Instrumental Methods of Analysis (Vi)

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UV-Visible Molecular

Absorption Spectrometry



Introduction and Background

• Involves absorption of ultraviolet,visible, or infrared radiation forquantitative purposes.

• Most common analytical technique inthe analytical laboratory

• Absorption commonly occurs with many– Organic molecules

– Metals

– Metal-organic complexes



Questions

• What is nature of light?

• Are there different types of light?

• How does light propagate?



What is Light?

• Light is a form of energy

• Light travels through space at extremely

high velocities

– The speed of light (c) ~ 3 x 1010 cm/sec or

186,000 miles per second

• Light is classified as electromagneticradiation (EMR)



Characteristics of Light

• Light behaves like a wave.– That is, it can be modeled or characterized with

wave like properties.

• Light also behaves like a particle.

• Today, we envision light as a self-contained packet of energy, a photon,which has both wave and particle likeproperties.



The Electromagnetic Spectrum





The Electromagnetic Spectrum



The EMR

SpectrumDifferent portions of the EMR spectrum anddifferent types of spectroscopy involvedifferent parts(quantum states) of theatom



EMR Wave Characteristics• Wavelength (λ ) is the distance from one wave crest to the

next.• Amplitude is the vertical distance from the midline of a wave

to the peak or trough.

• Frequency (v) is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s)



Wave Properties of Electromagnetic Radiation

• EMR has both electric (E) and magnetic (H) components that

propagate at right angles to each other.



Particle Properties of EMR

• The energy of a photon depends on

its frequency (v )

Ephoton = hv

h = Planck’s constant

h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js



V = Wave Number (cm

-1

)λ = Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

υ = Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Erg× sec)

V =C

E = h = hC C

=λ

υ C =

υ λ

Electromagnetic Radiation





How Light Interacts with Matter.

• An electron will interact with a photon.

• An electron that absorbs a photon will gain energy.

• An electron that loses energy must emit a

photon.• The total energy (electron plus photon) remains

constant during this process.




• Electrons bound to

atoms have discrete

energies (i.e. not all

energies are allowed).• Thus, only photons of

certain energy can

interact with the

electrons in a given

atom.




• Consider hydrogen, thesimplest atom.

• Hydrogen has a specific linespectrum.

• Each atom has its own specificline spectrum (atomicfingerprint).



Unique Atomic Signatures

Each atom has a specific set of energy levels, and thus a

unique set of photon wavelengths with which it can interact.



Molecular Absorption

• More complex than atomic absorption because many more potential transitions

exist – Electronic energy levels

– Vibrational energy levels

– Rotational energy levels

• Emolecule

= Eelectronic

+ Evibrational

+ Erotational

– Eelectronic

> Evibrational

> Erotational

• Result - complex spectra



Energy Level Diagram forMolecular Absorption



Molecular Absorption Spectraof Benzene in the Gas Phase

Electronic Transition

Vibrational Transition

Superimposed on the

Electronic Transition

Absorption Band –

A series of closelyshaped peaks



MolecularAbsorption

Spectra in theSolution Phase

• In solvents therotational andvibrational transitionsare highly restricted

resulting in broad band absorption spectra





Emission of EMR

• We distinguish several types of emission1. Atomic

2. X-Ray

3. Fluorescence

Involves molecules

Resonance and non-resonance modes

1. Phosphorescence

• Non-radiative relaxation• Similar to fluorescence only relaxation times areslower than fluorescence

• Involves metastable intermediates



Energy Level Diagrams of Excitation and Emission



Absorption byOrganic Compounds

Many common organic

compounds absorb in

the UV region



Absorption byInorganic Species

Many free metals and

inorganic metal

complexes absorb inthe visible region of

the spectrum



A few metal chlorides,

which fluoresce strongly in

the visible wavelengths,

are the basis for almost

all the colors in modern fireworks.

Barium chloride produces green;

strontium chloride produces red;

copper chloride produces blue



Absorption by ChargeTransfer Complexes

• Many inorganic and organiccomplexes form chargetransfer complexes

• A charge transfer complexconsists of an electron donorgroup bonded to an electronacceptor group

• Charge transfer complexesexhibit broad band

absorption in the visibleregion of the EMR spectrum

• Useful analytically because of the large molar absorption



Charge-Transfer Complex



Charge-Transfer Complex

•Nitrite can bedetermined analyticallyby adding reagents toform a colored chargetransfer complex.

• The complex exhibitsbroad band absorptionin the visible region of the EMR spectrum

• The wavelength of maximum absorption(λmax) can be determinedwith a wavelength scan

• Measurements are thenmade at λ

max

λλmaxmax



Choice of Solvents• Most absorption measurements are

conducted by dissolving the analyte in asolvent

• The solvent (and sample holder) shouldbe transparent in the region of thespectrum where the analyte absorbs



Single Beam Instruments



Double-Beam Instruments

• A double beam instrument is one inwhich the light source can be passed(simultaneously) through both areference and a sample cell

• Purpose and Approach

1. Adjust light output of the instrumentto 100% transmission (0 %absorbance)

2. Allows correction of the sampleabsorbance signal for non-analyteabsorbance





Dispersion of Polymagnetic Light with a Prism

Polychromatic

Ray

Infrared

Red

Orange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic

Ray

SLIT

PRISM

Polychromatic Ray Monochromatic Ray

Prism - Spray out the spectrum and choose the certain wavelength(λ ) that you want by slit.




• Example component layout for a double beam instrument

• Light beam is split using a “chopper”



Double Beam Instruments

Mechanical Chopper

• The rotating disk blocks

the transmission of light

(% T = 0)

• Putting a mirror on the

face of the rotating disk

will re-direct the light to

an alternate path




Used to provide areference and sample

path for the source

light

Used to correct for

non-analyte

absorption signals :

-Reflected or stray

light-Analyte in the

reagents

-Absorption by

reagents



Example of UV-Visible Instrument



Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is

dependent upon the electronic structure of the molecule.

So the ultraviolet spectrum is called electronic spectrum.



Electronic Excitation

The absorption of light energy by organic compounds in the

visible and ultraviolet region involves the promotion of electrons in σ , π , and n-orbitals from the ground state to

higher energy states. This is also called energy transition. These

higher energy states are molecular orbitals called antibonding.



E n e r g y

σ*

π*

n

π

σ

σ → σ *

π → π *

n

→ σ *

n

→ π *

Antibonding

Antibonding

Nonbonding

Bonding

Bonding



Electronic Molecular Energy Levels

The higher energy transitions (σ →σ *) occur a shorter

wavelength and the low energy transitions (π →π *, n→π *) occur at longer wavelength.





Chromophoric Structure

Group Structure nmCarbonyl > C = O 280

Azo -N = N- 262

Nitro -N=O 270

Thioketone -C =S 330

Nitrite -NO2 230

Conjugated Diene -C=C-C=C- 233

Conjugated Triene -C=C-C=C-C=C- 268

Conjugated Tetraene -C=C-C=C-C=C-C=C- 315

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Instrumental Methods of Analysis (Vi)