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Spectroscopy is a technique that is used to study the interaction of matter and

electromagnetic radiation. It involves the measurement of the energy absorbed
or emitted by a sample at different wavelengths. Spectroscopy can be used to
identify the composition of a sample, to study the physical and chemical
properties of a sample, and to determine the structure of a molecule

What is UV-Vis Spectroscopy?

UV-Vis spectroscopy is an analytical technique that measures the amount of

discrete wavelengths of UV or visible light that are absorbed by or transmitted
through a sample in comparison to a reference or blank sample. This property is
influenced by the sample composition, potentially providing information on what
is in the sample and at what concentration. Since this spectroscopy technique
relies on the use of light, let’s first consider the properties of light.

What is the main purpose of UV spectroscopy?

 Studying chemical reactions and reaction kinetics

 Analysis of biological macromolecules like proteins, DNA, and RNA
 Environmental analysis
 Pharmaceuticals and drug analysis
 Identifying and quantifying the concentration of a specific compound in a
mixture
 Characterizing the purity of a sample
 Determining the electronic structure of a compound, such as the presence of
conjugated double bonds or aromatic ring systems

 The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction
grating in a monochromator or a prism to separate the different wavelengths of light, and a
detector. The radiation source is often a Tungsten filament (300–2500 nm), a deuterium arc
lamp, which is continuous over the ultraviolet region (190–400 nm), Xenon arc lamp, which is
continuous from 160 to 2,000 nm; or more recently, light emitting diodes (LED)[1] for the visible
wavelengths. The detector is typically a photomultiplier tube, a photodiode, a photodiode array or
a charge-coupled device (CCD). Single photodiode detectors and photomultiplier tubes are used
with scanning monochromators, which filter the light so that only light of a single wavelength
reaches the detector at one time. The scanning monochromator moves the diffraction grating to
"step-through" each wavelength so that its intensity may be measured as a function of
wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of
these devices consist of many detectors grouped into one or two dimensional arrays, they are
able to collect light of different wavelengths on different pixels or groups of pixels simultaneously.


 Simplified schematic of a double beam UV–visible spectrophotometer
 A spectrophotometer can be either single beam or double beam. In a single beam instrument
(such as the Spectronic 20), all of the light passes through the sample cell.  must be measured
by removing the sample. This was the earliest design and is still in common use in both teaching
and industrial labs.
 In a double-beam instrument, the light is split into two beams before it reaches the sample. One
beam is used as the reference; the other beam passes through the sample. The reference beam
intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement displayed is
the ratio of the two beam intensities. Some double-beam instruments have two detectors
(photodiodes), and the sample and reference beam are measured at the same time. In other
instruments, the two beams pass through a beam chopper, which blocks one beam at a time.
The detector alternates between measuring the sample beam and the reference beam in
synchronism with the chopper. There may also be one or more dark intervals in the chopper
cycle. In this case, the measured beam intensities may be corrected by subtracting the intensity
measured in the dark interval before the ratio is taken.
 In a single-beam instrument, the cuvette containing only a solvent has to be measured first.
Mettler Toledo developed a single beam array spectrophotometer that allows fast and accurate
measurements over the UV/VIS range. The light source consists of a Xenon flash lamp for the
ultraviolet (UV) as well as for the visible (VIS) and near-infrared wavelength regions covering a
spectral range from 190 up to 1100 nm. The lamp flashes are focused on a glass fiber which
 drives the beam of light onto a cuvette containing the sample solution. The beam passes through
the sample and specific wavelengths are absorbed by the sample components. The remaining
light is collected after the cuvette by a glass fiber and driven into a spectrograph. The
spectrograph consists of a diffraction grating that separates the light into the different
wavelengths, and a CCD sensor to record the data, respectively. The whole spectrum is thus
simultaneously measured, allowing for fast recording. [15]
 Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of gases
and even of solids can also be measured. Samples are typically placed in a transparent cell,
known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width
of 1 cm. (This width becomes the path length, , in the Beer–Lambert law.) Test tubes can also be
used as cuvettes in some instruments. The type of sample container used must allow radiation
to pass over the spectral region of interest. The most widely applicable cuvettes are made of
high quality fused silica or quartz glass because these are transparent throughout the UV, visible
and near infrared regions. Glass and plastic cuvettes are also common, although glass and most
plastics absorb in the UV, which limits their usefulness to visible wavelengths. [1]

Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the

interaction of infrared radiation with matter by absorption, emission, or reflection. It is used to study
and identify chemical substances or functional groups in solid, liquid, or gaseous forms. It can be
used to characterize new materials or identify and verify known and unknown samples. The method
or technique of infrared spectroscopy is conducted with an instrument called an infrared
spectrometer (or spectrophotometer) which produces an infrared spectrum

There are four types of instruments for infrared absorption measurements available:
-Dispersive grating spectrophotometers for qualitative measurements
-Nondispersive photometers for quantitative determination of organic species in the
atmosphere
-Reflectance photometers for analysis of solids
-Fourier transform infrared (FT-IR) instruments for both qualitative and quantitative
measurements.

Infrared spectrometry - Infrared light sources

Instruments for measuring infrared absorption all require a source of continuous infrared
radiation and a sensitive infrared transducer, or detector.
Infrared sources consist of an inert solid that is electrically heated to a temperature
between 1,500 and 2,200 K. The heated material will then emit infra red radiation.

The Nernst glower

The Nernst glower is constructed of rare earth oxides in the form of a hollow cylinder.
Platinum leads at the ends of the cylinder permit the passage of electricity. Nernst
glowers are fragile. They have a large negative temperature coefficient of electrical
resistance and must be preheated to be conductive.

The globar source

A globar is a rod of silicon carbide (5 mm diameter, 50 mm long) which is electrically
heated to about 1,500 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 carbon dioxide laser

A tuneable carbon dioxide laser is used as an infrared source for monitoring certain
atmospheric pollutants and for determining absorbing species in aqueous solutions. 

Infrared Spectrometry - Detectors

The detectors can be classified into three categories, thermal detectors, pyroelectric
detectors and photoconducting detectors.

Thermal detectors
Thermal detectors can be used over a wide range of wavelengths and they operate at
room temperature. Their main disadvantages are slow response time and lower
sensivity relative to other types of detectors.

Thermocouple
A thermocouple consists 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. Several thermocouples connected in series are called a
thermopile.

Bolometer
A bolometer functions by changing resistance when heated. It is constructed of strips of
metals such as platinum or nickel or from a semiconductor.

Pyroelectric detectors
Pyroelectric detectors consists of a pyroelectric material which is an insulator with
special thermal and electric properties. Triglycine sulphate is the most common material
for pyroelectric infrared detectors. Unlike other thermal detectors the pyroelectric effect
depends on the rate of change of the detector temperature rather than on the
temperature itself. This allows the pyroelectric detector to operate with a much faster
response time and makes these detectors the choice for Fourier transform
spectrometers where rapid response is essential.
Photoconducting detectors
Photoconducting detectors are the most sensitive detectors. They rely on interactions
between photons and a semiconductor. The detector consists of a thin film of a
semiconductor material such as lead sulphide, mercury cadmium telluride or indium
antimonide deposited on a nonconducting glass surface and sealed into an evacuated
envelope to protect the semiconductor from the atmosphere. The lead sulphide detector
is used for the near-infrared region of the spectrum. For mid- and far-infrared radiation
the mercury cadmium telluride detector is used. It must be cooled with liquid nitrogen to
minimize disturbances. 

Infrared Spectrometry - Sample Handling

  

 Gas samples
The spectrum of a gas can be obtained by permitting the sample to expand into an
evacuated cell, also called a cuvette.
Solutions
Infrared solution cells consists of two windows of pressed salt sealed and separated by
thin gaskets of Teflon, copper or lead that have been wetted with mercury. The windows
are usually made of sodium chloride, potassium chloride or cesium bromide. Samples
that are liquid at room temperature are usually analysed in pure form or in solution. The
most common solvents are Carbon Tetrachloride (CCl 4) and Carbon Disulfide (CS2).
Chloroform, methylene chloride, acetonitrile and acetone are useful solvents for polar
materials.
Solids
Solids reduced to small particles can be examined as a thin paste or mull. The mull is
formed by grinding a few milligrams of the sample in the presence of one or two drops
of a hydrocarbon oil. The resulting mull is then examined as a film between flat salt
plates. In the reference beam path a window of the same thickness is placed. Another
technique is to ground a milligram or less of the sample with about 100 milligram
potassium bromide. The mixture is then pressed in an evacuable die to produce a
transparent disk. In the reference beam path a disk of pure potassium bromide is
placed. 

Dispersive IR Spectrometers

To understand the powerfulness and usefulness of FTIR spectrometer, it is essential

to have some background information of dispersive IR Spectrometer. The basic
components of a dispersive IR spectrometer include a radiation source,
monochromator, and detector. The common IR radiation sources are inert solids that
are heated electrically to promote thermal emission of radiation in the infrared region of
the electromagnetic spectrum. The monochromator is a device used to disperse or
separate a broad spectrum of IR radiation into individual narrow IR frequencies.

Generally, dispersive spectrometers have a double-beam design with two equivalent

beams from the same source passing through the sample and reference chambers as
independent beams. These reference and sample beams are alternately focused on the
detector by making use of an optical chopper, such as, a sector mirror. One beam will
proceed, traveling through the sample, while the other beam will pass through a
reference species for analytical comparison of transmitted photon wavefront
information.

After the incident radiation travels through the sample species, the emitted wavefront
of radiation is dispersed by a monochromator (gratings and slits) into its component
frequencies. A combination of prisms or gratings with variable-slit mechanisms, mirrors,
and filters comprise the dispersive system. Narrower slits gives better resolution by
distinguishing more closely spaced frequencies of radiation and wider slits allow more
light to reach the detector and provide better system sensitivity. The emitted wavefront
beam (analog spectral output) hits the detector and generates an electrical signal as a
response.
Detectors are devices that convert the analog spectral output into an electrical signal.
These electrical signals are further processed by the computer using mathematical
algorithm to arrive at the final spectrum. The detectors used in IR spectrometers can be
classified as either photon/quantum detectors or thermal detectors.

It is the absorption of IR radiation by the sample, producing a change of IR radiation

intensity, which gets detected as an off-null signal (e.g. different from reference signal).
This change is translated into the recorder response through the actions of synchronous
motors. Each frequency that passes through the sample is measured individually by the
detector which consequently slows the process of scanning the entire IR region. A block
diagram of a classic dispersive IR spectrometer is shown in Figure 4.3.1.

Figure 4.3.
1. Simplified representation of a dispersive IR spectrometer.