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Clinical Laboratory Measurements
Clinical Laboratory Measurements
1.) Colorimetry
An analytical technique in which the concentration of
an analyte is measured by its ability to produce or
change the color of a solution
- Changes the solution’s ability to absorb light
2.) Spectrophotometry
Any technique that uses light to measure
chemical concentrations
A colorimetric method where an instrument is
used to determine the amount of analyte in a
sample by the sample’s ability or inability to
absorb light at a certain wavelength.
Colorimetry
Instrumental Methods
4 (spectrophotometry)
Non-Instrumental Methods
Spectrophotometric Analysis
6
Fundamentals of Spectrophotometry
Properties of Light
E h
where: E = photon energy (Joules)
n = frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)
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Fundamentals of Spectrophotometry
Properties of Light
c c /
where: c = speed of light (3.0x108 m/s in vacuum))
n = frequency (sec )
-1
l) = wavelength (m)
Relationship between Energy and Wavelength
hc ~
E hc
where:
~ = (1/ l)) = wavenumber
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As wavelength (l) decreases, energy (E) of light increases
The radiation may be transmitted with
little absorption taking place, and
therefore, without much energy loss.
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Fundamentals of Spectrophotometry
Absorption of Light
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Fundamentals of Spectrophotometry
Absorption of Light
Energy required of
photon to give this
transition:
DE = E1 - Eo
Since different chemicals have different electron shells which are filled,
they will each absorb their own particular type of light
- Different electron ground states and excited states
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Fundamentals of Spectrophotometry
Absorption of Light
Increasing [Fe2+]
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Absorbance is directly proportional to concentration of Fe +2
Fundamentals of Spectrophotometry
Absorption of Light
P
%T 100
Percent transmittance
Po
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T has a range of 0 to 1, %T has a range of 0 to 100%
Fundamentals of Spectrophotometry
Absorption of Light
P
A log log(T) log(%T / 100)
Po
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A has a range of 0 to infinity
Fundamentals of Spectrophotometry
Absorption of Light
Abc
where: A = absorbance (no units)
e = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)
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Fundamentals of
Spectrophotometry
Absorption of Light
lA lB
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The absorption process
How does matter absorb radiation
hv1
E0 Ground state
20 v 1.2
•
This may occur upon absorption of far Ultraviolt as its energy is sufficiently high to
exceed the energy of formation of certain bonds.
• The total energy of a molecule is given by
Etotal=Eelectronic+Evibrational +Erotational
Fundamentals of
Spectrophotometry
Spectrophotometer
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Fundamentals of
Spectrophotometry
Spectrophotometer
- Filter:
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Fundamentals of
Spectrophotometry
Spectrophotometer
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Fundamentals of Spectrophotometry
Spectrophotometer
2. Quartz – ultraviolet
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Fundamentals of Spectrophotometry
Spectrophotometer
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Fundamentals of
Spectrophotometry
Spectrophotometer
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Fundamentals of Spectrophotometry
Chemical Analysis
1.) Calibration
To measure the absorbance of a sample, it is
necessary to measure Po and P ratio
- Po – the amount of light passing through the
system with no sample present
- P – the intensity of light when the sample is
present
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Multi channel calorimeter (photometer)
33 v 1.2
Microprocessor based spectrophotometer
34 v 1.2
Fundamentals of
Spectrophotometry
Chemical Analysis
4.) Example:
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Thank you
36 v 1.2