Spectrophotometry is a technique used to measure the amount of light absorbed by a chemical substance, with applications across various fields including chemistry and clinical diagnostics. It relies on the Beer-Lambert law, which correlates absorbance to concentration, and requires a spectrophotometer to analyze samples at specific wavelengths. Instruments include spectrometers and photometers, with different types utilized for ultraviolet and visible light measurements.

1. By - Priya Tamang
Spectrophotometry
CBSE NET JRF EXAM NOTES

2. Spectrophotometry
 Spectrophotometry is a method to measure how much a chemical substance absorbs
light by measuring the intensity of light as a beam of light passes through sample
solution.
 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.
 Spectrophotometry is one of the most useful methods of quantitative analysis in
various fields such as chemistry, physics, biochemistry, material and chemical
engineering and clinical applications.
 Every chemical compound absorbs, transmits, or reflects light (electromagnetic
radiation) over a certain range of wavelength.
 Spectrophotometry is a measurement of how much a chemical substance absorbs or
transmits. Spectrophotometry is widely used for quantitative analysis in various areas
(e.g., chemistry, physics, biology, biochemistry, material and chemical engineering,
clinical applications, industrial applications, etc).
 Any application that deals with chemical substances or materials can use this
technique.
 In biochemistry, for example, it is used to determine enzyme-catalyzed reactions.
 In clinical applications, it is used to examine blood or tissues for clinical diagnosis.

3.  A spectrophotometer is an instrument that measures the amount of photons
(the intensity of light) absorbed after it passes through sample solution.
 With the spectrophotometer, the amount of a known chemical substance
(concentrations) can also be determined by measuring the intensity of
light detected.
 Depending on the range of wavelength of light source, it can be classified
into two different types:
 UV-visible spectrophotometer: uses light over the ultraviolet range (185 -
400 nm) and visible range (400 - 700 nm) of electromagnetic radiation
spectrum.
 IR spectrophotometer: uses light over the infrared range (700 - 15000 nm)
of electromagnetic radiation spectrum.
 In visible spectrophotometry, the absorption or the transmission of a certain
substance can be determined by the observed color.
 For instance, a solution sample that absorbs light over all visible ranges (i.e.,
transmits none of visible wavelengths) appears black in theory.
 On the other hand, if all visible wavelengths are transmitted (i.e., absorbs
nothing), the solution sample appears white.
 If a solution sample absorbs red light (~700 nm), it appears green because
green is the complementary color of red.
 Visible spectrophotometers, in practice, use a prism to narrow down a
certain range of wavelength (to filter out other wavelengths) so that the
particular beam of light is passed through a solution sample.

4. Beer-Lambert Law
 Beer-Lambert Law (also known as Beer's Law) states that there is a linear
relationship between the absorbance and the concentration of a sample. For
this reason, Beer's Law can only be applied when there is a linear
relationship. Beer's Law is written as:
A=ϵlc
 where
 AA is the measure of absorbance (no units),
 ϵϵ is the molar extinction coefficient or molar absorptivity (or absorption
coefficient),
 ll is the path length, and
 cc is the concentration.
 The molar extinction coefficient is given as a constant and varies for each
molecule.
 Since absorbance does not carry any units, the units for ϵϵ must cancel out
the units of length and concentration. As a result, ϵϵ has the units: L·mol-
1·cm-1.
 The path length is measured in centimeters. Because a standard
spectrometer uses a cuvette that is 1 cm in width, ll is always assumed to
equal 1 cm.
 Since absorption, ϵϵ, and path length are known, we can calculate the
concentration cc of the sample.

5. Devices and mechanism
 A spectrophotometer, in general, consists of two devices; a
spectrometer and a photometer. A spectrometer is a device
that produces, typically disperses and measures light. A
photometer indicates the photoelectric detector that
measures the intensity of light.
 Spectrometer: It produces a desired range of wavelength
of light. First a collimator (lens) transmits a straight beam
of light (photons) that passes through a monochromator
(prism) to split it into several component wavelengths
(spectrum). Then a wavelength selector (slit) transmits
only the desired wavelengths, as shown in Figure 1.
 Photometer: After the desired range of wavelength of
light passes through the solution of a sample in cuvette,
the photometer detects the amount of photons that is
absorbed and then sends a signal to a galvanometer or a
digital display, as illustrated in Figure 1.

6. Figure 1 illustrates the basic structure of spectrophotometers. It
consists of a light source, a collimator, a monochromator, a
wavelength selector, a cuvette for sample solution, a photoelectric
detector, and a digital display or a meter. Detailed mechanism is
described below.
Figure 2: A single wavelenth spectrophotometer

7.  You need a spectrometer to produce a variety of
wavelengths because different compounds absorb best
at different wavelengths. For example, p-nitrophenol
(acid form) has the maximum absorbance at
approximately 320 nm and p-nitrophenolate (basic form)
absorb best at 400nm, as shown in Figure 3.
Figure 3: Absorbance of two different compounds

8.  Looking at the graph that measures absorbance and wavelength, an
isosbestic point can also be observed.
 An isosbestic point is the wavelength in which the absorbance of two or
more species are the same.
 The appearance of an isosbestic point in a reaction demonstrates that an
intermediate is NOT required to form a product from a reactant. Figure 4
shows an example of an isosbestic point.
Figure 4: An example of isosbestic point

9.  Referring back to Figure 1 (and Figure 5), 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:
 Transmittance(T)= It /Ito
 Where It is the light intensity after the beam of light passes through the
cuvette and Io is the light intensity before the beam of light passes through
the cuvette.
 Transmittance is related to absorption by the expression:
• Absorbance(A)=−log(T)=−log(It/ I0)
 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. Figure 5 illustrates transmittance of light through a sample. The
length ll is used for Beer-Lambert Law described below.
Figure 5: Transmittance (illustrated by Heesung
Shim)

10. Ultraviolet and visible spectroscopy

11. Introduction
 Many molecules absorb ultraviolet or visible light.
 The absorbance of a solution increases as
attenuation of the beam increases.
 Absorbance is directly proportional to the path
length, b, and the concentration, c, of the absorbing
species.
 Beer's Law states that A = ebc, where e is a constant
of proportionality, called the absorbtivity.
 Different molecules absorb radiation of different
wavelengths.
 An absorption spectrum will show a number of
absorption bands corresponding to structural groups
within the molecule.
 For example, the absorption that is observed in the
UV region for the carbonyl group in acetone is of the
same wavelength as the absorption from the carbonyl
group in diethyl ketone

12. Electronic transitions
 The absorption of UV or visible radiation corresponds to the excitation of
outer electrons.
 There are three types of electronic transition which can be
considered;Transitions involving p, s, and n electrons
 Transitions involving charge-transfer electrons
 Transitions involving d and f electrons (not covered in this Unit)
 When an atom or molecule absorbs energy, electrons are promoted from
their ground state to an excited state.
 In a molecule, the atoms can rotate and vibrate with respect to each other.
 These vibrations and rotations also have discrete energy levels, which can
be considered as being packed on top of each electronic level.

13. Absorbing species containing , π ,σ, and n electrons
 Absorption of ultraviolet and visible radiation in organic
molecules is restricted to certain functional groups
(chromophores) that contain valence electrons of low
excitation energy. The spectrum of a molecule containing
these chromophores is complex. This is because the
superposition of rotational and vibrational transitions on
the electronic transitions gives a combination of
overlapping lines. This appears as a continuous
absorption band.

14.  σ → σ* Transitions
 An electron in a bonding s orbital is excited to the corresponding antibonding
orbital.
 The energy required is large. For example, methane (which has only C-H
bonds, and can only undergo s ® s* transitions) shows an absorbance
maximum at 125 nm.
 Absorption maxima due to s ® s* transitions are not seen in typical UV-Vis.
spectra (200 - 700 nm)
 n→σ* Transitions
 Saturated compounds containing atoms with lone pairs (non-bonding
electrons) are capable of n ® s* transitions.
 These transitions usually need less energy than s ® s * transitions.
 They can be initiated by light whose wavelength is in the range 150 - 250
nm.
 The number of organic functional groups with n ® s* peaks in the UV region
is small.
 n→π* and π→π* Transitions
 Most absorption spectroscopy of organic compounds is based on transitions
of n or π electrons to the π* excited state. This is because the absorption
peaks for these transitions fall in an experimentally convenient region of the
spectrum (200 - 700 nm). These transitions need an unsaturated group in
the molecule to provide the p electrons.
 Molar absorbtivities from n→π* transitions are relatively low, and range
from 10 to100 L mol-1 cm-1 . π→π* transitions normally give molar
absorbtivities between 1000 and 10,000 L mol-1 cm-1 .

15.  The solvent in which the absorbing species is dissolved
also has an effect on the spectrum of the species.
 Peaks resulting from n→π* transitions are shifted to
shorter wavelengths (blue shift) with increasing solvent
polarity.
 This arises from increased solvation of the lone pair,
which lowers the energy of the n orbital. Often
(but not always), the reverse (i.e. red shift) is seen
for π→π* transitions.
 This is caused by attractive polarisation forces between
the solvent and the absorber, which lower the energy
levels of both the excited and unexcited states.
 This effect is greater for the excited state, and so the
energy difference between the excited and unexcited
states is slightly reduced - resulting in a small red shift.
 This effect also influences n→π* transitions but is
overshadowed by the blue shift resulting from solvation of
lone pairs

16. Charge - Transfer Absorption
 Many inorganic species show charge-transfer
absorption and are called charge-transfer
complexes.
 For a complex to demonstrate charge-transfer
behaviour, one of its components must have electron
donating properties and another component must be
able to accept electrons.
 Absorption of radiation then involves the transfer of
an electron from the donor to an orbital associated
with the acceptor.
 Molar absorbtivities from charge-transfer absorption
are large (greater that 10,000 L mol-1 cm-1).

17. Instrumental components
 Instruments for measuring the absorption of U.V. or visible radiation
are made up of the following components;Sources (UV and visible)
 Wavelength selector (monochromator)
 Sample containers
 Detector
 Signal processor and readout
 Sources of UV radiation
 It is important that the power of the radiation source does not change
abruptly over it's wavelength range.
 The electrical excitation of deuterium or hydrogen at low pressure
produces a continuous UV spectrum. The mechanism for this
involves formation of an excited molecular species, which breaks up
to give two atomic species and an ultraviolet photon. This can be
shown as;
D2 + electrical energy ® D2
* ® D' + D'' + hv
 Both deuterium and hydrogen lamps emit radiation in the range 160 -
375 nm. Quartz windows must be used in these lamps, and quartz
cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.

18.  Sources of visible radiation
 The tungsten filament lamp is commonly employed as a source of visible light.
 This type of lamp is used in the wavelength range of 350 - 2500 nm.
 The energy emitted by a tungsten filament lamp is proportional to the fourth power of
the operating voltage. This means that for the energy output to be stable, the voltage
to the lamp must be very stable indeed.
 Electronic voltage regulators or constant-voltage transformers are used to ensure this
stability.
 Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope"
which also contains the tungsten filament.
 The iodine reacts with gaseous tungsten, formed by sublimation, producing the volatile
compound WI2. When molecules of WI2 hit the filament they decompose, redepositing
tungsten back on the filament.
 The lifetime of a tungsten/halogen lamp is approximately double that of an ordinary
tungsten filament lamp.
 Tungsten/halogen lamps are very efficient, and their output extends well into the ultra-
violet. They are used in many modern spectrophotometers.
 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
 Polychromatic radiation (radiation of more than one wavelength) enters the
monochromator through the entrance slit. The beam is collimated, and then strikes the
dispersing element at an angle. The beam is split into its component wavelengths by
the grating or prism. By moving the dispersing element or the exit slit, radiation of only

19. Czerney-Turner grating monochromator

20.  Cuvettes
 The containers for the sample and reference solution must be transparent to
the radiation which will pass through them. Quartz or fused silica cuvettes
are required for spectroscopy in the UV region. These cells are also
transparent in the visible region. Silicate glasses can be used for the
manufacture of cuvettes for use between 350 and 2000 nm.
 Detectors
 The photomultiplier tube is a commonly used detector in UV-Vis
spectroscopy. It consists of a photoemissive cathode (a cathode which emits
electrons when struck by photons of radiation), several dynodes (which emit
several electrons for each electron striking them) and an anode.
 A photon of radiation entering the tube strikes the cathode, causing the
emission of several electrons. These electrons are accelerated towards the
first dynode (which is 90V more positive than the cathode). The electrons
strike the first dynode, causing the emission of several electrons for each
incident electron. These electrons are then accelerated towards the second
dynode, to produce more electrons which are accelerated towards dynode
three and so on. Eventually, the electrons are collected at the anode. By this
time, each original photon has produced 106 - 107 electrons. The resulting
current is amplified and measured.
 Photomultipliers are very sensitive to UV and visible radiation. They have
fast response times. Intense light damages photomultipliers; they are limited
to measuring low power radiation

21. Cross section of a photomultiplier
tube

22.  The linear photodiode array is an example of
a multichannel photon detector. These detectors are
capable of measuring all elements of a beam of
dispersed radiation simultaneously.
 A linear photodiode array comprises many small silicon
photodiodes formed on a single silicon chip. There can
be between 64 to 4096 sensor elements on a chip, the
most common being 1024 photodiodes. For each diode,
there is also a storage capacitor and a switch. The
individual diode-capacitor circuits can be sequentially
scanned.
 In use, the photodiode array is positioned at the focal
plane of the monochromator (after the dispersing
element) such that the spectrum falls on the diode array.
They are useful for recording UV-Vis. absorption spectra
of samples that are rapidly passing through a sample
flow cell, such as in an HPLC detector.
 Charge-Coupled Devices (CCDs) are similar to diode
array detectors, but instead of diodes, they consist of an

23. IR Atomic Absorption
 The term "infra red" covers the range of the
electromagnetic spectrum between 0.78 and 1000 mm.
 In the context of infra red spectroscopy, wavelength is
measured in "wave numbers", which have the units cm-1.
wave number = 1 / wavelength in centimeters
It is useful to divide the infra red region into three
sections; near, mid and far infra red;
Region Wavelength range (mm) Wave number range (cm-1)
Near 0.78 - 2.5 12800 – 4000
Middle 2.5 – 50 4000 – 200
Far 50 -1000 200 – 10
The most useful I.R. region lies between 4000 - 670cm-1.

24. Theory of infra red absorption
 IR radiation does not have enough energy to induce
electronic transitions as seen with UV.
 Absorption of IR is restricted to compounds with small
energy differences in the possible vibrational and rotational
states.
 For a molecule to absorb IR, the vibrations or rotations
within a molecule must cause a net change in the dipole
moment of the molecule.
 The alternating electrical field of the radiation (remember
that electromagnetic radation consists of an oscillating
electrical field and an oscillating magnetic field,
perpendicular to each other) interacts with fluctuations in
the dipole moment of the molecule.
 If the frequency of the radiation matches the vibrational
frequency of the molecule then radiation will be absorbed,
causing a change in the amplitude of molecular vibration.

25.  Molecular rotations
 Rotational transitions are of little use to the spectroscopist.
Rotational levels are quantized, and absorption of IR by gases
yields line spectra. However, in liquids or solids, these lines
broaden into a continuum due to molecular collisions and
other interactions.
 Molecular vibrations
 The positions of atoms in a molecules are not fixed; they are
subject to a number of different vibrations. Vibrations fall into
the two main catagories of stretching and bending.
 Stretching: Change in inter-atomic distance along bond axis

26.  Bending: Change in angle between two bonds.
There are four types of bend:
 Rocking
 Scissoring
 Wagging
 Twisting

27. Vibrational coupling
 In addition to the vibrations mentioned above, interaction
between vibrations can occur (coupling) if the vibrating
bonds are joined to a single, central atom. Vibrational
coupling is influenced by a number of factors;
 Strong coupling of stretching vibrations occurs when there
is a common atom between the two vibrating bonds
 Coupling of bending vibrations occurs when there is a
common bond between vibrating groups
 Coupling between a stretching vibration and a bending
vibration occurs if the stretching bond is one side of an
angle varied by bending vibration
 Coupling is greatest when the coupled groups have
approximately equal energies
 No coupling is seen between groups separated by two or
more bonds

28. Instrumental components
 Sources
An inert solid is electrically heated to a temperature in the range 1500-2000
K.
 The heated material will then emit infra red radiation.
 The Nernst glower is a cylinder (1-2 mm diameter, approximately 20 mm
long) of rare earth oxides. Platinum wires are sealed to the ends, and a
current passed through the cylinder.
 The Nernst glower can reach temperatures of 2200 K.
 The Globar source is a silicon carbide rod (5mm diameter, 50mm long)
which is electrically heated to about 1500 K.
 Water cooling of the electrical contacts is needed to prevent arcing.
 The spectral output is comparable with the Nernst glower, execept at short
wavelengths (less than 5 mm) where it's output becomes larger.
 The incandescent wire source is a tightly wound coil of nichrome wire,
electrically heated to 1100 K. It produces a lower intensity of radiation than
the Nernst or Globar sources, but has a longer working life.
 Detectors
There are three catagories of detector;
 Thermal
 Pyroelectric
 Photoconducting

29.  Thermocouples consist of a pair of junctions of different metals; for
example, two pieces of bismuth fused to either end of a piece of
antimony.
The potential difference (voltage) between the junctions changes
according to the difference in temperature between the junctions
 Pyroelectric detectors are made from a single crystalline wafer of a
pyroelectric material, such as triglycerine sulphate.
The properties of a pyroelectric material are such that when an
electric field is applied across it, electric polarisation occurs (this
happens in any dielectric material).
In a pyroelectric material, when the field is removed, the polarisation
persists. The degree of polarisation is temperature dependant. So,
by sandwiching the pyroelectric material between two electrodes, a
temperature dependant capacitor is made.
The heating effect of incident IR radiation causes a change in the
capacitance of the material. Pyroelectric detectors have a fast
response time. They are used in most Fourier transform IR
instruments.
 Photoelectric detectors such as the mercury cadmium telluride
detector comprise a film of semiconducting material deposited on a
glass surface, sealed in an evacuated envelope.
Absorption of IR promotes nonconducting valence electrons to a
higher, conducting, state.
The electrical resistance of the semiconductor decreases.

30. Types of instrument
 Dispersive infra red spectophotometers
These are often double-beam recording instruments, employing
diffraction gratings for dispersion of radiation.
 Radiation from the source is flicked between the reference and
sample paths. Often, an optical null system is used. This is when the
detector only responds if the intensity of the two beams is unequal. If
the intensities are unequal, a light attenuator restores equality by
moving in or out of the reference beam. The recording pen is
attached to this attenuator.
 Fourier-transform spectrometers
Any waveform can be shown in one of two ways; either in frequency
domain or time domain.

31.  Dispersive IR instruments operate in the frequency domain.
There are, however, advantages to be gained from
measurement in the time domain followed by computer
transformation into the frequency domain.
 If we wished to record a trace in the time domain, it could be
possible to do so by allowing radiation to fall on a detector and
recording its response over time.
 In practice, no detector can respond quickly enough (the
radiation has a frequency greater than 1014 Hz). This problem
can be solved by using interference to modulate the i.r. signal
at a detectable frequency.
 The Michelson interferometer is used to produce a new signal
of a much lower frequency which contains the same
information as the original IR signal. The output from the
interferometer is an interferogram.

32.  Radiation leaves the source and is split. Half is reflected to a stationary
mirror and then back to the splitter. This radiation has travelled a fixed
distance. The other half of the radiation from the source passes through the
splitter and is reflected back by a movable mirror.
Therefore, the path length of this beam is variable. The two reflected beams
recombine at the splitter, and they interfere (e.g. for any one wavelength,
interference will be constructive if the difference in path lengths is an exact
multiple of the wavelength.
If the difference in path lengths is half the wavelength then destructive
interference will result). If the movable mirror moves away from the beam
splitter at a constant speed, radiation reaching the detector goes through a
steady sequence of maxima and minima as the interference alternates
between constructive and destructive phases.
 If monochromatic IR radiation of frequency, f ( ir ) enters the interferometer,
then the output frequency, fm can be found by;
where v is the speed of mirror travel in mm/s
Because all wavelengths emitted by the source are present, the
interferogram is extremely complicated.

33.  The moving mirror must travel smoothly; a frictionless bearing is
used with electromagnetic drive. The position of the mirror is
measured by a laser shining on a corner of the mirror. A simple sine
wave interference paatern is produced. Each peak indicates mirror
travel of one half the wavelength of the laser. The accuracy of this
measurement system means that the IR frequency scale is accurate
and precise.
 In the FT-IR instrument, the sample is placed between the output of
the interferometer and the detector. The sample absorbs radiation of
particular wavelengths. Therefore, the interferogram contains the
spectrum of the source minus the spectrum of the sample. An
interferogram of a reference (sample cell and solvent) is needed to
obtain the spectrum of the sample.
 After an interferogram has been collected, a computer performs
a Fast Fourier Transform, which results in a frequency domain trace
(i.e intensity vs. wavenumber) that we all know and love.The detector
used in an FT-IR instrument must respond quickly because intensity
changes are rapid (the moving mirror moves quickly). Pyroelectric
detectors or liquid nitrogen cooled photon detectors must be used.
Thermal detectors are too slow.
 To acheive a good signal to noise ratio, many interferograms are
obtained and then averaged. This can be done in less time than it
would take a dipersive instrument to record one scan.

34. Advantages of Fourier transform
IR over dispersive IR;
 Improved frequency resolution
 Improved frequency reproducibility (older
dispersive instruments must be recalibrated for
each session of use)
 Higher energy throughput
 Faster operation
 Computer based (allowing storage of spectra and
facilities for processing spectra)
 Easily adapted for remote use (such as diverting
the beam to pass through an external cell and
detector, as in GC - FT-IR)