Introductory Lab Manual
For Electrogenerated
Chemiluminescence
Created By: Amy L. Hruska and Paul L. Walsh
Introduction to ECL: Background Information
Electrogenerated Chemiluminescence, also called electrochemiluminescence and
abbreviated as ECL, is the generation of light emitting species from electrochemical oxidation
and reduction at an electrodes surface.1 A voltage or voltage pattern applied to the electrode
causes electron transfer at the electrode’s surface to form intermediates. These intermediates
react to produce an excited state molecule near the electrode. As the excited molecule returns to
the ground state, energy is released in the form of light. This can be seen in the following general
mechanism for light emission by ECL:1
(1)
(2)
(3)
(4)
A + e-  A- (reduction potential)
D  D+ + e- (oxidation potential)
A- + D+  A* + D (or D* + A)
A* (or D*)  A (or D) + hv.
In this scheme, A is an electron acceptor, which is reduced at the electrode’s surface, and D is an
electron donor, which is oxidized at the electrode’s surface. A square wave potential pattern is
applied to the electrode with voltages chosen to reduce A and oxidize D. Note that the electron
donor and acceptor may be a different molecule or the same molecule.
ECL emission has been shown to occur by three main types of reaction schemes, which
fall into two larger groups: annihilation ECL and co-reactant ECL.1 The more specific relations
are called the S- Route and T-Route systems (annihilation), and oxidative-reductive or reductiveoxidative systems (co-reactant). In the S-Route, or energy sufficient system, the change in
enthalpy generated in creating the excited species that exhibits emission is greater than the
change in energy necessary to create the singlet species from the ground state.1,2 Unlike the other
ECL mechanisms, once the oxidized and reduced species of the luminophores react by the SRoute, the emitting species is created and has sufficient energy to emit a photon. A common
example of a luminophore which undergoes ECL by this mechanism is tris(2,2’-bipyridyl)
ruthenium (II) dichloride hexahydrate (Ru(bpy)32+) system. The reaction scheme for the
generation of ECL emission for this system is
(5)
(6)
(7)
(8)
Ru(bpy)32+ - e-  Ru(bpy)33+
E= +1.2V vs SCE
Ru(bpy)32+ + e-  Ru(bpy)3+
E= -1.4V vs SCE
3+
+
2+
Ru(bpy)3 + Ru(bpy)3  Ru(bpy)3 * + Ru(bpy)3
Ru(bpy)32+*  Ru(bpy)32+ + hv
(~2.1eV, 610nm).
Another example of a luminophore that undergoes ECL by the S-Route mechanism is
9,10-dephenylanthracene (DPA).1,3 The reaction scheme that DPA follows is
(9)
(10)
(11)
(12)
DPA + e-  DPA·DPA  DPA·+ + eDPA·+ + DPA·-  DPA* + DPA
DPA*  DPA + hv.
E = -1.6 V vs. Ag
E = +1.2 V vs. Ag
Above, the DPA·- and DPA·+ represent the reduced and oxidized free radical species,
respectively, and DPA* represents the excited species undergoing ECL emission. The oxidized
and reduced species are created at the surface of the electrode by applying a square wave that
cycles between the two potentials necessary to make DPA·- and DPA·+.3,4 The cycling of
potentials to create both a reduced and an oxidized species is true of all S-Route systems. These
two radicals then go on to react and create the excited emitting species that fall to the ground
state as emitted light.
While the S-Route is an “energy sufficient” system, some systems are known as “energy
deficient.” An energy deficient system proceeds by a slightly different mechanism known as the
T-Route or triplet-triplet annihilation pathway. The T-Route mechanism gets its name because
there are triplet states which are formed during the oxidation and reduction step that must
undergo an annihilation reaction to create the singlet excited species.1 The singlet state varies
from the triplet state because the electrons have paired spins in the singlet state (one spin up, the
other spin down), while they are the same spin in the triplet state. In the T-Route mechanism,
Equation 4 is replaced with the following emissive pathway:
(13)
(14)
3
1
A* + 3A* (or 3D* + 3D*)  1A* + 1A* (or 1D* + 1D*) + hv
A* (or 1D*)  A (or D) + hv.
Equation 3 from the scheme above only provides enough energy to excite the luminophore into
the first excited triplet state. This excited state may undergo non-radiative decay back to the
ground state, or it may react via Equation 13 to produce an excited singlet state of the
luminophore. This excited single state will then return to the ground state with the emission of
light. . DPA reacts with N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) via the T-Route
and ECL with tris-(2,2’-bipyridine)-ruthenium(II) demonstrates another example.
(15)
(16)
(17)
DPA + e-  DPA·- E = -1.89 V vs. SCE
TMPD  TMPD·+ + e- E = +0.24 V vs. SCE
DPA·- + TMPD·+  3DPA* + TMPD
(18)
(19)
3
DPA* + 3DPA*  1DPA* + DPA
1
DPA*  DPA + hv.
As shown above, the oxidized and reduced species react to form a triplet excited species
in the T-Route system that reacts with another triplet to form an excited species in the singlet
state; this excited singlet molecule has sufficient energy to undergo emission down to its ground
state. Similar to the S-Route system, a square potential waveform is applied to the surface of the
electrode in order to create both the oxidized and reduced species.1 Also, it is important to note
that many reactions proceed by both the T-Route and S-Route mechanisms to varying degrees.
ECL may also be achieved at a single potential by the oxidative-reductive system. In this
system, a co-reactant forms a strong reducing agent upon oxidation at the electrode. This
reducing agent then reacts with the luminophore to form an excited state. This system is the most
complicated because, instead of having a singlet reactant, the primary emitting species reacts
with a co-reactant to form the emitting species.1 An example of this system is Ru(bpy) 32+ and
tripropylamine (TPrA). The mechanism for it is
(20)
(21)
(22)
(23)
(24)
Ru(bpy) 32+  Ru(bpy) 33+ + eTPrA  [TPrA] + + e[TPrA] + + e-  TPrA + H+
Ru(bpy) 33+ + TPrA  Ru(bpy) 32+* + Products
Ru(bpy) 32+*  Ru(bpy) 32+ + hv.
Both the primary emitting species (Ru(bpy)32+) and the co-reactant (TPrA) are oxidized at the
surface of the electrode while applying a single potential, hence the reaction scheme name of
oxidative-reductive.1 If the primary emitting species and co-reactant are reduced at the surface of
the electrode, the system is called a reductive-oxidative system. The oxidized TPrA then
undergoes de-protonation and becomes a highly reactive free radical. The oxidized Ru(bpy)32+
reacts with the tripropylamine radical to form the excited species of Ru(bpy)32+* which decays to
its ground state by emitting a photon. The potential at which the co-reactant and primary emitting
species oxidize must be very close to one another so that when a single potential is applied to the
surface of the electrode both oxidations can occur simultaneously. Since Ru(bpy) 32+ is one of
the very few ECL emitting compounds which have an oxidation potential within the solvent
window for water, when combined with TPrA as a co-reactant, chemiluminescence is made
possible at a single potential applied in aqueous solutions (the potentials are between water’s
oxidation, +1.2V, and reduction, -1.2V which is why water can be used as a solvent).5
References
1.
Richter, Mark M. Electrochemiluminescence. Chemical Reviews, 2003, A – AH.
2.
Faulkner, L. R.; Glass, R. S. Electrogenerated Chemiluminescence from the Tris(2,2’bipyridine)ruthenium(II) System. An Example of S-Route Behavior. J. Phys. Chem.
1981, 85, 1160-1165.
3.
Bard, A. J.; Maloy, J. T. Electrogenerated Chemiluminescence. VI. Studies of the
Efficiency and Mechanisms of 9,10-Diphenylanthracene, Rubene, and Pyrene Systems at
a Rotating-Ring-Disk Electrode. J. Am. Chem. Soc. 1971, 93, 5968-5981.
4.
Wightman, R. M.; Collinson, M. M. High-Frequency Generation of
Electrochemiluminescence at Microelectrodes. Anal. Chem. 1993, 65, 2576-2582.
5.
Harvey, David. Modern Analytical Chemistry. McGraw Hill, Boston, 2000, Chapter 11.
Introduction to ECL Experiment #1: Electronics Review
Purpose:
This experiment seeks to review the concepts of electronics covered in University
Physics as well as to introduce new researchers to some more advanced electronics concepts and
materials not covered in prerequisite courses.
Materials:
Breadboard
Protoboard
Wiring kit
3 – Resistors
1 – 10 kΩ Variable Resistor
Multimeter
1 - 9V batteries with connecting ports
Background:
The use of electronics in the ECL lab is of the utmost importance. Not only is it necessary
for the generation of ECL emission, but it is also important for truly understanding how to take
the measurements of the ECL emission. Electronics has become an integral part of analytical
chemistry over the past 20 years with the advent of more specialized and sensitive
instrumentation. It is therefore necessary for all researchers to have an excellent background in
the subject beyond what is covered in the introductory physics classes. Before beginning this
experiment, make sure to review the basic concepts covered in those introductory physics classes
such as University Physics. Topics include: electric fields, electric potential differences, basic
circuits, Ohm’s Law, Kirchoff’s Current Law, Kirchoff’s Voltage Law, AC versus DC power
sources, resistors in series and parallel, oscilloscopes and function generators.
Procedure:
1.
Study the multimeter provided. Make sure that you understand what each setting
measures, and how to attach the leads to make the correct measurements. If there is something
you are unsure of, make sure to ask a fellow researcher or your instructor. Connect the leads to
make a resistance measurement and set the multimeter to do the same. Take three different
resistors and look up their resistance by the color code. Record the order of the colors and the
corresponding resistance. Now measure the resistance of each of the resistors with the
multimeter and record your results.
2.
Next, pick up the breadboard and study it. If there is an instruction manual for it, read
through it. On the breadboard, there are three knobs to one side. One is black and the other two
are red. The black knob is for connecting to electrical ground. Connecting to ground is much like
completing a circuit. It gives the electric current a place to flow, and if two ends of a circuit are
both attached to ground, it is the same as making a loop circuit like those introduced in
University Physics. The two red ports can be used if a common potential is to be applied
throughout the circuit. Along the two long sides of the breadboard, there are 4 sets of holes
stretching the length of the board. These are called power buses because each of the rows are
connected the length of the board. This allows for a common ground or potential to be applied to
any part of the board you want. In order to test to see how the various ports are connected, place
wires in various places and see where no resistance is measured. If the resistance between 2 ports
is about zero ohms, then they are connected. Make a sketch of the breadboard and label which
parts are connected in a way that makes sense to you in the space provided.
3.
First, measure the voltage of the battery. Then make a series connection between the
three resistors and one 9 V battery. Make sure that you have at least two 10 kΩ resistors before
connecting the batteries to the resistors. Next, measure the voltage drop across each of the
resistors and measure the current between each of the resistors as well as between the two end
resistors and the battery. Also, measure the voltage drop across all three resistors, and the current
before each of the resistors and after all the resistors. Record all measurements taken in the space
provided. Use the circuit diagram below to help you in creating the circuit.
4.
Next, create the same circuit, but instead of creating a complete circuit, use ground as
your area of low potential to allow the current to flow there. Measure the voltage drop across
each of the resistors and measure the current between each of the resistors as well as between the
two end resistors and the battery. Also, measure the voltage drop across all three resistors, and
the current before each of the resistors and after all the resistors. Record all measurements taken
in the space provided. Use the circuit diagram below to help you in creating the circuit.
5.
Then connect the three resistors in parallel with one another. Measure the voltage drop
across each of the resistors and measure the current between each of the resistors as well as
between the two end resistors and the battery. Also, measure the voltage drop across all three
resistors, and the current before each of the resistors and after all the resistors. Record all
measurements taken in the space provided. Use the circuit diagram below to help you in creating
the circuit.
6.
Then create the same circuit using ground as the area of low potential. Measure the
voltage drop across each of the resistors and measure the current between each of the resistors as
well as between the two end resistors and the battery. Also, measure the voltage drop across all
three resistors, and the current before each of the resistors and after all the resistors. Record all
measurements taken in the space provided. Use the circuit diagram below to help you in creating
the circuit.
7.
Once all the measurements of the series and parallel circuits have been completed, create
the series circuit from Step 4, except replace one of the resistors with a variable resistor. Make
sure you connect the correct lead to ground. If you are unsure, ask a fellow researcher or the
instructor. Measure the resistance of the variable resistor when the knob is turned completely
counter-clockwise as well as completely clockwise. Next, measure the voltage drop across the
resistor when the knob is turned completely counter-clockwise. Also measure the voltage drop
across the resistor when the knob is turned ¼, ½, ¾ of its full rotation as well as the entire
rotation clockwise. Also measure the resistance of the variable resistor. Record all measurements
taken in the space provided.
8.
Take the Proto-Board given and study it. If there is a user’s manual for it, read through it.
Along the top of the board, there is a ground connection much like on the Breadboard. The other
three red terminals are labeled +5V, +15V, and -15V. These are ports to which you can make
connections to supply these voltages to the circuit. There is also a power switch and an AC
power cord on the Proto-Board which are necessary to supply the voltages to the terminals.
There are power busses and terminals just as on the Breadboard. Make connections and test the
resistance between various ports to test which are connected and which are not. Make a
schematic sketch of the Proto-Board in the space provided.
9.
Now connect the +5V port to one of the terminals on the Proto-Board and connect the 10
kΩ variable resistor in series with a 5 kΩ resistor. Measure the potential at the end of the circuit
versus ground for when the variable resistor is turned completely counter-clockwise, as well as
¼, ½, ¾ of the way clockwise, and completely clockwise. Also be sure to measure the resistance
of the variable resistor. Record the data in the space provided.
Data/ Observations:
1.
Give the color code of each of the resistors provided, its expected resistance, and the
actual resistance measured by the multimeter.
2.
Draw the Breadboard, and schematically show how the terminals are connected to one
another, including the power buses.
3.
What is the true voltage of the 9 V battery?
4.
Report the values for the voltage drops, and current for the two series circuits.
5.
Report the values for the voltage drops and current for the two parallel circuits.
6.
Record the voltage drop across the variable resistor in the series circuit when the knob is
turned completely counterclockwise, and about 1/4, 1/2, 3/4, and all of the way clockwise.
7.
Draw a schematic representation of the Proto Board and show how the various ports are
interconnected.
8. Record the voltage drop across the entire circuit made in Step 9 when the variable resistor is
completely clockwise, as well as 1/4, 1/2, 3/4, and entirely clockwise. Also, record the resistance
of the variable resistor.
Calculations/ Questions:
1.
Using the measured values for the 9V battery and the resistors, calculate the theoretical
values for the current (I) and the voltage drops across each of the resistors for the series loop
circuit. Calculate percent errors for each of the values as well and report them below. What are
the possible sources of error? Do Ohm’s Law and Kirchoff’s Voltage Law remain true?
2.
Using the measured values for the 9V batter and the resistors, calculate the theoretical
values for the current (I) and the voltage drops across each of the resistors for the parallel loop
circuit. Calculate percent errors for each of the values as well and report them below. What are
the possible sources of error? Do Ohm’s Law and Kirchoff’s Current Law remain true?
3.
Are there any differences between the values obtained with the loop circuits versus the
circuits which use ground?
4. What might the circuit created in Step 9 of the procedure be used for?
Introduction to ECL Experiment #2: Cyclic Voltammetry
Purpose:
The purpose of this experiment is to give the student an understanding of the theory and
method of cyclic voltammetry. This will be accomplished by observing the effect of scan rate,
concentration, and supporting electrolyte on the CV scan.
Materials:
BAS CV-1B Voltammograph
Houston Instruments Omniographic 100 X-Y Recorder and pen
Multimeter
Lined graph paper
12 in ruler
4 - 25 mL volumetric flask
1 – 100 mL volumetric flask
1 – 200 mL volumetric flask
5 - 50 mL beaker
2 mm Pt working electrode
Pt auxiliary electrode
Ag/AgCl reference electrode
Chemicals:
Analytical Reagent Grade KNO3
Reagent Grade K3Fe(CN)6
Background:
Before beginning this laboratory, be sure to read the section on cyclic voltammetry in
your Analytical textbook. Only the highlights will be covered in this laboratory. Cyclic
voltammetry (CV) is an analytical technique used to detect electrochemically active species in
solution. CV is a little different from the techniques that are learned in General Chemistry and
Analytical Chemistry in that it is performed on single solutions as opposed to two solution
systems. When two solutions, which have the correct reduction potentials, are attached by a
circuit and salt bride, the REDOX reactions occur creating a current through the wire, and
therefore a potential difference is also created between the two solutions. In a single solution
sample, a potential is applied to the various electrodes in the solution to make electrochemically
reversible reactions occur.
A CV gives a plot of current versus potential. Recall from General Chemistry that
electrochemical reactions occur at the same potential no matter what the concentration of the
solution. The same is true for single solution systems. The shape and size of a CV depend upon a
few different variables. In completing the following experiment, you will learn about what
variables change the shape and size of voltammograms and what methods are best for making
the types of measurements used to obtain information from a CV. The peak current for a
reversible system at the electrode can be described by the Randles-Sevcik equation which states
ip= 2.69x105n3/2AD1/2C1/2 where ip = peak current (A), n= electron stoichiometry, A = electrode
area (cm2), D= diffusion coefficient (cm2/sec), C= concentration of solution (mol/cm3), and  =
scan rate (V/sec).
Procedure:
1.
Make 200mL of a 0.5 M solution of KNO3. This will act as your supporting electrolyte
for all of the other solutions.
2.
Make a 100 mL solution of 10 mM K3Fe(CN)6 using the supporting electrolyte as the
solvent. Dilute the 10 mM solution in the other 25 mL volumetric flasks to make 8, 5, 2, and 1
mM solutions.
3.
Study the BAS CV-1B and the Omniographic X-Y Recorder. On the CV-1B there are 4
sets of cords which are connected to various places. There is the power cord which is attached to
an AC power source (typical AC plug). There is a group of 3 wires also protruding from the back
which are the leads to be connected to the electrodes. The black lead attaches to the working
electrode, the red attached to the auxiliary, and the white attaches to the reference electrode. On
the front of the CV-1B, there are also two output sources. There is the “-E” and the “Output.”
The –E terminal goes to two places: the X-Y recorder (the X section of controls) and the
multimeter. The output terminal also goes to the X-Y recorder (the Y section of controls). Draw
a schematic representation of this in the space provided.
4.
Next, look at the controls on the CV-1B. On the very top there are two switches and a
knob. The right switch is to turn on and off the power and the light next to it tells whether the
power is on or off. The knob and switch on the left are the initial adjust. If the switch is set to off,
then the instrument will begin its scans at 0 V while adjusting the knob and switch accordingly
will change where the instrument beings its scans from. The next sets of knobs and switches are
for scanning. The offset changes where the maximum and minimum potentials are compared to
the initial position. The positive and negative knobs increase the maximum and minimum
potentials of the scan. The direction knob is used to change the direction of the scan and is useful
while initially setting up the instrument. The knob in the very middle is used to change the scan
rate and can be set anywhere from 0.00 to 999 mV/sec. The switch just to the right of this is the
multiplier of this setting and is typically left at 0 -103 mV/sec. Just below the scanning controls
are the attenuation and run controls. On the very bottom left of the CV-1B is the switch used to
turn on and off the potentials being applied to the working electrode. MAKE SURE THIS IS IN
THE OFF POSITION BEFORE CONTINUING. The large knob in the middle of the bottom is
the attenuation control for the instrument. The filter switch is to the right of this knob and is
typically left at 0.1 seconds. The switch on the upper left hand side of this section is used to tell
the instrument whether to be at the initial potential that has been set (init), to hold at a given
potential in a scan (hold), or to scan among the potentials that you have set. Draw a picture of
this labeling all parts in the space provided.
5.
Next, study the X-Y recorder. On the top right hand side, there are a few switches. The
important ones are the power switch, and the pen switch. Along the right hand side is where the
leads from the CV-1B attach to the recorder, and there are attenuation and zero knobs for each of
the settings (X and Y). There is also a large black switch next to where the paper is inserted
which can be adjusted to load the paper, or to hold it in place while the scan is being run.
6.
Adjust the controls so that the instrument is set to have an initial position of +0.69 V, and
should scan between +0.69 V and 0.00 V at a rate of 25 mV/sec. The attenuations should be set
to 20 mV per cm and 2.5 microA per cm.
7.
Place about 25 mL of your 10 mM K3Fe(CN)6 solution into a 50 mL beaker. Place all
three electrodes into the solution in close proximity to one another, but make sure no electrical
connections are made. Attach the appropriate leads to each of the electrodes making sure that the
CV-1B, recorder, and working electrode are in the OFF position.
8.
Turn both the CV-1B and the recorder on. Place a pen into the pen holder in the recorder
and remove its cap if one is not already in place, and place a piece of graph paper into the holder
and place the switch into the scan position to hold the paper down. Make sure that the paper is
completely flat on the surface of the recorder. Make sure that the zero is where you want it.
Remember, in CVs positive is to the left, and negative is to the right, so make your adjustments
appropriately.
9.
Turn the working electrode on before putting the pen down. The pen should jump
indicating that an electrical connection has been made through the electrolytic solution. Place the
pen down and switch the scan control switch on the CV-1B to scan. Allow the instrument to go
through three entire scans before stopping it. While the instrument is running, read ahead to step
10. *If at any time the pen goes off of the scale you have set for it, pick the pen up, turn the
working electrode off, place the instrument back to its initial position and adjust the attenuation
accordingly so you can restart the run.*
10.
Once the instrument has gone through three entire scans, pick the pen up, turn the
working electrode off, and put the instrument back to its initial potential IN THAT ORDER.
Once the pen has come to a stop, place the pen back down onto the paper and create a small X-Y
axis using the zero adjust knobs on the recorder. This axis will tell you where your initial
conditions were and can be used while making measurements. Pick the pen back up and remove
the paper from the recorder. Label the axes with the initial conditions of the scan. Also on the
back of the CV, label all electrodes used, the scan rate, both attenuations, and the concentrations
and molecular formulas of the analyte and supporting electrolyte.
11.
Determine the potentials of both of the reactions, and their respective currents taking the
attenuations into account for all three of the scans using a 12 in ruler. Record this data in the
space provided.
12.
Now change the scan rate to determine its affect on the shape of the CV. Take CVs of the
10 mM solution at scan rates of 10 mV/sec, 25 mV/sec, 50 mV/sec, 75 mV/sec and 100 mV/sec.
Record the potentials for each reaction as well as the respective currents for the third scan only
for each of the CVs. Make sure to change the attenuation to ensure that you can make the most
accurate measurement possible with the ruler. Set aside the 10 mM solution to be used later.
13.
Now change the scan rate back to 25 mV/ sec and make CVs for the 8, 5, 2, and 1 mM
solutions of K3Fe(CN)6 making sure to change the attenuation accordingly to get the best results
for taking measurements with the ruler. Also make sure to use a clean 50 mL beaker for each
solution separately. Record the potentials and currents for each reaction in the space provided of
the third scan of each CV.
14.
When finished, make sure to turn off all instruments and electrodes and remove the pen
from the holder and cap it while cleaning up. Electrodes should be stored properly.
Helpful Vocabulary List for the Instruments:
Direction:
Pressing this switch will manually change the direction of the scan in progress. This is
very useful for obtaining numerical values for the scanning limits.
Init. Adj.:
This setting adjusts the starting position for the scan. Switching to Init. on the
Init/Hold/Scan switch above will return to this specified potential. This potential does not have to
be at one of the limits for the scan.
Init/Hold/Scan:
Init: This setting moves the potential (and the pen) quickly back to a specified starting
position for the scan.
Hold: This stops a currently running scan immediately at the existing potential.
Scan: This begins a scan, adjusting the potential at a given rate. The scan continues in a
direction until it reaches a specified limit or the direction is manually changed.
Limits:
The two limit knobs alter the positive and negative limits for the scan. When a scan in
progress reaches a limit, the direction of the potential scan reverses. The left knob changes from
0 to positive potentials turning clockwise. The right knob changes from 0 to negative potentials
turning clockwise.
Microamps/V:
This knob controls the potentiostat attenuation. Using this value along with the X/Y
attenuation and division control on the recorder, the current generated by the cell can be
calculated.
Offset:
This 10-turn knob will shift the entire range of limits on the relative scale. For example, a
scan with limits of 0.5V to –0.5V could be offset to become 0.55V to –0.45V.
X/Y Attenuation:
The X and Y attenuation knobs on the recorder alter the scale on which the incoming data
is recorded. This attenuation is given in mV/Division. The division switch at the top of the
recorder can be used to set divisions of cm or inches. The incoming voltage is given by the
attenuation on the potentiostat.
Zero Adjust:
The X and Y zero adjust on the recorder will allow the scan to be centered on the page.
Data and Observations:
1.
Draw a schematic of the set up for the connections of the CV-1B, the Omniographic X-Y
recorder, the electrode leads and the multimeter.
2.
Draw and label all important controls of the CV-1B.
3.
Record all data for the first three scans of the K3Fe(CN)6 in the table below.
Scan
1
Eox
iox
Ered
ired
2
3
4.
Record all data for the third scan of the K3Fe(CN)6 at various scan rates.
Scan Rate
10 mV/sec
Eox
iox
Ered
ired
25 mV/sec
50 mV/sec
75 mV/sec
100 mV/sec
5.
Record all data for the third scan of the different concentrations of K3Fe(CN)6 at 25
mV/sec.
Concentration
1 mM
2mM
5 mM
8 mM
10 mM
Eox
iox
Ered
ired
Calculations and Questions:
1.
When studying the BAS CV-1B and the Omniographic X-Y Recorder, why are the 3 sets
of cords connecting to the instruments in the manner explained in the procedure?
2.
Why are there two peaks on a CV of K3Fe(CN)6? What does each of them represent?
3.
What kind of information can be gathered from a CV?
4.
Why is a CV taken of a single solution? What is the difference between single solution
electrochemistry and two solution electrochemistry you learned about in General Chemistry?
5.
What are the electrodes you used and what does each of them do?
6.
What is the purpose of the KNO3 in this experiment? What are its properties that make it
good at what it does?
7.
What is the electrochemically active species in this solution (what is the analyte)? Look
up the standard reduction potential in a textbook. How close if your value to that value?
8.
What is the effect of allowing the instrument to scan through the potentials 3 times?
9.
How does the scan rate affect the potential and current of the oxidation and reduction
peaks? Make a plot of the potential versus scan rate and the current versus scan rate for both the
oxidation and reduction peaks.
10.
How does the concentration affect the potential and current of the oxidation and
reduction peaks? Make a plot of the potential versus the concentration and the current versus the
concentration for both the oxidation and reduction peaks.
Introduction to ECL Experiment #3: Advanced Cyclic Voltammetry
Purpose:
This experiment is designed to introduce the experimenter to the ECL system which is
easiest with which to create emission. This is also the first ECL active system on which cyclic
voltammetry is performed.
Materials:
CH Instruments Electrochemical Analyzer
BAS CV-1B Voltammograph
Multimeter
2 mm Pt Working Electrode
Pt Wire Auxiliary Electrode
Ag/AgCl Reference Electrode
Au Working/ Ag Auxiliary Microelectrode
2 – 25 mL Volumetric Flask
Chemicals:
0.5 M KNO3
Tris(2,2’-bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3)
Tripropylamine (TPrA)
Background:
When analyzing any ECL system new to a researcher, cyclic voltammetry is the first
electrochemical method performed on the system. This allows for the potentials at which the
material reacts to be elucidated. This way, these potentials can be applied to the surface of an
electrode to allow for ECL emission to be obtained. Once a good CV is obtained for the system,
experimentation on emission of ECL can be performed, and various detection methods can also
be performed on the emission.
Procedure:
1.
Prepare a solution that is 1 mM in Ru(bpy)3 and 10 mM in TPrA solution using 0.5 M
KNO3 as the solvent.
2.
Turn on the electrochemical analyzer. The power switch is on the back right of the
instrument. Open the program “chi600c.exe” on the desktop. Perform a hardware test to check to
make sure all the connections between the electrodes are okay by selection “Setup  Hardware
Test.” If the connection fails, select “Setup  System…,” make sure COM 1 is selected and then
close the screen and retry the Hardware Test.
3.
Check and adjust, if necessary, the system setup. Select “Setup System…” and use the
following settings when taking a CV: Potential Axis should be selected as Positive Left, the
Current Axis should be selected as Positive Up, and the Current Polarity should be selected as
Cathodic Positive.
4.
Take a CV of this solution using a 2 mm Pt working electrode, a Pt auxiliary electrode
and a Ag/AgCl reference electrode. Set the parameters for the scan by selecting “Setup
Parameters…” Use the following settings when taking the CV: 0.025 V/sec, 1x10-5 A sensitivity,
initial E: 0 V, high E: +1.2 V, low E: 0 V cycles: 2. Print this CV.
5.
Record the values for the potentials and current for all of the peaks involved in the space
provided. Indicate whether the peak is an oxidation or a reduction and if any of the peaks are
related to one another. You can find this data by using the cursor on the CV on the computer
after completing a scan.
6.
Next, make a solution which is 1 mM in Ru(bpy)3 only (no TPrA) using the 0.5 M KNO3
as the solvent and repeat steps 4 and 5. Print this CV.
7.
Repeat steps 4-6 using the Au working/ Ag auxiliary microelectrode given as opposed to
the 2 mm Pt working and Pt auxiliary electrodes. Print these CVs.
8.
Attempt to create ECL emission with the 2 mm working electrode and the Pt auxiliary
electrode by applying a potential at the two peaks near +0.8 and 1.0 V. Note that the reference
electrode is not needed to perform this step. Do so by using the BAS CV-1B and monitoring the
potential with a multimeter. Place the initial scan at (+) and set it to zero by turning the zero
adjust knob. Slowly increase the potential until ECL emission is observed on the surface of the
working electrode. The solution may need to be stirred in order to observe the ECL emission
more easily. Record the potential where emission is observed in the space provided. Note that
ECL emission can also be observed when the electrochemical analyzer is scanning the solution.
9.
Now try to obtain ECL emission using the microelectrode as opposed to the 2 mm Pt
electrode. Note that the ECL emission will be much harder to see because the surface is so much
smaller on the microelectrode than on the 2mm Pt working electrode. Be sure to record the
potential at which you observe the emission in the space provided.
10.
Finally, using the 2 mm Pt working electrode and the Pt auxiliary electrode, apply the
opposite of the potential used in Step 7, but with the leads reversed (working lead to auxiliary
electrode and auxiliary lead to the working electrode). Note any observations you make in the
space provided (including any data such as a potential).
11. Repeat Step 10 with the microelectrode and note any observations you make in the space
provided (including any data such as a potential).
Data/ Observations:
1.
Record the potentials and currents evolved from the electrochemical reactions observed
when taking the CV of the Ru(bpy)3/ TPrA solution with the macroelectrodes.
2.
Record the potentials and currents evolved from the electrochemical reactions observed
when taking the CV of the Ru(bpy)3 solution with the macroelectrodes.
3.
Record the potentials and currents evolved from the electrochemical reactions observed
when taking the CV of the Ru(bpy)3/ TPrA solution with the microelectrode.
4.
Record the potentials and currents evolved from the electrochemical reactions observed
when taking the CV of the Ru(bpy)3 solution with the microelectrode.
5.
Record the potentials at which ECL emission was observed when applying the positive
potentials to the working electrodes in Steps 8 and 9.
6.
Record any observations you make while performing Steps 10 and 11.
Calculations/ Questions:
1.
Does the use of a microelectrode as opposed to the macroelectrode have an effect on the
potential or current of the peaks? What makes the change occur, if any?
2.
What conclusions can be reached when studying the data from Steps 4-7? Can you assign
individual peaks specific reactions for the analytes in solution? If so, write them here along with
the potentials of the reactions.
3.
How are the potentials applied to the working electrode that created ECL emission
related to those detected by cyclic voltammetry? What does this tell you about the system?
4.
Did you observe ECL emission when applying the negative potentials to the opposite
electrodes in solution? Why or why not? If so, were the potentials exactly opposite in sign? Why
or why not? What property of electric potential is indicated by this answer?
Introduction to ECL Experiment #4: Spectroscopy
Purpose:
The purpose of this laboratory is to make the researcher more comfortable with advanced
types of spectroscopy as well as the more advanced instrumentation used to take spectra of
solutions and emission.
Materials:
Ocean Optics USB 4000 Spectrometer
Ocean Optics Light Source (ask Dr. Sharpless to use this instrument)
Ocean Optics Fiber Optic Cable
Perkin-Elmer Lambda 40 UV/ Vis Spectrometer
Paired Quartz Cuvettes (ask instructor to show you which cuvettes are appropriate to use)
1 – 50 mL Volumetric Flask
5 – 25 mL Volumetric Flasks
Chemicals:
K3Fe(CN)6
Background:
Before beginning this lab, students should review spectroscopic methods of analysis from
Analytical Chemistry including Beer’s Law and the electromagnetic spectrum. Spectroscopy is a
way of determining the wavelength(s) which a solution or substance absorbs light. Light
absorption in the various portions of the electromagnetic spectrum lead to different chemical
characteristics changing over time. For instance, absorption of light in the ultraviolet and visible
regions excites electrons from their ground state while absorption in the infrared and microwave
regions allow for vibration and rotation, respectively. Since all ECL emission observed occurs in
the visible region, this will be the region of focus for this laboratory. Typically, solutions which
are colored (like Ru(bpy)3 and K3Fe(CN)6) also absorb in the visible region. The theory which
relates the energetics of the electrons to the colors is described thoroughly in Inorganic
Chemistry (Crystal Field Theory). UV/Vis spectra of molecules can lead to both its concentration
and often its chemical identity. The same principle can be applied to emission except instead of
studying the absorption of a solution, one would study the intensity of emission.
Beer’s Law is a scientific law which relates the absorption of light at a certain
wavelength to the concentration of the solution. Beer’s Law states that A=bc, where  = molar
absorptivity (L/mol*cm), b = cell length (cm), and c = concentration (mol/L). By making various
known concentrations of the solution, a calibration curve can be created in order to find the
concentration of unknown solutions.
Procedure:
Part I: Lambda 40 Spectrometer
1.
Make a 50 mL solution of 1 mM K3Fe(CN)6 using distilled water as the solvent.
2.
Make successive dilutions using 1, 2, 5, 7, and 10 mL amounts of the stock solution, and
diluting them to 25.0 mL. Calculate the concentrations of each of the solutions and record the
results in the space provided.
3.
On top of the Perkin-Elmer Lambda 40 UV/Vis Spectrophotometer should be a set of
directions on how to not only use the instrument, but also the computer program to analyze the
data from the spectrometer. Those same directions are given at the end of this experiment. Read
these directions before beginning, and refer to them as necessary throughout the experiment.
Turn on the spectrometer and open the computer program. The lamp takes about 5 minutes to
warm up. Following the directions, open the DEMOSCAN method. Use the following
parameters when performing the scan:
Start Wavelength: 750 nm
End Wavelength: 350 nm
Slit Width: 1 nm
Data Interval: 1 nm
Scan Rate: 240 nm/min.
4.
Open the top to the sample holder of the spectrometer. Inside there will be two places for
the square quartz cuvettes to fit. The one towards the back of the instrument is the reference cell
holder, while the one towards the front is the sample cell holder. In the reference cell slot, you
will place one of the paired cuvettes which has the solvent you used in it. In the sample cell, you
will place one of the paired cuvettes which has the sample you wish to analyze.
5.
Fill one of the paired quartz cuvettes 2/3 full with the solvent that was used to create the
solutions of K3Fe(CN)6. Fill the other 2/3 full with the same solvent. Make sure to wipe the
cuvette with a piece of lens paper. Press Scan Zero. This will take a background scan of the
solvent which it will subtract from each of your successive scans in case the cuvettes are not
perfectly matched.
6.
Once the background scan has been completed, take the sample cuvette out, empty it and
tap it face down on a Kimwipe. Be careful not to leave fingerprints on the glass, for this will
affect the readings. Then refill the cuvette with the lowest concentration solution of K3Fe(CN)6,
and press “Start.” As the scan is run, the spectrum will appear on the screen. When it is
completed, save it in a good place with a name which can be recognized later. Make sure to
record the name used.
7.
Repeat successive scans of each of the solutions from the lowest concentration to the
highest making sure to save each of the spectra.
8.
Once all of the scans have been completed, open each of them individually and determine
the wavelength of maximum absorbance using the Horizontal Peak Cursor tool. Record the
wavelength and absorbance for each of the scans in the space provided.
Part II: Ocean Optics Spectrometer
1.
Plug the USB 2000 spectrometer into the designated computer. Attach one end of the
fiber optic cable to the spectrometer and the other end to the light source. Plug the light source in
and switch it to the ON position.
2.
Open the Ocean Optics Software. It will prompt you to select a Spectrometer. Select the
2000 model and press okay.
3.
A spectrum of the lamp will appear on the screen. If it does not, check to make sure the
lamp is on and the fiber optic cable is connected. If they are and there is still no spectrum, close
the application and start again. Adjust the integration time (about 24-30 ms), averages (3), and
boxcar (1-3) settings to get a smooth spectrum that fits onto the screen. Then insert the wrench
used to attach the fiber optic cable into the sample chamber next to the light source. Select “Store
Dark” from the “Spectrum” menu.
4.
Fill a quartz cuvette with the solvent you are using and insert it into the sample chamber.
Select “Store Reference” from the “Spectrum” menu. Once this is completed, make sure not to
move the spectrometer, the light source, or the fiber optic cable as this will change the amount of
light which will reach the spectrometer, and will affect your results. Click on the “A” button on
the menu. This changes the spectrometer from measuring the intensity of the light to the
absorbance of your sample.
5.
Fill the quartz cuvette with the lowest concentration solution of K3Fe(CN)6. Save the
data. Repeat this step for the other solutions making sure to order the solutions from lowest
concentration to highest.
6.
Open the spectra using Excel. Make a graph of the spectrum by creating a scatter plot of
the data. Make sure to label and resize the axes. Using the graph as a reference, find the
wavelength where the spectrum shows a maximum in the absorbance between 350 and 750 nm
and record both the wavelength and absorbance data for this point in the space provided. Repeat
this step for all of the solutions.
Data/ Observations:
1.
Record the absorbance data for the maximum absorbance from the spectra acquired using
the Lambda 40 Spectrometer.
Concentration (mM)
λmax
Absorbance at λmax
2.
Record the absorbance data for the maximum absorbance from the spectra acquired using
the Ocean Optics Spectrometer.
Concentration (mM)
λmax
Absorbance at λmax
Calculations and Questions:
1.
Create a Beers-Lambert Plot of the data you acquired from the Lambda 40 Spectrometer
using Excel. What is the correlation coefficient for this plot? What does it mean?
2.
Create a Beers-Lambert Plot of the data you acquired from the Ocean Optics
Spectrometer using Excel. What is the correlation coefficient for this plot? What does it mean?
3.
How can you explain any differences in the plots between the two spectrometers? How
can you explain any differences between the λmax between different data points within the same
plots and between the two different plots?
Lambda 40 Brief Operating Instructions
1.
Turn on the Perkin Elmer Lambda 40 Spectophotometer (top right hand side) and allow
the lamps to warm up for approximately 5 minutes.
2.
Remove and cuvettes from the spectrometer.
3.
Turn on the computer next to the spectrometer and click on the LAMBDA 40 icon. The
METHODS window appears.
4.
Select SCAN and then DEMOSCAN.MSC. Use the tabs at the bottom of the window to
choose your wavelength range: 850-350 nm (default)
data interval: 1nm
ordinate range (A): 0.00-2.00
slit: 2nm
scan speed: 480 nm/min
5.
Place the cuvettes carefully back into the spectrometer with your reference solution (if
applicable)
6.
Press Autozero to perform a background correction.
The computer now asks you to insert the ‘Blank’ (you have already done this #5).
Press start (STOP AZ will be highlighted until the background finishes; nothing
else will appear).
7.
Press Start
8.
Insert your sample and click OK; the spectrum is displayed. To save the file, use FILE,
SAVE AS.
Gathering information from your spectrum or changing its appearance: to change the absorbance
range (ordinate), click on the Y-X ICON; this allows the scaling fro the ordinate (absorbance)
and abscissa (wavelength) to be altered.
To obtain wavelength information for peaks, select VIEW then VERT CURSOR CONT (the
second icon from the right also does this); drag the cursor to the desired position, information is
displayed below the spectrum.
To add text, use the ABC icon.
To overlay one or more saved spectra, choose VIEW, ADD SPECTRUM.
To remove a spectrum, click on the filename shown below the spectrum and hit DELETE.
9.
When you are finished using the instrument, remove the cuvettes, clean them out and
carefully store them in their boxes (in the drawers beneath the instrument). Shut down the
computer and turn off the spectrometer.
Introduction to ECL Experiment #5: Advanced Cyclic Voltammetry and
Emission Detection on a Non-Aqueous ECL System
Purpose:
This experiment is designed to familiarize the experimenter to a non-aqueous ECL
system using the cyclic voltammetry techniques learned thus far and to introduce a new emission
detection technique. It will also demonstrate to the researcher all the steps that are needed when
looking at a new ECL active system.
Materials:
Ch Instruments Electrochemical Analyzer
Ocean Optics USB 4000 Spectrometer
Ocean Optics Fiber Optic Cable
Beckman Industrial Function Generator
HAMEG 20Mhz Dual Trace Oscilloscope
Multimeter
4 Alligator Clips
2 mm Pt Working Electrode
Pt Wire Auxiliary Electrode
Ag Wire (Quasi Reference Electrode)
1-50 mL volumetric flask
1-50 mL beaker
1 gas tight cell
Chemicals:
Tris(2,2’-bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3)
Tetrabutylammonium hexafluorophosphate (TBAH)
Acetonitrile, spectrophotometric grade, 99.5% (MeCN)
Nitrogen gas or Argon gas
Procedure:
1.
Make a 50mL solution that is 1 mM Ru(bpy)3 and 10mM TBAH with acetonitrile as the
solvent. Make sure glassware is dry, in order to reduce oxygen contamination into the system.
2.
Using either Nitrogen gas or Argon gas bubble the solution in a gas tight cell for 20
minutes. Immediately affix the lid to the cell, with the three electrodes already attached through
it, when the solution is finished bubbling, in order to decrease the rate of oxygen contamination.
Note, since this is an organic system, rinse the electrodes with acetone before using to avoid
oxygen contamination.
Part 1: Take a CV
3.
Turn on the electrochemical analyzer. The power switch is on the back right of the
instrument. Open the program “chi600c.exe” on the desktop. Perform a hardware test to check to
make sure all the connections between the electrodes are okay by selection “Setup  Hardware
Test.” If the connection fails, select “Setup  System…,” make sure COM 1 is selected and then
close the screen and retry the Hardware Test.
4.
Check and adjust, if necessary, the system setup. Select “Setup System…” and use the
following settings when taking a CV: Potential Axis should be selected as Positive Left, the
Current Axis should be selected as Positive Up, and the Current Polarity should be selected as
Cathodic Positive.
5.
Take a CV of this solution using a 2 mm Pt working electrode, a Pt auxiliary electrode
and a Ag wire quasi reference electrode. Set the parameters for the scan by selecting “Setup
Parameters…” Use the following settings when taking the CV: 0.025 V/sec, 1x10-6 A sensitivity,
initial E: 0 V, high E: +1.8 V, low E: -1.7 V cycles: 2. Print this CV.
6.
Record the values for the potentials and current for all of the peaks involved in the space
provided. Indicate whether the peak is an oxidation or a reduction and if any of the peaks are
related to one another. You can find this data by using the cursor on the CV on the computer
after completing a scan.
Part 2: Make ECL
7.
Turn on the Function Generator and the Oscilloscope by pushing the red power buttons.
Set the function generator to 0.2 Hz. Connecting the function generator to the oscilloscope is a
black co-axial cable which has been stripped in the middle exposing a copper wire and a copper
screen. Attach one end of an alligator clip to the copper screen and the other to the multimeter.
Do the same for the copper wire. Set the multimeter to DC voltage and turn on the multimeter.
Configure the instrument to make a +/- 2V square wave by using the DC OFFSET knob to
show/move/place where the potential is jumping across, then use the amplitude to increase or
decrease the distance the potential is jumping across.
8.
Set the function generator to 2.0 KHz. Using an alligator clip connect the 2mm Pt
working electrode to the copper screen and using another alligator clip connect the Pt wire
counter electrode to the copper wire. Turn off all lights and look for light emission on the
working electrode. If light is not seen slowly increase the amplitude on the function generator
and slightly stir/shake the solution.
Part 3: Take a Spectrum of ECL
9.
Plug the USB 2000 spectrometer into the designated computer. Attach one end of the
fiber optic cable to the spectrometer and uncap the other end in the light tight box. Open the
Ocean Optics Software. It will prompt you to select a Spectrometer. Select the 2000 model and
press okay.
10.
Place the Ru(bpy)3 solution in the light tight box and align the working electrode with the
end of the fiber optic cable. Connect the alligator clips to the electrodes using the same
configuration as in part 2.
11.
After turning off the lights and closing the doors of the light tight box, set the integration
time to 10 seconds and the boxcar to 3. Then select “File Store Store Dark Spectrum” and
then select “File StoreStore Reference Spectrum.” Using the exact same settings as when
light was seen in part 2, turn the Function Generator and the Oscilloscope on.
12.
After a spectrum appears on the graph, select “Processing Processing Mode  Scope
Minus Dark.” This function will remove some of the background noise. Record the wavelength
of the emission peak in the space provided.
13.
Skip this step if a distinct peak is seen, but if a distinct peak does not appear on the graph
adjust the integration time increasing it by one to two seconds and then repeat steps 11 and 12. If
a peak is still not seen after an integration time of 15 seconds, check the settings on the Function
Generator and make sure light emission is seen with the naked eye, and if it is adjust the
alignment of the working electrode with the lens of the fiber optic cable.
Data/Observations:
1.
Label each peak (1-10) on the printed CV, and fill in the table according to your labeling.
What is the potential and the current at each peak? As well as what reaction is taking place at
each peak? And is that reaction an oxidation or reduction reaction?
Peak
1
Potential (V)
Current (A)
Reaction
2
3
4
5
6
7
8
9
10
2.
Record the potential at which ECL emission was observed.
Oxidation/Reduction
3.
Record the wavelength of the emission peak.
Calculations/Questions:
1.
Why is the TBAH added to the solution? And what is its purpose?
2.
Why is the solution bubbled with gas?
3.
Why is a Ag wire quasi reference electrode used instead of the Ag/AgCl reference
electrode?
4.
Why is a Function Generator used to supply a voltage in order to obtain light emission?
And what type of reaction scheme is taking place?
5.
Was the potential at which light emission observed, higher or lower than the expected +/2V? If so, what are some reasons for the increase or decrease in the expected potential?
6.
Compare the actual value of the wavelength for the solution’s emission peak to the
experimentally recorded value of the wavelength obtained. If there is a discrepancy, why might
that be?
Introduction to ECL Experiment #6: Electrochemical Flow Cell
Purpose:
The purpose of this experiment is to introduce the experimenter to an electrochemical
flow cell as well as the instrumentation used in taking measurements from the flow cell and the
significance that the flow cell has for ECL.
Materials:
Ch Instruments Electrochemical Analyzer
Ocean Optics USB 4000 Spectrometer
Ocean Optics Fiber Optic Cable
Electrochemical Flow Cell- Stainless Steel Top and Back, 4 Screws, 4 Nuts, Plastic Gasket
Rheodyne Injector
2 mm Pt working electrode
Pt auxiliary electrode
Ag/AgCl reference electrode
Au Working/ Ag Auxiliary Microelectrode
1-50mL Volumetric Flask
1-25mL Volumetric Flask
2-50mL Beakers
6 Alligator Clips
Chemicals:
0.5 M KNO3
Tris(2,2’-bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3)
Tripropylamine (TPrA)
Background:
Electrochemical flow cells are of the utmost importance when studying Electrogenerated
Chemiluminesence (ECL) for numerous reasons. The first of which is that it allows for the
replenishment of the analyte at the surface of the electrode. Note that this is especially important
when studying organic systems such as diphenylathracene (DPA) because any type of impurity
can quench the radicals being formed, thereby not allowing them to react and create ECL
emission. Another reason is that the smaller electrode allows for a reproducible alignment with
the measuring device used to quantify the ECL emission such as a PMT or spectrometer.
Procedure:
1.
Prepare a solution that is 1 mM in Ru(bpy)3 and 10 mM in TPrA using 0.5 M KNO3 as
the solvent.
Part 1:
2.
Turn on the electrochemical analyzer. The power switch is on the back right of the
instrument. Open the program “chi600c.exe” on the desktop. Perform a hardware test to check to
make sure all the connections between the electrodes are okay by selection “Setup  Hardware
Test.” If the connection fails, select “Setup  System…,” make sure COM 1 is selected and then
close the screen and retry the Hardware Test.
3.
Check and adjust, if necessary, the system setup. Select “Setup System…” and use the
following settings when taking a CV: Potential Axis should be selected as Positive Left, the
Current Axis should be selected as Positive Up, and the Current Polarity should be selected as
Cathodic Positive.
4.
Take a CV of this solution using a 2 mm Pt working electrode, a Pt auxiliary electrode
and a Ag/AgCl reference electrode. Set the parameters for the scan by selecting “Setup
Parameters…” Use the following settings when taking the CV: 0.025 V/sec, 1x10-5 A sensitivity,
initial E: 0 V, high E: +1.2 V, low E: 0 V cycles: 2. Print this CV.
5.
Still using the electrochemical analyzer, select “FileNew” to get a new window. Then
select “SetupTechniqueChronamperometry.” The chronamperometry parameters window
will appear on the screen, use the following settings: Init E (V): 0, High E (V): 1.2, Low E (V):
0, Initial Step Polarity: Positive, Number of Steps: 2, Pulse Width (sec): 1000, Sample Interval
(sec): 0.001, Quiet Time (s): 2, Sensitivity (A/V): 1e^-006.
6.
Attach both the counter and reference electrode connectors to the auxillary electrode, and
attach the working electrode connector to the working electrode, and have both electrodes in the
solution. Turn off all lights. Click “run” scan and check for visible light emission on the working
electrode.
7.
Repeat steps 5 and 6 in the light tight box using the Ocean Optics Spectrometer and Fiber
Optic Cable to detect the emission. Do this by first plugging the USB 2000 spectrometer into the
designated computer. Attach one end of the fiber optic cable to the spectrometer and uncap the
other end in the light tight box. Open the Ocean Optics Software. It will prompt you to select a
Spectrometer. Select the 2000 model and press okay.
8.
Set the integration time for the spectrometer at 20 seconds and the boxcar width at 3.
Align the detector of the fiber optic cable directly under the button on the working electrode.
Turn off all lights. Click “run” scan and wait for the detector to pick up the emission. Record the
wavelength of the ECL emission. Note, if no emission peak appears on the screen readjust the
alignment of the detector to the working electrode.
Part 2:
9.
A diagram for the set up of part 2 can be found below.
ECL Flow Cell
Rheodyne Injector
Electrochemical Analyzer
PEEK HPLC Tubing
To Computer
To Computer
USB Fiber Optic
Spectrometer
Light Tight Box
Fiber Optic Cable
10.
Assemble the electrochemical flow cell as shown below. Once the entire apparatus is
assembled, take note that the electrode does not have to sit flush against the bottom piece, so
make sure not to over tighten the screws, for over tightening will cause the glass on the top piece
to shatter.
Inlet
Rubber Gasket
11.
Arrange the flow cell in the light tight box, so that the glass window is aligned with the
fiber optic lens. Attach both the counter and reference electrode connectors to the auxillary wire
of the microelectrode, and attach the working electrode connector to the working wire of the
microelectrode. Keep the settings on the electrochemical analyzer the same as in step 5.
12.
Turn on the Rheodyne injector by flipping the switch on the back right of the instrument.
Place the inlet filter in the KNO3 solvent. Use the mode button to highlight the mL/min and set it
to 1 mL/min. Push the Prime button. Once it is finished priming, push the Run button and allow
the solvent to rinse the interior of the PEEK HPLC tubing. The tubing should not be connected to
the flow cell yet. After the solvent has flushed for a few minutes, push the Stop button. Now
place the inlet filter in the Ru(bpy)3 / TPrA solution and attach the tubing to the flow cell.
13.
Turn off the lights and close the doors of the light tight box. Set the spectrometer’s
integration time to 60 seconds and the boxcar to 3. Then select “File Store Store Dark
Spectrum” and then select “File StoreStore Reference Spectrum.” Push the Run button on
the Rheodyne injector and push the Run Scan button on the electrochemical analyzer.
14.
Once a spectrum appears on the graph, select “Processing Processing Mode  Scope
Minus Dark.” This function will remove some of the background noise. Record the wavelength
of the emission peak in the space provided.
Data/Observations:
1.
Record the potentials and currents evolved from the electrochemical reactions observed
when taking the CV of the Ru(bpy)3/ TPrA solution with the macroelectrodes.
2.
Record the wavelength of the emission peak using the macroelectrode.
3.
Record the wavelength of the emission peak using the microelectrode.
Calculations/Questions:
1.
How was part 1 applicable? Why were those steps necessary to complete before the
microelectrode in the flow cell was used?
2.
The function generator could have been utilized, cycling between 0.8V and 1.1V, instead
of the chronamperometry technique on the electrochemical analyzer. Why was
chronamperometry used?
3.
Why were the initial, high, and low voltages for the chronamperometry paratmeters set to
the values given? If those values had not been given, how would one have known them using the
previously gathered data in the experiment?
4.
Why was the pulse width for the chronamperometry parameters set to 1000 seconds?
What would happen to the data collected in the experiment exceeded 1000 seconds?
5.
Compare the wavelengths of the emission peaks for the system using the macroelectrode
and microelectrode. Are there any differences? If so, why?
6.
Why was the Rheodyne injector flushed with solvent?
Introduction to ECL Experiment #7: Fluorescence Spectroscopy
Purpose:
The purpose of this experiment is to introduce the experimenter to the phenomenon of
fluorescence as well as the instrumentation used in taking its measurements and the
interpretation of fluorescence spectra.
Materials:
SLM 48000 Spectrofluorimeter
Perkin-Elmer Lambda 40 Spectrometer
1 – 250 mL Volumetric Flask
4 - 100 mL Volumetric Flasks
1 – Fluorescence Quartz Cuvette
Paired Quartz Cuvettes
Chemicals:
Quinine Sulfate
Tris(2,2’-bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3)
0.05 M Sulfuric Acid
Background:
Before completing this experiment, researchers should read the fluorescence chapter from
an instrumental methods textbook as well as some selections from Nicholas Turro’s book
Modern Molecular Photochemistry. Fluorescence is the emission of energy from electronically
excited species in the form of a photon. The energy necessary to form the excited state can come
from a number of sources, but the most common is the absorption of a photon of light. Once the
energy from the photon is absorbed, energy transfers occur so that once the energy is emitted
from the excited species, the wavelength of the emission is higher than the wavelength of light
absorbed.
Procedure:
1.
Prepare a 100 mL solution of 0.05 M H2SO4 by diluting the 3 M stock solution found in
the Analytical Lab with ultrapure DI water.
2.
Prepare 50 mL of a 100 M solution of quinine sulfate using the 0.05 M H2SO4 as the
solvent.
3.
Prepare 50 mL of a 100 M solution of Ru(bpy)3 using ultrapure DI water as the solvent.
4.
Take an absorbance spectrum of the quinine sulfate solution using the Perkin-Elmer
Lambda 40 spectrometer by running the DEMOSCAN operation. Make sure to autozero the
instrument with your solvent. Use the following parameters when taking the spectrum:
Start Wavelength: 500 nm
Stop Wavelength: 200 nm
Data Interval: 1 nm
Scan Speed: 240 nm/min
Slit Width: 1 nm
Note the wavelength and absorbance of the peak near 350 nm. Save the spectrum and the data in
ASCII format to a floppy disc.
5.
Take an absorbance spectrum of the Ru(bpy)3. Make sure to autozero the instrument with
your solvent. Use the following parameters when taking the spectrum:
Start Wavelength: 750 nm
Stop Wavelength: 350 nm
Data Interval: 1 nm
Scan Speed: 240 nm/min
Slit Width: 1 nm
Note the wavelength and absorbance of the peak near 450 nm. Save the spectrum and the data in
ASCII format to a floppy disc.
6.
Clean the cuvettes and shutdown the spectrometer.
7.
For collecting fluorescence spectra, the instruction sheet for the instrument entitled “Brief
Operating Instructions for the SLM 48000 Spectrofluorimeter” should be used. The instruction
sheet can be found at the end of this experiment. Follow the instructions for “Power On & Start
Up” and “Setting Up to Collect A Spectrum” in order to prepare to collect an emission spectrum
of quinine sulfate. Be sure to set the slit widths on the fluorimeter to 4 nm. The signal from both
PMTs should be about 4. Be sure to record your excitation wavelength, emission wavelength,
and the voltages and gains of both PMTs in the space provided. The excitation wavelength
should be somewhere near the maximum of 350 nm and the emission wavelength should be
somewhere near the maximum of 450 nm. Push “Enter” and use the following settings for taking
an emission spectrum of the quinine sulfate:
360-650 nm
4 nm Intervals
1 Average
A/B Function ONLY
Save the data as “.txt” format onto a floppy after collecting the spectrum.
8.
Now, collect an excitation spectrum of quinine sulfate. Again, be sure to record the PMT
voltages and gains, emission wavelength, and excitation wavelength. The excitation wavelength
should be set somewhere near the maximum of 350 nm and the emission wavelength should be
somewhere near the maximum of 450 nm. Push “Enter” and use the following parameters to
collect the excitation spectrum:
230-400 nm
4 nm Intervals
1 Average
A/B Function ONLY
Save the data as “.txt” format onto a floppy after collecting the spectrum. Clean the cuvette
thoroughly.
9.
Next, an emission spectrum for Ru(bpy)3 will be collected. Be sure to record your
excitation wavelength, emission wavelength, and the voltages and gains of both PMTs in the
space provided. The excitation wavelength should be somewhere near the maximum of 450 nm
and the emission wavelength should be somewhere near the maximum of 620 nm. Push “Enter”
and use the following settings for taking an emission spectrum of the Ru(bpy)3:
500-800 nm
4 nm Interval
1 Average
A/B Function ONLY
Save the data as “.txt” format onto a floppy after collecting the spectrum.
10.
Finally, collect an excitation spectrum for Ru(bpy)3. Be sure to record your excitation
wavelength, emission wavelength, and the voltages and gains of both PMTs in the space
provided. The excitation wavelength should be somewhere near the maximum of 450 nm and
the emission wavelength should be somewhere near the maximum of 620 nm. Push “Enter” and
use the following settings for taking an emission spectrum of the Ru(bpy)3:
500-800 nm
4 nm Interval
1 Average
A/B Function ONLY
Save the data as “.txt” format onto a floppy after collecting the spectrum. Clean the cuvette
thoroughly and follow the instructions for “Shut Down & Power Off.”
Data/ Observations:
1.
What was the λmax for the quinine sulfate solution and what was the absorbance?
2.
What was the λmax for the Ru(bpy)3 solution and what was the absorbance?
3.
Record all of the settings used to collect the emission spectrum of the quinine sulfate
solution.
HVA
Gain on PMT A
HVB
Gain on PMT B
λem
λex
4.
Record all of the settings used to collect the excitation spectrum of the quinine sulfate
solution.
HVA
Gain on PMT A
HVB
Gain on PMT B
λem
λex
5.
Record all of the settings used to collect the emission spectrum of the Ru(bpy)3 solution.
HVA
Gain on PMT A
HVB
Gain on PMT B
λem
λex
6.
Record all of the settings used to collect the excitation spectrum of the Ru(bpy)3 solution.
HVA
Gain on PMT A
HVB
Gain on PMT B
λem
λex
7.
Be sure to attach copies of ALL the spectra collected in this experiment by making them
in EXCEL.
Calculations/ Questions:
1.
Why was the absorbance spectrum for quinine sulfate collected between 500 and 200 nm,
and the spectrum for Ru(bpy) collected between 750 and 350 nm? (Your answer shouldn’t just
be “because that is where the absorb,” it should include the reason(s) they absorb in this region.)
2.
Why were the emission spectra collected at higher wavelengths than the absorbance
spectra?
3.
Note any and all similarities you can note among the different spectra taken for the
various solutions. Explain why these similarities are seen (see Turro).
4.
Explain why there is a dip in the Ru(bpy)3 emission spectrum using Wood’s anomaly.
5.
What information can one gather from fluorescence emission spectra that would help
when analyzing a possibly new ECL emitter?
Brief Operating Instructions for the SLM 48000 Spectrofluorimeter
ALWAYS KEEP THE EMISSION SHUTTER CLOSED WHEN OPENING THE SAMPLE CHAMBER
Power On & Start Up
 Make sure the shutters are all closed.
 Turn on the fan to the lamp.
 Make sure the lamp power supply is as far from other equipment as it can get, then switch
it on.
 After the lamp lights, turn on the power to the control module and the computer.
 At the C: prompt, type “48000” and hit return.
 Run the monochromator calibration requested by the software.
Setting Up to Collect A Spectrum
 At the main menu, choose the type of measurement you will be taking (emission or
excitation).
o Note: the main menu can be accessed from anywhere in the program by hitting
“esc” a few times.
 Turn on the voltage (HV) to PMT B (the reference PMT) – this is done by using the arrow
keys to highlight the PMT B channel and then pressing H.
o Note: many commands are summarized on the screen under the settings tables.
 Set the voltage for 300V with a gain of x100.
o Note: To set the voltage, press F6, and then press F8 – this will allow you to
directly enter a value for the voltage. The gain is set by highlighting the channel
of interest and then pressing the F5 key.
 Set the excitation wavelength to 400 nm.
 Manually open the lamp shutter.
 While keeping an eye on the signal from channel B, open the shutter to the sample
chamber. It the signal goes off scale, close the shutter and lower the HV to the PMT. Raise
or lower the voltage until the signal is ~2 to 3.
o Note: The F9 (increase) and F10 (decrease) keys can be used to adjust the
voltage.
 Place a sample in the sample chamber.
 While keeping an eye on the signal from channel B, set the excitation wavelength to near
where you expect to see a maximum in the fluorescence signal. If necessary, adjust HV B to
get a signal near 3.
 Set the emission wavelength to near where you expect to see a maximum in the
fluorescence signal.
 Open the shutter to the emission monochromator.
 Without turning on the HV to PMT A, set the HV to 350 with a gain of x100.
 While keeping an eye on the signal from channel A, open the shutter to the sample
chamber. If the signal goes off scale, close the shutter and lower the HV to the PMT.
 If the signal does not go off scale, slowly increase the HV to channel A until you begin to
see a signal.
o Note: If you reach 700V without seeing a signal, yet you expect to see something,
alert your instructor.

When the signal is at ~3-4, adjust the excitation wavelength slightly up and down until you
find the wavelength at which the signal is a maximum. Repeat this for the emission
wavelength. If the PMT goes off the scale during these adjustments, close the shutter and
lower the HV slightly before proceeding.
 Once you have found the excitation/emission maximum, set the HV on channel A to
achieve a signal of approximately 7 to 8. You are now ready to acquire a scan.
 Hit “enter” and adjust the parameters on the next screen to the desired values.
 When you are ready, hit “enter,” and the scan will begin. Note that due to a software glitch,
the instrument will run a scan once without storing data and then will begin again. This is
an annoyance, but the data will get collected.
Shut Down & Power Off
 When you are finished:
o Make sure all the shutters on the instrument (including the lamp) are closed.
o Turn off the voltage to the PMTs.
o Copy your data to a floppy disk (your instructor will show you how to do this)
o Exit the software program.
o Turn off the computer.
o Turn off the control module.
o Turn off the power supply to the lamp, but leave the fan on for 5 minutes.
o After 5 minutes, turn off the fan.