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Spectroscopic Techniques

Absorption of light by molecules

What is light?

Lightis part of the electromagnetic spectrum, which ranges from radio waves to gamma rays.

Eelectromagnetic radiation from about 390 to 740 nm in wavelength is the visible light. It the

natural agent that stimulates sight and maes things visible.

Electromagnetic radiation is a form of energythat e!hibits wave"lie behavior as it travels

through space. It has both electric and magnetic fieldcomponents, which oscillate in phase

perpendicular to each other and perpendicular to the direction of energy propagation.

Electromagnetic radiation is classified according to the fre#uency of its wave. $he

electromagnetic spectrum, in order of increasing fre#uency and decreasing wavelength,consists

of radio waves, microwaves, infrared radiation, visible light, ultraviolet radiation, %"rays and

gamma rays

Internal energy of a molecule

& molecule possesses internal energy which may be subdivided into three classes.

" & molecule may be rotating about various a!es and possesses a rotational energy.

" &toms or group of atoms within the molecule may be vibrating, moving periodically with

respect to each other about their e#uilibrium positions, which shows the vibrational energy.

" & molecule possesses electronic energy. $his means a potential energy associated with the

distribution of negative charges 'electrons( about the positively charged nuclei of the atoms.

In a molecule, Eint ) Eelec*Eviv *Erot

& molecule can e!ist only in certain in certain +permitted energy state.

What happens when a molecule absorbs energy?

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y absorbing energy a molecule can be raised to a higher energy level. /or this, it absorbs

only a #uantity appropriate for the transition. 'It cannot absorb an arbitrary#uantity of energy

determined by us and cannot linger in an energy state intermediate its permitted levels(.

$he absorption of radiation can be from different regions 12, visible, I, microwave and

radiofre#uency.

owever, many substances only absorb the light photons in certain areas of the spectrum.

$he rotational energy levels of a molecule are #uietly closed spaced.

otational transitions re#uire relatively little energy and are produced by radiation of

very low fre#uency 'long wave length(. $his happens on far infrared region of spectrum.

5tudy in this region gives information on molecular structure. 2ibrational energy levels

are farther apart and they re#uire more energy photons to increase the vibrational energy.

&bsorption due to vibration is caused from 6000 to -0,000nm.

&bsorption of visible light and ultraviolet radiation ' ) -90"400nm and

400"800nm( can change the electronic energy levels 'increases the electronic energy( of a

molecule, i.e. the energy given by the photons help the electrons to overcome some of the

control of the nuclei and move out to new orbital of higher energy.

hen electrons present in the atoms of a molecule promoted from an

orbital of lower energy to one of higher energy, the atoms or ions absorbing the radiationare said to be changed from ground sate 'stable state( to an e!cited state.

:olecules tend not to remain in the e!cited state but rather to get rid of the

e!cess energy.

5ometimes energy is reemitted as radiation usually of longer wavelength than was

originally absorbed 'nown as fluorescence(.(luorescence is the emission of light by a substance that has absorbed light or other

electromagnetic radiation. It is a form of luminescence. In most cases, emitted light has a

longer wavelength, and therefore lower energy, than the absorbed radiation. owever,

when the absorbed electromagnetic radiation is intense, it is possible for one electronto

absorb twophotons;this two"photon absorptioncan lead to emission of radiation having

a shorter wavelength than the absorbed radiation. $he emitted radiation may also be of

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the same wavelength as the absorbed radiation, termed oined by different bonds electrovalent,

covalent bond, coordinate bond etc. In an atom, the electrons which are on the outer cell decide

the combining capacity 'valency of the element( and are called valence electrons. ithin the

molecule, atom may also have valence electrons in the form of a pair not evolved for the

formation of the bond.

In normal condition in a covalent binding there must be one sigma bond between the combining

atoms, In addition one or two ? bonds 'a single ? bond is weaer than the single sigma bond(.

In most compounds, when a radiation is passed, the valence electrons are more easily raised to

higher energy level than the electrons of inner cells. @f the valency electrons, those in single

bond 'A"electrons( as in, B"B or B" are the most difficult to e!icite, re#uiring radiation in the

far 12' much less than 600nm(. Electrons in non"bonding orbitals, the loan pairs, "@h, "Bl,

"C6 are easier to e!cites butstill re#uires radiation wavelength well below 600nm. ut the ?electrons of multiple bonds are much less tightly bound and re#uire energy corresponding to a

wavelength of about -8D nm to bring about transition.

5pectroscopic techni#ues of analysis are based on the measurement of the amount of radiation

absorbed, emitted or scattered by the sample. $hese methods use an instrument to measure the

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amount of radiation 'absorbed, emitted or scattered(. In this techni#ue, both absorption and

emission are perhaps the most widely used instrumental methods.

"ltra#iolet and #isible spectroscopic method

$% &asic information

efore dealing with the theoretical aspects of this method, the following points related to

electromagnetic spectrum are to be remembered

Electromagnetic radiationis a form of energythat e!hibits wave"lie behavior as it travels

through space 'e.g.all the different inds of energies released into space by stars such as the

5un(. It has both electric and magnetic field components, which oscillate in phase

perpendicular to each other and perpendicular to the direction of energy propagation.

Electromagnetic radiation is classified according to the fre#uency of its wave. $he

electromagnetic spectrum, in order of increasing fre#uency and decreasing wavelength,

consists of radio waves,microwaves,infrared radiation,visible light, ultraviolet radiation, %"

raysand gamma rays

$he figure shows the comparative wavelengths of three fre#uencies of visible light " ed, reen

and lue.

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$he electric field is in a vertical plane and the magnetic field in a horiFontal plane.

Gight is propagated in the form of transverse waves.

$hese waves may be described by wavelength, velocity and other parameter related to wavemotion.

ave length '( refers to the distance between two ad>acent crests 'troughs(

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ave number 'H; nu(

In spectroscopy, the wave number of electromagnetic radiationis defined as

H= c; where is thewavelength, H 'F=sec( is the fre#uency and c is the

velocity of the light 'at vacuum c is 3-0-0cm=sec(.

& beam of homogeneous radiation 'monochromatic( is characteriFed by its constant

wavelength '(.

/or wave length an angstrom and nanometer 'nm( are the commonly used units. '-

angstrom ) -.0 -0"-0meters(

&t wave lengths shorter than uv are !"ray, J"rays and cosmic rays, while beyond the I

are microwaves and radio"waves.

Guminous bodies such as sun or electric bulb emit a broad spectrum comprising many

wavelengths.

K

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2isible light forms a small part of the electromagnetic spectrum

e see ob>ects by means of either transmitted or reflected light.

& beam of light is a stream of particulate energy pacets called photons.

Each of these particles possesses a characteristic energy which is related to the fre#uencyof the light.

E ) h H, where h is LlanMs constant and H is fre#uency.

& molecule possesses internal energy which may be subdivided into three classes.

" & molecule may be rotating about various a!es and possesses a rotational energy.

" &toms or group of atoms within the molecule may be vibrating, moving periodically

with respect to each other about their e#uilibrium positions, which shows thevibrational energy.

" & molecule possesses electronic energy. $his means a potential energy associated

with the distribution of negative charges 'electrons( about the positively charged

nuclei of the atoms.

In a molecule, Eint ) Eelec*Eviv *Erot

& molecule can e!ist only in certain in certain +permitted energy state.

y absorbing energy a molecule can be raised to a higher energy level. /or

this, it absorbs only a #uantity appropriate for the transition. 'It cannot absorb an arbitrary

#uantity of energy determined by us and cannot linger in an energy state intermediate its

permitted levels(.

$he rotational energy levels of a molecule are #uietly closed spaced.

otational transitions re#uire relatively little energy and are produced by radiation of

very low fre#uency 'long wave length(. $his happens on far infrared region of spectrum.

5tudy in this region gives information on molecular structure. 2ibrational energy levels

are farther apart and they re#uire more energy photons to encrease the vibrational energy.

&bsorption due to vibration is caused from 6000 to -0,000nm.

&bsorption of visible light and ultraviolet radition increases the electronic

energy of a molecule, i.e. the energy given by the photons help the electrons to overcome

some of the control of the nuclei and move out to new orbitals of higher energy.

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:olecules tend not to remain in the e!cited state but rather to get rid of the

e!cess energy.

5ometimes energy is reemitted as radiation usually of longer wavelength than was

originally absorbed 'nown as fluorescence(.

(luorescence is the emission of light by a substance that has absorbed light or other

electromagnetic radiation. It is a form of luminescence. In most cases, emitted light has a

longer wavelength, and therefore lower energy, than the absorbed radiation. owever,

when the absorbed electromagnetic radiation is intense, it is possible for one electronto

absorb twophotons;this two"photon absorptioncan lead to emission of radiation having

a shorter wavelength than the absorbed radiation. $he emitted radiation may also be of

the same wavelength as the absorbed radiation, termed

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Light absorption

@f the light passing through a homogenous fluid 'li#uid, soln, vapours(, a portion of the incidentlight is reflected, some is absorbed within the medium, and the rest is transmitted.i.e. I0 ) Ir*Ia* It.

For air glass interface (in using glass cells) about 4% of the incident light is reflected. This Ir is

eliminated by the use of a comparison cell. In such condition

i.e. I0 ) Ia* It.

Theory of light absorption'

$he relationship that e!ists between the e!tent of light absorption and the #uantity of absorbing

material is e!plained by the eer"Gambert Gaw. It is the combination of two laws Gambert law

relates absorption to the length of the light path through the fluid. eerMs shows the effectproduced by the changes in concentration of the fluid. $he combined law may be written

&) log I0=I$) abc.

" here & is the absorbance 'optical density N or e!tinction E(

" I0 is the intensity of incident light

" I$is the intensity of the transmitted light

" c is the concentration of absorbing substance

" b is the length of light path 'length of absorbing solution(

" +a is the constant for the substance.

Lamberts Law

Gambert Gaw e!plains the relation between the changes in absorption of light and thicness of a

medium. It states that when monochromatic light passes through a transparent medium, the rate

of decrease in intensity with the thicness of medium is proportional to the intensity of the light.$he law may be e!pressed by the differential e#uation,

"dI=db )OI PPPPP.. 'i(, where I is the intensity of the incident light of wavelength '(, b is

the thicness of the medium, and O is the proportionality constant.

Integrating 'i(, Q " dI=db ) QOI

or, Q"dI=I ) QOdb

or, " ln I ) Ob * Ig, P.. 'ii(, where Ig is the integration constant9

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hen, b )0, I ) I0, 'ii( becomes,

" ln I0) O 0 * Ig

or, Ig ) " ln I0,putting this value on 'ii(,

"ln I ) Ob R lnI0

or, ln I ) lnI0"Ob PPPPPPP.. 'iii(

or, I ) I0 .e"b PPPPPPPPP.. 'iv(

/rom 'iii(, ln I=I0 ) " Ob PPPPP.'v(

hen the light reaches at the end of the thicness, the intensity at the thicness b can be written

as I$'i.e. I becomes I$(.

i.e. I$) I0e"Ob PPPPPPPPP'vi(

or, ln I0 =I$ ) Ob PPPPPPP 'vii(or, log I0 =I$ ) Ob =6.303

Absorbance and transmittance

In the above e#uation the #uantity lnI0=I$is called absorbance '&(.

i.e. & ) Ob =6.303 PPPPPP.'viii(

$hus, the above e#uations show that & is proportional to thicness 'b( of the length of the

solution absorbing the radiation.

$he older terms such as e!tinction, optical density and absorbency are now obsolete.

It is also evident that & is e#ual to the reciprocal of the logarithm of the transmittance.

&) -=$

i.e. & ) log I0=I$ ) Gog '-=$( ) "log $ )6"log 'S$( P.. 'i!(.

&eer Law

e have so far considered the light absorption and light transmission for monochromatic light as

a function of the absorbing layer only. In #uantitative analysis we are concerned with the

-0

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concentration of solution. eer studied the effect of concentration of a colored constituent in

solution upon the light transmission and absorption. e found that the intensity of a beam of

monochromatic light decreases e!ponentially as the concentration of the absorbing substances

increases arithmetically. $his may be written in the form,

I$ ) I0 e"OMc, where c is the concentration, and OM is the constant.

@r, ln I$ ) Gn I0R OMc

@r, ln I0= I$ ) OMc

@r, log I0 =I$ ) OMc=6.303

@r, & ) OMc=6.303 P.. '!(, where & is the absorbance of the solution

i.e. & is proportional to the concentration of the solution.

Bombining 'viii( and '!(, & ) log I0 =I$) abc PP '!i(, where +a is the new constant.

$his is the fundamental e#uation of colorimetry and spectrophotometry. It is nown as the eer"

GambertMs law or more recently eerMs law.

5pecific absorbance

$his is the absorbance of a specified concentration in a cell of specified length. $he most

common form in analytical chemistry is the '& -S-cm

(. $his is the absorbance of -g=-00mG

solution '-Sw=v( in a -cm cell. In this situation, $he eer"Gambert e#uation taes the form

& ) '& -S-cm (bc 'Sw=v(, where TbM is in cm and c is in g=-00ml

@r, & -S-cm ) &= b 'cm( c'Sw=v(.

5pecific e!tinction coefficient 'E -S-cm (

In the past & used to be described by E 'e!tinction(. If b is in cm and c is in S w=v, in e#uation &

) abc, +abecomes specific e!tension coefficient 'E -S-cm (.

i.e. 'E -S-cm ( ) E= Ub. c 'Sw=v(V.

$hus, & -S-cm andE -S-cm are same parameter.

:olar absorptivity 'molar e!tinction coefficient( or W

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In the e#uation, & ) abc, the value of +a depends on how the concentration is e!pressed. If c is

e!pressed in mol=lit and b in cm, the +aM is given as symbol +W and is called molar absorption

coefficient or molar absorptivity 'formerly molar e!tinction coefficient(.

$he molar absorptivity at a specified wavelength of a substance in solution is the absorbance at

that wavelength of a - mol=lit solution in a - cm cell 'light path(. $hus the units of +W are

-mol= lcm=lit

&gain, & ) abc

If TcM is e!pressed in moles=lit and TbM in cm,

&) W bc.@r, W ) & 'or E(= bc 'moles=lit(.

)elation between Specific absorbance (A$*

$cm orE$*

$cm and +olar absorpti#ity ,-

W ) '&-S-cm molecular weight(=-0

Instrumentation of spectrophotometer

5pectrophotometer is the instrument used for maing measurements of the monochromatic

radiation absorbed by a sample.

$he essential components of spectrophotometer are

'a( 5ource of electromagnetic radiation. It is a light source, which may be a hydrogen

discharge lamp for the range 600"340nm and a tungsten filament lamp emitting at wave

lengths from 300"7D0nm.

'b( Gight from the source pass to a monochromator'to give a particular wave length or

wave lengths(. :onochromator is a device for dispersing the light into a spectrum by means

of a prism.'c( $he monochromatic radiation then goes to the cells. $here are two cells for a sample

and solvent.

'd( &fter crossing the sample=solvent cells the radiation falls on a detector. $he detectors

'and associated electronics( measure the intensity of electromagnetic radiation. $he detector

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may be a photoelectric cell or a photomultiplier, coupled to a meter for recording the

response or displaying the response in a chart"paper.

Gight Gens 5lit :onochromator

5ample Netector Xuantitative &nalysis

5lits

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In double beam instrument, the beam of monochromatic radiation is split into two, one beam

being passed through the sample and the other through the reference cell. $he difference in

energy of the two emergent is then measured by an electronic device which records the

absorbance directly.

Some important points to remember

.lass strongly absorbs radiation below /01nm2 therefore2 prisms2 lens and cells

must be made of quart3 (a #ery hard mineral composed of silica2 Si4 5s found in

sandstone and granite etc%-

6ells are used in matched pairs' one containing the test substance in sol#ent (sample

sol%- and the other contains sol#ent alone (blan7 or reference cell-

Liquids used as sol#ent must be transparent down to the wa#elength of about

511nm%

" 6hloroform is opaque below about 508nm2 while carbon tetrachloride below

about 591nm%

" Some simple compounds li7e water2 saturated hydrocarbons2 alcohols2 and

ethers are transparent o#er 511:;81nm%

or I) the ma=- is used%

Application of colorimetric and spectrophotometric method$- etermination of a concentration by 6olorimetric method

In certain cases a compound or constituent can have a natural color or form a color with a

suitable reagent. $he absorbance due to the color can be used to find out the concentration of the

component. $his method is commonly termed as colorimetric analysis. In simple situation, the

intensity of the color of the constituent is compared with the intensity obtained by treating a

nown amount of the standard substance in the same manner.

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$he ratio i.e. &bsorbance for spl=&bsorbance for reference can be used to calculate the

concentration of the sample. /or this purpose the radiation of a specific wave length from the

visible range is used.

In classical instruments, sample solutions of a colored substance and its reference substance

'concentration c- and c6(are placed in an instrument that allows the thicness 'length( of the

solution to be altered and measured easily, and the instrument also allows a comparison of the

transmitted light. hen the two layers have the same color intensity 'i.e. when the system is

optically balanced(, then

b- c- ) b6 c6, where b- and b6are the lengths of the columns of the solutions 'sample and

reference( and c- and c6 are their respective concentrations. /rom the relation c- can be

determined.

5- etermination of the concentration by "@ Spectrophotometric method

hen using a spectrophotometer there are different possibilities for the determination of the

concentration of a sample solution.

-K