Course 7 Acquisition and treatment of experimental data

Complex techniques for electrochemical processes investigation using devices equipped

with microcontroller
1. Equipments controlled by PC including A/D and D/A conversion integrated systems and
microcontrolers

Most laboratory equipments and, in particular, the last generation of computer controlled devices
present the opportunity to retrieve the obtained experimental data. In order to assure this facility, the
equipments include in their structure one or more microcontrollers (or microprocessors) that facilitate data
transfer to/from the computer, control the equipments operation and coordinate the data acquisition.
Depending on the speed of the studied process and the data volume handled between device and computer,
different dedicated communication interfaces can be used: RS232, RS485, USB, GPIB etc.
A first example of such equipment is the REGLO Digital peristaltic pump (Ismatec, Switzerland),
which can be controlled either manually (through its control panel) and with the computer via a RS232
serial port. To enhance the attractiveness of the product, the manufacturer provides, in the product Operating
Manual, the codes that must be written on the serial port for programming the pump operation. Moreover,
for experienced users, the manufacturer offers, free of charge, a collection of LabView applications for easy
control of the pump through this software (collection available in the directory \ Resources \ Digital Reglo
LV drivers \).
A second example of such equipment is the optical fiber spectrometer model USB4000-VIS-NIR
(Ocean Optics, USA), which can be controlled by the computer via a serial port USB 2.0. In order to exploit
the product, the manufacturer provide, as indicated in the Installation and Operation Manual, a software
application (SpectraSuite software) able to control the operating parameters of the spectrometer and data
acquisition. Unfortunately, the software package is delivered for ~ $ 200 and it offers only a reduced set of
features for programming and operation of the spectrometer, the most annoying limitations being related to
the acquisition and saving of data.
In return, in order to increase the attractiveness of the product, the manufacturer, in collaboration
with National Instruments team, provides, for the experienced users guides, a wide collection of LabView
applications and drivers for easy control of the spectrometer, including a number of examples that can be the
starting point for the development of highly complex applications, able to maximize the capabilities of the
equipment. For example, the integration time used for the spectra acquisition can be easily controlled using
a LabView application that allows, also, the retrieving of acquired data for treatment and saving.

1
Course 7 Acquisition and treatment of experimental data

2. Examples of complex techniques for electrochemical processes investigation using devices equipped
with microcontroller
2.1. Spectroelectrochemistry

2.1.1. Introduction
Spectroelectrochemistry combines both electrochemical and spectroscopic monitoring and opens a
new way to investigate and unequivocally identify electroactive species or products of redox reactions.
Combining results from electrical and optical responses allows detailed insights in underlying mechanisms
and more precise studies. Under potential control, spectroscopic information about in situ electrogenerated
species can readily be obtained, including electronic absorption, vibrational modes and frequencies, light
emission and scattering, etc.
Spectroelectrochemical techniques are widely used in applications ranging from inorganic chemistry
and organic chemistry to biochemistry, in the studies of redox pathways and series, electrocatalytic
reactions, mixed-valence complexes, redox active coordination and organometallic compounds, electron
transfer in proteins and biomolecules (bioelectrochemistry), redox polymer processes, etc.
For a better understanding of the spectroelectrochemistry, it is necessary to review the basic terms of
spectroscopy, described next.

2.1.1.1. The electromagnetic spectrum

In spectroscopy, the matter is studied by investigating its interactions with electromagnetic radiation.
This form of energy can be described as an electromagnetic wave consisting of a coupled electric wave field
E and magnetic wave field B, described in the next figure.

Another important fact about light is its particle nature. When light contacts on a metal surface, light
particles transfer their energy to electrons which are ejected from the surface. This phenomenon. called
photoelectric effect, cannot be completely described by the light’s wave nature. Hence light also has to be

2
Course 7 Acquisition and treatment of experimental data

treated as flux of light particles, called photons. As described in the next equation, the energy E of photons
that is needed to eject electrons from the metal surface is inversely proportional to the light wavelength ():
E=h*=h*c*
where  is the frequency of the electromagnetic wave, h is the Planck constant (6.626*10 -34 J*s), and c is the
speed of light (about 300*106 m*s-1).
The entire electromagnetic radiation is summarized in the electromagnetic spectrum, described in the
next figure. Typically, it is divided into domains, according its wavelength and/or energy.

The most known part of the electromagnetic spectrum is the visible light (VIS), containing all colors
that human can see. However, the visible part, ranged from about 400 to 750 nm, is only a small portion of
the whole spectrum. Additionally to the VIS range, for the spectroelectrochemical measurements, the
infrared (IR, from about 750 nm to 1 mm) and ultraviolet (UV, from about 400 nm to 10 nm) light can be
also used.

2.1.1.2. Absorption spectroscopy

The spectroscopy can be classified into various groups depending on how the electromagnetic
radiation is interacting with the matter. The energy can be absorbed, emitted, reflected or dispersed. The
spectroelectrochemistry is based on the absorption spectroscopy, where the electromagnetic radiation which
is focused on matter is absorbed. Depending on the kind of electromagnetic radiation or energy content,
molecules and atoms interact with electromagnetic radiation in various ways, described in the next table.

3
Course 7 Acquisition and treatment of experimental data

MW Change of molecule orientation (rotational)

IR Change of molecule configuration (vibrational)

UV-VIS Change of electron distribution (outer shell)

X-rays Change of electron distribution (inner shell)

The amount and wavelength range of the absorbed energy depend on the molecular structure. Hence,
every molecular structure yields a spectroscopic fingerprint by measuring radiated energy passing the
sample.

2.1.1.3. Important parameters and equations

The spectrometer’s detector converts photo current from light into electric current. The output
depends mainly on the intensity of light that passes a sample.

2.1.1.3.1. Intensity (I)

When the light passes a transparent medium, the intensity I0 of the incident light drops exponentially.
The decay depends on the optical pathlength d through matter and its wavelength-dependent absorption
coefficient k, according with the next equation:
I = I0 * e^(-k * d)

2.1.1.3.2. Transmittance (T)

The transmittance (T, also called Transmission) is the ratio of light passing a transparent medium to
the incident light. It is typically represented as a percentage.
T = I / I0

2.1.1.3.3. Absorbance (A)

The absorbance (A, or also called absorption) is, similar to Transmittance, an expression for the
amount of energy that is absorbed by a medium. It is the negative logarithm of transmittance T.
A = - log(I / I0) = - log (T)
4
Course 7 Acquisition and treatment of experimental data

For example, if the absorbance has the value three, only one-thousandth of the incident light reaches
the detector. In general, a best accuracy is achieved for an absorbance around one.

2.1.1.3.4. Integration time (t)

The integration time (t) defines how long the detector of the spectrometer collects light in order to
record a single spectrum. Depending on the experimental conditions, it can range from several milliseconds
to seconds. In general, longer integration times lead to a stronger output signal and reduced signal to noise
ratio. However, if the integration time is set too long, the detector can get saturated.

2.1.1.3.5. Lambert Beer Bouguer law

P. Bouguer, J.H. Lambert and A. Beer proved that the absorbance is directly proportional to the
concentration c of a transparent medium, according to the next equation:
A=*c*d
where ε is the molar absorption coefficient and d is the optical pathlength of the cuvette. Due to the fact that
the absorption spectroscopy is a very sensitive technique, slightest changes in concentration of a sample can
be accurately measured.

2.1.1.3.6. Spectrum
In the absorption spectroscopy, the measured quantity of interest (i.e. absorbance A, transmittance T,
or just raw counts) is plotted versus wavelength . This plot is called absorbance, transmittance, or raw
count spectrum, respectively. The next figure shows an example for an absorbance spectrum, with peaks at
about 270, 380 and 630 nm.

5
Course 7 Acquisition and treatment of experimental data

Typically, the wavelength is given in nanometers (nm). For the IR-spectroscopy, the spectrum’s
x-axis is mostly given by the inverse of wavelength, so-called wavenumbers, represented commonly in cm-1.

2.1.2. Setup for spectroelectrochemical experiments

The typical setup for the absorption spectroelectrochemical experiments is shown in the next figure.

The main components of the experimental setup are presented below.

2.1.2.1. Light source

Usual, a beam of light is focused on the sample via a optical fiber. The common light sources include
but are not limited to Deuterium, Tungsten, or Halogen lamps, LEDs, and lasers.
The light output can have a wide spectral bandwidth or it can cover only narrow spectral regions, of
several nanometers. The latter one is mostly used for fluorescence measurements, where the matter is
irradiated by a laser and the induced fluorescence is measured.

2.1.2.2. Spectroelectrochemical cell

The studied sample is placed within a special designed cuvette. The wavelength range of the light
passing the sample is strongly limited by the material of the cuvette. It can be made of plastic (for cheaper
and disposable cuvettes) right up to fused silica quartz glass. Latter ones are more expensive but allow
spectroscopy measurements down to lower wavelengths in the UV range.
The cuvettes define also the pathlength of the light through the sample. For the classical absorption
spectroscopy, the typical value for the optical pathlength d is 1 cm. Shorter pathlengths, of several
millimeters, are used at higher analyte concentrations or if only small sample volumes are available. Longer
pathlengths (centimeters) can be used at lower analyte concentrations, when the absorbance is, in general,
lower, too.

6
Course 7 Acquisition and treatment of experimental data

For the spectroelectrochemical measurements, in order to increase the ratio between the length of
modified and unmodified sample, the cell requires a special design. Generally, different models of thin-
layer cells (TLC) are used, of which two examples are presented in the next figure.

Transmission TLC Reflection TLC

In addition, as indicated in the previous figure, for the spectroelectrochemical experiments, the
working, counter, and reference electrodes are immersed into the analyte. In the case of the transmission
TLC, only the working electrode, as fine metal meshes or thin coated glass substrates, is inserted within the
light path. For the reflection TLC, the working electrode is made by a noble metal (usual, Pt), with a mirror
like finished surface.

2.1.2.3. Spectrometer
After passing the cell, the beam of light is redirected, by an optical systems or an optical fiber, into
the spectrometer through its entry slit. The slit width defines how much light enters into the spectrometer
and strongly determines its optical resolution.
Another important part of a spectrometer is the grating, its fine structure being an important factor
for the optical resolution. Before passing the grating, the beam of light is a unity of radiated energy with
different wavelengths. The grating separates this unity and diffracts light into its monochromatic parts –
beams of light with a narrow band of wavelength.
The beams of light are redirected via mirrors within the spectrometer. In the end, they reaches the
detector which, for most modern spectrometers, is a CCD (charge coupled device) array. CCDs consist of

7
Course 7 Acquisition and treatment of experimental data

semiconductors with a photoactive region, it being able to convert photo current from light into electric
current.

2.1.2.4. Potentiostat
The potentiostat is used to perform electrochemical experiments within the cuvette, adjusting the
applied potential to the working electrode in concordance with the imposed value and measuring the
resulting current. On one side, the potentiostat is connected to electrodes, and, on the other side, it is
connected to and controlled by a computer.

2.1.2.5. Computer
The spectrometer and potentiostat are connected to a computer via dedicated data interfaces. The
experiments are set and controlled by using special computer software. After finishing an experiment, the
recorded data are saved on the computer and can be analyzed further.

2.1.3. Example of spectroelectrochemistry application

Spectroelectrochemistry can be successfully used to characterize optically active compounds whose
color depends essentially on their oxidation state. An example of this is represented by porphyrin
derivatives, which, depending on the nature and number of grafted ligands on the nucleus and the possibly
coordinated metal, exhibit a significantly different electrochemical and spectrometric behavior. The general
structure of these compounds is described in the following figure, and the composition and coding of the
name for a series of non-coordinated metal compounds are shown in the following table.

R1=R3 R2 R4 Me Compound code

Ph Ph Ph 2H TPP
Ph Ph PTZ 2H TP2A
Ph PTZ PTZ 2H TP2B
PTZ PTZ PTZ 2H TP2D

8
Course 7 Acquisition and treatment of experimental data

For example, as shown in the figures below for TP2B, the pure electrochemical measurements,
performed by cyclic voltammetry (VOC, Figure A) and square wave voltammetry (SWV, Figure B) in the
presence of ferrocene as internal standard, revealed two reversible redox processes in the cathodic domain
and 4 quasi-reversible redox processes in the anodic domain.

9
Course 7 Acquisition and treatment of experimental data

From the cyclic voltamograms recorded by the two techniques (VOC and SWV) it can be deduced
that the reversibility of the anodic processes depends essentially on the maximum value of the applied
potential, increasing it to +2.5 V causing significant attenuation of the reduction peaks.
The occurrence of intermediates with different oxidation states could be confirmed by
spectroelectrochemical measurements, the evolution of the absorption spectrum depending on the applied
anodic potential being presented in the following figure, in 3D format.

For a more rigorous processing of the results, the data were filtered for each spectrum, after which an
LabView application was used to detect the position and amplitude of the peaks. By the correlation of the
spectrometric and electrochemical data it was possible to confirm the occurrence of intermediates and
oxidation products, each having a specific absorption spectrum. A more eloquent comparison can be
achieved by a 2D representation, in which the positions and the magnitudes of the peaks, as well as the trend
of their evolution, can be better emphasized. Such a comparison and evolution for TP2B is exemplified in
the following figure.

10
Course 7 Acquisition and treatment of experimental data

The four curves highlighted by colors correspond to the following potentials: +0.6 V - black; +1.0 V
- red; +1.3 V - green; +1.8 V - blue. These values are perfectly consistent with the electrochemical
information obtained through VOC and SWV, confirming the occurrence of the intermediates shown in the
legend of the figure.

2.2. Potentiometric/spectroelectrochemical titration

Unfortunately, in the case of biological systems, although spectroelectrochemistry can provide
extremely valuable information on the species oxidation state, its applicability is limited by the very difficult
transfer of electrons between the metallic electrode and the biological material (proteins, enzymes, etc.). To
solve this problem, redox mediators (acetyl-ferrocene, iridate, ruhex, K ferricyanide, dyes etc.) are often
used, but, unfortunately, most of them are optically active exactly in the interest area of the biological
materials, which can significantly alter the accuracy of the obtained information.
The total elimination of this problem can be achieved by potentiometric/spectroelectrochemical
titration, a technique that allows the evaluation of the redox equilibrium potential for different species of
biological interest based on the change in the absorbance spectrum. An example of this is the determination
of the redox equilibrium potential for an Arabidopsis thaliana haemoglobin (encoded AtHb2), the oxidized
form of which is titrated, in the presence of a mixture of 6 mediators, with sodium dithionite, simultaneously
11
Course 7 Acquisition and treatment of experimental data

with the recording of the potential in the cell and absorption spectra. The evolution of the 3D absorption
spectrum (A) and the potential of the indicator electrode (E WE, Figure B) according to the volume of added
titrant are shown in the following figure.

A B

As it can be deduced from the previous figure, the significant change in spectrum and the most
pronounced decrease in EWE occurs around a titrant volume of about 17 μL. The 2D format representation
for a selection of absorption spectra depending on the titrant added volume reveals the movement of the
Soret absorption band from 409 to 424 nm, as illustrated in the following Figure.

12
Course 7 Acquisition and treatment of experimental data

For the rigorous evaluation of the formal redox potential (E0") of the studied protein, a more
laborious data processing is required, consisting in evaluating the evolution of absorbance at the two Soret
bands wavelengths and correlating these values with the titrant volume and EWE. To facilitate these
correlations, a LabView application (Spectrum 3D Titration Final.vi) can be used, which, after launching
and loading of the spectra set to be analyzed, allows the manual selection of the two wavelengths (when the
"Lambda mode" selector is in the "MANUAL" position) or automatic detection of the corresponding peaks,
by moving the "Lambda mode" selector to "AUTOMATIC". In the latter case, the user must indicate the
volumes at which the peak detection is desired and optimize the "Threshold" and "Width" parameters.
Subsequently, if the "Courve mode" selector is in the "NORMAL" position, the application identifies the
absolute absorbance values at the two selected wavelengths (manually or automatically) and saves them to
the HDD. The charts resulting from this dataset are shown in the following figure, correlated with titrant
volumes (A) and EWE (B).

A B

The absorbance evolutions in relation to EWE (previous B figure) can be fitted based on relations
deduced from the Nernst's equation. Thus, for the general process from the RE reagent to the PR product:
RE + n-  PR
and considering CRE and CPR the relative concentrations of RE and PR, respectively, it results:
RT C RE
E WE  E 0 ' ln
nF C PR

If the RE and PR species are optically active, then the ratio of their concentrations at a given
wavelength can be evaluated, based on the absorbance Ai, by the equation:
C RE A i  A PR

C PR A RE  A i

13
Course 7 Acquisition and treatment of experimental data

where ARE and APR are the absorbances of RE and PR pure species at that wavelength. Combining the last
two equations, it follows:
RT A i  A PR RT A  A PR
E WE  E 0 ' ln or E WE  E '2.303 *
0
log i
nF A RE  A i nF A RE  A i
Practically, starting from the previous equations, the nonlinear fit of the data was made using the
equation:

A min  A max *10^ [(E WE  E 0' ) / S]

Ai 
1  10^ [(E WE  E 0 ' ) / S]
where Amin and Amax represent the minimum and maximum absorption at a given wavelength, and the
sensitivity, S, corresponds to the equation:
RT
S  2.303 *
nF
The fitting results according to the previous equations, along with the evaluated regression
parameters, are shown in the following figures, the curves corresponding to data recorded at wavelengths of
409 nm (A) and 424 nm (B).

A B
For a more rigorous comparison of the behavior of the different chemical species, it is preferable, in
many scientific publications, to present these correlations with the normalized absorbance values, A N, i,
which can be calculated using the relation:
A i  A min
A N, i 
A max  A min
Thus, if the "Courve mode" selector is in the "NORMALIZED" position, the application identifies
the absolute values of the absorbances at the two selected wavelengths, the maximum and minimum values
in the value arrays, calculates the normalized absorbance values and saves them to the HDD. The charts
14
Course 7 Acquisition and treatment of experimental data

resulting from this dataset are shown in the following figure, correlated with titrant volumes (A) and E WE
(B).

A B

In this case, the Nernst's equation can be rewritten as:

RT 1  A N, i
E WE  E 0 '2.303 * log
nF A N, i

Practically, starting from the previous equation, the nonlinear fit of the data was made using, as a
model, the equation:
1
A N, i 
1  10^ [(E WE  E 0 ' ) / S]
The fitting results according to the previous equation, along with the evaluated regression
parameters, are shown in the following figures, the curves corresponding to data recorded at wavelengths of
409 nm (A) and 424 nm (B).

A B

15