Chapter 4

Analytical procedures and

Instrumentation

Clinical Chemistry I 1
 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 Clinical Chemistry I 2
 Outline

• 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

Clinical Chemistry I 3
Introduction to Colorimetry

Many colored solutions absorb light

Colorimetry: Measuring % transmitted light
through a colored solution

Clinical Chemistry I 4
 Colorimeter
• The instrument that produces
monochromatic light, transmits light
through a colored solution and measures
% Transmittance or Absorbance of light
• More accurate colorimeters are called
spectrophotometers

Clinical Chemistry I 5
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

Clinical Chemistry I 6
 Spectrophotometer Components

When you view the exterior of the instrument you notice a square cover on
the left. This is where the cuvette is placed. The knob on the right is used
to adjust the readout to 100% T or 0 Absorbance with the reference or blank
cuvette. The results, absorbance or %T are viewed on the top either as
analog or digital number.
Clinical Chemistry I 7
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

Clinical Chemistry I 8
 Schematic Diagram of a Single-
Beam UV-Vis. Spectrophotometer

In spectrophotometry, the light that is transmitted and measured is

monochromatic. In practice, however, monochromators allow a narrow range
of wavelengths to reach the specimen. This range of wavelengths is referred
to as the spectral bandwidth, or slit width, of the instrument, because the size
Clinical Chemistry I 9
of the slit affects the bandwidth.
Schematic Diagram of a Double-Beam
UV-Vis. Spectrophotometer

Clinical Chemistry I 10
 A. 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
Clinical Chemistry I 11
 B. Wavelength Selector
(Monochromator)
 Monochromators (provides one wavelength of
light) can be grating or prism or filter
 All monochromators contain the following
component parts:
a. Entrance slit admitting polychromatic light
b. Collimating lens or mirror collect the beam to strike prism
c. Prism or grating splits the beam into its component
d. Focusing lens
e. Exit slit which allows the monochromatic beam to escape.

Clinical Chemistry I 12
Monochromator

Clinical Chemistry I 13
Czerney-Turner Grating Monochromator

Clinical Chemistry I 14
 C. 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 for UV light, silica (quartz) cuvettes are used
below 320 nm and they must be clean and free of
scratches

Clinical Chemistry I 15
 D. Detector
• The photomultiplier tube
– Commonly used detector in UV-Vis spectroscopy
– Photomultiplier tubes are electron tubes that amplify
current (i.e, generated from photon striking the
cathode tube)
• 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 electrical energy when they are
struck by light

Clinical Chemistry I 16
 Photomultiplier tube (PMT)

• Used for low light intensity

applications, intense light
damages photomultipliers
• Photons strike electrode
• Electrons are emitted
• These strike secondary
surfaces
• Eventually a pulse of
electrons is produced

Clinical Chemistry I 17
 Photodiodes

• Photodiodes are
semiconductor light
sensors that generate a
current or voltage when
the P-N junction in the
semiconductor is
illuminated by light

Clinical Chemistry I 18
 Stray light

Light radiation outside the narrow band

nominally transmitted by the
monochromator, it is source of error
Scattering and diffraction inside the
monochromator introduce light of other
wavelengths into the exit beam.
Should be eliminated by
spectrophotometer using stray light filters

Clinical Chemistry I 19
 E. 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 analog 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.
Clinical Chemistry I 20
 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

Clinical Chemistry I 21
 Fluorometry
is the measurement of fluorescence. The
instrument used to measure fluorescence
is called a fluorometer or fluorimeter.
Fluorometer is a photometer that
measures the fluoresent light emitted
(relatively long wavelength) by a
substance that has been previously
excited by a source of short-wavelength
radiation.
Clinical Chemistry I 22
Fluorescence is the molecular absorption
of light energy at one wavelength and its
nearly instantaneous re-emission at
another, usually longer, wavelength.
Fluorescent compounds have two
characteristic spectra: an excitation
spectrum (the wavelength and amount of
light absorbed) and an emission
spectrum (the wavelength and amount of
light emitted).

Clinical Chemistry I 23
Clinical Chemistry I 24
 A fluorometer generates the wavelength of light
required to excite the analyte of interest; it
selectively transmits the wavelength of light
emitted, then it measures the intensity of the
emitted light. The emitted light is proportional to
the concentration of the analyte being measured
(up to a maximum concentration).

 The basic component of a spectroflorometer are:

excitation source, excitation monochromator,
sample cell, emission monochromator and
detector.

Clinical Chemistry I 25
Components of Fluorometer

Clinical Chemistry I 26
 Turbidimetry and
Nephelometry
 Both are used to measure Scattered Light
 Turbidity measures the decrease in the amount
of light as it passes through a particulate
solution.
 In turbidity the decrease in transmittance (increase in
absorbance) is measured at a 00 angle to the light
passing through the cuvette.
 Nephelometry is used to measure the amount
of light that is scattered as it passes through the
particulate solution.
 In nephelometry the particles scatter the light at a 900
angle and the rate of light scatter is measured.
 Both methods can be used to determine the
concentration of theClinical
particulate
Chemistry I
solution. 27
Clinical Chemistry I 28
 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.
Clinical Chemistry I 29
Measurement of scattered light

• Light scattering is 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 to measure scattered light.

Clinical Chemistry I 30
 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.
Clinical Chemistry I 31
• 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
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;
Clinical Chemistry I 32
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.
Clinical Chemistry I 33
 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

+ -

Clinical Chemistry I 34
The electrophoresis apparatus

Clinical Chemistry I 35
 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.
Clinical Chemistry I 36
 Summary, continued..
• Refractometry, turbidimetry, nephlometery,
and fluorometry 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 analytes

Clinical Chemistry I 37
 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?

Clinical Chemistry I 38
 References
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.

Clinical Chemistry I 39
 The next Chapter

Chapter 5

Measurement procedures and

calculations in spectrophotometer

Clinical Chemistry I 40