Key Points - 2º Cuatrimestre - Ruth

Key-points-second-term-2019-Ruth...
beasercer
Química Analítica II
3º Grado en Química
Facultad de Química
Universidad de Sevilla
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Introduction to Electroanalytical methods
1- Galvanic cell and electrolytic cell. Fundamentals, differences and applications.
2- Types of electrodes and examples.
3- Electrode processes. What is the double layer, what polarization is and what is its
importance.
4- Ideally non-polarizable electrode and ideally polarizable electrode.
5- Mechanisms of mass transfer.
Potentiometry
1- Characteristics of a potentiometric measurement instrument.

2- Reference and indicator electrodes in Potentiometry.
3- Ion selective electrodes. Examples and types.

4- What is junction potential?
5- Description of glass electrode, fluoride electrode.
Other electroanalytical methods.
1- What Voltammetry, Amperometry , Coulometry, Conductimetry consist on. Basic

principles.
2- Types of voltammetry techniques and what is the main difference between them.
3- Description of the part that a curve i-E presents, what happens in each part?
4- Working electrodes in polarography, detailed description.
5- Cyclic voltammetry is very useful to study mechanisms of electrodic reactions, why?
6- Types of stripping methods.
7- Clark electrode.
8- Glucose sensors.
9- Types of Coulometries. Differences between them
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Key-points-second-term-2019-Ruth...
beasercer
Química Analítica II
3º Grado en Química
Facultad de Química
Universidad de Sevilla

KEY POINTS SECOND TERM 2019.
A) Introduction to electroanalytical methods.
1. Galvanic cell and electrolytic cell: fundamentals, differences and applications.
A galvanic cell uses a spontaneous electrochemical reaction to generate electricity. To
accomplish this, one reagent must be oxidized and another must be reduced. The two cannot be
in contact, or electrons would flow directly from the reducing agent to the oxidizing agent.
Instead, the oxidizing and reducing agents are physically separated, and electrons are forced to
flow through an external circuit to go from one reactant to the other. It’s used on potentiometry.
An electrolytic cell is the inverse of the galvanic cell: it uses electricity (potential is applied)
to generated a non-spontaneous an electrochemical reaction. It’s used on voltammetry or
coulometry.
2. Types of electrodes and examples.

The reference electrode is an electrode whose potential is fixed. It provides a stable and
known potential, so that we can attribute any change in Ecell to the analyte’s effect on the
indicator electrode’s potential. Ej: Standard Hydrogen Electrode (SHE), Ag/AgCl electrode and
Hg/Hg2Cl2 (saturated calomel electrode or SCE).
The working electrode is the electrode in an electrochemical system on which the reaction
of interest is occurring. Common working electrodes can consist of inert metals such as gold,
silver or platinum, to inert carbon such as glassy carbon or pyrolytic carbon, and mercury drop
and film electrodes. Ej: metallic electrodes, mercury electrodes, carbon electrodes…
3. Electrode processes. What is double layer, what polarization is and what is its
importance.
a) Electrode processes.
ü Faradaic processes.
They are associated to the electronic transfer in the oxidation reaction that takes
place in one electrode and the reduction reaction occurring in the other electrode. These
processes originate the faradaic current. They follow the Faraday’s law and the amount of
product (Q) of reaction is:
Q = n · F · N; where:
n is number of electrons involved in the reaction.
F is the Faraday’s constant
N the number of moles.
ü Non-faradaic processes.
Under some conditions and due to thermodynamic or kinetic reasons cells show a
potential interval at which the faradaic processes are excluded. The surface of the
electrode behaves like capacitor. These processes originate the non-faradaic current. They
follow the Ohm’s law.
E=I·R
b) Double layer.
Origin of the double layer: a charge on the electrode originates a charge of opposite sign
in the solution close to the electrode. This is called double electric layer. If negative potential
is increased more positive ions flow to the surface of the electrode to neutralize the charge of
it, producing a transient charge current to attain the equilibrium.
Composition of the solution adjacent to the electrode is different to the bulk solution. We
can difference two parts: the compact layer, with an excess of positive charge, in which
potential linearly decreases with the distance to the surface of the electrode, and the diffuse
layer, in which potential is exponentially diminished. The compact layer has two parts. The first
one is the inner Helmholtz layer, that may include solvent and any adsorbed solute molecule as
neutral molecules, anions, or cations. The second one is the outer Helmholtz layer with solvated
cations.
c) Polarization.
Polarization is the variation of the linear relationship that exists between the intensity of
current of the cell (I) and the applied potential (E), due to a decrease of the current, by the
decrease in the speed of some stage of the electrical process. Separation of charges occurs.
The degree of polarization is measured with the overpotential, and is the deviation of the
electrode from its theoretical value at equilibrium. It is taken as a negative value.
4. Ideally non-polarizable electrode and ideally polarizable

electrode.

Ideally polarizable electrode (IPE) is characterized by an
absence of faradaic current between the electrode surface and the
electrolyte. Any transient current that may be flowing is considered
non-faradaic. The current does not change with the potential. There is
not IPE in all potential ranges.
Ideally non-polarizable electrode. Faradic current can freely pass. It can
maintain the potential at a constant value and independent to the current. Ej:
platinum electrode.
5. Mechanisms of mass transfer.
B) Potentiometry.
1. Characteristics of a potentiometric measurement instrument.
Potentiometry techniques are based on the measurement of the potential of an analyte in
the absence of current to determine the concentration of an analyte in a sample. The
potentiogram is the graph where the potential is represented as a function of the time.
The instrumentation required to perform these measurements is a galvanic cell made by a
reference electrode, and a standard electrode connected by a salt bridge, and a device capable
of measuring the potential.
Reference electrodes are electrodes whose potential (Eref) fixed, so they don’t change with
the temperature, the current that goes through the electrode, or the concentration of the
analyte or any other ion on the matrix of our sample.
The working electrode’s potential changes (Esam) with the analyte’s activity.
The salt bridge is the connection between both electrodes, which doesn’t allow the ions of
the reference electrode and the working electrode to mix by them. In each of the side of the
salt bridge may develop a potential. This potential, if the mobility of the anion and the cation on
the salt bridge are more-or-less the same, the potential may be same, so the difference in
potential through the bridge, the junction potential (Ej) is cancelled.
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At the end, the potential of the cell is defined as: 
E = Esam – Eref + Ej
From the Esam, we can obtain the analytes concentration. So, to perform a potentiometry
determination we need to measure the global potential of the cell, consider the reference and
the junction potential and from the resulting working electrode potential, to determine the
analyte’s concentration.
2. Reference and indicator electrodes in potentiometry.

a) Reference electrodes.
The reference electrode is an electrode whose potential is fixed. It provides a stable and
known potential, so that we can attribute any change in Ecell to the analyte’s effect on the
indicator electrode’s potential.
ü Standard Calomel Electrode (SCE).
It consists of an inner tube made with of Hg, Hg2Cl2, and KCl,
situated inside of a second tube that contains a saturated solution of
KCl. A small hole connects the two tubes, and a porous wick serves as a
salt bridge. It is based on the following redox reaction:
Hg2Cl2 (s) + 2e- ® 2Hg (l) + 2Cl-(aq)
ü Silver/Silver Chloride electrode (Ag/AgCl).

It consists of a Ag wire, the end of which is coated with a thin film

of AgCl, immersed in a solution of KCl. A porous plug serves as the salt
bridge. It is based on the following redox reaction:
AgCl (s) + 1e- ® Ag (s) + Cl-
b) Indicator or working electrodes.

ü Metallic electrodes.
Metal electrodes develop an electric potential in response to a redox
reaction in the metal surface, or to an anion that forms a precipitate or a stable complex.
ü Ion selective electrodes (ISE).

They are a class of electrodes based in a membrane which allow the pass to a selective ion.
There is not a redox reaction on the surface of the electrode. Potential is developed because
of the difference in concentration of the ion between both sides of the membrane, resulting
from a chemical interaction between the analyte and active sites on the membrane’s surface.
3. Ion selective electrodes. Examples and types.

a) Solid-State Ion-Selective Electrodes (SSISE): fluoride electrodes.
A SSISE has a membrane that consists in an inorganic crystal. The ion
sensitive material, for example for the fluoride SSISE, is a crystal made by
LaF3 doped with Eu2+. The internal part of the crystal is in contact with a
solution whose concentration of fluoride is constant. The outer part is the in
contact with the analyte solution, whose concentration is unknown. Due to Eu2+
provides only two positive charges (compared to the three F– ions in LaF3), each EuF2 produces
a vacancy in the crystal. Fluoride ions pass through the membrane by moving into adjacent
vacancies.

b) Liquid-Based Ion-Selective Electrodes (LBISE).
It uses a hydrophobic membrane containing a hydrophobic ion exchanger, the ionophore
(molecule soluble in lipids), that is selective for the analyte ion. Example, K+ LBISE, which uses
valinomycin as ionophore.
c) Compound electrodes: compound electrodes contain an electrode surrounded by a

membrane that isolates or generates a chemical substance to which the electrode responds.
ü Gas-sensing electrodes: CO2 electrodes.
They consist in a glass pH electrode surrounded by a thin layer of
electrolyte solution, with a Ag/AgCl reference electrode immersed
inside, enclosed in a semipermeable membrane. When CO2 diffuses
through the semipermeable membrane, it lowers the pH in the
electrolyte solution. The response of the glass electrode to the
change in pH is a measure of the CO2 concentration outside the
electrode. Other acidic or basic gases, including NH3, SO2, NOx can
be detected in the same manner.
ü b) Biocatalytic sensors.
They respond biochemically to an important species. The most
common are enzyme electrodes, in which an enzyme is trapped in a gel or immobilized by
adsorption on a porous layer, at the surface of a potentiometric electrode. The analyte’s

reaction with the enzyme produces a product whose concentration is monitored by the
electrode.
4. What is junction potential?

Junction potential is the difference in potential in the interface of two solutions with
different concentration, which are in contact through a membrane. This difference in potential
is due to the ions of the most concentrated solution tend to diffuse towards the more diluted
solution, at different speeds, generating a double layer of ions with different charge. Consider
the interface between NaCl and water solution.
Na+ and Cl- ions diffuse from the NaCl solution into
the water. However, Cl- ions has a greater mobility than
Na+. That is, Cl- diffuses faster than Na+. As a result, a
region rich in Cl-, with excess negative charge, develops
at the front. Behind it is a positively charged region
depleted of Cl-. The result is an electric potential
difference at the junction of the NaCl and H2O phases.
5. Description of glass electrode and fluoride electrode.

ü Glass electrode.
Glass electrodes are a type of ion-selective electrodes that selectively respond to the H+
concentration. They are generally combined electrodes, that consist on a tube that ends in a
glass membrane (made by Na2O, CaO and SiO2), with a Ag/AgCl internal reference electrode
inside a second internal tube, and a Ag/AgCl external reference electrode surrounding that
second internal tube. Both tubes have a reference HCl saturated with KCl solution inside them,
plus the electrode. Finally, the tube is introduced in the solution with unknown pH.
The glass has a tetrahedral structure of silicate groups, whose negative charge is
compensated by cations, preferably Na+, which are placed in the interstices, having the
possibility of moving through the crystalline structure and conducting the electricity.

We can consider the glass as three layers: an inner
one, a hygroscopic gel layer, hydrated by the reference
solution; the intermediate one or “dry sheet”, where Na+
cations conduct the electricity; and the outer one, the
same that the inner one, but hydrated by the external
analyte’s solution, and not by the internal reference
solution. So then, the conduction depends on the
movement of H+ ions through the hygroscopic layers.
The potential difference between internal and external Ag/AgCl reference electrodes
depends on the |Cl-| concentration in each electrode compartment, and on the potential
difference across the glass membrane. Due to |Cl-| is fixed in each compartment, and due to |H+|
is fixed on the inside of the glass membrane, the only variable is the pH of analyte solution
outside the glass membrane.
ü Fluoride electrode.

A fluoride electrode is a type of Solid State Ion Selective Electrode (SSISE). Its
fundament is similar to the glass electrode. It is based on the use of mono-crystals, such as LaF3
doped with Eu2+, in order to create fluoride vacancies (anionic vacancies) to compensate the
deficiency of positive charge produced by the introduction of Eu2+ (it has one less positive charge
than La3+). Fluoride ions pass through the membrane by moving into adjacent vacancies, creating
a difference in potential that is measured. This glass is placed at the end of a plastic tube
containing a solution of NaF and NaCl (whose concentration is constant), and an internal
reference electrode. The outer part is in contact with the analyte solution, whose concentration
is unknown.
C) Other analytical methods.

1. What voltammetry, amperometry, coulometry and conductimetry consist on. Basic
principles.
• Voltametry: the current is measured when a voltage is applied to the working electrode. A
variable potential is applied.
• Amperometry: electric current is measured between a pair of electrons that perform an
electrolysis reaction. A reagent is the analyte and the measured current is proportional to the
analyte concentration.
• Coulometry: it is based on the measurement of the amount of electricity necessary to complete
a reaction. No standardization is necessary to calculate the amount of analyte.
• Conductivity: the conductivity measures the resistance of the solution between two flat or
cylindrical electrodes separated by a fixed distance in a solution. Alternating current is used
to prevent electrolysis.
2. Types of voltammetry techniques and what is the main difference between them.
3. Description of the parts that a curve i-E presents. What does happen in each part?
The voltammogram is the i-E sigmoid curve obtained (voltammetric
wave), where the current is represented as a function of the potential,
when it is sweep. Three parts can be considered: 
a) Initial, with zero current. Potential is not negative enough to
produce reduction reaction.
b) Reaction takes place, and cathodic current is measured. E1/2: half-
wave potential. It is the potential at which half the maximum current is
reached.
c) At a more negative potential current becomes constant.
4. Working electrodes in polarography: detailed description (only
DME)
It is formed by a capillary connected to a reservoir containing Hg. Suspend a drop of Hg from
the bottom of the capillary, and then the current and voltage are measured and the drop is
dislodged. Then another drop is suspended and the next measurement is made. The drop acts
like an electrode. Constant dropping occurs while potential is increased. Current increases while
size of the drop increases, but it decays when drop falls. Very quick increasing is produced with
the new drop. As a consequence, oscillation of current is produced. Mechanism of mass transfer
is only diffusion.
5. Cyclic voltammetry is very useful to study mechanisms of electrodic reactions, why?

Cyclic voltammetry is very useful to study mechanisms of electrodic reactions because:
1. Ratio ipa/ipc is 1 in reversible reactions. Values ≠ 1 indicate a more complicated mechanism
(several intermediate steps, kinetic effects).
2. Difference between Epa and Epc is 0.059/n V in reversible systems.
3. Average of Epa and Epc is the standard redox potential of the redox system to the reference
electrode.
4. In irreversible reactions, cathodic and anodic peaks are very separated.
6. Types of stripping methods.

Stripping methods are voltammetric techniques and are given in two stages.
In a first stage or deposit stage, the working
electrode is immersed in a stirred solution containing the
analyte, a potential is applied and the analyte is deposited
on the electrode. This step is equivalent to a
preconcentration of the analyte, which means that the
concentration of the analyte on the surface of the
electrode is greater than in the bulk solution. Therefore,
stripping methods are the most sensitive voltammetric
techniques. The sensitivity increases with the control of
the electrode potential, electrode dimensions, deposition time and agitation speed.
In a second stage or stripping stage, the deposited analyte is removed by stripping, hence
the name of the method. The potential is reversed and the analyte returns to its original state.
The current measured in this stage is proportional to the analyte deposited.
There are two different types of stripping methods, depending on the mechanism that
follows the deposit stage (first stage).
1. Electrochemical stripping methods: for electroactive species. The deposit on the surface of
the electrode takes place by electrolytic deposit. This can happen by two ways:

a) Anodic stripping voltammetry: the working electrode behaves as a cathode in the
deposition stage and as an anode in the stripping stage (first reduces and then
oxidizes).
b) Cathodic stripping voltammetry: the working electrode behaves as an anode in the
deposition stage and as a cathode in the stripping stage (first oxidizes and then
reduces).
2. Adsorptive stripping methods: for species with a tendency to be adsorbed. The deposit on the
surface of the electrode takes place by physical adsorption.
7. Clark electrode: measurement of O2 in solution.
It is based on an amperometric measurement of the current produced between a pair of
-
electrodes that are driving the reduction reaction of O2 to OH , and is proportional to the
-
concentration of OH , in consequence, also to the analyte (O2). The cathode, a Pt working
electrode, is held at -0.6 V with respect to the anode, a Ag/AgCl reference electrode. The cell is
covered by a semipermeable membrane, across which O2 can diffuse.
8. Glucose sensors: measurement of glucose in solution.

It is based on an amperometric measurement of the current produced in the oxidation of
glucose.
The system is form by strip, that has three electrodes. One carbon working electrode (WE1),
that is coated with glucose oxidase, that catalyzes the oxidation of glucose by O2, giving as a

product H2O2. A second one carbon working electrode (WE2), that is not coated with the enzyme so
glucose isn’t oxidized here, but it measures the signals produced by other molecules found in blood
acetaminophen that produce interferences. And finally, a third one Ag/AgCl reference electrode.
A small volume of blood is deposited into the hole of the strip, and reaches them. Then, glucose is
oxidized on WE1, but it is not oxidized on WE2. A potential is applied vs Ag/AgCl reference
electrode, and H2O2 is oxidized. The current produced is proportional to the concentration of H2O2,
so, in consequence, also to the analyte (glucose). Difference between the current measured in WE1
and WE2 is the analytical signal.
9. Types of coulometries.
ü Coulometry at a constant potential.
Potential is applied during enough time to oxidize
or reduce completely the analyte. Because analyte is
consumed, current decreases with the time in an
exponential form. Q is calculated from a graph of i vs
t (area under the curve).
But it is not necessary to complete the curve. Current is zero when 100% of electrolysis
$
is accomplished, and then 𝑄 = %. Constant of the cell, k, can be obtained plotting Ln i vs t.
&
𝑖 = 𝑖( · 𝑒 +&, → 𝐿𝑛 𝑖 = 𝐿𝑛 𝑖( − 𝐿𝑛 𝑒 · 𝑘𝑡 = 𝐿𝑛 𝑖( − 𝑘𝑡

Once we know Q, we can relate it with any other
variable with Faraday’s equation:
Q = i · t = n · F · N = n · F · V · C; where:
n = number of electrons per mol of analyte;

F = Faraday’s constant.
N = mol of analyte; V = solution volume;
C = solution concentration.
ü Coulometry at constant current.
This coulometric method is also called coulometric titration. A reagent, produced
by an electrolytic process, is used in the procedure to react with the analyte. When
the end-point is reached keeping constant the current, the charge is obtained by
multiplying the current by time. An indicator system is necessary to detect the end-
point.
D) Introduction to analytical separations.

1. Citing some separation technique according to the physico-chemical process that takes
place.
Molecular size: exclusion chromatography, Adsorption: liquid-solid chromatography.

Density difference: centrifugation. Electric field: electrolysis.
Change of state: crystallization. Chemical reaction: precipitation.
2. Classification of chromatographic techniques according to the support of the stationary

phase used.
1. Column chromatography.
2. Planar chromatography: thin layer chromatography and paper chromatography.
3. Describe the mechanisms of interaction that can take place on a chromatographic

separation.
ADSORPTION
Stationary phase is a solid that adsorbs the molecules of analyte. Fresh portions of mobile
phase dissolve the retained molecules taking them further in the stationary phase
Stationary phases: silica gel...
PARTITION
Separation is due to the “distribution” of the solute between the mobile and stationary
phases (relative solubility)
Stationary phases: bonded silica…
ION INTERCHANGE
Separation is due to the electrostatic attraction between the analyte ions and the opposite
charge ions located at the surface of an ion exchanger resin which acts as stationary phase
(polystyrene divinylbenzene)

SIZE EXCLUSION
Analyte separation is mainly based on its molecular size (sieving). Also known as gel filtration
chromatography
AFFINITY CHROMATOGRAPHY
Separation is based on highly specific interactions between the analyte and the called
affinity ligands, bounded to the stationary phase.
4. What are the causes of asymmetric band shapes? Could they be eliminated?
Asymmetry in band shapes can be be of two types: overloading and tailing.
The center isotherm is the ideal one, producing to a
symmetric peak.
The upper isotherm arises from an overloaded column in
which too much solute has been applied to the column. It
produces a long front of gradually increasing concentration

before the peak, and then, it ends abruptly. As the
concentration of solute increases, the solute becomes more
and more soluble in the stationary phase. There is so much
solute in the stationary phase that the stationary phase
begins to resemble to the solute. The peak front it produces
can be eliminated injecting diluted sample.
The lower isotherm arises when small quantities of
solute are retained more strongly than large quantities. It produces a long tail of gradually
decreasing concentration after the peak. Sites that bind solute strongly, like silica surfaces that
have hydroxyl polar groups that form hydrogen bonds with polar solutes, produce tailing. Tailng
can be eliminated by sylanization, that reduces it by blocking the hydroxyl groups with nonpolar
trimethylsilyl groups.
5. Describe the kinetic theory of plates, contributions and meaning of them.

The smaller the plate height is, the narrower the band. The van Deemter equation tells us
how some processes affect to the band broadening, consequently, the plate height. Some are
3
proportional to flow rate (A), or inversely proportional to flow rate ( ), or independent of flow
45
rate (C · 𝑢7 ).
𝐵
𝐻 ≈ 𝐴 + + C · 𝑢7
𝑢7
• Multiple flow paths (A).

In packed columns, there are many possible pathways available to mobile phase, and
some of them are longer than others. Molecules entering the column at the same time on
the left are eluted at different times on the right. This enables that some molecules elute
more or less, faster. For open tubular columns, the multiple path term, A, is zero, so
bandwidth decreases. The effect of this factor is related to particle size, dp, and
homogeneity, λ, of the particles (A = 2 · λ · dp).
3
• Longitudinal diffusion ( ).
45
Molecules tend to diffuse ahead and behind of the
chromatographic band, according to a concentration gradient.
The band slowly broadens as molecules diffuse from the high
concentration zones to lower concentration zones on the edges of
the band. The faster the flow, the less time is spent on the column
and the less longitudinal diffusion occurs. It is called longitudinal
diffusion because it takes place along of the column while the band
is transported by the mobile phase.
It depends on Dm (diffusion coefficient), that is higher in a
gas than in a liquid, and then, this effect is bigger in gas
chromatography (B = 2 · 𝛾 · Dm).
• Finite equilibration time between phases (𝐶 · 𝑢7 ).

The term 𝐶 · 𝑢7 comes from the finite time required for solute to equilibrate between
mobile and stationary phases. Although some solute is stuck in the stationary phase, the
remainder in the mobile phase moves forward, thereby resulting in spreading of the overall
zone of solute. The slower the linear flow, the more complete equilibration is and the less
zone broadening occurs
E) HPLC.

1. Draw a scheme of a typical HPLC instrument indicating all its parts. Indicate how it
works.
The HPLC system consists on a phase
mobile supply (made of plastic or glass), a
solvent delivery system (pump), a sample
injection valve (injector), a microfilter
(to remove any particle that clogs the
column), a degasser (for instance, N2, to
remove air bubbles that introduce
changes on the pressure), a high-
pressure column, a detector, and a
computer to control the system and
display results.
Many systems may include an oven for temperature control of the column. Between pump
and the detector there is a very high pressure. Consequently, all the tubing connecting the
components of this part of the instrument, are made of stainless steel or other material capable
to endure the high pressures.
Working method: in one hand, the mobile phase is introduced on a chamber, and then it goes
to a degasser that delete the gases out of the solution to not enter on the column and interfere
in the analysis. Then, it goes to a reciprocate pump, that boost it to the injector and then, to the
column. In the other hand, the sample is injected on a six-valve injector, by a syringe with a non-
beveled needle (because there is not a septum like in GC), and direct to the pre-column by the
mobile phase. The pre-column has the same composition that the column, to saturate the mobile
phase with the stationary phase, to minimize the loss of it in the column, avoiding the “bleeding”.
Then, the mobile phase saturated with the stationary phase, plus the sample, goes to the column,
and it reaches the detector out of it. The signal, will be catch by a register, obtaining a
chromatogram.

2. Draw a recriprocating pump and indicate how it works.
Piston is driven in and out of the solvent chamber by an eccentric cam which
is driven by an electric engine. Solvent chamber is emptied and filled with mobile
phase and inlet and outlet check valves are alternatively closed and opened.
Double reciprocating pump is used to assure a constant flow without pulses.
3. Kinds of elution in HPLC: explain differences between them.

Isocratic elution is performed with a constant composition mobile phase, that is a single
solvent or solvent mixture.
ADVANTAGES: no reconditioning, shorter time of analysis and reduced solvent consumption.
DISADVANTAGES: low resolution for difficult samples.
Gradient elution is performed by changing the composition of the mobile phase in a

programmed form to create a continuous gradient. This composition changes are made to
increase or decrease the eluent strength of the mobile phase, due to the mobile phase interacts
with the analytes. It is equivalent to a temperature gradient in GC.
ADVANTAGES: high resolution.
DISADVANTAGES: reconditioning, longer time of analysis and solvent consumption. 
4. Draw an injection valve and explain how it works.

This injection system consists of a six-

door valve, two of which are connected by a
loop whose volume is known, and measures
the volume of sample that we introduce in
the column.
The introduction of the sample is
carried out in two stages. In a first stage,
the sample is previously microfiltered and
loaded in a syringe in the injector at atmospheric pressure. In the second stage, the valve is
rotated, introducing the mobile phase in the loop, and dragging the previously loaded sample to
the column.
5. Common stationary phases for liquid-liquid partition chromatography. How are they
synthetized and their main mechanism of action according to its structure? Most
common types.
The most common support is silica, that is permeable to
solvent and have a huge specific area. Bare silica has a surface of
silanol (Si-OH) polar groups. Above pH 3, silanol groups are
dissociate to negative siloxane groups (Si-O-). This causes that
some polar compounds are retained, producing the presence of
important tails in the peaks of the chromatogram, which increases
the retention time of them, and getting worse its resolution.
To solve this problem, we use a technique called ENDCAPPING.
This technique consists in prepare "bonded silica", which is the
silica that is bound by the free O- of siloxane groups, to the Cl of a ClSiMe3, blocking the free
O- of siloxane groups. The polarity of the bound silica can be modified by bonding it with
different R groups, and the most common is C18.

6. Draw a scheme of a UV detector for HPLC explaining how it works.
Eluate from the column is passed through a flow cell held in the radiation
beam of an UV-Vis spectrophotometer. A reference beam comes out from the
beam splitter, and analytical signal is obtained as the difference between the
sample cell and reference. In this way fluctuations of the lamp are
compensated. In case of ultraviolet diode array detector is used, the
polychromatic radiation, after passing through the sample, is dispersed by a
fixed grating and then falls on to an array of photodiodes. Each diode measures
a narrow band of wavelengths in the spectrum, thus the DAD measures
absorbance at all wavelengths simultaneously.
7. Draw a scheme of a refractive detector for HPLC explaining how it works.

It senses the difference in refractive index between the
column eluent and a reference stream of pure mobile phase. A cell
with two compartments is used. Visible light passes through the
cell, and is directed to a photodiode array by the deflection plate.
While mobile phase passes through both compartments there is no
change in the signal. When solute with a different refractive index
enters the cell, the beam is deflected and different pixels of the
array are irradiated. It is useful for compounds that cannot be
detected with other detectors, like aliphatic hydrocarbons,

triglycerides. It is useless in gradient elution because it is very
difficult to match exactly the sample and the reference while the solvent composition is
changing.
8. Draw a scheme of an evaporative light scattering detector for HPLC explaining how it
works.
It responds to any solute that is significantly less volatile than the
mobile phase. Eluate enters the detector at the top. In the nebulizer,
the eluate is mixed with nitrogen and an aerosol (dispersion of droplets)
is produced. Solvent evaporates in the heated drift tube, leaving a fine
mist of solid particles to enter the detection zone at the bottom. The
particles are detected by the light that they scatter from a diode laser
to a photodiode
The evaporative light scattering detector response is related to the
mass of the solute, not to the structure or molecular mass of the solute.
It is universal and is compatible with gradient elution.

9. Explain schematically how it works an electrospray interface (ESI).
It is an ionization technique at
atmospheric pressure where the solvent-
analyte flow from the LC passes through a
positively charged and very narrow capillary,
and gets nebulized as microscopic, positively
charged solvent-analyte droplets. These
droplets fly towards the negatively-charged
faceplate, with solvent evaporating on the way,
until they disintegrate in a Coulomb explosion,
when the repulsive charge of their ionized
components exceeds their surface tension. The
individual ionized analyte molecules then pass through the faceplate entry hole into the mass
spectrometer.

10. Explain schematically how it works the atmospheric pressure chemical ionization
interface (APCII).
In APCII, the solvent-analyte stream from the LC is
vaporized by heated nebulizer gas. Then, the solvent
molecules vapor is ionized by a discharge of a Corona
needle. Then, the ionized solvent molecules vapor
transfers their charge to the ionizable vapor analyte-
molecules, which pass through the faceplate entry hole
into the mass spectrometer.
11. How it works a quadrupole analyzer.

It consists in four tube electrodes (paired in
diagonal) which apply an electrical field inside of
them. The fragments go through the four tubes, and
because of the field, their straight trajectories
start to bounce one side and another, until they hit
one of the quadrupoles. This happen with all the
fragments, excepts the ones, with a determined value
of m/z, that keep their trajectories reaching the
detector at the end of the quadrupole. A scan of all
the frequencies of the applied field allow us to detect
the fragments for each mass-charge ratio.
12. How it works an ion trap analyzer.

The ion trap analyzer is formed by
three electrodes; of them, the central
electrode, which is annular, the upper
electrode and the lower electrode.
During mass analysis, a field is
applied electromagnetic inside the
electrodes, where the ions are confined
with a stable oscillating trajectory. To
detect the ions, the electromagnetic
field is changed, which results in the destabilization of the trajectory of an ion with a specific
m/z ratio, being expelled from the trap. Changes in the electromagnetic field, will cause the ions
to be expelled in increasing order of their m/z ratio, giving rise to a mass spectrum.
13. How it works a time of flight analyzer.

They separate the ions by the time
they need to cover the distance between
the ion fragmentation and the detector.
This also means that the starting moment
has to be well defined, because it can be
notable source of error. To minimize this
time, this system has to employ electric
fields to enclose the ions, and then release
them all at the very same time.
Another important fact would be the distance covered by the fragments. A further distance
would result on a higher resolution (because they take longer to reach the detector). To increase
it without make much bigger the analyzer, we use a reflectron, an optical device through which
the ions pass through and their path is reversed.
F) Other chromatographic techniques.

1. Stationary phases in ion chromatography. How are they synthetized and their main
mechanism of action according to its structure? Most common types.  

The stationary phases in ion chromatography have charged sites bound to it, acting as an ion
exchangers. The most common stationary phases are resins. Polystyrene resins are made by
copolymerization of styrene and divinylbenzene.
The benzene rings are modified to produce a cation-exchange resin, containing sulfonate
groups (-SO3-), or anion-exchange resin, containing ammonium groups (-NR3+).

2. Factors affecting the retention in ion chromatography.  
a) pH.
C6H5COOH ⇆ C6H5COO- + H+; pKa = 4.2
Benzoic is a weak acid, and according to the pH of the mobile phase, it will be in a
different species: benzoic acid, benzoate or a mixture of both.
• If pH < pKa – 1, benzoic acid is the predominant species. It is the neutal form and the
retention decreases.
• If pH > pKa + 1, benzoate is the predominant species.
• If pKa – 1 > pH < pKa + 1 a mixture of benzoic acid + benzoate exists in solution and a tailed
peak is obtained.
In a mixture of weak acids, that of a higher pKa elutes before.
b) Buffer concentration.
When concentration of buffer in mobile phase increases, retention diminishes
c) Temperature.
Temperature decreases retention, because equilibrium is attained more rapidly.
d) Organic solvents.
Organic solvents in the mobile phase may reduce retention but it is difficult to predict.
3. Explain in detail what suppressed ion chromatography and ion chromatography without
suppression are.  

1. Suppressed ion chromatography.
The conductivity of the ions is very high due to the high ionic concentration necessary to
elute most ions; this makes it very difficult to detect small concentrations of analyte. The
key feature of suppressed ion chromatography is removal of unwanted electrolyte prior to
conductivity measurement by a suppressor placed between the exit of the column and the
detector.
2. Nonsuppressed ion chromatography.

If the ion-exchange capacity of the separator is sufficiently low and if dilute eluent is
used, ion suppression is unnecessary. It is based on the small differences in conductivity
between the ions in the sample and those of the eluent. To increase these differences, low
capacity exchangers are used that make elution possible with solutions containing low
concentrations of electrolyte. In addition, eluents with low conductivity are used It is
conducted with diluted HNO3 as eluent for monovalent ions, and with ethylenediammonium
salts for divalent ions.

4. Common applications of size exclusion chromatography: molecular mass determination.
Gel filtration is used mainly to separate molecules of
significantly different molecular size. For each stationary
phase, we construct a calibration curve, which is a graph of
log (molecular mass) vs. elution volume. We estimate the
molecular mass of an unknown by comparing its elution
volume with those of standards.
Molecules with the same molecular mass but different
shapes exhibit different elution characteristics.
For proteins is important to use an ionic strength high
enough (0.05 M) to eliminate electrostatic adsorption of
solute by occasional charged sites on the gel.


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1- Characteristics of a potentiometric measurement instrument.

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Other electroanalytical methods.

1- What Voltammetry, Amperometry , Coulometry, Conductimetry consist on. Basic

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2. Types of electrodes and examples.

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4. Ideally non-polarizable electrode and ideally polarizable

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5. Mechanisms of mass transfer.

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2. Reference and indicator electrodes in potentiometry.

ü Silver/Silver Chloride electrode (Ag/AgCl).

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b) Indicator or working electrodes.

ü Ion selective electrodes (ISE).

3. Ion selective electrodes. Examples and types.

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c) Compound electrodes: compound electrodes contain an electrode surrounded by a

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4. What is junction potential?

5. Description of glass electrode and fluoride electrode.

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C) Other analytical methods.

5. Cyclic voltammetry is very useful to study mechanisms of electrodic reactions, why?

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6. Types of stripping methods.

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8. Glucose sensors: measurement of glucose in solution.

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n = number of electrons per mol of analyte;

D) Introduction to analytical separations.

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2. Classification of chromatographic techniques according to the support of the stationary

3. Describe the mechanisms of interaction that can take place on a chromatographic

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5. Describe the kinetic theory of plates, contributions and meaning of them.

• Multiple flow paths (A).

• Finite equilibration time between phases (𝐶 · 𝑢7 ).

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3. Kinds of elution in HPLC: explain differences between them.

Gradient elution is performed by changing the composition of the mobile phase in a

4. Draw an injection valve and explain how it works.

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7. Draw a scheme of a refractive detector for HPLC explaining how it works.

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11. How it works a quadrupole analyzer.

12. How it works an ion trap analyzer.

13. How it works a time of flight analyzer.

F) Other chromatographic techniques.

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2. Nonsuppressed ion chromatography.