2-UV-Vis Molecular Spectrometry

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UV-Vis Molecular

Spectrophotometry
Review

 Ultraviolet-visible (UV-Vis) molecular spectrochemical methods utilize


light in the ultraviolet and visible region of the electromagnetic
spectrum to analyze laboratory samples for molecular compounds
and complex ions.
 Qualitative analysis (identification of unknown and detection of
impurities in knowns) is accomplished by comparing adsorption or
transmission spectra (molecular fingerprints) with known spectra.
 Quantitative analysis is accomplished with the use of Beer’s law.
 Since vibrational levels are superimposed on the electronic levels,
many electronic transitions are possible, resulting in continuous spectra
rather than the line spectra that characterize spectra of atoms.
UV-Vis Instrumentation

 The fundamental instrument is shown in this figure


 There are:
1. Sources
2. Wavelength selection
3. Sample compartment
4. Detectors
Sources

 An ideal light source is one that emits an intense continuous


spectrum of light across an entire region of the spectrum.
 A light source that can be used in UV-Vis spectrophotometer:
1.1. Tungsten filament lamp
1.2. Deuterium lamp
1.3. Xenon arc lamp
1.1. Tungsten Filament Lamp

 For the visible region, many instruments


utilize a light bulb with a tungsten
filament
 Such a source is very bright and emits
light over the entire region and into the
near infrared region (350-2500 nm)
 However, the intensity of the light varies
dramatically across this wavelength
range
 Tungsten/halogen lamp (240-2500 nm)
1.2. Deuterium Lamp

 Deuterium is the name given to the isotope of hydrogen that has


one neutron in the atomic nulcleus (as opposed to zero neutrons
for hydrogen and two neutrons for tritium, the other hydrogen
isotopes)
 The lamp contains deuterium at a low pressure.
 Electricity applied to electrodes in the lamp results in a continuous
UV emission due to the presence of the deuterium.
 Its wavelength output ranges from 185 to 375 nm, satisfactory for
most UV analyses
1.3. Xenon Arc Lamp

 This lamp contains xenon at a fairly high pressure and the light is
formed via a discharge across a pair of electrodes.
 A continuous UV and Vis emission is emitted due to the presence of
the xenon.
 Its wavelength output ranges from 180-2500 nm
 The intensity varies with wavelength
2. Wavelength selection

 In order to plot the absorption spectrum of a compound or


complex ion, we must be able to carefully control the wavelength
from a broad spectrum of wavelength emitted by the source, so
that we can measure the absorbance at each wavelength.
 Additionally, in order to perform quantitative analysis by Beer’s law,
we need to be able to carefully select the wavelength of
maximum absorption, also from this broad spectrum of
wavelength, in order to plot the proper absorbance at each
concentration.
 These facts dictate that we must be able to filter out the unwanted
wavelengths and allow only the wavelength of interest to pass.
2.1. Absorption filters

 The most inexpensive way to isolate a wavelength band is with the use of
absorption filters.
 For a visible light, such a filter would consist of colored glass, the color of the glass
indicating what region of the visible spectrum is passed.
 Thus, if the wavelength called for a given method is in the red region of the visible
spectrum, a red colored glass filter is chosen
 Absorption filters have the lowest
resolution, having effective
bandwidths varying
from 30-250 nm.
2.2. Monochromators

 It is more sophisticated than an absorption filter


that isolates the narrow band of wavelength
from visible and ultraviolet sources
 A monochromator is made up of three parts:
 An entrance slit
where light enters the monochromator from the source.
Its purposes is to create a unidirectional beam of light of
appropriate intensity from the multidirectional light
emanating from the source.
 A dispersing element
the dispersing element disperses the light into its
component wavelength
 An exit slit
the narrow band of spectrum is then selected by the
exit slit
3. Sample compartment

 Cuvettes used for UV-Vis spectrophotometry must be transparent to all


wavelengths of light for which it is used.
 If visible light is used, the material must ideally be completely clear and
colorless, which means that inexpensive materials, such as colorless
plastic and ordinary colorless glass, are perfectly suitable.
 However, ordinary colorless glass and plastic are not transparent to
light in the ultraviolet region. For UV spectrophotometry, the cuvettes
must be made of quartz, which is more expensive.
 If two or more different cuvettes are used in an analysis, one should be
sure that they are matched.
 Avoid any scratches, fingerprints, water spots on the cuvette surfaces
4. Detectors

 Photomultiplier tubes
 A photomultiplier tube is a light sensor combined with a signal amplifier.
 The light emerging from the sample compartment strikes the photosensitive
surface and the resulting electrical signal is amplified

 Photodiodes (light sensors)


 Photodiodes make use of the unique properties of semiconductors, such as
silicon
Single beam spectrophotometer

 The monochromatic light beam created by the monochromator


passes directly through the sample solution held in the cuvette and
then proceeds to the detector.
 This is especially useful for routine absorbance measurements for
which the wavelength of maximum absorbance is known in
advance without having to scan a particular wavelength range to
determine it.
Double beam instruments

 The light beam emerging from the


monochromator is split into two beams at
some point between the
monochromator and the detector
 Another double beam design is one in
which the beam emerging from the
monochromator is split with a
beamsplitter.
Deviations

 Deviations from Beer’s law are evidence when the Beer’s law plot is
not linear.
 This is probably most often observed at the higher concentrations
of the analyte.
 Such deviations can be:
 chemical
 instrumental
Instrumental deviations

 Instrumental deviations occur because it is not possible for an instrument to be


accurate at extremely high or extremely low transmittance values
 The normal working range of transmittance is between 15 and 80%
 The absorbance values between 0.10 and 0.82.
 It is recommended that standards be prepared to
measure in this range and that unknown samples be
diluted if necessary.
Chemical deviations

 Deviations due to chemical interferences occur when a high or low


concentration of the analyte causes chemical equilibrium shifts in
the solution that directly or indirectly affect its absorbance.
 This means that, unknown samples may also need to be further
diluted, as in the instrumental deviation case
Absorbing species

 A band consists of a large number of closely spaced vibrational


and rotational lines.
 The energies associated with these lines differ little from one
another
 There are two kind of absorbing species in UV-Vis
Spectrophotometry:
 Absorption by organic compounds
 Absorption by inorganic species
Absorption by organic compounds

 The wavelength of absorption of an organic molecule depends on how tightly its


electrons are bound.
 The shared electrons in carbon-carbon or carbon-hydrogen single bonds are so firmly
held that their excitation requires energies corresponding to wavelength in the
vacuum ultraviolet region below 180 nm.
 Electrons in double and triple bonds of organic molecules are not as strongly held and
therefore more easily excited by electromagnetic radiation.
 Unsaturated organic functional groups that absorb in the ultraviolet or visible regions
are known as chromophores.
 Most transitions above 200 nm are π  π* or n π*
Absorption by inorganic species

 Absorption occurs when electrons make transitiononal between


filled and unfilled d-orbitals with energies that depend on the
ligands bonded to the metal ions.
 The energy differences between these d-orbitals depend on the
position of the element in the periodic table, its oxidation state,
and the nature of the ligand bonded to it.
Qualitative applications of ultraviolet/visible
spectroscopy

 Spectrophotometric measurements with ultraviolet radiation are


useful for detecting chromophoric groups.
 Because large parts of even the most complex organic molecules
are transparent to radiation longer than 180 nm, the appearance
of one or more absorption band in the region from 200-400 nm is
clear indication of the presence of unsaturated groups of atoms
such as sulfur or halogens.
 Usually, UV spectra do not have sufficient fine structure to permit an
analyte to be identified, it must be supplemented with IR, NMR, MS,
solubility and melting/boiling point information.
Quantitative applications

 UV-Vis Spectrophotometry is one of the most useful tools available


for quantitative analysis. It is:
 Wide applicability (inorganic, organic, biochemical species)
 High sensitivity (typical detection limits range from 10-4 to 10-5 M
 Moderate to high selectivity
 Good accuracy
 Ease and convenience
Procedural details

 Wavelength selection
in order to realize maximum sensitivity, spectrophotometric absorbance
measurements are usually made at the wavelength of maximum absorption.

 Variables that influences absorption


Common variables that influence the absorption spectrum are the nature of
solvent, the pH of the solution, the temperature, high electrolyte concentrations,
the presence of interfering substances

 The relationship between absorbance and concentration


a calibration curve of absorbance vs the concentration of several standard is
usually obtained to evaluate the relationship.
Quantitative calculation

 A sample in 1.0 cm cell transmit 80% light at a certain wavelength .


If the absorptivity of this substances at this wavelength is 2.0, what is
its concentration?
Quantitative calculation

 a solution containing 1.00 mg iron (as the thiocyanate complex) in


100 mL was observed to transmit 70.0% of the incident light
compared to an appropriate blank.
a. What is the absorbance of the solution at this wavelength?
b. What fraction of light would be transmitted by a solution of iron
four times as concentrated?
Quantitative calculation

 Chloroaniline in a sample is determined as the amine picrate. A


0.0265 g sample is reacted with picric acid and diluted to 1 L. the
solution exhibits an absorbance of 0.368 in a 1 cm cell. What is the
percentage chloroanline in the sample?

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