Instrumental Methods of Analysis

An analytical method involves the use of an analytical

technique, operated within specific and appropriate
measurement parameters, for solving a problem.
For examples:
- The analysis of styrene-acrylonitrile copolymers
using infrared spectrophotometry (IR), and
- The determination of lead in drinking water using
ICP-AES
 Instrumental Methods of Analysis

• Instrumental methods of chemical analysis have become the

principal means of obtaining information in diverse areas of
science and technology.
• The speed, high sensitivity, low limits of detection,
simultaneous detection capabilities, and automated operation of
modern instruments, when compared to classical methods of
analysis, have created this predominance.
• A variety of instrumental techniques are available for the
application to chemical problems. They include Spectroscopy,
Photometry, Spectrophotometry, Mass- Spectrometry, NMR-
Spectroscopy, Polarography, Chromatography (GC, HPLC ),
Nepleometry, Polarimetry, and Coulometry.
Instrumental Methods of Analysis (Continue)

• There are very few, if any, methods for chemical

analysis that are specific for a single chemical species.
• At best, analytical methods are selectiv for a few species or a
class of species.
• As a result, the separation of the analyte from potential
interferences is quite often a vital step in analytical
procedures.
• Until the middle of the twentieth century, analytical separations
were largely carried out by such classical methods as
precipitation, distillation, and extraction.
• Today, however, analytical separations are most commonly
carried out by chromatography and
electrophoresis, particularly with samples that are
multicomponent and complex.
5
Definition of chromatography

IUPAC definition (International Union of pure and

applied Chemistry) (1993):
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary while the
other moves in a definite direction.
History of Chromatography

Chromatography, literally "color writing",

was first employed by Russian
scientist Mikhail Tswett in 1903/1906.
Tswett used to chromatography to separate
plant pigments
He called the new technique chromatography
because the result of the analysis was 'written
in color' along the length of the adsorbent
column
Chromatography is a combination of two
words;
* Chromo – Meaning color
* Graphy – representation of
something on paper
 History of Chromatography (continue)
• Some 25 years after Tswetts experiments, Kuhn, Lederer and
Winterstein [45] rediscovered the technique. From that time on,
chromatography became considerably important, particularly in
the fields of organic chemistry and biochemistry.
• The first thorough theoretical work on chromatography was done
in 1941 by Martin and Synge.
• Instead of liquid-solid systems they used a liquid-liquid
technique to measure partition coefficients of liquids and were in
1954 awarded the Nobel-Prize for their groundbreaking work.
Their finding that paper strips could replace the columns was an
astonishing result and lead to the technique of paper
chromatography.
History of Chromatography (continue)

• In their original paper, Martin and Synge also pointed out that a gas
stream could replace the flowing liquid. In 1947,
• Glueckauf mentioned the possibility of determining adsorption isotherms
from the breakthrough curves of gas-solid chromatography.
• Not much work was done in the field of chromatography until 1952 when
James and Martin [48] reported their work on gas liquid chromatography.
This was the beginning of a rapid development of both gas liquid and
gas-solid forms of the technique. Within the next years, chromatography
became a powerful method in analytical, physicochemical, and
preparative applications
• James and Phillips were the first who used the chromatographic method
to measure gas-solid adsorption isotherms.
 Fantastic advances in
chromatography over 35 years

Chromatography has application in every branch

of the physical and biological sciences
12 Nobel prizes were awarded
between 1937 and 1972 alone for work
in which chromatography played a vital role
General Description of Chromatography
• Chromatography encompasses a diverse and important group of methods
that allow the separation, identification, and determination of closely
related components of complex mixtures;
• many of these separations are impossible by other means.
• In all chromatographic separations the sample is dissolved in a mobile
phase, which may be a gas, a liquid, or a supercritical fluid.
• This mobile phase is then forced through an immiscible stationary phase,
which is fixed in place in a column or on a solid surface.
• The two phases are chosen so that the components of the sample
• distribute themselves between the mobile and stationary phases
• to varying degrees.
 PRINCIPLES and USES OF CHROMATOGRAPHY

It is a physical separation method of separation in which the components of a

mixture are separated by differences in their distribution between two phases,
one of which is stationary (stationary phase) while the other (mobile phase)
moves through it in a definite direction. The substances must interact with the
stationary phase to be retained and separated by it. Those components
strongly retained by the stationary phase move only slowly with the flow of
mobile phase. In contrast, components that are weakly held by the stationary
phase travel rapidly. Because of these differences in migration rates, sample
components separate into discrete bands, or zones, that can be analyzed
qualitatively and quantitatively.
 Analyze
Separate
• Identify
• Purify

Components • Quantify
Mixture
PRINCIPLES OF CHROMATOGRAPHY (Continue)

Mobile Phase:
The Phase that travels through the column
(gas or liquid) – transport sample through
the column.
Stationary Phase:
Immiscible solid or liquid phase that fixed
in place in the column or on a solid support
– retain analytes within the column.

Band or Zone:
-Area across which analyte is distributed on
column
-Zones of different analytes gradually
separate as bands progress down column
13
 Sample Mobile

Figure:
Schematic diagram showing the separation of compounds
A and B. and the output of the detector response at
various stages of elution

The process of:

 Addition of sample
 Mobile elution process
 Separation mechanism
 Retention time ?
 Detection by, UV lamp, UV detector,
other detectors.
 Eluted bands / collection
A
 Chromatogram? (function of retention B
Response
time versus detector response)
 Partition coefficient K’
 k’ = Cs/CM time 14
Classification of chromatography
according to mobile phase:

1- Liquid chromatography:
mobile phase is a liquid. (LLC, LSC).
2- Gas chromatography :
mobile phase is a gas. (GSC, GLC).
Classification according to the packing
of the stationary phase:
1- Thin layer chromatography (TLC):
the stationary phase is a thin layer supported
on glass, plastic or aluminium plates.

2- Paper chromatography (PC):

the stationary phase is a thin film of liquid
supported on an inert support.

3- Column chromatography (CC):

stationary phase is packed in a glass column.
Classification according to
the force of separation:

1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.
01/14/21 19
Thin layer chromatography (TLC)

A method for identifying substances and

testing the purity of compounds.

TLC is a useful technique because it is

relatively quick and requires small
quantities of material.
Separations in TLC involve distributing a mixture of two or
more substances between a stationary phase and a mobile
phase.

The stationary phase:

is a thin layer of adsorbent (usually silica gel or alumina)
coated on a plate.

The mobile phase:

is a developing liquid which travels up the stationary phase,
carrying the samples with it.

Components of the samples will separate on the stationary

Thin Layer Chromatography (TLC)
Preparing the Chamber
To a jar with a tight-fitting lid add enough of
the appropriate developing liquid so that it is
0.5 to 1 cm deep in the bottom of the jar.
Close the jar tightly, and let it stand for about
30 minutes so that the atmosphere in the jar
becomes saturated with solvent.
 Preparing the Plates for Development

With a pencil, etch two small notches into the adsorbent about
2 cm from the bottom of the plate.

The notches should be on the edges of the plate, and each

notch should be the same distance up from the bottom of the
plate.

The notches must be farther from the bottom of the plate than
the depth of the solvent in the jar.

Using a drawn-out capillary tube, spot the samples on the

plate so that they line up with the notches you etched.
 Developing the Plates
After preparing the development chamber and spotting the
samples, the plates are ready for development.

Be careful to handle the plates only by their edges, and try to

leave the development chamber uncovered for as little time
as possible.

When the plates are removed from the chamber, quickly trace
the solvent front (the highest solvent level on the plate) with
a pencil.
KHROMATOGRAFI LAPISAN TIPIS
Identifying the Spots (visualization)
If the spots can be seen, outline them with
a pencil.

If no spots are obvious, the most common

visualization technique is to hold the
plate under a UV lamp.
Many organic compounds can be seen
using this technique, and many
commercially made plates often
contain a substance which aids in the
visualization of compounds.
 Visualizing Agents
Alkaloids: Dragendorff’s reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin

 Interpreting the Data

The Rf (retention factor) value for each spot should be

calculated.
It is characteristic for any given compound on the same
stationary phase using the same mobile phase for
development of the plates.

Hence, known Rf values can be compared to those of

unknown substances to aid in their identifications.
(Note: Rf values often depend on the temperature and the
solvent used in the TLC experiment.
the most effective way to identify a compound is to spot
known substances – authentic - next to unknown
substances on the same plate.)
In addition, the purity of a sample may be estimated from the
chromatogram.
An impure sample will often develop as two or more spots,
while a pure sample will show only one spot
 Two-dimensional chromatography

When large numbers of substances are to be separated on a single

chromatogram.

Development in a direction perpendicular to the first, and with a solvent

system different from that used initially is often necessary.

The sample is applied on one corner of a TLC and after development with
the first solvent, the TLC is dried , rotated 90o and developed in the
second direction.

Usually, different types of solvents systems are used in each direction.

 Paper Chromatography

A method of partition chromatography using filter paper

strips as carrier or inert support.
The factor governing separation of mixtures of solutes on filter
paper is the partition between two immiscible phases.
One is usually water adsorbed on cellulose fibres in the paper
(stationary phase).
The second is the organic solvent flows past the sample on the
paper (mobile phase).
General Procedure

1- Choice of paper and solvent to be used.

2- Application of the sample.
3- Equilibration of paper.
4- Development.
5- Detection.
6- Identification of substances.
Techniques of development with
various flow directions

Ascending development

Descending development