3 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,florometry 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
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.
Factorsto 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 measure 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
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
Assay techniques in clinical
chemistry
Basically there are different types of analytical
techniques:
End point,
Differential,
Kinetics method-fixed kinetics
- continuous monitoring
End point method
Reagent mixed with sample, and Abs is measured
after a predetermined incubation time interval.
In enzymatic assays, it does not indicate substrate
depletion or presence of inhibitors in reaction system.
best for batch runs (multiple samples ran
simultaneously)
Absorbance reading can take place at any time after
incubation, and during pre determined result reading
time, and analyte concentration calculated by using
appropriate absolute or competitive method
Differential method
Absorbance reading take place at two fixed points:
absorbance of the analyte (Aa), and absorbance at
reference point(Ar)
Then the corrected absorbance (Ac) value
calculated by subtracting Ar from Aa
Then analyte concentration is calculated by using
Ac and appropriate methods mentioned on slide 1.
Kinetics method
Is a method where the reaction rate is monitored
frequently at fixed predetermined reading times.
Two types:
- Fixed kinetics
- Continuous monitoring method
/atleast 3 data points/
Fixed-Time Assays
Substrate is added and Abs is measured after a
predetermined incubation period, at fixed time
intervals.
C A-B=lag phase
Absorbance B-C=reading
D time
B
A Point B and
C= fixed times
at which
Time/sec
reading taken
C-D= Substrate
depletion
Continuous-monitoring
method
In this method, multiple absorbance readings
are recorded continuously over the entire
reading time interval.
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
