UV-VIS Spectroscopy

PINSET

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Introduction

Utilize the sample source of light in the UV or VIS region of spectrum

Ultraviolet (UV) and visible (VIS) spectroscopy

This is the earliest method of molecular spectroscopy.

A phenomenon of interaction of molecules with ultraviolet and visible lights.

Absorption of photon results in electronic transition of a molecule, and electrons are promoted from ground state to higher electronic states.

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Intro continued…

These energy absorbed will then be used to determine the presence of functional molecules in a sample.

Prediction of molecules in sample is then done via UV-VIS spectroscopy.

In structure determination : UV-VIS spectroscopy is used to detect the presence of chromophores like dienes, aromatics, polyenes, and conjugated ketones, etc.

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Chromophores

Term originated from Chromophorus (Greek origin) which means color carrier

Specific conjugated groups in the molecules of sample under analysis is the factor that is responsible for the absorption of specific i.eUV radiation in the compounds

Such groups responsible for specific absorption of radiation are chromophores

Initially it was used for groups imparting color Any functional group that absorbs

electromagnetic radiation

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Energy of molecules

There are different energies in a molecule that affect the nature of absorbing radiations.

Etotal=Eelec+Evib+Erot+Enucl

Eelec: electronic transitions (UV, X-ray)

Evib: vibrational transitions (Infrared)

Erot: rotational transitions (Microwave)

Enucl: nucleus spin (nuclear magnetic resonance) or (MRI: magnetic resonance imaging)

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Auxochromes

Groups that augment color imparting properties of chromophore in the same molecule

When present in combination with a chromophoreit Effects wavelength and intensity of absorption

Nitro benzene group imparts light yellow colour to compounds

If NH2 is attached at Para position to nitro it enhances its color

Chromophore?

Auxochrome?

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Terms describing UV

absorptions Bathochromic shift: shift to longer λ, also called red

shift. It is due to change in medium or Auxochrome.

Hypsochromic shift: shift to shorter λ, also called blue

shift. It might occur due to removal of electron pair

from conjugated specie.

Hyperchromism: increase absorption intensity in ε of a

band.

Hypochromism: decrease in absorption intensity or ε of

a band.

Graphical representation of shifts

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Interaction of light and sample

Scattering of light

Refraction at interfaces

Scatter in solution

Large molecules

Air bubbles

Normalized by comparison to reference cell

Contains only solvent

Measurement for transmittance is compared to results from reference cell

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Principle

Beer lamberts law

Is applicable for the clear solutions only with chemical deformation and reformations

Lamberts law is the relation between the total absorption of light and the path length through which the light traverse

Beers law is the relation between the absorption of light to the concentration of the absorbing medium

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Beer lambert’s law

log10 𝐼. 𝐼 = e.c.l

A = e.c.l

A = log10 𝐼. 𝐼

A= absorbance

I.= intensity of the incident light

I= intensity of the transmitted light

e= molecular extinction constant

c= concentration of solution in

mol/dm3

l= path length of sample in cm 11

Path length

/ cm 0 0.2 0.4 0.6 0.8 1.0

%T 100 50 25 12.5 6.25 3.125

Absorbance 0 0.3 0.6 0.9 1.2 1.5

External Standard and the

Calibration Curve

Spectroscopic instrument

Names of both

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Single and Double Beam

Spectrometer

Single-Beam: There is only one light beam or

optical path from the source through to the

detector.

Double-Beam: The light from the source, after

passing through the monochromator, is split

into two separate beams-one for the sample

and the other for the reference.

UV-VIS spectrophotometer

Instrumentation

Light Source

Deuterium Lamps－a truly continuous

spectrum in the ultraviolet region is produced

by electrical excitation of deuterium at low

pressure. (160nm~375nm)

Tungsten Filament Lamps－the most

common source of visible and near infrared

radiation. Wavelength more than 375nm.

Sample cells

Composition is silica Why silica? Transparency of material and passage of

light easy Glass cells are not used because absorbs

radiation above 300nm Path length 1cm 0.1cm and 10cm cells are also there Path length of reference an sample cells are

kept same in double beam spectrometer

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Monochromators

Used as a filter: the monochromator

will select a narrow portion of the

spectrum (the bandpass) of a given

source

Used in analysis: the monochromator

will sequentially select for the detector

to record the different components

(spectrum) of any source or sample

emitting light.22

Monochromator

Czerny-Turner design

Grating

Detector

Barrier Layer/Photovoltaic

Principle of Barrier

Layer/Photovoltaic Detector

This device measures the intensity of photons

by means of the voltage developed across the

semiconductor layer.

Electrons, ejected by photons from the

semiconductor, are collected by the silver layer.

The potential depends on the number of

photons hitting the detector.

Detector

Photomultiplier

Principle of Photomultiplier Detector The type is commonly used.

The detector consists of a photo-emissive

cathode coupled with a series of electron-

multiplying dynode stages, and usually called

a photomultiplier.

The primary electrons ejected from the photo-

cathode are accelerated by an electric field so

as to strike a small area on the first dynode.

Principle of Photomultiplier

Detector

The impinging electrons strike with enough

energy to eject two to five secondary electrons,

which are accelerated to the second dynode to

eject still more electrons.

A photomultiplier may have 9 to 16 stages,

and overall gain of 106~109 electrons per

incident photon.

Standard Addition Method

Standard addition must be used whenever the

matrix of a sample changes the analytical

sensitivity of the method.

In other words, the slope of the working

curve for standards made with distilled water

is different from the same working curve.

So standards could be used for concentration

determination of similar samples

Prepare the Standards

The concentration and volume of the stock solution

added should be chosen to increase the

concentration of the unknown by about 30% in

each succeeding flask.

Calculation of concentrations

You should now know the concentration making numerical

Via formula like C1V1=C2V2

What would be the concentration of chlorophyll pigment in sample solution of plant extract. If the standard was 1uM concentrated with chlorophyll in 1 mL.calculate sample concentration in 1uL.

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Limits to Beer’s Law

Chemical Deviations

－absorbing undergo association,

dissociation or reaction with the solvent

Instrumental Deviations

－non-monochromatic radiation

－stray light

Limits to Beer’s Law

Chemical Deviations

high concentration－particles too close

Average distance between ions and molecules

are diminished to the point.

Affect the charge distribution and extent of

absorption.

Cause deviations from linear relationship.

Limits to Beer’s Law

Chemical Deviations

chemical interactions－monomer-dimer

equilibria, metal complexation equilibria,

acid/base equilibria and solvent-analyte

association equilibria

The extent of such departure can be predicted

from molar absorptivities and equilibrium

constant. (see p561 ex 21-3)

Limits to Beer’s LawInstrumental Deviations

non-monochromatic radiation

Limits to Beer’s LawInstrumental Deviations

Stray light

(Po' + Po")Am = log --------------

(P' + P")

Applications of UV-VIS

Wavelength range is 180nm ~400 nm Has limited applications though used in

identification of species in a sample Differentiation for the conjugated and

unconjugated species. Extent of conjugation Detection of chromospheres or

chromophores in a compound Detection of functional groups in a compound Detection of geometric isomers

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Thanks

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