Spectrophotometr
y
Dr. Ahmed Jabbar Abbas
PhD. CLINICAL BIOCHEMISTRY
Introduction
Photometry is defined as the measurement of light
spectrophotometry is defined as the measurement of the
intensity of light at selected wavelengths.
Spectrophotometric analysis is a widely used method of
quantitative and qualitative analysis in the chemical and
biological sciences.
The method depends on the light-absorbing properties of the
substance or a intensity of the transmitted light beam.
Beer’s Law—Relationship Between
Transmittance, Absorbance, and Concentration
Beer’s law (also known as the Beer-Lambert
law)
states that the concentration of a substance is
directly proportional to the amount of light
absorbed
or inversely proportional to the logarithm of
the transmitted
Principle of Spectrophotometer
A spectrophotometer is based on the Beer-Lambert law,
A∝ CL
A= Absorbance
C= concentration of the sample
L= path length
OR,
A= εCL
ε= molar extinction coefficient, the value of which is constant
for a specific molecule.
Components of a
Spectrophotometer
Light Source
The most common source of light for work in the visible and near-infrared regions is the
incandescent tungsten or tungsten-iodide lamp.
Often, a heat-absorbing filter is inserted between the lamp and the sample to absorb the
infrared radiation.
The lamps most commonly used for ultraviolet (UV) work are the deuterium discharge
lamp and the mercury arc lamp. Deuterium provides continuous emission down to 165
nm.
Low-pressure mercury lamps emit a sharp line spectrum, with both UV and visible lines.
Medium and high-pressure mercury lamps emit a continuum from UV to the mid-visible
region.
The most important factors for a light source are range, spectral distribution within the
range, the source of radiant production, stability of the radiant energy, and temperature.
Monochromators
Isolation of individual wavelengths of light is an
important and necessary function of a
monochromator.
The degree of wavelength isolation is a function
of the type of device used and the width of
entrance and exit slits.
Numerous devices are used for obtaining
monochromatic light.
1. The least expensive are colored glass filters.
2. The prism is another type of monochromator
3. Diffraction gratings are most commonly used as
monochromators
Sample Cell
The next component of the basic spectrophotometer is
the sample cell or cuvette, which may be round or square.
The light path must be kept constant to have absorbance
proportional to concentration. This is easily checked by
preparing a colored solution to read midscale when using
the wavelength of maximum absorption.
Fill each cuvette to be tested, take readings, and save
those that match within an acceptable tolerance (e.g.,
±0.25% T).
Because it is difficult to manufacture round tubes with
uniform diameters, they should be etched to indicate the
position for use.
Square cuvettes have plane-parallel optical surfaces and
a constant light path.
Square cuvettes have an advantage over round
cuvettes in that there is less error from the lens
effect, orientation in the spectrophotometer, and
refraction.
Cuvettes with scratched optical surfaces scatter
light and should be discarded.
Inexpensive glass cuvettes can be used for
applications in the visible range, but they absorb
light in the UV region.
Quartz cuvettes must, therefore, be used for
applications requiring UV radiation.
Photodetector
The purpose of the detector is to convert the
transmitted radiant energy into an equivalent
amount of electrical energy.
The detector has many types
1. Photocell
2. Phototube
3. The photo multiplier tube
Types of Spectrophotometers
UV-Visible Spectrophotometer: measure the
absorption or transmission of light in the
ultraviolet and visible regions of the
electromagnetic spectrum.
Infrared (IR) Spectrophotometer: measure
the absorption or transmission of light in the
infrared region of the electromagnetic spectrum.
Fluorescence Spectrophotometer: These
instruments are commonly used in biochemistry
and molecular biology to detect and quantify
Uses of Spectrophotometers
Quantitative analysis: measure the concentration of a
sample.
Qualitative analysis: used to identify specific substances in
a sample based on their unique absorption or transmission
spectra.
Kinetic studies: monitor the progress of a chemical
reaction over time.
Biochemical assays: like measure enzyme activity or to
detect specific biomolecules such as proteins, nucleic acids,
and carbohydrates.
THA
YOU N K
SO
MUC
H
