Biochemistry Spectrophotometer Beer`s

Dentistry Department; 2nd grade
Dijlah University college
Biochemistry
Lab. 2
Ass. Lec. Anas Hashim
Spectrophotometer
An instrument designed to detect the amount of radiant light energy
absorbed by molecules in specified wavelength, this method is used as a
quantitative way to determine concentration of an absorbing species
(unknown) in solutions, using the Beer-Lambert law.
The spectrophotometer allows selection of wavelength pass through
the solution. Usually the wavelength (measured in nanometer nm) has
chosen which corresponds to the absorption maximum of the solute.
Colorimetry: methods for quantitative analysis of blood, urine and
other biological materials based upon the production of a coloured
compounds in solution, the intensity of which is used as a measure of
concentration.
Beer's-Lambert law:
Lambert Beer's law means how light is absorbed by matter.
The absorbance of light is directly proportional to the thickness of the
media through which the light is being transmitted multiplied by the
concentration of absorbing chromophore:
A = ɛbc
where:
A
absorbance
ɛ
wavelength-dependent molar absorptivity coefficient
b
length of light path
c
concentration of the solute
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Dentistry Department; 2nd grade
Dijlah University college
when we compare the concentration of an unknown test solution
with a standard solution measured in the same way we use the following
relation:
A test = ɛbC test
A standard = ɛbC standard
If the blank reading is significant then :
Absorbance (A): the spectrophotometer reading for the quantity of the
light that absorbed by the sample molecules at a specified wavelength .
A = - log T
= log (1/T)
= log (100/%T)
= log (100) – log T
= 2 - log %T
%T
per cent transmittance
Transmittance (T): is defined as the ratio of transmitted light to incident
light :
T=I/Io
Where:
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Dentistry Department; 2nd grade
I
Dijlah University college
light intensity after it passes through the sample and
Io
the initial light intensity. The relation between A and T is:
A = -log T
= - log (I / I o(
Design
There are two major classes of devices: single beam and double
beam. A double beam spectrophotometer compares the light intensity
between two light paths, one path containing a reference sample and
the other the test sample. A single-beam spectrophotometer measures
the relative light intensity of the beam before and after a test sample is
inserted. Although comparison measurements from double-beam
instruments are easier and more stable.
Major components of spectrophotometer
1. Light source: two kinds of lamps, a Deuterium for measurement in
the ultraviolet range and a tungsten lamp for measurement in the
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Dentistry Department; 2nd grade
Dijlah University college
visible and near-infrared ranges, are used as the light sources of a
spectrophotometer.
2. Wavelength selector Monochromator: All monochromators
contain the following component parts;
An entrance slit
 A collimating lens
 A dispersing device (usually a prism or a grating)
 A focusing lens
 An exit slit
3. Sample cell: a container that contains a sample is usually called
"cell"; three types are available: plastic, glass and quartz cells.
Since light in the ultraviolet range with a wavelength of 340 nm or
less hardly passes through a glass cell, it is used for measurement
in the visible range of 340 nm or more.
On the other hand, although a quartz cell allows passage of
light in the entire wavelength in the ultraviolet and visible ranges,
it is mainly used for the measurement in the ultraviolet range due
to its high price.
4. Detector: measure the light intensity.
5. Signal processor and readout

Solution required for photometric measurements:
1. Test: made from any unknown specimen (blood, plasma
and serum)
2. Blank: contain all reagents used except the substance.
3. Standard: made from a known quantity of the substance.
4. Control: make in enzyme assay.
5. Quality control: made from pooled serum of known
chemical constituents.
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Dentistry Department; 2nd grade
Dijlah University college
Calibration (standard) curve
A calibration curve is a general method for determining the
concentration of a substance in an unknown sample by comparing the
unknown to a set of standard samples of known concentration.
The operator prepares a series of standards across a range of
concentrations near the expected concentration of analyte in the
unknown. The concentrations of the standards must lie within the
working range of the technique they are using. Analyzing each of these
standards using the chosen technique will produce a series of
measurements. For most analyses a plot of instrument response vs.
concentration will show a linear relationship. The operator can measure
the response of the unknown and, using the calibration curve, can
interpolate to find the concentration of analyte.
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