06523-83 20molecular 20spectroscopy 20uv
TRANSCRIPT
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Module 06523/83
Intermediate Physical and Analytical Chemistry 2
Atomic Spectroscopy
• Interaction of radiation with ATOMS• Only discrete energy levels available for
transitions. Line spectra obtained
Molecular Spectroscopy
• Interaction of radiation with MOLECULES
• Multiple energy levels and transitions
200 nm 800 nm
E
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So although transitions still between discrete energy levels,the steps are much smaller
200 nm 800 nm
A
Actually made up of finer
transitions. For molecular
spectroscopy mainly absorption
Electronic Vibrational Rotational
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Electromagnetic Radiation – Light!
• Described in terms of both
particles and waves
• Wavelength (λ) is crest-to-crest
distance between waves
• Frequency (ν), the number ofcomplete oscillations that the
wave makes each second
νλ = c
• c is speed of light (2.998 × 108
ms-1 in vacuum)
λ
Magnetic Field
Electric Field
• With regards to energy it is convenient to think of light as particles called photons. Each photon has an energy E
• E = hν [h = 6.626 × 10-34 Js ( Planck’s constant )]
• E = hc/λ = hcν [ν (1/λ) is called the wavenumber (IR)]˜ ˜
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EXAMPLE – Photon Energies
By how many kilojoules per mole is the energy of O2 increased when it absorbs
ultraviolet radiation with a wavelength of 147 nm? How much is the energy of CO2
increased when it absorbs infrared radiation with a wavenumber of 2300 cm-1?
Solution – For UV radiation, the energy increase is:∆E = hν = h(c/λ)
= 6.626 × 10-34 J·s [(2.998 × 108 m/s) / (147 × 10-9 m)]
= 1.35 × 10-18 J/molecule
(1.35 × 10-18 J/molecule)(6.022 × 1023 molecules/mole) = 814 kJ/mol
That is enough energy to break the O=O bond in oxygen.
For CO2 the energy increase is:∆E = hν = h(c/λ) = hcν
= (6.626 × 10-34 J·s) (2.998 × 108 m/s) [(2300 cm-1) (100cm/m)]
= 4.6 × 10-20 J/molecule = 28 kJ/mol
Infrared absorption increases the amplitude of the vibrations of the CO2 bonds
˜
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Electromagnetic Spectrum
• Names of the regions are historical in nature. Visible light – thekind of electromagnetic radiation we see – represents only asmall part of the electromagnetic spectrum
400 500 600 700
Wavelength / nm
Visible
γ-rays X-rays Ultraviolet
Infrared Microwave
Radio
λ
E
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Absorption of light and what is colour?
E0
E*
E n e r g y
GreenPurple680-780Blue-GreenRed620-680
BlueOrange580-620
Violet-BlueYellow550-580
VioletYellow-Green520-550
PurpleGreen500-520
RedBlue-Green470-500
OrangeBlue440-470
YellowViolet-Blue420-440
Green-YellowViolet380-420
Colour observedColour absorbedWavelength ofmaximum
absorption (nm)
A b s o r b a n
c e
Wavelength / nm
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Relationship between Transmittance and Absorbance
P0 P
b
The diagram shows a beam of monochromatic radiation of radiant power P0,
directed at a sample solution. Absorption takes place and the beam of
radiation leaving the sample has radiant power P.
The amount of radiation absorbed may be measured
in a number of ways:
Transmittance: T = P / P0 (%T = 100 T)
Absorbance: A = log10 (P0 / P) = -log T
A = log10 1 / T
A = log10 100 / %TA = 2 - log10 %T
When no light is absorbed, P = P0 and A = 0. If 90% of the light is absorbed
10% is transmitted and P = P0/10. This ratio gives A =1. If only 1% of the light
is transmitted, A = 2.
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Beer’s Law
A =ε
bc
Relates concentration to absorbance and is the heart of analytical spectroscopy
Where A is absorbance which is dimensionless (though sometimes noted as
absorbance units (a.u.)
ε is the molar absorptivity with units of L mol-1
cm-1
b is the path length of the sample - that is, the path length of the cuvette in
which the sample is contained. Usually expressed in centimetres.
c is the concentration of the compound in solution, expressed in mol L-1
Molar absorptivity is the characteristic of a substance that tells how much
light is absorbed at a given wavelength
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Limitations of Beer’s Law
• Deviations in absorptivity coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in close
proximity
• Shifts in chemical equilibria as a function of concentration
• Changes in refractive index at high analyte concentration
• Fluorescence or phosphorescence of the sample
• Non-monochromatic radiation, deviations can be minimized by using
a relatively flat part of the absorption spectrum such as the maximum
of an absorption band
• Scattering of light due to particulates in the sample• Stray light
I t t ti
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Instrumentation
Light
source
Wavelength
selector Sample
Light
detector
Light source – Needs to cover region from 200-800 nm with sufficient power
and must be constant over the time taken to make the measurement. Twosources used; tungsten lamp and deuterium lamp.
Tungsten (Incandescent) Lamp
Measurement from 350 nm – NIR (2-3 µm)Contain coiled filament (tungsten), heated by electrical current to 2700K
Filament is contained in a glass (quartz) envelope.
The lamp is operated in a vacuum or with iodine gas in the envelope.
Deuterium Discharge Lamp
Operate at low pressure (0.2-0.5 torr) and at low voltage (40V)
Continuous discharge between cathode and anode
Strong continuum of radiation over UV region (160 – 360 nm) with fused
silica casing
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Emission profile of a tungsten filament and a deuterium arc lamp
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Wavelength Selectors
Beer’s Law - discreet wavelength of light is used in the measurement
FiltersAbsorption filters
Short pass filter Long pass filter
%T
λ
%T
λ
Transmit λ’s of light, 50-80%
efficient. Can’t scan so need to
change filters
Interference Filters
Bandwidth 10-20 nm
FWHM – width at half maximum (height)
Monochromators
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Monochromators
• Entrance slit that provides a narrow optical
image of the radiation source
• A collimator that renders the radiation
emanating from the entrance slit parallel• A grating or prism (dispersing element) for
dispersion of the incident radiation
• A second collimator re-forms the image of tentrance slit and focus it onto the exit slit
• The exit slit to isolate the desired spectral
band by blocking all of the dispersed radiati
except that in the desired range
A monochromator disperses light into its component wavelengths and selects a
narrow band of wavelengths to pass on to the sample or detector
Polychromatic radiation (radiation of more than one wavelength) enters the
monochromator through the entrance slit. The beam is collimated, and then strikes
he dispersing element at an angle. The beam is split into its component wavelengthsby the grating or prism. By moving the dispersing element or the exit slit, radiation
of only a particular wavelength leaves the monochromator through the exit slit.
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Dispersion Elements
Light from
source
Prism
Grating
A prism separates polychromatic
radiation into its component parts by a
process of refraction. Prisms are found in
older instruments.
A grating is a reflective or transmissive
optical component with a series of
closely spaced, parallel, ruled grooves.The grating is coated with Al to make it
reflective. A thin protective layer of
silica (SiO2) on top of the Al protects the
metal surface from oxidising, whichwould reduce its reflectivity.
Choosing the Monochromator Bandwidth
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Primary function of a monochromator is to provide a beam of radiant energy of a
given wavelength and spectral bandwidth.
Wide Slits
Large spectral bandwidth and
deviations from Beer’s Law
Narrow Slits
Low light throughput and
decreasing S/N ratio
Choosing the Monochromator Bandwidth
A secondary function is the adjustment
of the energy throughput – the light
emerging from the exit silt can bevaried by adjusting the slit width.
Cuvettes
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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 or plastics can be used for the manufacture of
cuvettes for use between 350 and 2000 nm.
REMEMBER: Keep the clear surfaces of the cuvette clean – fingerprints scatterand absorb light
Detectors
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Detectors
The photomultiplier tube (PMT) 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.
Photomultipliers are very sensitive to UV and visible radiation. They have fastresponse times. Intense light damages photomultipliers; they are limited to
measuring low power radiation.
Diagram of a photomultiplier
tube with 9 dynodes.
Amplification of the signaloccurs at each dynode, which
is approximately 90 volts
more positive than the
previous dynode.
A photon of radiation entering the tube strikes the cathode causing the emission of
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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 foreach 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 amplifiedand measured.
Photodiodes
Photodiodes are constructed from a layer of p-type silicon on a n-type silicon
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substrate, creating a p-n junction diode, which is overlaid with a protective a SiO2
layer. A reverse bias is applied, drawing electrons and holes away from the junction.
There is a depletion region at each junction, in which there are few electrons and
holes. The junction acts as a capacitor, with charge stored on either side of the
depletion region. At the start of each measurement the diode is fully charged. When
radiation hits the semiconductor, free electrons and holes are created and migrate to
regions of opposite charge, partially discharging the capacitor. The more radiationthat strikes the diode the less charge remains at the end of the measurement. The
state of the capacitor at the end of the measurement is determined by measuring the
current required to recharge the capacitor.
Linear photodiode arrays
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Linear photodiode arrays are an example of a multichannel photon detector.
These detectors are capable of measuring all elements of a beam of dispersed
radiation simultaneously (In contrast to conventional dispersive spectrometers,where only a narrow band of wavelengths reaches the detector at any time).
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 achip, 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 entire 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)
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g p ( )
CCDs are similar to diode array detectors, but instead of diodes, they consist of an
array of photocapacitors. They are extremely sensitive and are often used for
imaging.
CCD Operation - When light is absorbed in the p-doped region an electron is
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CCD Operation - When light is absorbed in the p-doped region, an electron is
introduced into the conduction band and a hole is left in the valence band. The
electron is attracted to the region beneath the positive electrode, where it is stored.
The hole migrates to the n-doped substrate where it combines with an electron.The stored charge is proportional to the number of incident photons, with a
capacity of ~105 electrons per electrode.
CCD readout - Data is read and digitized one pixel (picture element) at a time.After the desired observation time, electrons stored in each pixel of the top row are
moved into the serial register at the top, and then moved, one pixel at a time, to the
top right position, where the stored charge is read out. Then the next row is moved
up and read out, and the sequence is repeated until the entire array has been read.Binning: Pixels can be combined into “superpixels” trading spatial resolution for
dynamic range and quiet operation.
Complete Spectrophotometers
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Complete Spectrophotometers
• Single light path
• Must account for variations in detector response and source output for each λ
• Best when working with single λ and individual analytes
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Ph t di d A S t t
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Photodiode Array Spectrometer
• Photodiode array is able to measure a range of λ at once
• Typically have a trade-off between resolution and λ range
• Resolution of 1 nm is possible
• Entire spectrum can be measured in less than 1 second
Applications and correlation to str ct re
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Applications and correlation to structure
The absorption of UV or visible radiation corresponds to the excitation ofouter electrons. There are three types of electronic transition which can be
considered:
•Transitions involving π, σ, and n electrons
•Transitions involving charge-transfer electrons•Transitions involving d and f electrons
E0
E*
E n e r
g y
When an atom or molecule absorbsenergy, 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 topof each electronic level.
Absorbing species containing π σ and n electrons
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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 ofoverlapping lines. This appears as a continuous absorption band.
Possible electronic transitions of π, σ, and n electrons are
and non-bonding electrons
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σ π and non bonding electrons
Sigma- & pi-bondingelectrons are those found in
single & multiple bonds
respectively. Non-bonding
(n) electrons are lone-pairelectrons which do not take
part in bonding but are still
outer electrons which are
easily excited.
For each bonding orbital there is a corresponding antibonding orbital designatedwith a * e.g. sigma*. It is to this higher energy antibonding orbital that the
electron is promoted on absorption of UV-visible light. So, for example, an
electron is said to undergo a sigma-sigma* transition. Electrons may only be
promoted to antibonding orbitals of the same “type” i.e. pi-electrons may onlyundergo the transition pi-pi* and NOT pi-sigma*. Non-bonding electrons may be
promoted to EITHER antibonding orbital i.e. n-sigma* or n-pi*.
s → s* Transitions
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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 maximadue to s → s* transitions are not seen in typical UV-Vis. spectra (200 - 700 nm)
n → s* 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 → p* and p → p* Transitions
Most absorption spectroscopy of organic compounds is based on transitions of n or p
electrons to the p* 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 absorptivities from n → p* transitions are relatively low, and range from 10 to100 L
mol-1 cm-1 . p → p* transitions normally give molar absorptivities between 1000 and
10 000 L mol-1 cm-1 .
The solvent in which the absorbing species is dissolved also has an effect on the
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spectrum of the species. Peaks resulting from n → p* 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 notalways), the reverse (i.e. red shift) is seen for p → p* 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 isslightly reduced - resulting in a small red shift. This effect also influences n → p*
transitions but is overshadowed by the blue shift resulting from solvation of lone pairs.
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 absorptivities from charge-transfer absorption are large (greater than 10,000 L
mol-1 cm-1).
Absorption Involving d- and f-Electrons
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Many transition metal ion solutions are coloured as a result of their
incomplete d-levels, which allows promotion of an electron to an excited state by the absorption of relatively low energy visible light. The bands are broad &
strongly influenced by the chemical environment, because of the spatial shape
& orientation of the d-orbitals. Such bands are rarely intense enough to use
directly for quantitative analysis. The sensitivity of the analysis may be
greatly augmented by complexing the metal ion with some suitable organic
chelating agent to produce a charge-transfer complex. This type of complex
typically has very high molar absorptivity in excess of 10,000 L mol-1
cm-1
.There are numerous chelating agents available which may or may not
complex selectively where there is more than one type of metal ion present.
1,10-phenanthroline is a common chelate for the analysis of Fe(II).
Lanthanides & actinides have incomplete f-levels & give rise to absorption
bands in a similar fashion to transition metals. In contrast to transition metal
ion spectra, those of the lanthanides/actinides contain narrow, well-defined
bands which are little affected by ligands & the local chemical environment.
Analysis of a Two Component Mixture
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Analysis of a Two Component Mixture
λ1 λ2
A1
A2 If a mixture contains two species to be
analysed then there is a high
probability that the absorption bands
from each species will overlap. Thus,
the absorption at a particularwavelength will not be due to one
component alone.
An accurate quantitative analysis requires that the overlap is corrected for. It is
assumed that the measured absorbance at a wavelength (the peak maximum for
one of the components) is additive, i.e, A = A1 + A2, where A1 is the absorbance
due to species 1 etc. On applying Beer’s Law:
A = ε1c1 b + ε2c2 b
where A is measured and ε1, ε2 are obtained from appropriate calibration graphs.
This equation may not be solved because it contains two unknowns (the two
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y
concentrations). However, if we now make a measurement at the other
components peak maximum, then we will obtain a second equation:
A' = ε1' c1 b + ε2' c2 b
We thus have a pair of simultaneous equations which we can solve for the twounknown concentrations if we know the molar absorptivities of both
components at both wavelengths (need to carry out four calibrations).
This technique may be extended to three components (i.e. make measurementsat three wavelengths) but beyond this it becomes unreliable. Multicomponent
analysis requires complex pattern recognition software to analyse the spectra
(chemometrics).
Isosbestic Points
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Absorption spectrum of 3.7×10-4 M methyl red as a function of pH between pH 4.5 and 7.1
If one absorbing species, X, is converted to another absorbing species, Y, in a
chemical reaction, then the characteristic behaviour shown in the figure below isobserved. If the spectra of pure X and pure Y cross each other at any wavelength,
then every spectrum recorded during this chemical reaction will cross at the same
point, called an isosbestic point. The observation of an isosbestic point during a
chemical reaction is good evidence that only two principal species are present.