UV-visible spectroscopy
Table of Contents
- History
- Introduction
- Principle
- Instrumentation
- Applications
History
For the most part, humans cannot see ultraviolet
photons. In Latin, the word "ultra" means "beyond," and the
word "violet" refers to the colour of the highest frequencies of
visible light. Compared to violet light, ultraviolet has a greater frequency
and a shorter wavelength.
John Wilhelm Ritter, a German physicist, first
discovered UV radiation by observing that the paper soaked in silver
chloride browned more quickly when exposed to UV radiation than when it was
exposed to violet light itself in 1801. The impact of UV light on DNA was
identified in 1960.
Victor Schumann, a German physicist, made the
discovery of ultraviolet light with wavelengths below 200 nm in 1893. This
light is known as "vacuum ultraviolet" because it is substantially
absorbed by the oxygen in the air.
In 1940, the first UV-Vis gadgets became a commercial
reality. In 1941, the Beckman UV-Vis spectrophotometer made the bolder claim
that it would complete analyses faster than its rivals. Since then, the history
of UV-Vis spectrophotometers has seen a number of developments. In 1941, the
Beckman UV-Vis spectrophotometer made the bolder claim that it would complete
analyses faster than its rivals. Since then, the history of UV-Vis
spectrophotometers has seen a number of developments.
Introduction
The concentration of a chemical within a bigger
substance can be determined using the straightforward yet effective laboratory
technique known as UV-Visible spectroscopy (UV-Vis). The device that enables
this operation, a spectrophotometer, measures the amount of light that chemical
compounds absorb and logs the wavelengths that the compounds transmit to
produce different spectra. To accomplish this, a cuvette (a transparent
container) containing the desired material is sent a beam of light by the
machine. Based on the solution's concentration, the cuvette only absorbs
certain wavelengths. The Light wavelengths that are not absorbed travel
through the sample, through a tiny aperture, and are then diffracted onto a
photoluminescence detector, producing a specific pattern that varies with each
molecule. The amount of time needed
to set up a UV-VIS spectrometer for use is its principal drawback. While
employing UV-VIS spectrometers, setup is essential. The area must be cleared of
any outside light, electrical noise, or other impurities that can affect the
spectrometer's measurement.
Reusable quartz cuvettes were formerly needed for
measurements in the ultraviolet region since glass and the majority of polymers
absorb ultraviolet light.
In UV-Vis spectroscopy, photomultiplier tubes are a
common detector. It is made up of an anode, many dynodes, and a photo emissive
cathode, which emits electrons when exposed to radiation photons.
Beer-Lambert Law
The Beer-Lambert law states that the rate at which the
intensity of a monochromatic light beam decreases along the thickness of a
solution that contains an absorbent of that monochromatic light is directly
proportional to the concentration of the absorbing substance in the solution
and is also directly proportional to the intensity of the incident
monochromatic radiation.
According to the Beer-Lambert equation, the amount of
radiation absorbed increases with the number of absorbing molecules (molecules
with the capacity to absorb light of a particular wavelength).
Principle
The UV-Visible Principle The foundation of
spectroscopy is the absorption of ultraviolet or visible light by chemical
substances, which generates distinctive spectra. The basis of spectroscopy is
the interaction of light and matter. A spectrum is created when the substance
absorbs the light through excitation and de-excitation processes.
The electrons existing in matter experience excitation
when it absorbs UV energy. As a result, they move abruptly from their ground
state—an energy condition with a negligible amount of energy—to their excited
state (an energy state with a relatively large amount of energy associated with
it). It is significant to remember that
the amount of ultraviolet or visible radiation absorbed by an electron is
always equal to the energy difference between its ground state and excited
state.
Instrumentation
Single and Double Beam Spectrometer
Single-Beam, From the source to the detector, there is
just one light beam or optical path. Double-beam is one in which the beam follows passage through the monochromator, the source's light is divided into two
distinct beams, one for the sample and the other for the reference.
Spectrophotometer with a single beam
The reference cell, which is used to put the
absorbance scale at zero for the wavelength under study, is exposed to a single
beam of light. The sample cell is then used to measure the sample's absorbance
at that wavelength. This was the original design, and it is still in use in
research and teaching labs.
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Double beam spectrophotometer
A UV/Vis spectrophotometer is the device used in
ultraviolet-visible spectroscopy. It measures the amount of light that enters a
sample and compared it with the amount of light that exits the sample as shown below. The
transmittance, also known as the ratio, is often given as a percentage (%T). On
the transmittance, the absorbance, (A), is based.
A= -log10
(1/T)
a) Light source
A spectrophotometer light source must be bright across
a broad wavelength range, stable over time, have a long service life, and be
inexpensive.
Both a high level of brightness and uniform brightness
across the measuring wavelength range are necessary for "a) Bright across
a wide wavelength range" (uniform brightness distribution).
Halogen lamp
A halogen lamp's filament heats up and creates light
when a current pass through it, much like a regular incandescent bulb does.
High temperatures cause the tungsten used as the filament material to vaporise.
As a result, an inert gas is used to fill the bulb that houses the filament of
a typical incandescent lamp in order to stop the tungsten from evaporating.
A deuterium lamp is a discharge light source that
contains a bulb of several hundred Pa deuterium. It takes roughly 10 seconds
for the cathode to preheat before the discharge can begin since it uses a hot
cathode to create stable and dependable arc discharge.
Deuterium lamp
A deuterium lamp is more expensive than a halogen lamp
because it needs a big, complicated power supply. It is one of the few sources
of continuous spectrum light that is stable in the ultraviolet region, though. The
short emission wavelength of the deuterium lamp is 400 nm or less. Its
application is restricted at the short wavelength end by the window material.
Xenon lamp
A xenon lamp is a discharge light source that contains
xenon gas inside a bulb. Depending on the illumination technique, xenon lamps
are classified as either direct current or alternating-current varieties. The
tungsten electrode material may vaporise and adhere to the tube wall if the
electrodes get too hot, which will reduce brightness. A direct-current type
xenon lamp's anode is built larger than the cathode to boost its thermal
capacity as the anode gets exceptionally hot. An alternating-current type
electrode has two identical electrodes that alternately serve as the cathode
and anode. As a result, tungsten evaporates more quickly than it would with
direct current. The alternating-current type, however, enables the employment
of a small, affordable lighting equipment since current rectification is not
necessary.
In terms of price and production fluctuations, the
xenon lamp falls below the halogen light and the deuterium lamp overall. In
typical spectrophotometers, halogen lamps are frequently employed, however
because of their extreme brightness, xenon lamps are used in situations when a
high light intensity is necessary (such as spectrofluorophotometers).
Flashing Xenon
lamp
Due to pulsed ignition, this tiny xenon light produces
low heat. There are both straight and U-tube options depending on the
application. In a quartz glass tube (or high-silica glass tube) that is filled
with xenon gas, the electrodes are sealed. However, integration of the output
data is necessary to acquire consistent data due to its poor repeatability,
which is a result of bigger output fluctuations than an arc lamp. As a result,
it is employed in automated equipment (like colorimeters) in conjunction with
an array detector to quickly acquire continuous spectra.
Mercury Lamp with
Low Pressure
When lit, the low-pressure mercury lamp has a low
mercury vapour pressure (100 Pa max.), which effectively emits the mercury
resonance lines (254 nm or 185 nm).
b) Dispersing device
or wavelength selector
A wavelength selector is a part of an instrument that
either transmits one or more lines from a discrete wavelength source or chooses
and transmits a limited band of wavelengths coming from a broad band optical
source. There are two types of wavelength selectors: fixed wavelength and
scanning.
Diffraction gratings, prisms, or coloured filters are
employed as three different kinds of wavelength pickers. Currently, gratings
are employed in the majority of instruments because they perform significantly
better, have a wide wavelength range, and are simple to adjust.
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| Diffraction Grating |
c) Sample
compartment
Test tubes in the shape of a cuvette. They serve the
same purpose as regular test tubes: to hold aqueous solutions. Chemical
reactions can be aided by regular test tubes. Cuvettes, on the other hand, are
employed in UV-Vis spectrophotometers or fluorometers to measure the
transmittance or absorption of light at a specific wavelength.
A cuvette is a small, clear, rectangular vessel that
can be found in various materials, qualities, and dimensions for
spectrophotometric studies. For measurements in the visible spectrum between
320 and 2500 nm, glass cuvettes are employed. Quartz cuvettes provide accurate
findings throughout the entire visible and UV spectrum between 200 and 2500 nm.
The measurement is better and more repeatable the smaller the manufacturing
tolerance.
d) Detectors
1. Photocells
These are composed of cadmium sulphide, silicon, and
selenium are used in UV-VIS detectors. Silver film is first applied to the
steel basis, and then a thin layer of selenium. Selenium allows electrons to
travel through to silver, which serves as the collecting electrode and steel
plate as a second electrode. A micro-ammeter is then used to measure the
current flowing between the two electrodes.
2. Phototube
The energy of
the photon is transferred to the loosely bound electrons of the cathode surface
in phototubes, which have a glass envelope with a quartz window, a metal wire
in the middle that serves as an anode, and a semicircle that serves as a
cathode. The excited electrons flow in the circuit as they go towards the
anode. As phototube currents are extremely tiny, they must first be amplified
before being recorded.
3. Photomultiplier
This device, which consists of an evacuated glass tube
into which the cathode and anode are sealed and extra intermediary electrodes
known as dynodes, is intended to enhance the initial photoelectric action and
is suited for usage at very low light intensities. The applied potential
difference drives the freed electrons towards the first dynode as the radiation
strikes the cathode. Each dynode after
that has a larger electrical potential and functions as an amplifier.
4. Photodiodes
These are semiconductors that, when exposed to
radiation, charge their charged voltage, which is then converted to current and
measured.
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| Instrumentation |
Applications
In the discipline of analytical chemistry, UV-Visible
spectroscopy is frequently employed, particularly when performing a
quantitative examination of a particular analyte. For instance, UV-visible
spectroscopy can be used to quantitatively analyse transition metal ions.
Moreover, UV-Visible spectroscopy can be used to quantitatively analyse
conjugated organic molecules. It should be mentioned that under certain
circumstances, this form of spectroscopy can also be used to analyse solid and
gaseous analyte.
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