4.

Analytical procedures and

Instrumentation
 Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-
Ethiopia
 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 and
electrophoresis
 Outline of analytical procedures and
instrumentation lecture
• Introduction to colorimetry
• Colorimetry and spectrophotometry
• Basic components of spectrophotometers
• General principles of refractometry
• General principles of fluorometry
• General principles of turbidimetry,and
nephlometery,
• General principles of electrophoresis
 Introduction to Colorimetry

Many colored solutions absorb light

Colorimetry: Measuring % transmitted light
through a colored solution

P
 Colorimeter
• The instrument that produces monochromatic
light, transmits light through a colored
solution and measures % Transmittance or
Absorbance of light

• More accurate colori-

meters are called
spectrophotometers
 Colorimetry and Spectrophotometry
• The spectrophotometer is commonly used for
manual analysis of many clinical chemistry
tests
– It is often used as the back-up technique when the
automated system is temporarily not performing
well
• The principle behind analysis of many clinical
chemistry tests is spectrophotometric
Spectrophotometer Components
 Basic Instrument Components
(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
 Schematic Diagram of a Single-
Beam UV-Vis. Spectrophotometer

e-

Light Entrance Monochromator Exit Cuvette Detector Readout

Source Slit Slit Device
Schematic Diagram of a Double-
Beam
UV-Vis. Spectrophotometer
 Light Sources
• 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
 Wavelength Selector
(Monochromator)
• All monochromators contain the following
component parts:
– Entrance slit
– Collimating lens
– Prism or grating
– Focusing lens
– Exit slit
Prism
Czerney-Turner Grating
Monochromator
 Sample Containers (Cuvettes)
• Cuvettes can be round, square or rectangular
– Constructed from glass, silica or plastic
– Square or rectangular cuvettes have a constant light path, the most
usual being 1 cm in length
• Glass cuvettes are suitable for use between 320 and 950 nm
– But- UV light, silica (quartz) cuvettes are used below 320 nm and they
must be clean and free of scratches
 Detector
• The photomultiplier tube
– Commonly used detector in UV-Vis spectroscopy
– Photomultiplier tubes are electron tubes that amplify current
• Photodiode arrays
– Example of a multichannel photon detector. These detectors are
capable of measuring all elements of a beam of dispersed radiation
simultaneously
– Diodes discharge energy when they are struck by light
Cross-Section of Photo-Multiplier Tube
 Stray light
• Light radiation outside the narrow band
nominally transmitted by the monochromator.

• Scattering and diffraction inside the

monochromator introduce light of other
wavelengths into the exit beam.

• Should be eliminated by spectrophotometer

 Signal Processor/ReadOut
• 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 units (optical density), 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.
Manual Spectrophotometer

Manual
Spectrophotometer
 Refractometry
• Measures the change in the refractive index of
sample and relates it to the concentration of
total dissolved solutes
• It is a quick alternative to chemical analysis for
serum total protein when a rapid estimate is
required.
• Instrument used: refractometer
 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.
• The basic component of a spectroflorometer
are: excitation source, excitation
monochromator, sample cell, emission
monochromator and detector.
 Turbidimetry and Nephelometry:
Scattered Light
• Light meets a particle, an oscillating dipole is
induced in the particle by the incident light.
• Magnitude of this dipole moment is proportional
to the electric field strength of incident light.
• Polarizability of the electron cloud surrounding
the particle
• Oscillating dipole becomes a source of
electromagnetic radiation, re-radiating light at the
same wave length as the incident light in all
directions
 Scattered Light

• Factors to consider and understand

about the light scattering:
– the effect of partcle size
– wavelength dependence
– distance of observations
– effect of polarization of incident light
– the concentration of particles
– the molecular size of particles.
Measurement of scattered
light
• Light scatterings a physical phenomena
resulting from the interaction of light with a
particle in solution.
• the phenomena should not be confused with
turbidity and nephlometry, which are
methods used tomeasure scattered light.
 Turidimetry and nephelometry
• Turidimetry is the measurement of turbidity; generally
performed through use of an instrument
(spectrophotometer or photometer) that measure the
ratio of the intensity of the light transmitted through
dispersion to the intensity of the incident light

• Nephelometry: A technique that uses a nephelometer

to measure the number and size of particles in
suspension; measures the intensity of light scattered
by the particles with a detector at an angle to the
incident light beam.
• 1=Incident light
• 2=Excitation optics
• 3=Excitation filter 4
• 4=Sample cell 1
2
• 5=Light scattering optics
• 6=Detector filter 3
• 7=Detector

A
6
0o turbidometer
Io

B C
90 nephelometer
o
30o forward scatter nephelometery
Fig. Schematic diagram of light scattering instrumentation showing, A,the optics
position for a turbidometer;B, the optics position for a forward scattering nephelometer;
and C, the optics position for a right angle nephelometer
 Electrophoresis
• The migration of charged colloidal particles or
molecules through a solution under the
influence of an applied electric field usually
provided by immersed electrodes.
• A method of separating substances, especially
proteins, and analyzing molecular structure
based on the rate of movement of each
component in a colloidal suspension while
under the influence of an electric field.
 A schematic diagram of a typical electrophoresis apparatus
showing two buffer boxes with baffle plates, electrodes,
electrophoretic support (gel), wicks, cover,and power supply

-ve +ve

V A

+ -
The electrophoresis
apparatus
 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.
 Summary, continued..
• Refractometry, turbidimetry, nephlometery,
and florometry are methods that are used in
clinical chemistry laboratories to measure
cocnentartion of analte in the sample
• Electrophoresis is versatile and powerful
analytical technique used to separate and
analyze a diverse range of ionized analytes
 Review Questions
• What are the main components of a
spectrophotometer and the function of each?
• What is scattered light?
• What two types of spectrophotometry
measure scattered light?
• What is the use of electrophoresis in Clinical
Chemistry?
 Reference
1. Burtis, Carl A., and Ashwood, Edward R. Tietz:
Fundamentals of Clinical Chemistry. WB
Saunders, Co., Philadelphia, 2001.
2. Arneson, W and J Brickell: Clinical Chemistry:
A Laboratory Perspective 1st ed. FA Davis Co.,
Philadelphia, 2007
3. Burtis, Carl A., and Ashwood, Edward R..
Tietz: textbook of Clinical Chemistry. WB
Saunders, Co., Philadelphia, 1999.
 The next Chapter

Chapter 5

Measurement procedures and

calculations in spectrophotometer