Chapter 4

Analytical procedures and

Instrumentation

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 Objectives

Upon completion of this lecture the student

will be able to
• List basic components of spectrophotometers
• Describe spectrophotometer component parts
with respective functions
• Explain general principles of refractometry,
turbidimetry, nephlometery, fluorometry,
atomic absorption spectroscopy, flame
emission spectroscopy, electrophoresis
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 Outline

• Colorimetry and spectrophotometry

• Basic components of spectrophotometers
• General principles of refractometry
• General principles of fluorometry
• General principles of turbidimetry,and
nephlometery
• Principles of AAS and FES
• General principles of electrophoresis

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 Introduction
 Spectroscopy is the use of the absorption, emission, or
scattering of electromagnetic radiation by atoms or
molecules (or atomic or molecular ions) to qualitatively or
quantitatively study the atoms or molecules.

 The interaction of radiation with matter can cause

redirection of the radiation and/or transitions between
the energy levels of the atoms or molecules.
 A transition from a lower level to a higher level with
transfer of energy from the radiation field to the atom or
molecule is called absorption.
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 Introduction Cont..
 A transition from a higher level to a lower level
is called emission where energy is transferred to
the radiation field

Redirection of light due to its interaction with

matter is called scattering and may or may not
occur with transfer of energy, i.e., the scattered
radiation has a slightly different or the same
wavelength.
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 Colorimetry and Spectrophotometer
• Spectrophotometer
– the instrument that produces monochromatic light, transmits
light through a colored solution and measures %
Transmittance or Absorbance of light
– an instrument that measures the intensity of the light entering
a sample and the light exiting a sample and compares the two
intensities.
– Similar to colorimeters but It provide narrow range of light
(closer to monochromatic) with the use of diffraction gratings
– More accurate and precise photometers

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 Spectrophotometer cont..

 Used to measure the light transmitted by a

solution to determine the concentration of the
light-absorbing substance in the solution

 It is based on two principles:

i. Substances absorb light at unique wavelengths and
ii.The amount of light absorbed is proportional to the
amount of substance that is present

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Colorimetry

Colorimetry: Measuring % transmitted light through

a colored solution

Many colored solutions absorb light

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• Spectrophotometers are similar to colorimeters but provide narrow
range of light (closer to monochromatic) with the use of diffraction
gratings and have more accurate and precise photometers.
• A spectrophotometer may have a double beam of light to measure the
reference and sample light absorbance at the same time.

• Absorbance cannot be measured directly by a spectrophotometer but

rather is mathematically derived from % T

• Spectrophotometry: the process of measuring the amount of light

absorbed by a substance and relating to the concentration of that
substance.
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Schematic Diagram of a Double-
Beam
UV-Vis. Spectrophotometer

By switching the monochromatic light to a reference cell and

then a sample cell

This may require 2 separate photodetectors, making this a

more sophistacated instrument.

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 Spectrophotometer Components

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 Essential Instrumentation
(Spectrophotometer)
Basic spectrophotometer components include:
1. Light sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers (cuvettes)
4. Detector
5. Signal processor and readout

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 1. Light Sources
 Light source : provides radiant energy which is absorbed
by the compound under investigation.
 Light source must fulfil the following precondition:
It must produce a beam of sufficient power,
should be able to emit a spectrum which contain all
wavelengths (polychromatic light), and
must be stable
 Therefore, care must be taken in choosing a light source for
a particular analysis, because the amount of light emitted at
the desired wavelength may be too little or too much.

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 Light Sources cont..
• Tungsten filament lamp common source of
visible light
– Used in the wavelength range of 350 - 2500 nm.
• Deuterium and hydrogen lamps common source of
UV light
– emit radiation in the range 160 - 375 nm
• Tungsten/halogen lamps are very efficient, and their
output range extends into the ultra-violet
– Used in many modern spectrophotometers

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 2.Wavelength Selector
(Monochromator)
• It is a system for isolating radiant energy of a desired
wavelength and excluding that of other wavelengths
• All monochromators contain the following
component parts:
– Entrance slit
– Collimating lens
– Prism or grating
– Focusing lens
– Exit slit
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 Monochromator cont’d
Slits
• There are two types of slits present in monochromators:
– The first, at the entrance, focuses the light from the light
source into the monochromator
– The second slit determines the band width of light.
• The overall purpose of slits in photometers is to make
the light parallel and reduce the stray light.

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 Monochromator cont’d
• There are various ways of selecting the desired wavelength,
including the use of filters, prisms, and diffraction gratings.
• Filter :Glass filters are not good monochromator since they
transmit light over a relatively wide range of wavelengths/poor
spectral purity (have wide band pass, 50 nm)
• Prisms: separate white light into a continuous spectrum of light
by refraction with shorter wavelength that are bent, or refracted,
more than longer wave lengths. A bandwidths of 0.5 nm, or
less, can be obtained.

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 Diffraction grating

• The gratings act as scattering centers for the rays of light,

with each groove producing diffracted waves of light.
• The grating is rotated and different parts of the spectrum are
allowed to fall on the photocell.
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 (3)Sample Containers (Cuvettes)
• A small vessel used to hold a liquid sample to be analyzed in the light
path of a spectrometer
• May be round, square, or rectangular
• Constructed from glass, silica (quartz), or plastic.
• Square or rectangular cuvets have plane parallel optical surfaces and
a constant light path.
• Most have a 1.0-cm light path.
• Ordinary borosilicate glass cuvets are suitable for measurements in
the visible portion of the spectrum.
• For readings below 340 nm, quartz cells are usually required
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 (4)Detector
• Devices that convert light beam into an electric signal
– The electric signal is proportional to the number of photons striking
its photosensitive surface.
• The detector in a spectrometer must produce a signal related to the
intensity of the radiation falling on it.
• Photosensors are not equally sensitive to all wavelengths.
• All photosensors have a frequency below which they will not respond.
This is termed the excitation frequency (Fex).
• No matter how intense a beam of light striking the photosensor is, if
its frequency is below Fex, the device will not respond.
• Include: Photomultiplier tubes and Photodiode arrays
 
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 a) Photodiodes
 Photodiodes are semiconductors generate potential
difference (usually 5V) or discharge energy upon being
struck by light.
 It is example of a multichannel photon detector.
 Photodiodes detectors are capable of measuring all
elements of a beam of dispersed radiation simultaneously
• The changes are converted to current and are
measured.
• Each diode is scanned, and the resultant electronic
change is calculated to be proportional to
absorption.
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 b) Photomultiplier tube
• Photomultiplier tubes are the most sensitive components of
all photo sensors to UV and visible radiation.
• They have fast response times.
• The photomultiplier tube consists three components:
1)cathode which emits electrons when struck by photons of
radiation),
2) several dynodes (which emit several electrons for each
electron striking them) and
3) an anode (where electron eventually collected ).
• A photon of radiation entering the tube strikes the cathode,
causing the emission of several electrons.
• These electrons are accelerated towards the first dynode
(which is 90V more positive than the cathode).
• The electrons strike the first dynode, causing the emission
of several electrons for each incident electron.
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• These electrons are then accelerated towards the
second dynode, to produce more electrons which are
accelerated towards dynode three and so on.
• Eventually, the electrons are collected at the anode.
By this time, each original photon has produced 106
- 107 electrons.
• The resulting current is amplified and measured.

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 (5) Signal Processor/Read Out
• Electrical energy from the detector is displayed on some type of
meter or read out systems.
• The result is usually presented in transmittance units, absorbance
unit, or a direct concentration units.
• A meter reading device displays the analogue signal by reflecting a
needle along a scale or digitally.
• On a spectrophotometer, the readout will be in %Transmittance or
Absorbance.
• The user will have to record the value on paper and then perform the
appropriate calculations before reporting out the control or patient
result.
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 Concept of Lamda max (Λmax)
• The wavelength at which there is maximum absorbance
for that particular solution is referred as lamda max,
• Symbolically represented as “λmax”
• This spectrum will help to determine the best
wavelength for the spectrophotometric analysis.
– improves the sensitivity as well as specificity of the
measurement
• The optimum wavelength for a specific analysis
depends on several factors,
– the absorption maxima of the chromogen
– the absorption spectra of possible interfering chromogens.
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 Concept of Lamda max (Λmax) cont’d

• According to Beer's law, the higher the molar absorptivity,

the greater the absorption at a given concentration and
wavelength and the higher the sensitivity of the analysis.
• A general rule for selecting optimum wavelength at
which to monitor a spectrophotometric reaction includes:
– (1) Choose absorption peak with the greatest possible
molar absorptivity.
– (2) Choose a peak that is as far as possible from the
absorption peaks of commonly interfering chromogens.

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 Refractometry
• Refractometry is the method of measuring substances refractive
index to assess their composition or purity
• Refractometer: instrument used to measure refractive index
• Principles of Refraction
– The degree of refraction of light beam depends on the
difference in the speed of light between two different media
– the ratio of the two speeds has been expressed as the index of
refraction, or refractive index.

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 Fluorometry
• A Fluorometer is a photometer that measures the light
emitted (relatively long wavelength) by a substance that
has been previously excited by a source of short-
wavelength radiation
Principle of Fluorescence Emission
• When light impinges on matter, it can simply pass
through, or can be scattered, or it can be absorbed.
• When light is absorbed by the matter, the absorber
molecule gets excited and spontaneously returned back
to the ground state emitting long wavelength light
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 Turbidimetry and Nephelometry
• Light scattering is a physical phenomenon resulting from the
interaction of light with particles in solution.
• Two analytical methods are based on light scattering:
• Turbidimetry:
– the measurement of decrease in intensity of light as the incident
beam of light passes through a solution of particles
– Turbidity is measured at 180° from the incident beam
• Nephelometry
– It is the detection of light energy scattered or reflected toward a
detector that is not in the direct path of the transmitted light.
– Turbidity is measured at 90° from the incident beam
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 Atomic Absorption Spectrophotometry(AAS)
• It measures light to determine the concentration of an analyte
• It is used for determination of calcium, magnesium, lithium, lead,
copper, zinc and other metals.
• Highly specific: use line spectra as the incident energy source
• 100 times more sensitive than flame emission methods
• Measurement principle
– Vaporized ground state atoms suspended in a flame will absorb
radiation at a very narrowly defined wavelength (line
spectra=spectrum with only certain color)characteristic wavelength of
light originates from a hollow cathode lamp.
– Unabsorbed radiation is transmitted through the monochromator to
the detector.
– The amount of absorbed light is directly proportional to the
concentration of atoms in the flame.
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 Flame Emission spectrophotometer
• A photometer with a carefully controlled flame for a light source
• Alkali metals are relatively easy to excite in a flame.
– Lithium produces a red emission; Sodium, a yellow emission; and
potassium, a red-violet color in a flame.
– These colors are characteristic of the metal atoms that are present
as cations in solution.
• Measurement principle
– Based on the characteristic emission of light by atoms of many
metallic elements when given sufficient energy (hot flame)
– The intensity of the characteristic wavelengths of radiant energy
produced by the atoms in the flame is directly proportional to the
number of atoms excited in the flame, which is directly
proportional to the concentration of the substance of interest in
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the sample.
 Electrophoresis
• Electrophoresis is a Greek word coined from
electricity and phoresis, Phoresis means to carry
• Hence electrophoresis is a process by which charged
particles or solutes travel in electric field.
• It also defined as the migration of charged solutes or
particles of different size and charge in a liquid
medium under the influence of an electrical field.
• A method of separating substances, especially
proteins, before analyzing molecular structure based
on the rate of movement of each component in a
colloidal suspension while under the influence of an
electric field.
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 Summary
• Spectrophotometers and filter colorimeters differ in the way in
which light of specific wavelength is selected.;
spectrophotometers use prisms and diffraction gratings while
colorimeters use colored filters.
• Spectrophotometer do have component parts including: light
source, Entrance slit, monochromator, exit slit, cuvette holder,
detector and read out devices.
• Refractometry, turbidimetry, nephlometery, and florometry are
methods that are used in clinical chemistry laboratories to
measure concentration of analyte in the sample
• Electrophoresis is versatile and powerful analytical technique
used to separate and analyze a diverse range of ionized analyses

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