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

The stationary phase is a

viscous liquid coated onto

a solid support material.

Separating principle

depends on the difference

in the partition coefficients

between the liquid and gas

phases of the constituents

of a mixture.

Gas solid Chromatography

In this adsorption

chromatography the

column

packing is a solid material

with surface active

properties.

The separation depends to

the extent to which the

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

(SOP)

The Standard operating

procedures are detailed

description of all the

activities performed by a

Laboratory.

Quality Assurance Unit

(QAU)

The Quality assurance unit

is the unit that is

responsible for instituting

quality, controlling and

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.