as the values of k (apply integrated rate laws, page 9).

[NaOH]eff (M) [NaOH]eff2 (M2) k

Table 4. Data of k and effective NaOH concentration

Use your data to create and interpret a graphical math model to get order of reaction with respect to
[NaOH]. Plot [NaOH]eff vs. k and [NaOH]eff2 vs. k. Determine the trend lines and read the R2 values.
Compare the R2 values. Determine which is the better fit and from that, the reaction order with respect to
NaOH.

In your Lab Report:

Collect the slope values from all of the reactions and record them as the values of k. Use your data to
create and interpret a graphical math model to get order of reaction with respect to [NaOH]. Attach the
graphs that you generate on your computer.

10