STC 212 Power Point

STC 212
INSTRUMENTAL ANALYTICAL
CHEMISTRY AND QUALITY CONTROL
Properties of light
Definition of light: light is a form of energy that visible to the human eye which is radiated by moving charge
particles.
 Technically, light or visible light is electromagnetic radiation of a wavelength that is visible to eye.
 light is composed of elementary particles called photons.
 Common sources of light are lamp,lantern,electric lighting ,Sun ,illumination from moon light etc.
 When there is a need to separate light of different wave length with high resolution then a diffraction grating is
most often the tool of choice.
 It acts as super prism, separating the different colors of light much more than the dispersion effect in prism.
The spectrum
 Spectrum is a rain bow like series of colours ,in the
order of violet ,blue ,green ,yellow, orange and red .
 This is produced by splitting a composite light into
component colours.
Electromagnetic spectrum
 Electromagnetic spectrum is the distribution of
electromagnetic radiation according to energy or
equivalently, by virtue of the relations to frequency
or wavelength.
Basic principles of light absorption and emission
Laws of light absorption
 Lamberts law-states that the same proportions of incident light is absorbed per unit
thickness irrespective of its intensity.
 l0 = incident light, I = emergent light, l = path length, k = constant. Log10 l0 ̸ l is called
either absorbance (A) or optical density (O.D).
A is directly proportional to path length.
 Beer’s Law- The law states that the absorption of light is directly proportional to the
number of absorbing molecules (Transmittance decreases with the number of absorbing
molecules)
 limitation: Lambert’s law holds for all cases, but Beer’s law is usually only obeyed for
dilute solutions.
Principles of UV spectrophotometer
 Spectrophotometer consist of two instruments, namely spectrometer for
producing light of any selected colour (wave length) and a photometer
for measuring the sensitivity of light. The instruments are arranged so
that liquid in a curvette can be placed between the spectrometer beam
and the photometer. The amount of light passing through the tube is
measured by the photometer. The photometer delivers voltage signed to a
display device, normally galvanometer. The UV-Visible
spectrophotometer uses two light sources, a deuterium (D2), lamp for
ultraviolet light and a tungsten (W) lamp for visible light. The intensities
of the light beams are then measured at the end.

Principles of atomic spectroscopy(flame photometer)
 The principle is that atoms of element in an excited
state emit radiation at a specific wave length as
they return to the ground state. The amount of
light that is emitted is proportional to the
concentration of the material in solution to be
tested. In this method heat radiation is applied
instead of light.
 A flame photometer, is designed to cause atomic
excitation of the analyte from the sample to be
tested and subsequently to measure the emitted
radiation. The flame temperature must be high
enough to excite the atom under investigation. The
hotter the flame, the greater the proportion of the
atoms to be excited.
Fig. 3 A schematic diagram of a flame photometer
Principles of atomic absorption spectrophotometry (AAS)
 In atomic absorption analysis, the absorption of light by

free atomic species is measured. This is done by the use of
an instrument called Atomic absorption spectrophotometer
(AAS). In this process, a flame system is generally employed
to dissociate elements from their chemical bonds. The atoms
absorb light at characteristic wave length when present in
their ground state. The sample is sprayed into the flame.
 The source of light is a hollow cathode lamp containing the

element under examination. The lamp produces radiation at
the appropriate wavelength for absorption by the free atoms
of the sample.
Fig.4, A schematic diagram of AAS
Applications of AAS
 Atomic absorption can be used to determine the
concentration of metallic elements such as copper,
Arsenic, lead, cadmium etc, in varieties of samples.
 Atomic absorption is widely used in food analysis.
 widely used in food analysis , environmental impact

analysis ,etc.
Principles of mass spectroscopy
 This technique is based on the fact that charged particles in
motion is detected by a magnetic field . The amount of
deflection on the momentum of the particle .The mass
spectrometer uses differences in mass to charge ratio to
diffentiate ionized molecules or fragment of molecules from
one another. Each molecule has distinctive fragmentation
pattern that provides structural information .The MS is
useful in determining the structure of unknown molecule.

Components of mass spectroscopy
 The sample inlet system and ion source.
 Mass analyzer.
 Detector system.
 Control and signal processing electronics

Fig.5, Schematic diagram of mass spectroscopy
The application of mass spectrometry
 In many cases, the relative molecular mass can be

found quickly and with a very high degree of
precision by mass spectrometry.
 It is also applied in the identification of structural

fragments of unknown compound etc.
Gas Chromatography (GC)
 Gas chromatography is a technique for carrying
out the separation and measurement of mixtures
of materials that can be volatilized.
 In gas chromatography, mixture is separated by

adsorption between two immiscible phases, one of
which is a mobile (carrier gas) and One of which is
stationary (column packing).
Types of gas chromatography
Gas Liquid Chromatography Gas solid Chromatography

 The stationary phase is a  In this adsorption
viscous liquid coated onto chromatography the
a solid support material. column
 Separating principle packing is a solid material
depends on the difference with surface active
in the partition coefficients properties.
between the liquid and gas  The separation depends to
phases of the constituents the extent to which the
of a mixture. components of a mixture
are adsorbed by the solid,
i.e silica.
GC column
 The column is the most important feature of a GC.
 It contains the stationary phase and has a great

influence on the separation of mixture.
 The column is made of glass or metal.

The stationary phase
 The stationary phase for GC needs to be:
 thermally stable
 unreactive
 negligible volatility
 should have a reasonable column life over the
operating range.
NOTE: The choice of stationary phase depends on
the selectivity and degree of polarity of the
compounds.
The detectors for GC
 The purpose of a detector is to monitor the GC column
effluent and to measure the variations in the
composition of the eluted components.
 Detectors used in GC are:
 Flame ionization (FID)
 Nitrogen phosphorous (NPD)
 Flame photometric
 Kathermeter (Thermal conductivity – TCD)
 Electron capture (ECD)
Applications of GC
 GC can be used for separating, identifying and

estimating volatile compounds of fatty acids,
steroids, other liquids, vitamins, sugars and amino
acids.
 It is also extensively used in the analysis of drugs,

pesticides, petroleum products and many other
substances.
High performance liquid Chromatography(HPLC)
 High performance liquid chromatography is the
type of column chromatography in which the
mobile phase is a liquid material Separation of
mixture is achieved by differential distribution
of the sample components between the
stationary and the mobile phase.
 For effective separation, it is essential to have a
very small and regular shaped support media, a
supply of mobile phase pumped at a pressure
that is adequate to give suitable constant flow
rate through the column and a convenient
efficient detector system .
Basic components of HPLC
 HPLC instrument consist of five sections:
 the solvent reservoir

 pump
 chromatographic column and oven
 detector unit
 Amplifier and signal processing unit or recorder.
Figure 6. schematic diagram of HPLC
The mobile phase and the column
 The liquid which can be used for HPLC separations
may comprise of water, aqueous buffer solutions,
organic solvents such as methanol, aceto nitrile, etc.
 The column for analytical HPLC are typically 10-25cm

long and 4 – 6mm internal diameter. The columns are
made of stainless steel to cope with the operating high
pressure.
Detectors used in HPLC
 These are classified as:

 Detectors which monitors a specific property of
the solute, e.g. UV absorbance and fluorescence.
 Detecting system which monitors a bulk property
of the eluant, e.g. refractive index.
 Detectors which function by separating the solvent
from the eluant, e.g. flame ionization (FID) or mass
spectrometry (MS) detectors.
Application of HPLC
 HPLC is used for both qualitative and quantitative

analysis of variety of substances ,such as drugs,
pesticides, herbicides, vitamins, natural products,
e.t.c.
 NOTE: The choice of detector is governed by the

properties of the solute and the sensitivity required
from the analysis.
The principles of quality control
 Quality control is the analytical process by which we

measure the “fitness for purpose” of a particular
product or process. It ensures that a product is
manufactured within certain defined limits of purity or
that an analytical process is working within a
particular range of accuracy. This is to make the
products fit for the purpose.
 So, quality can be defined as the body of
characteristics, properties, attributes or abilities of an
entity that make it fit for purpose.
Quality control in the laboratory
 Quality control involves the examination of laboratory

and its results.
 But quality assessment involves inspecting quality

control, the laboratory, the results it supplies and their
relationship to the solution to the analytical problem.
 Quality assurance activities should lead to the

implementation of corrective actions, which should
initially focus on the laboratory.
Good laboratory practices (GLP)
 Good Laboratory practice can be defined as a body
of rules, operating procedure and practices
established by a given organization that are
considered to be mandatory with a view to ensuring
quality and correctness in the results produced by a
Laboratory.
Unique elements of GLP
Standard operating procedures Quality Assurance Unit
(SOP) (QAU)
 The Standard operating  The Quality assurance unit

procedures are detailed is the unit that is
description of all the responsible for instituting
activities performed by a quality, controlling and
Laboratory. assessing it, and proposing
actions to enhance it.
Laboratory accreditation
 Laboratory accreditation with respect to quality is

defined as the formal written acknowledgment
that a Laboratory is fit and competent to perform
one or more type of analysis. This type of
accreditation is provided by a public or private
national organization and relies on internationally
established standards.

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STC 212

 light is composed of elementary particles called photons.

most often the tool of choice.

 Spectrum is a rain bow like series of colours ,in the

order of violet ,blue ,green ,yellow, orange and red .

 This is produced by splitting a composite light into

electromagnetic radiation according to energy or

equivalently, by virtue of the relations to frequency

thickness irrespective of its intensity.

 l0 = incident light, I = emergent light, l = path length, k = constant. Log10 l0 ̸ l is called

either absorbance (A) or optical density (O.D).

A is directly proportional to path length.

number of absorbing molecules (Transmittance decreases with the number of absorbing

producing light of any selected colour (wave length) and a photometer

for measuring the sensitivity of light. The instruments are arranged so

that liquid in a curvette can be placed between the spectrometer beam

measured by the photometer. The photometer delivers voltage signed to a

display device, normally galvanometer. The UV-Visible

spectrophotometer uses two light sources, a deuterium (D2), lamp for

of the light beams are then measured at the end.

 In atomic absorption analysis, the absorption of light by

 The source of light is a hollow cathode lamp containing the

 Atomic absorption is widely used in food analysis.

 widely used in food analysis , environmental impact

 This technique is based on the fact that charged particles in

motion is detected by a magnetic field . The amount of

deflection on the momentum of the particle .The mass

spectrometer uses differences in mass to charge ratio to

diffentiate ionized molecules or fragment of molecules from

one another. Each molecule has distinctive fragmentation

pattern that provides structural information .The MS is

useful in determining the structure of unknown molecule.

 The sample inlet system and ion source.

 Control and signal processing electronics

 In many cases, the relative molecular mass can be

 It is also applied in the identification of structural

 In gas chromatography, mixture is separated by

Gas Liquid Chromatography Gas solid Chromatography

 It contains the stationary phase and has a great

 The column is made of glass or metal.

 GC can be used for separating, identifying and

 It is also extensively used in the analysis of drugs,

 HPLC instrument consist of five sections:

 the solvent reservoir

 The column for analytical HPLC are typically 10-25cm

 These are classified as:

 HPLC is used for both qualitative and quantitative

 NOTE: The choice of detector is governed by the

 Quality control is the analytical process by which we

 Quality control involves the examination of laboratory

 But quality assessment involves inspecting quality

 Quality assurance activities should lead to the

 Good Laboratory practice can be defined as a body

of rules, operating procedure and practices

established by a given organization that are

considered to be mandatory with a view to ensuring

quality and correctness in the results produced by a

 The Standard operating  The Quality assurance unit