SPECTROPHOTOMETRY
INTRODUCTION
• Spectrophotometry is a scientific technique
used to measure how much light a chemical
substance absorbs or transmits as a function of
wavelength.
• It is an essential analytical method in various
scientific fields, including chemistry, biology,
physics, and environmental science, allowing for
the quantitative analysis of substances in a
sample.
• The basic principle is that each compound absorbs
or transmits light over a certain range of
wavelength. This measurement can also be used
to measure the amount of a known chemical
substance.
ELECTROMAGNETIC RADIATION
• The electromagnetic radiation is energy that is
transmitted at the speed of light through
oscillating electric and magnetic field. It is a form
of energy that is all around us such as radio
waves, microwaves, X-rays and gamma rays.
Sunlight is also a form of electromagnetic
radiation.
• The distribution of electromagnetic radiation
according to frequency or wavelength is known as
the Electromagnetic Spectrum.
• A small fraction of the Spectrum can be detected
by the naked eyes and is known as the Visible
Light Spectrum.
Electromagnetic Spectrum
SPECTROPHOTOMETER
• This EM spectrum emitted by a source is analyzed
by spectroscopy through a special device known
as spectroscope.
• The spectroscope or spectrophotometer is an
instrument which is used to analyze EM radiation
spectrum especially of ultraviolet, visible light and
infrared ranges.
• Spectrophotometer transmits and receives light for
the analysis of spectrum and therefore it is used
to evaluate samples of test materials by passing
light by means of the sample and studying the
intensity of the wavelengths.
• It becomes possible due to the fact that different
samples absorbs the light differently and modify
the light that is emitting from the test materials
and therefore, by viewing the change in light
conduct as it passes by way of the test materials;
an investigator is able to obtain facts about the
test materials like measurement of solute
concentration.
• It was first developed by a Scientist Arnold J.
Beckman
at
the
National
Technologies
Laboratories (NTL) in 1940 and the device was
then called as Beckman DU Spectrophotometer.
• The Spectrophotometer is used in many studies in
biology, chemistry, physics and industrial
laboratories.
KEY PRINCIPLE OF
SPECTROPHOTOMETER
1. Interaction of Matter with Light
• It is based on the photometric technique which
states that when a beam of incident light of
intensity ‘Lo’ passes through a solution, a part of
the incident light is reflected ‘Lr’, a part is
absorbed ‘La’ and rest of the light is transmitted
‘Lt’.
Lo = Lr + L a + Lt
• The specific wavelengths absorbed depend on the
molecular structure of the substance.
• The amount of photons that goes through the
cuvette and into the detector is dependent on the
length of the cuvette and the concentration of the
sample.
• Once you know the intensity of light after it passes
through the cuvette, you can relate it to
transmittance (T).
• Transmittance is the fraction of light that passes
through the sample. This can be calculated using
the equation:
𝑙𝑡
Transmittance (T) = log𝑙0
• Where lt is the light intensity after the beam of
light passes through the cuvette and lo is the light
intensity before the beam of light passes through
the cuvette.
• Transmittance is related to absorption by the
expression:
𝑙𝑡
Absorbance(A) = -log(T) = -log 𝑙0
• Where absorbance stands for the amount of
photons that is absorbed. With the amount of
absorbance known from the above equation, you
can determine the unknown concentration of the
sample by using Beer-Lambert Law.
2. Beer-Lambert Law
• The Beer-Lambert Law (or Beer’s Law) is a
fundamental principle in spectrophotometry,
which states that the absorbance (A) of a
sample is directly proportional to the
concentration (c in mol l-1) of the absorbing
substance, the path length (l in cm) of the
light through the sample, and the molar
absorptivity (ε in L mol-1 cm-1) of the
substance.
A = ϵ⋅c⋅l
ϵ = A/cl
• This Law was derived from the individual Laws of
Lambert (1728 – 1777) that states that the
amount of light absorbed (Absorbance) is directly
proportional to the length and thickness of the
solution under analysis and
• Beer (1825 – 1863) that states that the amount of
light
absorbed
(Absorbance)
is
directly
proportional to the concentration of the solute in
the solution.
BASIC INSTRUMENTATION OF A
SPECTROPHOTOMETER
• A spectrophotometer is basically composed of the
following specialized units;
• A stable and cheap radiant energy source of light
e.g tungsten lamps for visible light, hydrogen and
deuterium lamps for UV light, Nernst Glower for IR
radiations.
• Wavelength selectors such as filters and a
monochromator to break the polychromatic
radiation into component wavelength (or) bands of
wavelengths. It does this by using a prism,
diffraction grating, or filters, allowing only light of
a specific wavelength to pass through to the
sample.
• Sample holders (cuvettes) which holds the sample,
typically in a transparent container made of glass
or quartz, depending on the wavelength range
being analyzed.
• A photosensitive detector which measures the
intensity of the light that passes through the
sample. Common detectors include photodiodes,
photomultiplier tubes, or charge-coupled devices
(CCDs).
• Readout device which converts the detected light
intensity into an absorbance or transmittance
value and displays the results, often as a digital
readout or graphical plot.
Components of a Spectrophotometer
TYPES OF SPECTROPHOTOMETER
• According to different wavelengths and application
fields, spectrophotometers can be divided into
visible spectrophotometer, ultraviolet visible
spectrophotometer, infrared spectrophotometer,
fluorescence spectrophotometer and atomic
absorption spectrophotometer.
• They can also be divided into two types based on
the number of beams of light used. It is either a
Single Beam Spectrophotometer and a Double
Beam Spectrophotometer.
VIS spectrophotometer
• The measured wavelength range of the VIS
spectrophotometer is the visible light region of
400~1000nm.
• The visible spectrophotometer is a single beam
spectrophotometer.
• It is an instrument used to measure the
absorbance of the substance to be tested to
visible light and to perform qualitative and
quantitative analysis on it.
• The instrument is widely used in the fields of
medicine
and
health,
clinical
testing,
environmental monitoring, food production and so
on.
UV VIS spectrophotometer
• The measured wavelength range of the ultraviolet
visible spectrophotometer is between 200 and
1100nm in the ultraviolet region.
• Based on the principle of ultraviolet-visible
spectrophotometry, the UV VIS spectrophotometer
is an analytical instrument that utilizes the
absorption of radiation in the ultraviolet-visible
spectrum region by substance molecules for
analysis.
• This instrument is used to measure the
absorbance of the sample to visible light or
ultraviolet light, and perform qualitative and
quantitative analysis on it.
• The optical path of the product can be divided into
single beam, pseudo double beam and double
beam.
• At present, this instrument is one of the most used
analytical instruments with the widest coverage.
• It is widely used in many fields such as life
science, material science, environmental science,
agricultural science, chemistry and chemical
engineering for the quantification of nucleic acids,
proteins, and other biological molecules, as well as
in chemical analysis and quality control.
Infrared Spectrophotometer
• Infrared spectrophotometer exactly refers to the
light emitted by the light source, and it is divided
into two beams of equal energy and symmetry.
• One beam is the sample light passing through the
sample, and the other beam is the reference light
as the reference.
• After the two beams of light enter the photometer
through the sample chamber, they are modulated
by the fan-shaped mirror at a certain frequency to
form an alternating signal.
• The general infrared spectrum refers to the
infrared spectrum greater than 760nm.
• This is the most commonly used spectral region
for studying organic compounds, so it can be used
to analyze samples in various states (gas, liquid,
and solid).
• The spectrum has the characteristics of fast
speed,
small
sample
amount,
strong
characteristics, and no damage to the sample.
Fluorescence Spectrophotometer
• Measures the intensity of fluorescent light emitted
by a sample after it has absorbed light, usually of
a shorter wavelength.
• Widely used in biochemistry and molecular biology
to study fluorescently labeled biomolecules.
Atomic Absorption
Spectrophotometer
• Atomic Absorption Spectrophotometer analyzes
the metal elements based on the effect of the
atomic vapor of the ground state of the substance
on the characteristic radiation absorption.
• The light source emits the characteristic spectral
radiation to be measured, which is absorbed by
the ground state atoms of the measured element
in the sample vapor after passing through the
atomizer.
• The content of the measured element can be
obtained by measuring the size of the
characteristic radiation absorbed.
• It can sensitively and reliably determine trace or
trace elements, so it has become a powerful tool
for material analysis and quality control
departments to analyze major and trace metals
(semi-metals).
APPLICATIONS OF
SPECTROPHOTOMETRY
• Quantitative Analysis:
– Spectrophotometry is widely used to determine
the concentration of a substance in solution,
such as proteins, DNA, or chemicals in industrial
processes.
• Identification of Compounds:
– By analyzing the absorption spectrum, scientists
can identify unknown compounds based on
their unique spectral fingerprints.
• Kinetic Studies:
– Used to monitor the rate of chemical reactions by
measuring changes in absorbance over time.
• Environmental Monitoring:
– Spectrophotometry is used to analyze pollutants in
water, air, and soil, such as measuring the
concentration of nitrates or phosphates in water
samples.
• Clinical Diagnostics:
– Employed in medical laboratories to measure blood,
urine, and other biological fluids for diagnostic
purposes, such as determining glucose levels or
enzyme activities.