In potentiometry we measure the potential of an electrochemical cell under

static conditions. Because no current—or only a negligible current—flows

through the electrochemical cell, its composition remains unchanged. For
this reason, potentiometry is a useful quantitative method of analysis. The
first quantitative potentiometric applications appeared soon after the
formulation, in 1889, of the Nernst equation, which relates an
electrochemical cell’s potential to the concentration of electroactive species
in the cell [Stork, J. T. Anal. Chem. 1993, 65, 344A–351A].

For an on-line introduction to much of the material in this section, see Analytical
Electrochemistry: Potentiometry by Erin Gross, Richard S. Kelly, and Donald M. Cannon, Jr., a
resource that is part of the Analytical Sciences Digital Library.

Potentiometry initially was restricted to redox equilibria at metallic

electrodes, which limited its application to a few ions. In 1906, Cremer
discovered that the potential difference across a thin glass membrane is a
function of pH when opposite sides of the membrane are in contact with
solutions that have different concentrations of H 3O+. This discovery led to the
development of the glass pH electrode in 1909. Other types of membranes
also yield useful potentials. For example, in 1937 Kolthoff and Sanders
showed that a pellet of AgCl can be used to determine the concentration of
Ag+. Electrodes based on membrane potentials are called ion-selective
electrodes, and their continued development extends potentiometry to a
diverse array of analytes.

Potentiometric Measurements

As shown in Figure 11.1.3, we use a potentiometer to determine the

difference between the potential of two electrodes. The potential of one
electrode—the working or indicator electrode—responds to the analyte’s
activity and the other electrode—the counter or reference electrode—has a
known, fixed potential. In this section we introduce the conventions for
describing potentiometric electrochemical cells, and the relationship
between the measured potential and the analyte’s activity.

In Chapter 6 we noted that a chemical reaction’s equilibrium position is a

function of the activities of the reactants and products, not their
concentrations. To be correct, we should write the Nernst equation in terms
of activities. So why didn’t we use activities in Chapter 9 when we calculated
redox titration curves? There are two reasons for that choice. First,
concentrations are always easier to calculate than activities. Second, in a
redox titration we determine the analyte’s concentration from the titration’s
end point, not from the potential at the end point. The only reasons for
calculating a titration curve is to evaluate its feasibility and to help us select
a useful indicator. In most cases, the error we introduce by assuming that
concentration and activity are identical is too small to be a significant
concern.

In potentiometry we cannot ignore the difference between activity and

concentration. Later in this section we will consider how we can design a
potentiometric method so that we can ignore the difference between activity
and concentration. See Chapter 6.9 to review our earlier discussion of
activity and concentration.

Potentiometric Electrochemical Cells

A schematic diagram of a typical potentiometric electrochemical cell is

shown in Figure 11.2.1 . The electrochemical cell consists of two half-cells,
each of which contains an electrode immersed in a solution of ions whose
activities determine the electrode’s potential. A salt bridge that contains an
inert electrolyte, such as KCl, connects the two half-cells. The ends of the salt
bridge are fixed with porous frits, which allow the electrolyte’s ions to move
freely between the half-cells and the salt bridge. This movement of ions in
the salt bridge completes the electrical circuit.

Figure
11.2.1 . Example of a potentiometric electrochemical cell. The activities of Zn 2+ and
Ag+ are shown below the two half-cells.

By convention, we identify the electrode on the left as the anode and assign
to it the oxidation reaction; thus
 Zn(s)⇌ Zn2+(aq)+2e−Zn(𝑠)⇌ Zn2+(𝑎𝑞)+2𝑒−

The electrode on the right is the cathode, where the reduction reaction
occurs.

Ag+(aq)+e−⇌Ag(s)Ag+(𝑎𝑞)+𝑒−⇌Ag(𝑠)

The potential of the electrochemical cell in Figure 11.2.1 is for the reaction

Zn(s)+2Ag+(aq)⇌2Ag(s)+Zn2+(aq)Zn(𝑠)+2Ag+(𝑎𝑞)⇌2Ag(𝑠)+Zn2+
(aq)

We also define potentiometric electrochemical cells such that the cathode is

the indicator electrode and the anode is the reference electrode.

The reason for separating the electrodes is to prevent the oxidation reaction
and the reduction reaction from occurring at one of the electrodes. For
example, if we place a strip of Zn metal in a solution of AgNO 3, the reduction
of Ag+ to Ag occurs on the surface of the Zn at the same time as a potion of
the Zn metal oxidizes to Zn2+. Because the transfer of electrons from Zn to
Ag+ occurs at the electrode’s surface, we can not pass them through the
potentiometer.

pH is a measure of hydrogen ion concentration, a measure of

the acidity or alkalinity of a solution. The pH scale usually ranges
from 0 to 14. Aqueous solutions at 25°C with a pH less than 7
are acidic, while those with a pH greater than 7 are basic
or alkaline. A pH level of 7.0 at 25°C is defined as "neutral" because
the concentration of H3O+ equals the concentration of OH− in pure
water. Very strong acids might have a negative pH, while very strong
bases might have a pH greater than 14.
H = -log[H+]
where log is the base-10 logarithm and [H+] stands for the hydrogen
ion concentration in units of moles per liter solution.
pH meter, electric device used to measure hydrogen-ion activity
(acidity or alkalinity) in solution. Fundamentally,
a pH meter consists of a voltmeter attached to a pH-
responsive electrode and a reference (unvarying) electrode. The
pH-responsive electrode is usually glass, and the reference is
usually a silver–silver chloride electrode, although a mercury–
mercurous chloride (calomel) electrode is sometimes used. When
the two electrodes are immersed in a solution, they act as
a battery. The glass electrode develops an electric
potential (charge) that is directly related to the hydrogen-ion
activity in the solution (59.2 millivolts per pH unit at 25 °C [77 °F]),
and the voltmeter measures the potential difference between the
glass and reference electrodes.
A pH meter is consisted of three different parts: an internal electrode, a reference
electrode, and a high input impedance meter. Glass probe often contains the two
electrodes -- internal electrode and reference electrode. The internal electrode is a
Silver wire covered with Silver Chloride (Ag/AgCl wire), and reference electrode is often
made up of the same materials. Inside the probe is a buffer solution at pH of 7.
Measured pH is the difference in [H+] between the reference buffer inside the probe and
the sample solution.
The pH measurements are made by comparing the pH reading of a sample solution to
that of a reference solution with defined pH, such as buffers. Therefore, it is important to
calibrate the instrument with appropriate buffer solutions before making any
measurements. The figure on the right is a simple depiction of a glass electrode used
with pH meters.

1. Wastewater treatment
During wastewater treatment, heavy metals, organic compounds, and other toxic substances are
removed from water; pH levels need to be adjusted during this process by adding chemicals to
the water to separate dissolved waste from the liquid. Managers at facilities that process sewage
or recycle water used in manufacturing need to monitor pH levels to ensure that water can safely
pass to the next phase of treatment so that they end up with pure, safe water at the end of the
cleaning process.

2. Aquaculture

Aquaculture is a multibillion-dollar industry that is responsible for supplying nearly half of the
world’s seafood. To keep aquatic creatures alive and ensure they’re healthy enough to be
consumed, companies in this space need to monitor changes in pH levels on a regular basis.
Generally speaking, fish thrive in pH levels between 6.5 and 9.0.

3. Food and beverage

It is important to monitor pH levels in the production of any food or beverage product. For
example, when water used in beverage manufacturing is too acidic, consumers can
potentially damage their dentition. Companies also need to monitor pH levels in food
production to ensure their products are safe to eat and of high quality. For example, pork with a
pH of 5.6 to 6.0 indicates that a pig was raised well, while a lower pH reading indicates the pig
might have lived a stressful life. What’s more, a meat product like salami should have a pH
under 5.3 to protect against bacteria growth.

4. Pool and spa water

Whether you’re managing a large community swimming pool or a private spa, you need to
measure pH levels to determine how many sterilizing agents (e.g., chlorine) you need to put in
the water to maintain a safe swimming environment. When pH levels are too high, water gets
cloudier, and swimmers’ skin and eyes may get irritated. When pH levels are too low,
swimmers’ skin and eyes may get irritated, too, and your pool or spa’s plaster can get damaged
as well. The ideal pH reading for a spa is between 7.2 and 7.8.

5. Aquariums and fish tanks

To keep fish and other aquatic creatures alive—and to keep fish tanks and aquariums clean—
many water composition characteristics need to be proactively monitored, including pH.
Otherwise, at best, the water in the aquarium or fish tank will look cloudy and, at worst, fish may
struggle to survive.
6. Research

Water plays an integral role in many research projects. While the requirements of each
experiment are unique, lab science demands highly accurate results. In experiments, researchers
need to monitor pH levels to ensure high-quality findings that can be repeated. For example,
different types of bacteria thrive in environments with different pH levels. To reach accurate
conclusions, it is, therefore, critical to monitor pH levels in any experiment involving bacteria.

7. Hydroponics

The practice of growing plants in a nutrient-enriched water-based solution instead of soil is

called hydroponics. If the nutrient balance or pH level of the water swings too much in one
direction, plants can die rather quickly. To this end, managers at hydroponic facilities keep a
close eye on pH levels at all times.

When water is involved in an industrial application, it’s likely that water quality characteristics—
including pH levels—need to be measured to ensure safety and compliance.

Electrophoresis is a technique used to separate macromolecules in a fluid or gel based

on their charge, binding affinity, and size under an electric field. In the year 1807,
Ferdinand Frederic Reuss was the first person to observe electrophoresis. He was from
Moscow State University. Anaphoresis is the electrophoresis of negative charge
particles or anions whereas cataphoresis is electrophoresis of positive charge ions or
cations. Electrophoresis has a wide application in separating and analysing
biomolecules such as proteins, plasmids, RNA, DNA, nucleic acids.
Principle: When charged molecules are placed in an electric field, they migrate toward either the
positive or negative pole according to their charge. In contrast to proteins, which can have either a net
positive or net negative charge, nucleic acids have a consistent negative charge imparted by their
phosphate backbone, and migrate toward the anode.

An ion placed in such an electric field will experience a force:

Where,
F = electrophoretic force
K = a constant
q = net charge on the protein (atomic charges/protein molecule)

Types of Electrophoresis
The different forms of electrophoresis have been modified into several types of
electrophoresis, which are used to separate various types of biomolecules, analyze their
characteristics, and study their interaction with a molecule of interest. The following are
selected electrophoresis methods, based on different formats of electrophoresis.
1. Gel Electrophoresis
Gel electrophoresis is a form of ZE that uses gel, a non-fluid cross-linked polymer
network, as a support medium to maintain stable pH value in the solution buffer, acting
as an anti-convective stabilizer. It also serves as a separation matrix, due to its porous
nature that filters large particles and hinders smaller ones during electrophoretic
separation (Jorgenson, 1986).

The gel is cast into strips or slabs with slots or sample wells. Once it is completely
polymerized, the gel is submerged in an electrophoresis solution buffer, and samples
are loaded into each well before the electric current is applied to initiate electrophoretic
separation (Walker, 2010). At the end of the gel electrophoresis, components in the
samples will be separated based on their mass (Westermeier, 2005).

As mentioned earlier, gel electrophoresis is one of the most used types of

electrophoresis in research and routine diagnosis due to its ease-of-use and their
versatility. It can be adapted to separate a variety of biomolecules by changing the type
of polymers used to cast the gel and by adjusting the composition of the polymer,
altering the pore size of the gel (Xu, 2008; Chung, et al., 2014).

Gel types

 Polyacrylamide is a clear and transparent gel made from the co-polymerization of

acrylamide monomers in the presence of a crosslinking agent, N, N-methylene-
bisacrylamide, commonly referred to as ‘bis-acrylamide’. The polymerization reaction is
catalyzed by ammonium persulphate (APS) and N, N, N’, N’-tetramethylene diamine
(TEMED). The pore size of polyacrylamide gels is determined by the concentration of
acrylamide, which must be in proportion with its crosslinking agent. Generally, a low
percentage of acrylamide gel (3%-15%) is used to separate DNA and proteins. A higher
percentage of acrylamide gel (10%-20%) is commonly used in sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), in which proteins are separated
in denatured conditions, according to their size (Walker, 2010; Chung, et al., 2014).
 Agarose, a natural linear polysaccharide made of galactose and 3, 6-anhydrogalactose
chains, extracted from agar isolated from red seaweeds (Westermeier, et al., 2005).
Agarose, like agar, is stored as a dry powder. To cast agarose gel, the agarose powder
is dissolved in a relevant solution buffer, heated, and allowed to cool to room
temperature. Similar to polyacrylamide gel, the concentration of the agarose in the
solution buffer determines the pore size of the gel. Agarose gel is commonly used at
0.8% (w/v) to 5% (w/v) to separate DNA and RNA molecules (Walker, 2010). Agarose
can be used in combination with SDS to separate high-molecular-weight proteins, which
can be problematic when separated using SDS-PAGE (Greaser et al., 2012).

2. Pulsed-field Gel Electrophoresis (PFGE)

Pulsed-field Gel Electrophoresis (PFGE) is a variation of gel electrophoresis, in which
two electrical fields are periodically applied, in rotation, to the gel electrophoresis at
different angles. This type of electrophoresis is specifically designed for the separation
of chromosomes, which are high-molecular-weight DNA molecules of over 20 kilobases.

Unlike smaller DNA molecules, high-molecular-weight DNA molecules are in a

compressed conformation, causing them to move in a size-independent manner. During
the application of the first electric field, the coiled DNA molecules are stretched and will
move through the gel. The termination and the alteration in the direction of the electric
field, however, force these large DNA molecules to reorient themselves before migration
can resume.

Larger high-molecular-weight molecules will be slower in the process of reorientation

and migration renewal, compared to smaller high-molecular-weight molecules due to
their longer viscoelastic relaxation time. Repeated application of two alternating electric
fields from different angles will eventually reveal further migration distance for smaller
high-molecular-weight DNA molecules and shorter migration distance for larger high-
molecular-weight DNA (Westermeier, 2005; Westermeier et al., 2005; Walker,
2010).

3. Capillary Electrophoresis (CE)

Capillary electrophoresis, also known as High-Performance Capillary Electrophoresis
(HPCE), is a type of electrophoresis performed in a narrow capillary immersed in an
electrolyte buffer. It is the only type of electrophoresis capable of performing all four
types of electrophoreses (Heiger, 2000). The capillary is typically 20-30 centimeters
long and possesses a 25-75 micrometer inner diameter.

Electrophoretic separation is initiated when a sample is injected into the capillary, either
by high voltage or by pressure, and high electric fields are applied across the capillary
(Heiger, 2000; Westermeier, et al, 2005). Components in the sample are separated
along the length of the capillary, based on the format of electrophoresis performed.
Towards the other end of the capillary, separated components are detected at
the detector, where the time of detection or retention time is automatically recorded.

Because CE is performed in a narrow capillary, only a very small sample volume is

required for the separation. Another major advantage of CE is the automated
instrumentation system, enabling high-throughput analysis (Heiger, 2000).

4. Immunoelectrophoresis
Immunoelectrophoresis is a type of electrophoresis that separates antigens, including
proteins and peptides, based on their reaction and specificity to antibodies,
or immunoglobulins (Ig). The binding of antigen to its corresponding antibody at a
specific antigen/antibody ratio, or the equivalent point, will result in the precipitation of
antigen-antibody complex. Thus, antigens in a sample of interest can be separated
based on their ability to bind to a given antibody (Nowotny, 1979).
Contemporary settings for immunoelectrophoresis are based on modifications of zone
electrophoresis and gel electrophoresis. Immunoelectrophoresis can be performed in a
one-dimensional or two-dimensional mode (Westermeier et al., 2005; Walker, 2010).

5. Affinity Electrophoresis
Affinity Electrophoresis is a type of electrophoresis that separates a biomolecule that
interacts with or binds to another molecule for which it has an affinity. It makes use of
the phenomenon that the electrical mobility (µ) changes when a biomolecule, including
nucleic acids, proteins, peptides, and polysaccharides, binds to another molecule, and
this change in the electrical mobility will be reflected in the electrophoretic pattern.

Thus, biomolecules of interest in a sample can be singled out based on their affinity to
another molecule—whether or not the biomolecules of interest tend to bind to another
molecule more than the other unwanted biomolecule. Contemporary settings for affinity
electrophoresis are based on either gel electrophoresis or capillary electrophoresis
(Kinoshita et al., 2015).

Instrumentation
All types of electrophoresis separate charged particles while they are submerged in a
solution buffer. All forms of electrophoresis require a power supply and
an electrophoresis unit, commonly referred to as an electrophoresis chamber. The
power supply provides the electric current to the chamber that propels the
electrophoretic separation. The chamber is composed of two opposite electrodes,
cathode and anode, and of a buffer solution reservoir, in which the samples and the
separation thereof take place (Walker, 2010).

The use of a power supply and an electrophoresis chamber is also required by all types
of electrophoresis. However, each format necessitates specific equipment for the setting
up of the separation process. For example, caster trays, glass plates, and combs are
prerequisites for gel electrophoresis. Capillary electrophoresis requires an internal
cooling system that effectively suppresses excessive heating and provides a thermally
stable condition during electrophoresis.

Factors influencing Electrophoresis:

Movement of proteins depends on various aspects. Within the gel the molecules must pass through as
they are moving from one pole to another. The smaller molecules can weave in and out of the matrix
of the gel with more ease, compared with larger molecules. As a general rule, the molecules move
rapid if it has more net charge, has a shape of ball and shorter diameter

Size, shape, and net charge of the molecule

 Mobility is inversely proportional to the size of the molecule and directly proportional to the net
charge of the molecule. Globular proteins have compact structures and faster mobility than
fibrous proteins of similar molecular weight.[2]
  Particles with a negative charge (anions) always move in the direction of the positive pole,
whereas particles with a positive charge (cations) always move in the direction of the negative
pole. When performing gel electrophoresis, the positive pole refers to the anode, while the
negative pole refers to the cathode. As a result, charged particles move to the nodes that are
appropriate for them [e.g., anions migrate from cathode (-) to anode (+)].[2]

Strength of the electrical field

 Mobility is proportional to the potential gradient (voltage) and inversely proportional to
resistance.

Buffer
 Buffer functions to carry the current and maintain the pH of the medium. The optimum ionic
strength of the buffer is necessary as higher ionic strength increases the share of current carried
by buffer ions & slows down the sample migration. It also produces high heat, leading to
increased diffusion of separation bands. The low ionic strength of the buffer also reduces
resolution due to reduced overall current passing through the medium.
 The ionization of molecules, such as proteins, amino acids, etc., depends on the pH of the
medium. Alteration in the pH of the medium can alter the direction and velocity of migration.
[2]

Supporting medium
 A medium having affinity for the molecules in samples can hinder the rate of migration and can
decrease the resolution of separation. The pore size in the support medium is inversely
proportional to the gel concentration. Adjusting pore size according to the properties of a
molecule of interest is necessary for optimum resolution.
 Fixed groups such as sulfate get ionized and acquire a negative charge at alkaline or neutral pH.
Applying the electrical field, HO ions associated with these negatively charged groups start
migrating toward the cathode. This movement hinders sample movement towards the anode
and can reduce separation resolution. This effect is known as electroendosmosis. It can be
reduced by using ultrapure agarose gel with low sulfate content

Detecting electrophoretic separation

When electrophoresis is completed, the resulting product in all forms and types of
electrophoresis, except capillary electrophoresis, is the separation of particles or
components in the sample that are oftentimes invisible to human eyes. Therefore,
additional detection and analytical methods are imperative for the interpretation of
results obtained from electrophoresis (Jorgenson, 1986; Walker, 2010).

Figure 3: An example of a Gel Document System, or Gel Doc equipped with a UV

light source, suitable for the dye that emits ultraviolet fluorescence when stained
targeted biomolecules are exposed to UV light. The Gel Doc can be connected to
a computer unit, which allows imaging of stained gels. (Taken from SynGene)
In the case of gel electrophoresis, for example, the separated particles remain
embedded in the gel, invisible under normal light. To visualize the pattern of migration,
the gel is stained and then visualized using a Gel Documentation System, which
provides the necessary light source. The gel documentation system is frequently
equipped with a camera, which can image the gel visualization for quantification or
downstream analysis.

Detection methods

There are several approaches to detect and visualize the result of electrophoresis, each
with a different degree of sensitivity and complexity. Common detection methods are as
follows (Jorgenson, 1986):

 Staining uses a variety of dyes that can bind to the molecules of interest. For
example, Coomassie Brilliant Blue or silver staining can bind to proteins. Ethidium
bromide is an example of a dye that can insert itself between the nucleobases and
fluoresces strongly under short-wave ultraviolet (UV) light. This approach is a common
detection method for gel electrophoresis.
 Autoradiography can be used for detection if the molecules of interest are labeled with
radioisotopes. Compared to staining, autoradiography is more sensitive.
 Online detection, which more often than not, is a part of automated instrumentation.
Several detection mechanisms can be employed for online detection such as detection
of UV absorption, fluorescence, electric field, and conductivity. Capillary electrophoresis
and isotachophoresis employ this detection approach.

Apart from autoradiography, the quantification of separated components is relative to

known standards subjected to the same electrophoretic separation. In the case of
autoradiography, direct quantification can be achieved using a densitometer.

Gel Electrophoresis Steps

The broad steps involved in a common DNA gel electrophoresis
protocol:

1. Preparing the samples for running

The DNA is isolated and preprocessed (e.g. PCR, enzymatic

digestion) and made up in solution with some basic blue dye to help
visualize the movement of the sample through the gel.

2. An agarose TAE gel solution is prepared

TAE buffer provides a source of ions for setting up the electric field
during electrophoresis. The weight-to-volume concentration of agarose
in TAE buffer is used to prepare the solution. For example, if a 1%
agarose gel is required, 1g of agarose is added to 100mL of TAE. The
agarose percentage used is determined by how big or small
the DNA is expected to be. If one is looking at separating a pool of
smaller size DNA bands (<500bp), a higher percentage agarose gel
(>1%) is prepared. The higher percentage of agarose creates a denser
sieve to increase the separation of small DNA length differences. The
agarose-TAE solution is heated to dissolve the agarose.

3. Casting the gel

The agarose TAE solution is poured into a casting tray that, once
the gel solution has cooled down and solidified, creates a gel slab with
a row of wells at the top.

4. Setting up the electrophoresis chamber

The solid gel is placed into a chamber filled with TAE buffer. The gel is
positioned so that the chamber wells are closest to the negative
electrode of the chamber.

5. Loading the gel

The gel chamber wells are loaded with the DNA samples and usually,
a DNA ladder is also loaded as reference for sizes.

6. Electrophoresis
The negative and positive leads are connected to the chamber and to a
power supply where the voltage is set. Turning on the power supply
sets up the electric field and the negatively charged DNA samples
will start to migrate through the gel and away from the negative
electrode towards the positive.

7. Stopping electrophoresis and visualizing the DNA

Once the blue dye in the DNA samples has migrated through the gel
far enough, the power supply is turned off and the gel is removed and
placed into an ethidium bromide solution. Ethidium bromide
intercalates between DNA and is visible in UV light. Sometimes
ethidium bromide is added directly to the agarose gel solution in step
2. The ethidium bromide stained gel is then exposed to UV
light and a picture is taken. DNA bands are visualized in from each
lane corresponding to a chamber well. The DNA ladder that was loaded
is also visualized and the length of the DNA bands can be estimated.
An example is given in the figure below.
Chromatography is the most widely used separation technique in chemical laboratories, where it
is used in analysis, isolation and purification, and it is commonly used in the chemical process
industry as a component of small and large-scale production. In terms of scale, at one extreme
minute quantities of less than a nanogram are separated and identified during analysis, while at
the other, hundreds of kilograms of material per hour are processed into refined products. It is the
versatility of chromatography in its many variants that is behind its ubiquitous status in
separation science, coupled with simplicity of approach and a reasonably well-developed
framework in which the different chromatographic techniques operate.
Chromatography is essentially a physical method of separation in which the components of a
mixture are separated by their distribution between two phases; one of these phases in the form
of a porous bed, bulk liquid, layer or film is generally immobile (stationary phase), while the
other is a fluid (mobile phase) that percolates through or over the stationary phase. A separation
results from repeated sorption/desorption events during the movement of the sample components
along the stationary phase in the general direction of mobile-phase migration. Useful separations
require an adequate difference in the strength of the physical interactions for the sample
components in the two phases, combined with a favourable contribution from system transport
properties that control sample movement within and between phases. Several key factors are
responsible, therefore, or act together, to produce an acceptable separation. Individual
compounds are distinguished by their ability to participate in common intermolecular
interactions in the two phases, which can generally be characterized by an equilibrium constant,
and is thus a property predicted from chemical thermodynamics. Interactions are mainly physical
in type or involve weak chemical bonds, for example dipole–dipole, hydrogen bond formation,
charge transfer, etc., and reversible, since useful separations only result if the compound spends
some time in both phases. During transport through or over the stationary phase, differential
transport phenomena, such as diffusion and flow anisotropy (complex phenomena discussed
later), result in dispersion of solute molecules around an average value, such that they occupy a
finite distance along the stationary phase in the direction of migration. The extent of dispersion
restricts the capacity of the chromatographic system to separate and, independent of favourable
thermodynamic contributions to the separation, there is a finite number of dispersed zones that
can be accommodated in the separation.
Basic Terms and Definitions Chromatography
Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (stationary phase) while the other (the
mobile phase) moves in a definite direction.
Chromatogram A graphical or other presentation of detector response, concentration of analyte
in the effluent or other quantity used as a measure of effluent concentration versus effluent
volume or time. In planar chromatography "chromatogram" may refer to the paper or layer with
the separated zones. Chromatograph (verb) To separate by chromatography.
Chromatograph (noun) The assembly of apparatus for carrying out chromatographic separations.
Stationary Phase The stationary phase is one of the two phases forming a chromatographic
system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid. This solid
may or may not contribute to the separation process. The liquid may also be chemically bonded
to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase). The expression
Chromatographic Bed or Sorbent may be used as a general term to denote any of the different
forms in which the stationary phase is used. Note: Particularly in gas chromatography where the
stationary phase is most often a liquid, the term Liquid Phase is used for it as compared to the
Gas Phase, i.e., the mobile phase. However, particularly in the early development of liquid
chromatography, the term "liquid phase" had also been used to characterize the mobile phase as
compared to the "solid phase" i.e., the stationary phase. Due to this ambiguity, the use of the
term "liquid phase" is discouraged. If the physical state of the stationary phase is to be expressed,
the use of the adjective forms such as Liquid Stationary Phase and Solid Stationary Phase,
Bonded Phase or Immobilized Phase is proposed.
Bonded Phase A stationary phase which is covalently bonded to the support particles or to the
inside wall of the column tubing.
Immobilized Phase A stationary phase which is immobilized on the support particles, or on the
inner wall of the column tubing, e.g., by in situ polymerization (cross-linking) after coating.
Mobile Phase A fluid which percolates through or along the stationary bed, in a definite
direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a
supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression
Carrier Gas may be used for the mobile phase. In elution chromatography the expression
Eluent is also used for the mobile phase. Elute (verb) To chromatograph by elution
chromatography. The process of elution may be stopped while all the sample components are
still on the chromatographic bed or continued until the components have left the
chromatographic bed. Note: The term "elute" is preferred to the term Develop used in former
nomenclatures of planar chromatography.
Effluent The mobile phase leaving the column. Sample The mixture consisting of a number of
components the separation of which is attempted on the chromatographic bed as they are carried
or eluted by the mobile phase.
Sample Components The chemically pure constituents of the sample. They may be unretained
(i.e., not delayed) by the stationary phase, partially retained (i.e., eluted at different times) or
retained permanently. The terms Eluite or Analyte are also acceptable for a sample component.
Solute A term referring to the sample components in partition chromatography.
Solvent A term sometimes referring to the liquid stationary phase in partition chromatography.
Note: In liquid chromatography the term "solvent" has been often used for the mobile phase.
This usage is not recommended.
Zone A region in the chromatographic bed where one or more components of the sample are
located. The term Band may also be used for it.
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-
volatile mixtures.[1] Thin-layer chromatography is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary
phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as
the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend
the TLC plate at different rates, separation is achieved.[2] The mobile phase has different
properties from the stationary phase. For example, with silica gel, a very polar substance, non-
polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing
chemists to fine-tune the bulk properties of the mobile phase.
After the experiment, the spots are visualized. Often this can be done simply by
projecting ultraviolet light onto the sheet; the sheets are treated with a phosphor, and dark spots
appear on the sheet where compounds absorb the light impinging on a certain area. Chemical
processes can also be used to visualize spots; anisaldehyde, for example, forms colored adducts
with many compounds, and sulfuric acid will char most organic compounds, leaving a dark spot
on the sheet.
To quantify the results, the distance traveled by the substance being considered is divided by the
total distance traveled by the mobile phase. (The mobile phase must not be allowed to reach the
end of the stationary phase.) This ratio is called the retardation factor (Rf). In general, a substance
whose structure resembles the stationary phase will have low Rf, while one that has a similar
structure to the mobile phase will have high retardation factor. Retardation factors are
characteristic, but will change depending on the exact condition of the mobile and stationary
phase. For this reason, chemists usually apply a sample of a known compound to the sheet before
running the experiment.
Thin-layer chromatography can be used to monitor the progress of a reaction, identify
compounds present in a given mixture, and determine the purity of a substance. Specific
examples of these applications include: analyzing ceramides and fatty acids, detection
of pesticides or insecticides in food and water, analyzing the dye composition of fibers
in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification
of medicinal plants and their constituents [3]
A number of enhancements can be made to the original method to automate the different steps,
to increase the resolution achieved with TLC and to allow more accurate quantitative analysis.
This method is referred to as HPTLC, or "high-performance TLC". HPTLC typically uses
thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of
resolution due to diffusion.

Plate preparation[edit]
TLC plates are usually commercially available, with standard particle size ranges to
improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a
small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a
thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The
resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The
thickness of the absorbent layer is typically around 0.1–0.25 mm for analytical purposes and
around 0.5–2.0 mm for preparative TLC.[4]

Technique[edit]
The process is similar to paper chromatography with the advantage of faster runs, better
separations, and the choice between different stationary phases. Because of its simplicity and
speed, TLC is often used for monitoring chemical reactions and for the qualitative analysis of
reaction products. Plates can be labeled before or after the chromatography process using a
pencil or other implement that will not interfere or react with the process.
To run a thin layer chromatography plate, the following procedure is carried out:[5]

 Using a capillary tube, a small spot of solution containing the sample is applied to a plate,
about 1.5 centimeters from the bottom edge. The solvent is allowed to completely evaporate
off to prevent it from interfering with sample's interactions with the mobile phase in the next
step. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in
a vacuum chamber. This step is often repeated to ensure there is enough analyte at the
starting spot on the plate to obtain a visible result. Different samples can be placed in a row
of spots the same distance from the bottom edge, each of which will move in its own
adjacent lane from its own starting point.
 A small amount of an appropriate solvent (eluent) is poured into a glass beaker or any other
suitable transparent container (separation chamber) to a depth of less than 1 centimeter. A
strip of filter paper (aka "wick") is put into the chamber so that its bottom touches the solvent
and the paper lies on the chamber wall and reaches almost to the top of the container. The
container is closed with a cover glass or any other lid and is left for a few minutes to let the
solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate
the chamber will result in poor separation and non-reproducible results.)
 The TLC plate is then placed in the chamber so that the spot(s) of the sample do not touch
the surface of the eluent in the chamber, and the lid is closed. The solvent moves up the plate
by capillary action, meets the sample mixture and carries it up the plate (elutes the sample).
The plate should be removed from the chamber before the solvent front reaches the top of the
stationary phase (continuation of the elution will give a misleading result) and dried.
 Without delay, the solvent front, the furthest extent of solvent up the plate, is marked.
 The plate is visualized. As some plates are pre-coated with a phosphor such as zinc sulfide,
allowing many compounds to be visualized by using ultraviolet light; dark spots appear
where the compounds block the UV light from striking the plate. Alternatively, plates can be
sprayed or immersed in chemicals after elution. Various visualising agents react with the
spots to produce visible results.
Separation process and principle[edit]
Different compounds in the sample mixture travel at different rates due to the differences in their
attraction to the stationary phase and because of differences in solubility in the solvent. [6] By
changing the solvent, or perhaps using a mixture, the separation of components (measured by
the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to
estimate the separation of a flash chromatography column. (A compound elutes from a column
when the amount of solvent collected is equal to 1/Rf.)[7] Chemists often use TLC to develop a
protocol for separation by chromatography and use TLC to determine which fractions contain the
desired compounds.

Separation of compounds is based on the competition of the solute and the mobile phase for
binding sites on the stationary phase.[3] For instance, if normal-phase silica gel is used as the
stationary phase, it can be considered polar. Given two compounds that differ in polarity, the
more polar compound has a stronger interaction with the silica and is, therefore, better able to
displace the mobile phase from the available binding sites. As a consequence, the less polar
compound moves higher up the plate (resulting in a higher Rf value).[6] If the mobile phase is
changed to a more polar solvent or mixture of solvents, it becomes better at binding to the polar
plate and therefore displacing solutes from it, so all compounds on the TLC plate will move
higher up the plate. It is commonly said that "strong" solvents (eluents) push the analyzed
compounds up the plate, whereas "weak" eluents barely move them. The order of
strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel-
coated TLC plates, the eluent strength increases in the following
order: perfluoroalkane (weakest), hexane, pentane, carbon
tetrachloride, benzene/toluene, dichloromethane, diethyl ether, ethyl
acetate, acetonitrile, acetone, 2-propanol/n-butanol, water, methanol, triethylamine, acetic
acid, formic acid (strongest). For C18-coated plates the order is reverse. In other words, when the
stationary phase is polar and the mobile phase is nonpolar, the method is normal-phase as
opposed to reverse-phase. This means that if a mixture of ethyl acetate and hexane as the mobile
phase is used, adding more ethyl acetate results in higher Rf values for all compounds on the
TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order
of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in
selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar
stationary phase should be used, such as C18-functionalized silica.

Analysis[edit]
As the chemicals being separated may be colorless, several methods exist to visualize the spots:

 Fluorescent analytes, like quinine, may be detected under blacklight (366 nm)
 Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate,
is added to the adsorbent that allows the visualization of spots under UV-C light (254 nm).
The adsorbent layer will thus fluoresce light-green by itself, but spots of analyte quench this
fluorescence.
 Iodine vapors are a general unspecific color reagent
 Specific color reagents into which the TLC plate is dipped or which are sprayed onto the
plate exist.[8][9][10]
o Potassium permanganate – oxidation
o Bromine
o Acidic vanillin
o Phosphomolybdic acid

 In the case of lipids, the chromatogram may be transferred to a polyvinylidene

fluoride membrane and then subjected to further analysis, for example mass spectrometry, a
technique known as far-eastern blot.
Once visible, the Rf value, or retardation factor, of each spot can be determined by dividing the
distance the product traveled by the distance the solvent front traveled using the initial spotting
site as reference. These values depend on the solvent used and the type of TLC plate and are not
physical constants.

Applications[edit]
Characterization[edit]
In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a
capillary tube are placed on the plate: a spot of starting material, a spot from the reaction
mixture, and a cross-spot with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to
run. The analysis is qualitative, and it will show if the starting material has disappeared, i.e. the
reaction is complete, if any product has appeared, and how many products are generated
(although this might be underestimated due to co-elution). Unfortunately, TLCs from low-
temperature reactions may give misleading results, because the sample is warmed to room
temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC
is not the same as what is in the low-temperature flask. One such reaction is
the DIBALH reduction of ester to aldehyde.
In one study TLC has been applied in the screening of organic reactions,[11] for example in the
fine-tuning of BINAP synthesis from 2-naphthol. In this method, the alcohol and catalyst
solution (for instance iron(III) chloride) are placed separately on the baseline, then reacted, and
then instantly analyzed.
A special application of TLC is in the characterization of radiolabeled compounds, where it is
used to determine radiochemical purity. The TLC sheet is visualized using a sheet of
photographic film or an instrument capable of measuring radioactivity. It may be visualized
using other means as well. This method is much more sensitive than the others and can be used
to detect an extremely small amount of a compound, provided that it carries a radioactive atom.