06523-83 20molecular 20spectroscopy 20uv

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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



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



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)]˜ ˜



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

˜



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



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



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.



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



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



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



Emission profile of a tungsten filament and a deuterium arc lamp



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



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.



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



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



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



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



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



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



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)



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



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



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



Ph t di d A S t t



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



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



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



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



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



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



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



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



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



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.

06523-83 20molecular 20spectroscopy 20uv

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