U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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Intro to Spectroscopy
a) explain the nature of electromagnetic radiation
b) know the approximate wavelength ranges of the X-ray, UV/VIS, IR
and radiofrequency regions of the EM spectrum
c) appreciate the relative energies and dangers associated with exposure
to high energy wavelengths
d) perform calculations using the equation E = hν = hc / λ
e) appreciate that energy levels in atoms and molecules are quantised
UV-VIS Spectroscopy
a) explain the origin of absorption in UV/VIS spectroscopy
b) explain why some species will absorb light in the uv/vis region but
some others will not
c) describe the basic steps involved in analysing samples by uv/vis
spectroscopy (also mention use of complexing agents to form coloured
compounds, as well as sensitivity and detection limits)
d) state the Beer-Lambert Law and be able to use the equation to
calculate the concentration of a given species in solution (implies the use
of standards and calibration curves)
e) list examples of the use of uv/vis spectroscopy in the quantitation of
substance (refer to iron tablets, glucose and urea in blood, cyanide in
water)
Introduction to spectroscopy
Electromagnetic radiation can be described in terms of a stream of photons,
which are massless particles each travelling in a wave-like pattern and
moving at the speed of light. Each photon contains a certain amount (or
bundle) of energy, and all electromagnetic radiation consists of these
photons.
The electromagnetic (EM) spectrum is just a name that scientists give a
bunch of types of radiation when they want to talk about them as a group.
Radiation is energy that travels and spreads out as it goes-- visible light that
comes from a lamp in your house and radio waves that come from a radio
station are two types of electromagnetic radiation.
Other examples of EM radiation are microwaves, infrared and ultraviolet
light, X-rays and gamma-rays. Hotter, more energetic objects and events
create higher energy radiation than cool objects. Only extremely hot objects
or particles moving at very high velocities can create high-energy radiation
like X-rays and gamma-rays.
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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EM waves are typically described by any three of the following physical
properties: the frequency,
, and wavelength, λ, and photon energy, E.
Frequencies range from about a million billion Hertz (gamma rays) down to
a few Hertz (radio waves). Wavelength is inversely proportional to the
wave frequency, so gamma rays have very short wavelengths that are
fractions of the size of atoms, whereas radio wavelengths can be as long as a
several thousand kilometers. Photon energy is directly proportional to the
wave frequency, so gamma rays have the highest energy around a mega
electron volt and radio waves have very low energy around femto electron
volts (femto = 10 − 15).
These relations are illustrated by the following equation E = h
variations below:or
or
Where: c = 3 x 108 m s-1 (speed of light in vacuum) &
h = 6.626 ×10−34 Js-1 (Planck's constant).
and its
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
Checkpoint A
1.
2.
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U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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High energy EM radiation e.g. ultraviolet rays, X-rays and gamma rays are
very dangerous to living tissue. These energetic rays have to ability to
disrupt and destroy DNA in tissue which can result in mutations or even
death. All three types of rays would result in electronic transitions and even
ionisation.
In atoms, energy levels do NOT form a continuum. In fact, these energy
levels are discrete or quantised. For example, energy levels may be at
5J, 10 J, 20 J but never 5.1J, 5.2 J, 10.3 J etc. There are no “in
between” energy levels or values.
UV-VIS Spectroscopy
The absorption of ultraviolet or visible radiation corresponds to the
excitation of outer electrons and thus electronic transitions.
When an atom or molecule absorbs energy, electrons are promoted from
their ground state (lowest energy level) to an excited state.
Absorption of ultraviolet and visible radiation in organic molecules is
restricted to certain functional groups (chromophores) that contain
valence electrons of low excitation energy. Chromophores generally
contain either П or lone pair(s) of electrons
Electronic transitions generally seen are n* and * transitions.
In essence molecules containing lone pairs of electrons OR  electrons
would absorb uv-vis radiation.
All other transitions either do NOT occur or are TOO SMALL to be
considered. Below is a diagram that shows the various electronic
transitions.
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
Checkpoint B
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Underline the molecules in the list below which would absorb uv-vis
radiation. O2, C6H6, NH3, CH4, C2H4, HCl
Sample preparation
UV-VIS samples are usually liquids or solutions. They are placed in a
“cell” or cuvette which is a rectangular block, with two sides frosted and
the other two sides clear and one end open and the other closed. The
frosted sides can be touched but not the clear sides.
Cells are made of fused synthetic silica.
Solutions or liquids are placed in the cell in increasing
concentrations. A “blank” (which contains everything else but
the analyte in question) is first used to use as a reference point.
Sometimes, complexing agents must be added to provide colour
to the analyte which allows it to be analysed via uv-vis more
easily. For example iron present in aqueous samples can be
determined spectrophotometrically by complexation with a
suitable complexing agent. The absorbance of the metal-ligand
complex is usually measured in the visible region and is related
to metal ion concentration. Colorimetric determination of iron
can be done using several known complexing agents. Among
the routinely used is 1,10-phenanthroline (phen) which reacts
with Fe2+ to form an orange-red complex. Also
dimethylglyoximeis is a complexing agent using to
determine the amount of nickel present.
The procedure depends on the construction of a calibration
curve from standard Fe2+, followed by measurement of the
unknown Fe2+ concentration from the curve.
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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Using absorbance to determine the concentration of an analyte
The Beer-Lambert Law
ε = molar absorbtivity
ℓ = cell length in cm (usually 1 cm in length)
c = molar concentration of solution
A = absorbance
Standards and calibration curves are
used for UV-VIS spectroscopy
coupled with Beer-Lambert Law as
concentration of the analyte MUST
show a linear signal response for the
Law to be of any worth.
NB
Io = incident light,
I = transmitted light
Spectrophotometer
The basic parts of a spectrophotometer are a light source, a holder for the
sample, a diffraction grating or monochromator 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), and more recently
light emitting diodes (LED) and Xenon Arc Lamps for the visible
wavelengths. The detector is typically a photodiode. Photodiodes are
used with monochromators, which filter the light so that only light of a
single wavelength reaches the detector.
The dual-beam design greatly simplifies this process by simultaneously
measuring P and Po of the sample and reference cells, respectively. Most
spectrometers use a mirrored rotating chopper wheel to alternately direct
the light beam through the sample and reference cells. The detection
electronics or software program can then manipulate the P and Po values
as the wavelength scans to produce the spectrum of absorbance or
transmittance as a function of wavelength.
Schematic of a dual-beam uv-vis spectrophotometer
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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Applications of UV-VIS spectroscopy
UV/VIS spectroscopy is routinely used in the quantitative determination
of solutions of transition metal ions and highly conjugated organic
compounds, of iron in iron tablets, glucose and urea in blood and cyanide
in water
Solutions of transition metal ions which are too pale to give a reasonable
absorbance can be complexed with various ligands to form more intense
colours (i.e., absorb visible light) because d electrons within the metal
atoms can be excited from one electronic state to another. The colour of
metal ion solutions is strongly affected by the presence of other species,
such as certain anions or ligands. For instance, the colour of a dilute
solution of copper sulphate is a very light blue; adding ammonia
intensifies the colour and changes the wavelength of maximum
absorption (λmax)
U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
Checkpoint C
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U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
2.
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U2 M2 Intro. to spectroscopy & uv-vis spectroscopy
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