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UV-Vis Spectroscopy

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Molar Absorptivity

We have seen earlier that validation of Beer’s law is dependent on the nature of the molar absorptivity. It was found that the molar absorptivity is influenced by:

1. The wavelength of radiation

2. The refractive index and is thus indirectly related to concentration

3. Electrostatic interactions taking place in solution; and thus electronic distribution

4. In rare cases, like methylene blue, the molar absorptivity is directly dependent on concentration

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Molar absorptivities

e = 8.7 x 10 19 P A

–A: cross section of molecule in cm2

(~10-15)

–P: Probability of the electronic transition (0-1)

–P>0.1-1 allowable transitions

–P<0.01 forbidden transitions

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The molar absorptivity, however, is supposed to be constant for Beer’s law to be valid. The molar absorptivity is a measure of the ability of an analyte to absorb light at a specified wavelength. Therefore, the value of the molar absorptivity is crucial for the sensitivity of an analysis. The value of the molar absorptivity can usually range from zero (for a nonabsorbingspecies) to 105 (for highly absorbing species). For quantitative analysis, a value of at least 103

is necessary for a reasonable quantitative analytical determination of an analyte

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Molecules Containing p, s, and n Electrons

1. Molecules with s Bonds Only

All bonds in methane are s bonds and the only transition possible is the s-s* transition. However, the s-s* transition requires very high energy which occurs in vacuum UV. It is not wise to think of doing UV measurements on molecular species in the vacuum UV region (125-185 nm) for five important reasons:

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1. The high energy required can cause rupture of the s bonds and breakdown of the molecule

2. Air components absorb in vacuum UV which limits the application of the method

3. Working in vacuum UV requires special training and precautions which limit wide application of the method.

4. Special sources and detectors,, must be used

5. All solvents contain s bonds

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2. Molecules with n Electrons

Electrons in the valence shell that are not used up in chemical bonds are referred to as nonbonding electrons (n electrons). Consider a molecule like ammonia:

The line on nitrogen is a symbol for two nonbonding electrons. Now, the type of transitions observed in this molecule can be listed as:

a. s-s*

b. n-s*

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We have seen earlier that the s-s* transition is not useful in

practical UV-Vis spectroscopy but the other transition (n-s*)

is of lower energy . The absorption wavelength for a n-s*

transition occurs at about 185 nm where, unfortunately, most

solvents absorb. For example, the most important solvent is,

undoubtedly, water which has two pairs of nonbonding

electrons that will strongly absorb as a result of the n-s*

transitions; which is use of this transition for studies in

aqueous and other solvents with nonbonding electrons. In

summary, it is also impractical to think of using UV-Vis

absorption spectroscopy to determine analytes based on a

n-s* transition.

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3. Molecules with p Bonds

Absorption of radiation by an alkene, containing a double bond, can

result in s-s* or p-p* transitions. We have seen that a s-s* transition

is not useful but on the other hand, the p-p* turned out to be very

useful since it requires reasonable energy and has good absorptivity. A

molecule having s, p, and n electrons can show all types of transitions

possible in UV-Vis spectroscopy. For example, an aldehyde molecule

shows all these transitions since it contains s, p, and two pairs of n

electrons.

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Effect of Solvent Polarity on Absorption Wavelength

The molar absorptivity for a n-p* transition is rather small (10-100 L mol-1 cm-1) and the energy required for transition is affected by solvent polarity. In presence of a polar solvent, nonbonding electrons will interact with protic solvents to form hydrogen bonds. The solvation of n electrons is the result; which lowers the energy of the orbitals holding the n electrons. Partial stabilization of the polar p* is also observed but to a much lower extent than the n electrons.

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A net increase in energy required for a n-p* transition is thus

observed in protic solvents; like water or alcohols. Therefore,

an increase in energy will reflect a decrease in transition

wavelength, or what is called hypsochromic shift or blue shift.

On the other hand, a p-p* transition is affected in an opposite

manner with solvent polarity. In presence of a polar solvent, the

more polar p* orbital will be more stabilized than the p orbital

leading to a net decrease in the transition energy. This results in

an increase in transition wavelength or what is called a

bathochromic shift (red shift).

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Spectral nomenclature of shifts

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Optically clear solvent concept

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Conclusions on Electronic Transitions

There are four different types of electronic transitions which

can take place in molecules when they absorb UV-Vis

radiation. A s-s* and a n-s* are not useful for reasons

discussed earlier. The n-p* transition requires low energy but

the molar absorptivity is also low and transition energy will

increase in presence of polar solvents. The n-p* transition is

seldom used in quantitative UV-Vis spectroscopy.

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The most frequently used transition is the p-p* transition for the

following reasons:

a. The molar absorptivity for the p-p* transition is high allowing

sensitive determinations.

b.The energy required is moderate, far less than dissociation

energy.

c.In presence of the most convenient solvent (water), the energy

required for a p-p* transition is usually smaller.

Conclusions on Electronic Transitions

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Organic Chromophores

Molecules having unsaturated bonds or free nonbonding electrons that can absorb radiation of relatively low energy are called chromophores. Examples include alkenes, alkynes, ketones, aldehydes, phenyl and other aromatic species, etc.

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Examples of

UV-Visible Absorptions

LOW!

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UV-Visible Absorption Chromophores

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Typical UV Absorption Spectra

Chromophores?

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The effects of substitution

Auxochrome is a functional group that

does not absorb in UV region but

has the effect of shifting chromophore

peaks to longer wavelength as well

As increasing their intensity.

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Effect of Conjugation and Aromaticity of Chromophores

As conjugation is increased in a molecule, more delocalization

(stability) of the p electrons results. The effect of this

delocalization is to decrease the p* molecular orbital. The result

is a decrease in transition energy from p-p* and thus a red or

bathochromic shift. The molar absorptivity will increase in this

case and better quantitative analysis will be achieved.

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If greater then one single bond apart

- e are relatively additive (hyperchromic

shift)

- l constant

CH3CH2CH2CH=CH2 lmax= 184 emax = ~10,000

CH2=CHCH2CH2CH=CH2 lmax=185 emax = ~20,000

If conjugated

- shifts to higher l’s (red shift)

H2C=CHCH=CH2 lmax=217 emax = ~21,000

Rule of thumb for conjugation

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In cases of introduction of more unconjugated double bonds, the

molar absorptivity will increase as well depending on the

number of the double bonds. For example, at 185 nm,1-hexene

has a molar absorptivity of about 10,000 L mol-1 cm-1 but hexa-

1,4-diene has a molar absorptivity of twice as much as 1-

hexene. However, when the double bonds are conjugated as in

hexa-1,3-diene the molar absorptivity is about 21,000 L mol-1

cm-1.

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On the other hand, aromaticity results in extraordinarily high

degree of delocalization of electrons and thus stabilization

of the p*. If we assume a molar absorptivity of about

10,000 L mol-1 cm-1 for each double bond, we expect the

sum of the three double bonds in benzene to be just above

30,000 L mol-1 cm-1 (at 185 nm) but actually the value is

about 60,000 L mol-1 cm-1 due to increased delocalization

as a result of aromaticity. It is therefore advantageous to

use UV-Vis absorption spectroscopy for determination of

compounds having aromatic character.

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Absorption by Inorganic Groups

Inorganic groups containing double bonds absorb in the UV-Vis

region. The most transitions are a result of n-p* transitions as in

nitrate (313 nm), carbonate (217 nm), nitrite (280 and 360 nm) and

azide (230 nm)

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Absorption Involving d and f Orbitals

Many transition metals have colored solutions and are also colored

in the solid state. The transition metals have some of their d

orbitals empty where a d-d transition can occur. The d-d

transitions require excitation energy in the UV-Vis region. The

direct interaction of the d electrons with ligands around the

transition metal results in a spectrum of broad band nature. On

the other hand, inner transition elements show transitions by

absorption of UV-Vis radiation (f-f transitions).

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Since the electrons in the f orbitals are far inside the

metal orbitals and are screened by electrons in orbitals

of higher principal quantum numbers, f-f electronic

transitions will not be affected by the nature of ligands

or solvent around the inner transition metals. Therefore,

the spectra of inner transition metals have narrow

bands.

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The crystal field theory is usually used to explain splitting of the d orbital

energy so that a transition from a lower energy d orbital electron can be

excited to a higher energy d orbital. The theory will be described for a

transition metal with six ligands or molecules of water around it. An

octahedral arrangement of these ligands is most appropriate where ligands

will be located at the z axis and at the x and y axis (will repel electronic

cloud in the dz2 and dx2-y2 orbitals and thus will make these two orbitals to

have higher energies). The other four ligands will be arranged in between

axis (dxy, dxz, and dyz) which will increase their energies but to a lower

extent.

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Charge Transfer Absorption

When a ligand permanently donates an electron to a metal ion, a

charge transfer is said to take place. The net outcome of the

process is an oxidation reduction phenomenon occurring within

the complex. An example is the reaction of Fe3+ with thiocyanate

where the product is an excited species with neutral thiocyanate

and Fe3+.

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Applications of Absorbance

Measurement to Qualitative Analysis

As seen earlier, the broad band absorption spectra obtained in UV-Vis absorption spectroscopy is usually featureless and lacks details that can be used in qualitative analysis. Therefore, this technique is mainly a quantitative technique.

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Solvents

We have seen earlier that solvent polarity affects the absorptivity

of the analyte molecules; due to change in transition energies.

Usually, polar solvents are used when possible. However, polar

solvents like water or alcohol tend to liberate the fine spectral

details. Therefore, in cases where the fine spectral details are

really needed (as in qualitative analysis) a non polar solvent

like hexane should be used. In addition, the solvent must be

optically clear (does not absorb incident radiation), well

dissolve the sample, and chemically pure

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Quantitative Analysis

The basis for quantitative analysis in the UV-Vis relies on Beer’s law.

Several characteristics of quantitative measurements using UV-Vis

absorption spectroscopy can be rationalized:

1. Applicability to all types of analytes as far as they absorb in the UV-

Vis region.

2. Moderate sensitivities in the range from 10-4 to 10-6 with possibility

to extend this range under certain conditions

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3. The relative standard deviation occurs within 1-3% which

reflects good precision.

4. Easy to perform and convenient.

Can be used for quantitative analysis in liquid chromatographic

separations.

5. Non absorbing species can also be determined if they are

derivatized with an absorbing species as the case of metal

ions when complexed to ligands.