UV / Visible Spectroscopy

Presented By Nandakumar V.G Mphil student Dept.of Opto electronics. Kerala University,

Kariavattom .

Contents.

• Introduction• Electromagnetic Radiation• Basic Principle• Electronic Transitions• Instrumentation• Interpretations• Advantages and Disadvantages• Applications• Conclusion.

Introduction

• The history of spectroscopy began with Isaac Newton's optics experiments (1666–1672). Newton applied the word "spectrum" to describe the rainbow of colors that combine to form white light and that are revealed when the white light is passed through a prism. During the early 1800s, Joseph von made experimental advances with dispersive spectrometers that enabled spectroscopy to become a more precise and quantitative scientific technique. Since then, spectroscopy has played and continues to play a significant role in chemistry, physics and astronomy.

Spectroscopy

• It is the branch of science that deals with the study of interaction of matter with light.

OR• It is the branch of science that deals with the

study of interaction of electromagnetic radiation with matter.

Four common spectroscopic techniques use to determine structure:

• UV/Visible Spectroscopy

• Infrared Spectroscopy (IR)

• Nuclear Magnetic Resonance Spectroscopy (NMR)

• Mass Spectrometry (MS or Mass Spec)

Electromagnetic Radiation

Electromagnetic Radiation

• Electromagnetic radiation consist of discrete packages of energy which are called as photons.

• A photon consists of an oscillating electric field (E) & an oscillating magnetic field (M) which are perpendicular to each other.

Electromagnetic Radiation

• Frequency (ν):– It is defined as the number of times electrical field

radiation oscillates in one second.– The unit for frequency is Hertz (Hz).

1 Hz = 1 cycle per second

• Wavelength (λ):– It is the distance between two nearest parts of the

wave in the same phase i.e. distance between two nearest crest or troughs.

Electromagnetic Radiation

• The relationship between wavelength & frequency can be written as:

c = ν λ• As photon is subjected to energy, so

E = h ν = h c / λ

Electromagnetic Radiation

Electromagnetic Radiation

Violet 400 - 420 nm Yellow 570 - 585 nm

Indigo 420 - 440 nm Orange 585 - 620 nm

Blue 440 - 490 nm Red 620 - 780 nm

Green 490 - 570 nm

Principles of Spectroscopy

Principles of Spectroscopy

• The principle is based on the measurement of spectrum of a sample containing atoms / molecules.

• Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (ν) or wavelength (λ).

• Spectrometer is an instrument design to measure the spectrum of a compound.

Principles of Spectroscopy

1. Absorption Spectroscopy:• An analytical technique which concerns with

the measurement of absorption of electromagnetic radiation.

• e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15 μm)

Principles of Spectroscopy

2. Emission Spectroscopy:• An analytical technique in which emission

(of a particle or radiation) is dispersed according to some property of the emission & the amount of dispersion is measured.

• e.g. Mass Spectroscopy

Interaction of EMR with

Matter

Interaction of EMR with matter1. Electronic Energy Levels:• At room temperature the molecules are in the

lowest energy levels E0.

• When the molecules absorb UV-visible light from EMR, one of the outermost bond / lone pair electron is promoted to higher energy state such as E1, E2, …En, etc is called as electronic transition and the difference is as:∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)

∆E = 35 to 71 kcal/mole

Interaction of EMR with matter

2. Vibrational Energy Levels:• These are less energy level than electronic

energy levels.

• The spacing between energy levels are relatively small i.e. 0.01 to 10 kcal/mole.

• e.g. when IR radiation is absorbed, molecules are excited from one vibrational level to another or it vibrates with higher amplitude.

Interaction of EMR with matter

3. Rotational Energy Levels:• These energy levels are quantized & discrete.

• The spacing between energy levels are even smaller than vibrational energy levels.

∆Erotational < ∆Evibrational < ∆Eelectronic

Energy levels

Lambert’sLaw

Lambert’s Law• When a monochromatic radiation is passed

through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light.

• Let I be the intensity of incident radiation.x be the thickness of the solution.

Then

Lambert’s Law

Idx

dI

So, KIdx

dI

Integrate equation between limit I = Io at x = 0 and

I = I at x=l,We get,

KlI

I

0

ln

Lambert’s LawKl

I

I

0

log303.2

lK

I

I

303.2log

0

Where, AbsorbanceAI

I0log

EK

303.2

lEA .

Absorption coefficient

Lambert’s Law

Beer’sLaw

Beer’s Law• When a monochromatic radiation is passed

through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light as well as concentration of the solution.

• Let I be the intensity of incident radiation.x be the thickness of the solution. C be the concentration of the solution.

Then

Beer’s Law

ICdx

dI.

So, ICKdx

dI.'

Integrate equation between limit I = Io at x = 0 and

I = I at x=l,We get,

lCKI

I.'ln

0

Beer’s Law

lCKI

I..log303.2 0

lCK

I

I.

303.2log 0

Where, AbsorbanceAI

I0log

EK

303.2

lCEA ..

Molar extinction coefficient

Beer’s Law

Beer’s LawlCEA ..

0I

IT OR A

I

IT

0

loglog

From the equation it is seen that the absorbance which is also called as optical density (OD) of a solution in a container of fixed path length is directly proportional to the concentration of a solution.

PRINCIPLES OF UV - VISIBLE SPECTROSCOPY

Principle• The UV radiation region extends from 10 nm

to 400 nm and the visible radiation region extends from 400 nm to 800 nm.

Near UV Region: 200 nm to 400 nmFar UV Region: below 200 nm

• Far UV spectroscopy is studied under vacuum condition.

• The common solvent used for preparing sample to be analyzed is either ethyl alcohol or hexane.

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• In order to obtain a UV-VIS spectrum the sample is ideally irradiated with the electromagnetic radiation varied over a range of wavelength.

• A monochromatic radiation i.e., a radiation of a single wavelength is employed at a time.

• This process is called scanning. • The amount of the radiation absorbed at each wavelength is

measured and plotted against the wavelength to obtain the spectrum.

• Thus, a typical UV spectrum is a plot of wavelength or frequency versus the intensity of absorption.

CHARECTERISTICS OF SPECTRUM

04/18/2023 UV-VISIBLE SPECTROSCOPY 37

• The UV spectra of substances are characterised by two major parameters, namely, the position of the maximum of the absorption band called λmax, and the intensity of the bands.

• The λmax refers to the wavelength of the most absorbed radiation and is a measure of the difference in the electronic energy levels involved in the transition.

• The intensity on the other hand is indicative of the probability of the transition i.e., whether the transition is allowed or not.

• It is also is a measure of the concentration of the absorbing species.

CHARECTERISTICS OF SPECTRUM

04/18/2023 UV-VISIBLE SPECTROSCOPY 38

• The wavelength of the radiation absorbed by an organic molecule is determined by the difference in energy between the ground state and the various excited electronic states of the molecule.

• In organic molecules that the constituent atoms are bonded through σ and π bonds.

• In addition, these have nonbonding electrons on the atoms like, N,O,S and halogens etc.

• There are a number of transitions possible involving the bonding and the nonbonding electrons.

ORGANIC ABSORPTION

Electronic Transitions

The possible electronic transitions can graphically shown as:

• σ → σ* transition1

• π → π* transition2

• n → σ* transition3

• n → π* transition4• σ → π* transition5• π → σ* transition6

The possible electronic transitions are

• σ electron from orbital is excited to corresponding anti-bonding orbital σ*.

• The energy required is large for this transition.

• e.g. Methane (CH4) has C-H bond only and can undergo σ → σ* transition and shows absorbance maxima at 125 nm.

• σ → σ* transition1

• π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.

• Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π* transitions.

• e.g. Alkenes generally absorb in the region 170 to 205 nm.

• π → π* transition2

• Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens are capable of n → σ* transition.

• These transitions usually requires less energy than σ → σ* transitions.

• The number of organic functional groups with n → σ* peaks in UV region is small (150 – 250 nm).

• n → σ* transition3

• An electron from non-bonding orbital is promoted to anti-bonding π* orbital.

• Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions.

• n → π* transitions require minimum energy and show absorption at longer wavelength around 300 nm.

• n → π* transition4

•These electronic transitions are forbidden transitions & are only theoretically possible.

•Thus, n → π* & π → π* electronic transitions show absorption in region above 200 nm which is accessible to UV-visible spectrophotometer.

•The UV spectrum is of only a few broad of absorption.

• σ → π* transition5• π → σ* transition 6&

Components of spectrophotometer Source Monochromator Sample compartment Detector Recorder

INSTRUMENTATION

Block Diagram

RADIANT SOURCE

WAVELENGTHSELECTOR SOLVENT PHOTO-

DETECTORREADOUT

SAMPLE

Fig.-block diagram of instrumentation of UV-spectrophotometer

Tungsten filament lamps and Hydrogen-Deuterium lamps are

most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

Problem-• Due to evaporation of tungsten life period decreases.• It is overcome by using tungsten-halogen lamp.• Halogen gas prevents evaporation of tungsten.

RADIATION SOURCE

- Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism..

Monochromator

All Monochromators contain the following component parts;

• An entrance slit• A collimating lens• A dispersing device (a prism or a grating)• A focusing lens• An exit slit

Filters – a)Glass filters- Made from pieces of colored glass which transmit limited wave length range of spectrum. Wide band width 150nm.

b)Gelatin filters- Consist of mixture of dyes placed in gelatin & sandwiched between glass plates. Band width 25nm.

c)Inter ferometric filters- Band width 15nm

Prisms- -Prism bends the monochromatic light. -Amount of deviation depends on wavelength -They produce non linear dispersion.

A variety of sample cells available for UV region. The choice of sample cell is based ona) the path length, shape, sizeb) the transmission characteristics at the desired wavelengthc) the relative expense

• One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region. The thickness of the cell is generally 1 cm. cells may be rectangular in shape or cylindrical with flat ends.

Sample and reference cells-

Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.Three common types of detectors are used.

I. Barrier layer cell II. Photo cell detector III. Photomultiplier cells

DETECTORS

1. Barrier layer cells

It consist of flat Cu or Fe electrode on which semiconductor such as selenium is deposited. on the selenium a thin layer of silver or gold is sputtered over the surface.

2.

3. Photomultiplier

Photomultipliers have an internal amplification that gives them great sensitivity and a wide spectral range. Light causes emission of electrons from a photocathode which accelerate past a series of dynodes maintained at progressively increasing potentials.

Amplifier

• Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.

Recording Device

•

• Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

Over all arrangements

Terms used in UV / Visible Spectroscopy

ChromophoreThe part of a molecule responsible for imparting color, are called as chromospheres.

ORThe functional groups containing multiple bonds capable of absorbing radiations above 200 nm due to n → π* & π → π* transitions.

e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

ChromophoreTo interpretate UV – visible spectrum following points should be noted:

1. Non-conjugated alkenes show an intense absorption below 200 nm & are therefore inaccessible to UV spectrophotometer.

2. Non-conjugated carbonyl group compound give a weak absorption band in the 200 - 300 nm region.

Chromophoree.g. Acetone which has λmax = 279 nm

and that cyclohexane has λmax = 291 nm.

When double bonds are conjugated in a compound λmax is shifted to longer wavelength.e.g. 1,5 - hexadiene has λmax = 178 nm

2,4 - hexadiene has λmax = 227 nm

CH3

CCH3

OO

CH2

CH2CH3

CH3

Chromophore3. Conjugation of C=C and carbonyl group shifts

the λmax of both groups to longer wavelength.

e.g. Ethylene has λmax = 171 nm

Acetone has λmax = 279 nm

Crotonaldehyde has λmax = 290 nm

CH3

CCH3

O

CH2 CH2

CCH3

O

CH2

AuxochromeThe functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light , altering the wavelength or intensity of absorption.

ORThe functional group with non-bonding electrons that does not absorb radiation in near UV region but when attached to a chromophore alters the wavelength & intensity of absorption.

Auxochromee.g. Benzene λmax = 255 nm

Phenol λmax = 270 nm

Aniline λmax = 280 nm

OH

NH2

Absorption & Intensity Shifts

1 • Bathochromic Shift

(Red Shift)

2 • Hypsochromic Shift

(Blue Shift)

3 • Hyperchromic Effect

4 • Hypochromic Effect

• When absorption maxima (λmax) of a compound shifts to longer wavelength, it is known as bathochromic shift or red shift.

• The effect is due to presence of an auxochrome or by the change of solvent.

• e.g. An auxochrome group like –OH, -OCH3 causes absorption of compound at longer wavelength.

• Bathochromic Shift (Red Shift)1

• In alkaline medium, p-nitrophenol shows red shift. Because negatively charged oxygen delocalizes more effectively than the unshared pair of electron.

p-nitrophenolλmax = 255 nm λmax = 265 nm

• Bathochromic Shift (Red Shift)1

OH

N+ O

-O

OH-

Alkaline

mediumO

-

N+ O

-O

• When absorption maxima (λmax) of a compound shifts to shorter wavelength, it is known as hypsochromic shift or blue shift.

• The effect is due to presence of an group causes removal of conjugation or by the change of solvent.

• Hypsochromic Shift (Blue Shift)2

• Aniline shows blue shift in acidic medium, it loses conjugation.

Anilineλmax = 280 nm λmax = 265 nm

• Hypsochromic Shift (Blue Shift)2

NH2H

+

Acidic

medium

NH3+Cl

-

• When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift.

• If auxochrome introduces to the compound, the intensity of absorption increases.

Pyridine 2-methyl pyridine

λmax = 257 nm λmax = 260 nmε = 2750 ε = 3560

• Hyperchromic Effect3

N N CH3

• When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift.

Naphthalene 2-methyl naphthaleneε = 19000 ε = 10250

CH3

• Hypochromic Effect4

Wavelength ( λ )

Abso

rban

ce (

A )

Shifts and EffectsHyperchromic shift

Hypochromic shift

Redshift

Blueshift

λmax

Solution too concentrated Diluted five-fold

NH2

NO2

Solvent: Ethanol

Concentration: 15.4 mg L-1

Pathlength: 1 cm

UV-visible spectrum of 4-nitroanaline

How does a spectrophotometer work.mp4

UV Vis spectroscopy.mp4

Advantages and disadvantages

APPLICATIONS OF UV / VISIBLESPECTROSCOPY

Applications• Qualitative & Quantitative Analysis:

– It is used for characterizing aromatic compounds and conjugated olefins.

– It can be used to find out molar concentration of the solute under study.

• Detection of impurities:– It is one of the important method to detect impurities in organic

solvents.• Detection of isomers are possible.• Determination of molecular weight using Beer’s law. • Detection of unknown compound.

REFERENCES

Reference Books

• Introduction to Spectroscopy– Donald A. Pavia

• Elementary Organic Spectroscopy– Y. R. Sharma

• Physical Chemistry– Puri, Sharma & Pathaniya

Resources• http://www2.chemistry.msu.edu/faculty/reusch/Vir

tTxtJml/Spectrpy/UV-Vis/spectrum.htm

• http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy

• http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm

Thank You