Lecture 19

Partition Chromatography (HPLC)
The most widely used HPLC technique
• Liquid-liquid partition chromatography (was developed first)

Stationary phase: liquid-coated solid support; liquid is retained on the
support surface due to physical adsorption.
• Bonded-phase partition chromatography (at present, dominates)

Stationary phase is bound to the solid support covalently.
The most popular bonded-phase coatings – siloxanes:
CH3
Si O Si R
CH3
R = Alk
siloxanes
The main advantage of the bonded-phase method over liquid-liquid partition:

it eliminates partial loss of stationary phase due to dissolution in the liquid
mobile phase.
Bonded-phase partition chromatography by the type of column packing and
appropriate elution mode is divided into:
♪ Normal-phase (historic name): polar, hydrophylic stationary phase
(triethyleneglycole or other polar groups on SiO2 or Al2O3) and relatively
non-polar mobile phase (such as hexane, ethylacetate, i-propylether, etc.)
The least polar component is eluted first (due to better solubility in
non-polar mobile phase). Increase in the eluent polarity decreases the
elution time.
♫ Reversed-phase: non-polar, hydrophobic stationary phase (such as hyrocarbons
or siloxanes with R=C8 (n-octyl) or C18(n-octyldecyl)) and polar mobile phase
(such as water, methanol, acetonitrile, etc.).
The most polar component is eluted first (due to better solubility in
polar mobile phase). Increase in the eluent polarity increases the
elution time.
Chiral recognition chromatography : Separation of enantiomers
(A particular application of reversed-phase partition HPLC)
Dr. Shulamit Levin, Medtechnica

The most selective application of HPLC: Separation of
such analytes as proteins and enzymes

ELECTROCHEMICAL METHODS OF ANALYSIS:
Fundamentals of Electrochemistry (Chapter 18)
A Quick Review of Reduction-Oxidation Concepts
Reduction-oxidation reactions (or redox reactions) are the reactions in which

elements change their oxidation numbers due to electron transfers between the
participating atoms.
!! Please review how to find oxidation numbers of atoms.
Oxidation of an element in a redox reaction is always accompanied by

reduction of the other element.
Oxidation is a loss of electrons. Therefore, oxidation of an atom leads to an
increase in its oxidation number:
0 +2
Zn → Zn2+ + 2e–; Zn → Zn.
Reduction is a gain of electrons. Therefore, reduction of an atom decreases
its oxidation number:
0 -1
Cl + 1e– → Cl– ; Cl → Cl.
A compound (an element) that loses electrons is called the reducing agent.
A Reducing agent is oxidized in a redox reaction.
A compound (an element) that gains electrons is called the oxidizing
agent. An Oxidizing agent is reduced in a redox reaction.
The number of electrons gained by the oxidizing agent is always equal to the
number of electrons lost by the reducing agent. (Recall Law of conservation
of matter).
An overall redox reaction may be broken into two parts or half-reactions:
-- one of them describes oxidation part of the process,
-- the other one describes the reduction part.
For example:
The overall (unbalanced) redox reaction
Ag+(aq) + Zn(s) Ag(s) + Zn2+(aq)
consists of the following half-reactions:
Oxidation: Zn(s) → Zn2+(aq) + 2 e–
Reduction: Ag+(aq) + e– → Ag(s) × 2
To obey the Law of conservation of matter,

2 Ag+(aq) + Zn(s) 2 Ag(s) + Zn2+(aq)
(balanced overall eqn)
!! Please review:
-- writing redox half-reactions and
-- balancing redox reactions by half-reaction method.
Electric charge (q):
The unit for electric charge is coulomb (C).
q=nF
where
n is the number of moles of electrons transferred in the reaction;
F is the Faraday constant:
F is equal to the electric charge (in coulombs) of 1 mole of electrons,
F = 96,485 C/(mol e–).
Electric current (I):

Electric current is the quantity of charge flowing per second through a circuit:
I = q/t
The unit for electric current is ampere (A):
ampere = coulomb/second.
Two types of electrochemical cell:
• A voltaic cell (or galvanic cell) uses a spontaneous redox reaction (G < 0)
to generate electrical energy.
• An electrolytic cell uses electrical energy from an external source to drive a
non-spontaneous redox reaction (G > 0).
Any electrochemical cell consists of two half-cells (an electrode dipping in
the electrolyte solution) connected into a circuit via wire and a salt bridge
filled with an inert electrolyte (often it is saturated KCl solution).
An electrode (and the corresponding half-cell) is identified as
the anode or the cathode, according to the half-reaction that takes place
at it.
In both voltaic and electrolytic cell:
• The oxidation half-reaction takes place at the anode.
• The reduction half-reaction proceeds at the cathode.
However, the direction of electron flow and the signs of the electrodes
in the voltaic and the electrolytic cells are reversed.
Principal differences between the voltaic and the electrolytic cell
Consider a redox-reaction Cu2+ + Zn Zn2+ + Cu
• In a voltaic cell, electrons are generated at the anode (so it is
negative) and consumed at the cathode (so it is positive).
• In an electrolytic cell, the electrons come from the external
power source, which supplies them to the cathode (so it is
negative) and removes them from the anode (so it is positive).
Voltaic cell Electrolytic cell

The reaction: Cu2+ + Zn → Zn2+ + Cu Zn2+ + Cu → Cu2+ + Zn
Rxn spontaneity: Spontaneous Non-spontaneous
Anode half-rxn: Zn → Zn2+ + 2 e- Cu → Cu2+ + 2 e-
Cathode half-rxn: Cu2+ + 2 e- → Cu Zn2+ + 2 e- → Zn
Electron flow: anode → cathode cathode → anode
Anode charge: _ +
Cathode charge: + _
Cu(s) + 2 Ag+(aq) Cu2+(aq) + 2 Ag(s)
Based on the same redox-reaction,

(1) voltaic cell;
(2) electrolytic cell.
(1)
(2)
Notation for a Voltaic Cell
Note: By a convention, the components of the anode compartment
(oxidation half-cell) are written on the left and components of the cathode
compartment (reduction half-cell) are shown on the right.
For example:
Zn(s) Zn2+(aq) Cu2+(aq) Cu(s)
Anode Cathode
A double vertical line
separates the half-cells
(denotes the salt bridge)
A vertical line represents a phase
boundary
This cell is based on the redox-rxn: Zn + Cu2+ Zn2+ + Cu

Oxidation (anode) half-rxn: Zn → Zn2+ + 2 e- ; Notation: Zn(s)Zn2+(aq)
Reduction (cathode) half-rxn: Cu2+ + 2 e- → Cu; Notation: Cu2+(aq)Cu(s)
Note: In the cell notation, half-cell components appear in the same order as
they appear in the half-rxn.
Electrodes are written at the far left and far right of the notation.
Half-cell components in the same phase are separated by a comma:
(aq) I–I(aq)
C(s) II2-(s) 2(s) H+(aq), MnO4–(aq), Mn2+(aq) C(s)
When required, concentrations (activities) of the dissolved components are

given in parenthesis:
Cu(s) │ Cu2+(aq)(1.0
Cu(s) Cu 2+
M) ║ Cl2M)
(1.0 (g)(1Cl
atm) Cl–(1.0 M) │C(s)
│ Cl-(aq)(1.0
2(1 atm) C(s)
Problem 1:
Write a balanced equation for the overall cell reaction of a galvanic
cell represented by the following notation:
Pb(s) Pb2+(aq) Br2(l) Br–(aq) Pt(s)
Solution:
The half-reactions are:
Note: Pt(s)-plate and carbon C(s) rode
Anode: Pb(s) → Pb2+(aq) + 2e–
do not participate in the redox-process.
Cathode: Br2(l) + 2e– → 2 Br–(aq)
They just serve as inert support
Overall reaction:
for gas/liquid/solution components of
Pb(s) + Br2(l) → Pb2+(aq) + 2 Br–(aq)
the electrodes.
Cell Potential
A voltaic cell converts the free energy of a spontaneous redox reaction
into the kinetic energy of electrons moving through the circuit
(in other words, into electric energy).
■ The difference in electric potential between two electrodes is

called the cell potential (Ecell) or electromotive force (emf).
• For a spontaneous process, Ecell > 0.

• For a non-spontaneous process, Ecell < 0.
• If Ecell = 0, the cell reaction is at equilibrium and the cell cannot
do work any more.
The cell potential is measured in volts:

1 V = 1 J/C (V = volt, J = joule, and C = coulomb).
Standard Cell Potentials
The measured cell potential varies with concentrations of the
components, temperature, etc.
In order to compare the output of different cells, standard cell

potentials (Ecell) are used.
A standard cell potential is the potential at 25 C of a cell with all

components (reactants and products) present in their standard states
(1 atm pressure for gases and 1.0 M solutions).
For example,
for a Zn/Cu2+ cell operated under standard conditions,
Zn(s) + Cu2+ (aq, 1 M) → Zn2+ (aq, 1 M) + Cu(s),
the standard cell potential Ecell = + 1.10 V.
Standard Electrode (Half-Cell) Potentials
The standard electrode (half-cell) potential (Ehalf-cell) is the
potential associated with a given half-reaction when all the
components are in their standard states.
● By IUPAC convention, a standard electrode potential always

refers to the half-reaction written as a reduction.
Therefore, the standard electrode potentials are usually presented as

standard reduction potentials (SRP).
For any voltaic cell,

the standard cell potential is the difference between the standard
electrode potential of the cathode half-cell and the standard electrode
potential of the anode half-cell:
Ecell = Ecathode – Eanode.
The standard reduction potential (E or SRP) is the half-cell potential
measured under the std.-state conditions against the standard hydrogen
electrode (SHE).
See in Fig. 18-6
The standard hydrogen electrode :
a Pt-electrode in contact with H2(gas)
and aqueous H+ at standard-state
conditions:
H+(1 M)H2(1 atm)Pt(s) (25 C).
SHE
The standard hydrogen potential,
which is the E for the half-reaction:
2 H+(aq) + 2 e– → H2(g),
is assigned 0.00 volts.

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Partition Chromatography (HPLC)

The most widely used HPLC technique

• Liquid-liquid partition chromatography (was developed first)

• Bonded-phase partition chromatography (at present, dominates)

The main advantage of the bonded-phase method over liquid-liquid partition:

Dr. Shulamit Levin, Medtechnica

Dr. Shulamit Levin, Medtechnica

Reduction-oxidation reactions (or redox reactions) are the reactions in which

!! Please review how to find oxidation numbers of atoms.

Oxidation of an element in a redox reaction is always accompanied by

To obey the Law of conservation of matter,

Electric current (I):

Voltaic cell Electrolytic cell

Based on the same redox-reaction,

This cell is based on the redox-rxn: Zn + Cu2+ Zn2+ + Cu

When required, concentrations (activities) of the dissolved components are

■ The difference in electric potential between two electrodes is

• For a spontaneous process, Ecell > 0.

The cell potential is measured in volts:

In order to compare the output of different cells, standard cell

A standard cell potential is the potential at 25 C of a cell with all

● By IUPAC convention, a standard electrode potential always

Therefore, the standard electrode potentials are usually presented as

For any voltaic cell,

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