Spectrophotometry

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Photometric techniques inPhotometric techniques in
clinical laboratoriesclinical laboratories
Muhammad Saiedullah, BSc (Hons in
Biochemistry, DU), MSc (Biochemistry, DU),
MPhil (Biochemistry and Molecular Biology, DU)
Assistant Professor and Head
Department of Physiology and Molecular
Biology, Bangladesh University of Health
Sciences (BUHS)
70th NITUB

2. • Light is a form of electromagnetic radiation.
• When visible/ordinary light (a group of
electromagnetic radiations) is passed
through a prism, it is split up into seven
colors which correspond to definite
wavelength. (This phenomenon is called
dispersion.)
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If shines a beam of light on a sample,
• The molecules in the sample interact
with the light waves in 3 ways:
–Absorb the energy
–Reflect the energy
–Transmit the energy between and
through the atoms and molecules of
the sample.

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6. • I0 = Ia + It + Ir
• For air-glass interfaces, Ir = 4% of the
incident light, and hence eliminated by the
use of control cell.
• I0 = Ia + It
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7. Absorption of light
• Excitation and transition of valence/lone pair
electrons from ground state to excited state.
• A molecule can only absorb radiation of
certain frequency, if the magnitude of an
energy transition within a molecule (ΔE) is
equal to h. c/ λ ie sufficient to promote
ground state electrons to excited higher
energy state (anti-bonding orbital).
• So, ΔE depends on λ
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Beer-Lambert’s Law
• A = ε. c. l
When l = 1cm
• A = ε. c
• Thus, ε = A/C
• The amount of light absorbed is proportional
to the concentration of a solute when
monochromatic light passes through a
transparent medium at constant path length.

9. Calculation
Where: Cs = concentration of standard
Cu = concentration of unknown
A(s) = absorbance of standard
A(u) = absorbance of unknown

10. Calibration Curve
Abs Conc
0 0
0.184 3.125 mmolL
0.3855 6.25 mmol/L
0.7045 12.5 mmol/L
1.473 25 mmol/L

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Enzyme activity = moles of substrate converted
to product per unit time.
The rate of appearance of product or the rate
of disappearance of substrate
IU (International Units) / SI units = amount
of enzyme that will convert 1 μmol of
substrate per minute in specified conditions.
(Usually reported in IU per liter (IU / L)
Calculation of enzyme activity:
U/L = (ΔA/min) x (1/ ε in μM) x Dilution Factor
= (ΔA/min) x Factor

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16. i. When different forms of the absorbing
molecules are in equilibrium as in keto-
enol tautomers.
ii. When fluorescent compounds are present.
iii. When solute and solvent form complexes
through some sort of association.
iv. When the concentration is high (>0.01M).
Limitations of Beer-Lambert Law:
This law is not obeyed
Limitations of Beer-Lambert Law:
This law is not obeyed
At higher concentrations (>0.01M), the distance between molecules diminish thus affecting the charge distribution to its neighbor.
Thus possibily diminishing the ability of the neighbor to absorb a given wavelength of radiation. The higher concentration may also
change the refractive index of the medium . 16

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v. When wide-band radiation is used / when
monochromatic light is not used.
vi. Presence of stray light.
vii. Presence of electrolytes (high
concentration)
viii. Physically unstable solution.
ix. Presence of bubbles in the light path.
Both positive and negative deviation may
occur.

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• For non-color substances, a suitable colored
complexes are produced that can absorb light
• Or to produce molecules that specifically absorb
light at specific wavelength

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Colorimeter
• Colorimeter is the traditional name of an
instrument that isolates specific wavelength
of light with interchangeable filters
(usually materials in the form of plates of
colored glass, gelatin etc.) for the visible
portions of the spectrum.
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Spectrophotometer
• Uses continuously adjustable
monochromator (prism or grating)
capable of producing definite wavelength
(1-10 nm).
• Can often measure the intensity of light
from the UV through the visible.
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Components of spectrophotometers
/ Instrumentation of UV or Visible
Spectroscopy
• Power supply
• Sources (UV and visible) light
• Filter or monochromator
• Sample containers (sample holder) or
sample cells (cuvet)
• Photo detector
• Meter or other readout devices
Instruments for measuring the absorption of U.V. or visible
radiation are made up of the following components;

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Power supply
• A simple transformer- to convert line
voltage to a constant low voltage for the
lamp
• To provide current (both AC and DC)
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28. • Quartz windows must be used in these
lamps, and quartz cuvettes must be used,
because glass absorbs radiation of
wavelengths less than 350 nm.
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UV radiation sources
(<350 nm)
Visible radiation
sources
Deuterium lamp
Hydrogen lamp
Tungsten lamp Tungsten lamp
Xenon discharge lamp
Mercury arc lamp Mercury vapour lamp
Halogen lamp Halogen lamp
Carbonone lamp

30. All monochromators contain the following
component parts
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit
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Filters or Monochromator
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• Interference filters: consist of a thin layer of
magnesium fluoride crystal with a
semitransparent coating of silver on each side.
The crystal transmits only light for which an
exact multiple of the wavelength is equal to the
thickness of the crystal. All other wavelengths
are blocked. Interference filters have a bandpass
of 5 to 8 nm.
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• Bandpass: The width of the segment of the
spectrum that will be isolated by a
monochromator
• It is the range of wavelength between the
points at which the transmittance is equal to
one-half the peak transmittance.
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• The monochromator consists of an entrance
slit to exclude unwanted or stray light and a
prism or diffraction grating preceded by a
series of light focusing lenses.
• The exit slit allows only a narrow fraction
of the spectrum to reach the sample cuvet.
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• For visible wavelength – glass prism and
lenses are suitable
• For UV region (<340 nm) – quartz or fused
silica is essential
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• Diffraction gratings – consist of a series of
parallel grooves cut into a surface, all at
exactly the same angle (10,000 – 50,000
grooves per inch)
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42. Sample containers or sample
cells
A variety of sample cells available for UV
region. The choice of sample cell is based
on
• a) the path length, shape, size
• b) the transmission characteristics at the
desired wavelength
• c) the relative expense
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• For accurate and precise readings – the
cuvet must be clean, there must be no
fingerprints, or any spills on the optical
surface, there should be no bubbles
adhering to the inner surface filled cuvet, no
moisture condenses on the exterior optical
surface
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• Additional components – flow-through or
flush-out cuvet
• Water jacket or thermostatically-controlled
heating coils
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Detectors
In order to detect radiation, three types of photosensitive
devices are -
These are light-sensitive surfaces capable to release electrons
in numbers proportional to the intensity of the light
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Readout devices
• The magnitude of the current generated by
the detector can be measured by a
galvanometer or ammeter. The signal may
be transmitted to a computer or printout
device
• The information from the readout may be
presented as %transmittance, absorbance
units or as concentration of the constituent
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Double beam spectrophotometers
• Is designed to compensate the possible
variations in the intensity of the light
source.
• Accomplished by splitting the light beam
from the lamp and directing one portion to a
reference cuvet and the other to the sample
cuvet

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50. Applications of a spectrophotometer
• Determines the presence and
concentrations of samples.
• Determines the purity of a sample.
• Look at the change of samples over
time.