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UV/VIS Spectroscopy

Humera Naz

PHC 403

By

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Introduction to UV-Visible Spectroscopy

• It deals with the measurement of energy absorbed whenelectrons are promoted to higher energy levels.

• It provides information about the length and structure ofthe conjugated part of molecule.

The excitation process

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• A UV spectrum is a plot of wavelength (or frequency) oflight absorbed versus the absorption intensity (absorbanceor transmittance).

• The range of this absorption spectroscopy is 190 800 nm.

• The UV spectrum may be divided into three regions.

Far (or vacuum) ultraviolet (10-200 nm)Near or Quartz ultraviolet (200-380 nm) Visible region (380-780 nm)

Types of electronic transition

• The total energy of a molecule is the sum of Eelec, Evib, and Erot.

• Energy absorbed in the UV region produces changes in theelectronic energy of the molecule resulting from transitions ofvalence electrons in the molecule.

• Excitation of an electron from bonding or nonbondingmolecular orbital to antibonding orbital.

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Electronic energy levels and transitions

Unoccupiedlevels

Occupiedlevels

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Some possible transitions in different compounds

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Relative energies of the MO’s of ethene and 1,3-butadiene

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• * and * transitions:

High-energy

Accessible in vacuum UV (max <150 nm)

Not usually observed in UV-Vis spectroscopy

• n * transition:

Non-bonding electrons (lone pairs)

wavelength (max) is in the 150-250 nm region

• n * and * transitions:

most common transitions in UV-Vis spectroscopy

observed in compounds with lone pairs and multiple bonds

with max = 200-600 nm.

The UV Absorption process

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Principles of Absorption Spectroscopy

The measurement of light absorption by a solution of molecules isgoverned by the Beer-Lambert Law, which is written as follows:

A = log (I /I ) = b c

A = absorbance

I = intensity of light incident upon sample cell

I = intensity of light leaving sample cell

c = molar concentration of solute in moles per literb = length of sample cell in cm

= molar absorptivity (0 – 106)or molar extinction coefficient

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The Molar absorptivity of a compound is a constant that is

characteristic of the compound at a particular wavelength. The size

of the absorbing system and the probability that the electronic

transition will take place control the absorptivity. It ranges from 0 to

106. (value above 104 are termed high intensity absorption,

while value below 103 are low intensity absorption)

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UV-visible Spectrum

• Plot of absorbance versus wavelength.

• Spectra often displayed (after simple calculation) as aplot of either or log versus wavelength.

max = 230 nm log = 4.2

= 272 nm = 3.1

= 282 nm = 2.9

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Choice of solvents

• It should be transparent over the desired range ofwavelength.

• It should not contain conjugated system.

• Water, 95% ethanol and hexane are most commonly usedsolvent.

• Polarity of solvent

Non polar solvent clear spectrum (fine structure)Polar solvent fine structure

disappear

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Important Terms in UV spectroscopy

Chromophores

It is a group of atoms (part of molecule) that gives risean electronic absorption.

OR

The colored substances have their color due to the

presence of one

or more unsaturated groups responsible for electronic

absorption.

(C C, C C, C N, C N, C O, N N)

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• Auxochrome

Substituents that increase the intensity of the absorption,

and possibly the wavelength, are called auxochrome.

For e.g. CH3, OH, OR, X, and NH2 groups)

Auxochrome Unsubstituted

chromophore

max

(nm)

Substituted chromophore max

(nm)

CH3

OR

Cl

H2CCH CH CH2

H3CCH CHCOOH

H2CCH2

217

204

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H2CCH CH CHCH3

H3CC(OCH3) CHCOOH

H2CCH2 Cl

224

234

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Auxochromic effects

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• Bathochromic shift (red Shift)

• Shift of an absorption maximum to longer wavelength due to

substitution or solvent effect.

• transitions undergo bathochromic shift with a increase

in the polarity of the solvent or

When an auxochrome is attached to a C=C.

Benzene, max = 256 nm Aniline, max = 280 nm

• The bathochromic shift is progessive as the number of

alkyl group increases.

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• Hypsochromic shift (blue shift)

• A shift of absorption maximum to higher energy or shorter

wavelength.

• This is caused by the removal of conjugation or change in the

polarity of the solvent.

• n transitions undergo this shift with an increase in the

polarity of solvent.

Aniline, max = 280 nm

Anilinium ion, max = 254 nm

CH3H3C

O max = 279 nm in hexane,

max = 264.5 nm in water

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OR

When an auxochrome is attached to C=O where n electrons are available.

H

C

H

O

NH2

C

H

O

OH

C

H

O

Cl

C

H

O

max (nm) 295 205 205 235

• Hyperchromic effect

An increase in absorption intensity max

The introduction of an auxochrome usuallycauses hyperchromic effect. For examplebenzene shows B-band at 256 nm, max 200,whereas aniline shows B-band at 280 nm,max 1430.

E = 180 nm B = 250 nm

K = 200 nm

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• Hypochromic effect

• A decrease in absorption intensity max.

• This is caused by the introduction of a group which distorts the chromophore.

H3C

H3C

max = 252 nm

max = 19,000

max = 270 nm

max = 800

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The Effect of Conjugation

In the presence of conjugated double bonds, The electronic energylevels of a chromophore move closer together. As a result, the energyrequired to produce a transition from an occupied electronic energylevel to an unoccupied level decreases, and the wavelength of the lightabsorbed becomes longer.

CH3-(CH=CH)n-CH3 of dimethylpolyenes(A) n = 3; (B) n = 4; (C) n = 5

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The Effect of Conjugation on Alkenes

• Increase in conjugation decreases the energy required forelectronic excitation.

A comparison of the energy gap in a series of polyenes of increasing chain length

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• Auxochromes increase wavelength of absorption byextension of the length of conjugating system. Resonanceinteraction of lone pair with double bond(s) increase thelength of conjugated system.

• As a result of this interaction, the nonbonded electronsbecome part of the system of molecular orbitals.

• -OH, -OR, -X, or NH2 are the examples of auxochromicgroup.

C C B C C B

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Aromatic Compounds

Primary bands184 nm

Primary bands202 nm

Secondary bands255 nm

Substitution on benzene ring can cause bathochromic and hyperchromic shifts

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A. Substituents with Unshared Electrons:

Substituents that carry nonbonding electrons (n electrons)can cause shifts in the primary and secondary absorptionbands to longer wavelength (extended conjugation). Thenonbonding electrons can increase the length of the

system through resonance.

The more available these n electrons for interaction withthe e system of the aromatic ring, the greater will be theshift.

Presence of n electrons in these compounds gives thepossibility of n transitions.

Y Y+

Y+

Y+

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In compounds that are acids or bases, pH changes canhave very significant effects on the position of the primaryand secondary bands.

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B. Substituents Capable of -Conjugation:

Substituents that are themselves chromophores usuallycontain electrons, interaction of the benzene-ring electronsand the electrons of the substituents can produce a newelectrons transfer band.

C O

R

C O

R

C O

R

C O

R

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C. Electron-releasing and Electron-WithdrawingEffects:

e-Releasing or e-withdrawing substituents have differingeffects on the position of absorption maxima.

Any substituent, regardless of its influence on the electrondistribution elsewhere in the aromatic molecule, shift theprimary absorption band to longer wavelength.

e-Withdrawing gps have essentially no effect on theposition of the secondary absorption band unless, they arecapable of acting as chromophore.

e-Releasing gps increase both the wavelength and intensityof the secondary absorption band.

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D. Disubstituted Benzene Derivatives:

The effect of each two substituents to be considered.

For para-disubstituted benzene, two possibilities exist:

• Both groups are e-releasing or e-withdrawing(They exert effects similar to those observes with monosubstituted

benzenes)

• If one of the gps is e-releasing while the other is e-withdrawing

(The magnitude of the shift of the primary band is greater than the shifts due to the individual gp, the enhanced shifting is due to resonance interactions)

H2N N

O

O

H2N N

O

O

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If the two groups of a disubstituted benzene derivativeare either ortho or meta to each other, the magnitude ofthe observed shift is approximately equal to the sum of theshifts caused by the individual group.

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Added conjugation of benzene

The added conjugation in naphthalene, anthracene

and tetracene -> bathochromic shifts of absorptionbands.

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UV spectra of some representative drug molecules

Steroid enones:

• Steroids usually have absorbance maxima at around 240 nm.• The extra double bond in betamethasone as compared with

hydrocortisone does not make a great difference to the max sinceit does extend the original chromophore linearly.

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Ephedrine: The benzoid chromophore

Ephedrine has the simplest type of benzene ring chromophore.

Its spectrum is similar to benzene spectrum.

There are no polar groups involved in the chromophore so that its fine structure is preserved.

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Ketoprofen: extended benzene chromophore

The benzoid chromophore has been extended by four double bonds

and thus the symmetry of the benzene ring has been altered.

The strong absorbance band present in benzene at 204 nm hasundergo bathochromic shift and giving max for ketoprofen at 262nm.

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Procaine: amino group auxochrome

The benzene chromophore has been extended by addition of a C=O

group.

The molecule also contains an auxochrome in the form of aminogroup.

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Phenylephrine: hydroxyl group auxochrome

The chromophore of phenylephrine is not extended but its structure

includes a phenolic hydroxyl group.

The phenolic group function as an auxochrome under acidic andalkaline condition.

UV Spectrum of phenylephrine under acidic and basic condition

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Application of UV / visible spectroscopy in pharmaceutical quantitative analysis

• Pharmacopoeial methods rely heavily on simple analysis byUV/visible spectrophotometry to determine activeingredients in formulations. These methods are usuallybased on the use of a standard A (1%, 1 cm) value for theactive ingredient being assayed. Such method alsopresume that there is no interference from excipients(preservatives, colorants, etc.) present in formulation andthat the sample is free of suspended matter, which willcause light scattering.

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The ultraviolet spectrum of benzonitrile shows a primary absorptionband at 224 nm and a secondary band at 271 nm.

a. If a solution of benzonitrile in water, with a concentration of 1 x 10-4 M,is examined at a wavelength of 224 nm, the absorbance is determinedto be 1.30. The cell length is 1 cm. What is the molar absorptivity ofthis absorption band?

b. If the same solution is examined at 271 nm, what will be theabsorbance reading ( = 1000)? What will be the intensity ratio, I0/I ?

Ans. = 13000I0/I = 1.26

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Recommended Book:

• Introduction to Spectroscopy, 4th Edition 2001, Pavia, D. L.,Lampman, G. M., Kriz, G. S. & Vyvyan, J. R., Brooks/ColeCengage Learning.