1.
Lecture 3 – Analytical Techniques
and Instrumentation

2.
Learning outcomes
At the of this session the student shall be able to:
• Apply Beer’s law to calculate the concentration of a substance in
solution
• Explain the basic concepts and clinical laboratory applications of
photometry, fluorescence, nephelometry and turbidimetry
• Compare clinical uses of Capillary Microchip, Disc Two-dimensional
and Isoelectric focusing electrophoresis
• Discuss clinical applications of Ion exchange, Affinity and
Adsorption chromatography
• Describe clinical applications of mass spectrometry including the
hyphenated-MS techniques
• Discuss advantage and quality control issues in the use of point-of -
care testing (POCT).

3.
Introduction
• Analytic techniques used in clinical
laboratory fall into four categories
Method Technique
Spectrometry spectrophotometry, atomic absorption,
and mass spectrometry (MS)
Luminescence fluorescence, chemiluminescence, and
nephelometry
Electroanalytic electrophoresis, potentiometry, and
amperometry
Chromatography gas, liquid, and thin-layer

4.
SPECTROPHOTOMETRY AND PHOTOMETRY
• Spectrophotometers measure absorption or emission of
radiant energy to determine concentration of atoms or
molecules.
• Energy is emitted when excited valence electron falls back
to ground state.
I = transmitted energy
I0 = incident energy
%𝑇 = 𝐼 𝐼0 × 100

5.
Absorbance
• Absorbance (𝐴) = − log 𝐼 𝐼0
where I0 = incident light, I = transmitted light
But 𝐼 𝐼0 = %𝑇 100
∴ 𝐴 = log 100 − 𝑙𝑜𝑔%𝑇
= 2 − 𝑙𝑜𝑔%𝑇

6.
Beer’s Law
• states that: concentration of a
substance is directly proportional to
the amount of light absorbed
A = εlc
where ε = molar absorptivity,
l = path length, c = concentration
A ≈ C (ε and l are constants)
• Thus concentration of unknown
solution can be determined from a
calibration curve of a standards of
known concentration.

7.
A spectrophotometer
Component Description
Light Source lamps emitting UV-visible light . e.g. tungsten, deuterium
and mercury lamp.
Monochromator isolate individual wavelengths of light. e.g. prisms and
Diffraction gratings
Sample cell glass or Quartz (glass not suitable for UV region)
Photodetectors convert transmitted radiation into electrical energy
Analog to-digital (A/D) converters the signal to a voltage and digital.

8.
Photodetectors
Photodiode array
(PDA) use pn-junction
diode. less sensitive
than PMT, but
excellent linearity,
speed, and small size
give them advantage
Photocell on
illumination generates
current that is
proportional to
incident radiation.
Disadvantage: no
amplification.
temperature sensitive ,
nonlinear at very low
and very high
illumination.
Photomultiplier tube (PMT)
detects and amplifies radiant
energy by dynodes, that
produce a successively higher
positive voltage. PM T is >
200 times more sensitive
than the phototube.
cathode
Incident
light

9.
Double beam spectrophotometer
• Double-beam spectrophotometers permit automatic
correction of sample and reference absorbance. The
system performs continuous zeroing electronically

10.
Atomic Absorption Spectrophotometer
• In atomic absorption spectrophotometer radiation is
detected by atoms
• It is routinely used to measure concentration of trace
metals in samples

11.
Fluorometry
• It is the measurement of
emitted fluorescence light.
• Fluorescence occurs when
a molecule absorbs light at
one wavelength and
reemits light at a longer
wavelength.
• Loss of energy is due to
vibrational equilibration
• The difference btw max of
the excitation light and
max of the the emitted
fluorescence light is
referred to as the Stokes
shift.

12.
Fluorometers
Source: Gas-discharge lamps (mercury and xenon).
Attenuator controls light intensity.
Primary
filter
selects the wavelength that is best
absorbed by the solution.
Cuvet sample cell
Detector PMT (placed at right angles to prevent
incident light from striking the
photodetector
Secondary
filter
passes only the longer wavelengths of
fluorescent.
In spectrofluorometers, the filters are replaced by prisms or grating monochromators.

13.
Concentration and Fluorescence Intensity
 =0εlc
Where
 = fluorecence intensity
 = fuorescence efficiency
0 = initial excitation intensity,
ε = molar absorptivity
l = path length
c = the concentration in mol/L.
• In dilute solutions with instrument parameters
held constant, fluorescence is directly
proportional to concentration

14.
Fluorescence Polarization
Fluorescence polarization, 𝑃 =
I𝑣− 𝐼ℎ
𝐼𝑣+ 𝐼ℎ
– Where, Iv = intensity of emitted fluorescence in the vertical plane
– Ih = intensity of emitted fluorescence in the horizontal plane
• A large fluorophor emits polarized light if radiant energy is
polarized.
• A small molecule can only emits polarized light if bound to large
molecule.
• fluorescence polarization will change if antibody bound to a
fluorescent-labeled analyte is mixed with another analyte
competing with the fluorescent ligand
• The change is inversely proportional to the amount of analyte
contained in a given sample
• It can thus be used to determination concentration of the analyte
contained in a given sample.

15.
Advantages of Fluorometry
• Advantages
– Fluorometry increases specificity by selecting the optimal
wavelength for both absorption and fluorescence.
– Fluorometry is approximately 1,000 times more sensitive than most
spectrophotometric methods
• emitted radiation is measured directly and can be increased simply by
increasing the intensity of the exciting radiant energy.
• Disadvantages
• fluorescence can decrease due to quenching due to
– Changes in pH (affect availability of electrons)
– temperature (loss of energy by collision rather than fluorescence).
– Contaminants or a change of solvents (may change the structure).
– UV light used for excitation can cause photochemical changes.

16.
Chemiluminescence
• Is a process where excitation event is caused
by a chemical reaction, and not by photo
illumination.
• The excitation event is caused by a oxidation
reaction with compound such as luminol,
acridinium esters, and dioxetanes

17.
Instrumentation – Chemiluminescence
• are known as luminometers.
• The basic components of a luminometer include
– sample cell housed in a light-tight chamber
– injection system for adding reagents to the sample cell
– the PMT detector
• Advantages : Fast, ultrasensitive and simple instrumentation.
• Disadvantage : impurities can cause a background signal that degrades
sensitivity and specificity

18.
Turbidity and Nephelometry
• Nephelometry and turbidimetry are analytical techniques that
measure scattered light due to interaction of light with particles
in solution.
• These techniques determine concentration of particulate matter
in a sample such as serum proteins.
• The amount of light scattered by a suspension of particles
depends on:
– wavelength, concentration and size.

19.
Instrumentation
• Instrument operation is the same as for any
spectrophotometer.
• Common nephelometers measure scattered
light at 90o or other angle to take advantage of
the increased forward scatter.

20.
Laser Applications
• Light amplification by stimulated emission of
radiation (LASER) is produced by interaction of
radiant energy and suitably excited atoms or
molecules.
• Laser has same wavelength, propagation,
phase, and plane of polarization as the incident
radiation.
• Laser light can serve as the source of incident
energy in a spectrometer or nephelometer.
• Laser powered spectrometers are 3 – 6 orders
more sensitive than conventional ones