4.
Analytical procedures and
Instrumentation
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
Prism
Exit slit
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 2
1
5=Light scattering optics
3
6=Detector filter
7=Detector
5
A
6
0o turbidometer
Io
7
B C
90o nephelometer
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?
