Assignment.

Topic: Ultraviolet-Visible spectroscopy

Course: Advanced Spectroscopy

Submitted by: Anisa Batool

Submitted to:Dr. Mehdi Hassan

Level: MS-Chemistry

Date: 2-6-2022
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Contents:
Ultraviolet-Visible spectroscopy OR Electronic Spectroscopy.
Section#1:

Introduction.

Section#2:

Theoretical

Aspects.

A) Principle.

B) Absorption Laws, Beer’s and Lambert’s Laws.

c) Types of

transitions.

Section#3:

Instrumentation.

Section#4 Sample

Preparation.
Section#5 Interpretation of Spectra.
A) Presentation of data.

B) Different terms

used in UV

Section#6:

References
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Ultraviolet-Visible spectroscopy OR Electronic Spectroscopy:

1- Introduction: An analytical technique in which electromagnetic radiation of ranges UV/visible is
used for the detection of different types of electrons present in a molecule is known as ultraviolet-visible
(UV/visible) spectroscopy. This technique is also known as electronic spectroscopy due to the
involvement of excitation of electrons from ground state to excited state.

The electronic spectroscopy provide information about the different types of electron such as sigma, pi
and non-bonding electrons because the energy required for excitation of different type of electrons is
quantized.

Ranges of UV/Visible: The photons which have energy between 10-400nm are ultraviolet and
photons of energy ranges from 400-750nm are visible. The ultraviolet region is sub-divided into near
ultraviolet (quartz) and far ultraviolet (vacuum ultraviolet) regions. The near UV extends from 220-400
nm while vacuum UV extends from 10-200nm.

In UV/visible spectroscopy we normally use the photons of 200-750nm because the photons of vacuum
ultraviolet are more energetic and excite the electrons of atmospheric oxygen and nitrogen. The
excitation of electrons  fall in this region and we are compelled to use evacuated
spectrophotometer instead of ordinary spectrophotometer.

2- Theoritical Aspects:
Principle of UV/Visible spectroscopy: In ultraviolet and visible spectroscopy, the transitions that
result in the absorption of electromagnetic radiation in this region of the spectrum are transitions
between electronic energy levels. As a molecule absorbs energy, an electron is promoted from an
occupied orbital to an unoccupied orbital of greater potential energy. Generally, the most probable
transition is from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular
orbital (LUMO) or Lower energy molecular orbitals (LMOs) to higher energy molecular orbitals
(HMOs).In addition to these two we can also say that electrons are excited from bonding molecular
orbitals (BMOs) to antibonding molecular orbitals (ABMOs).This process is shown in fig.1. The energy
differences between electronic levels in most molecules vary from 125 to 650 kJ/mole (kilojoules per
mole).

Fig.1

Mostly, the highest occupied molecular orbitals (HOMO) are the sigma orbitals, which correspond to
sigma bonds. The pi orbitals lie at somewhat higher energy levels, and orbitals that hold unshared pairs,
the nonbonding (n) orbitals, lie at even higher energies. The lowest unoccupied molecular orbitals
(LUMO), or antibonding orbitals ( * and  *), are the orbitals of highest energy. This is shown in fig.2
given below.
 4

Fig.2
NOTE: There are many restrictions for the transitions called selection rules and many transitions are not
allowed which are known as forbidden transitions which will explain later.
Absorptions Laws: There are two laws which govern the absorption of light by the molecules. These
are:
(1) Lambert’s Law.
(2) Beer’s Law.
(1) Lambert’s Law: It states that,
“When a beam of monochromatic radiations passes through a homogenous absorbing medium,
the rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to the
intensity of the incident radiation.”
Mathematically the law is expressed as,
𝑑𝐼
− ∝𝐼
𝑑𝑥

−dI
= kI (1)
dx
Where I= Intensity of radiation after passing through a thickness x of the medium.

dI= Infinitesimally small decrease in the intensity of radiation on passing through infinitesimally
small thickness dx of the medium.
−𝑑𝐼
= Rate of decrease of intensity of radiation with thickness of the absorbing medium.
𝑑𝑥
K = Proportionality constant or absorption co-efficient. Its value depends upon the nature of the
absorbing medium.
Let I0 be the intensity of radiation before entering the absorbing medium (x=0). Then the intensity of
radiation after passing through any thickness, say x of the medium can be calculated as,
𝐼 𝑥=𝑥
𝑑𝐼
∫ = −𝑘 ∫
𝐼 𝑑𝑥
𝐼0
𝑥=0
ln I
I  k x x
Io o

ln I  ln I 0  k(x  0)
I
ln
I  kx
0

Taking anti-log on both sides of the above equation.

I kx
e
I0

I  I e kx
0
(2)

Equation (2) is known as Lambert’s equation.

(2) Beer’s Law: This law can be stated as,

“When a beam of monochromatic radiation is passed through a solution of an absorbing substance, the
rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the
intensity of incident radiation as well as the concentration of the solutions.”
 5

Mathematically, this law can be stated as,

−𝑑𝐼
∝ 𝐼𝑐
𝑑𝑥
𝑑𝐼
= −𝑘𝐼𝑐
𝑑𝑥
Where c= Concentration of solution in moles per liter.

K=Molar absorption co-efficient and its value depends upon the nature of the absorbing
substance.

Suppose I0 be the intensity of the radiation before entering the absorbing solution (when x=0), then the
intensity of radiation, I after passing through the thickness x of the medium can be calculated,
I
dI
 x x

I0
I
 k c  dx
x0

I
ln I
I0  k c xox

ln I  ln I 0  k c(x  o)
I
ln
I  k cx
0

Taking anti-log on both sides.

I k cx
e
I0
(1)

The equation (1) is known as Beer’s equation.

Beer-Lambert Equation: The mathematical statements of Beers and Lamberts law are given below,

 dI
dx  I (Lamberts law)

 dI
dx
c (Beers law)

Combining the above two laws,

 dI
dx  cI
dI
  kcI
dx
(A)

Equation (A) is known as Beer-Lambert equation.

But, Now suppose the I0 is the intensity of radiation when the thickness of the solution x=0 and I is the
intensity of radiation when the thickness is x=x.

Now integrate the above equation, by applying boundary conditions.

I x
dI


I0
I
 kc dx
o

I
ln I  kx x ox
I0

ln I  ln I 0  kc(x  o)
ln I
 kcx
I0
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Taking anti-log on both sides.

I kcx
I0 e
(B)

In form of common logarithm.

I 1
log  kcx
I 2.303
0

𝑘
=
2.303
Where  = 𝑀𝑜𝑙𝑎𝑟 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜 − 𝑒𝑓𝑓𝑖𝑐𝑒𝑛𝑡 or Molar absorption permittivity.

I
log
I  cx..........(C)
0

Equation (C) is the combined form of Beer-lambert law.

THE ORIGIN OF UV BAND STRUCTURE:

For an atom that absorbs in the ultraviolet, the absorption spectrum sometimes consists of very sharp
lines, as would be expected for a quantized process occurring between two discrete energy levels. For
molecules, however, the UV absorption usually occurs over a wide range of wavelengths because
molecules (as opposed to atoms) normally have many excited modes of vibration and rotation at room
temperature. In fact, the vibration of molecules cannot be completely “frozen out” even at very low
temperatures. Consequently, a collection of molecules generally has its members in many states of
vibrational and rotational excitation. The energy levels for these states are quite closely spaced,
corresponding to energy differences considerably smaller than those of electronic levels. The rotational
and vibrational levels are thus “superimposed” on the electronic levels. A molecule may therefore
undergo electronic and vibrational–rotational excitation simultaneously, as shown in Figure 3. Because
there are so many possible transitions, each differing from the others by only a slight amount, each
electronic transition consists of a vast number of lines spaced so closely that the spectrophotometer
cannot resolve them. Rather, the instrument traces an “envelope” over the entire pattern. What is
observed from these types of combined transitions is that the UV spectrum of a molecule usually
consists of a broad band of absorption centered near the wavelength of the major transition.

Figure 3
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Types of Electronic Transitions: The electrons in a molecule can be classified as sigma (), pi ()
and non-bonding (n) electrons. On the basis of these types of electrons following types of transitions are
possible:

a) 

b) n

c) →

d) n→
The energy required for various transitions obey the following order:

> n> →> n→

a)  Transition: This transition can occur in compounds in which all the electrons are involved in
the formation of single bonds and there is no lone pair of electrons. Example of such transitions are
saturated hydrocarbons like methane, ethane etc.

Such transition requires radiation of very short wavelength lesser than 150nm (vacuum UV region) and
evacuated spectrophotometer is needed. Overall this region is less informative.

Methane (CH4) which contains only C-H, -bond can undergo  transition exhibiting absorption
peak at 125nm. Ethane has absorption peak at 135nm which also must arise from the same type of
transition but here electrons of C-C bond appear to be involved. Since the strength of the C-C bond is
less than that of C-H bond, less energy is required for excitation, as a result absorption occurs at higher
wavelength. Thus organic molecules in which all the valence shell electrons are involved in the
formation of  bonds do not show absorption in the normal UV region i.e. 200nm to 400nm.

b) n Transition: this type of transition occurs in saturated compounds containing one hetro atom
with unshared pair of electrons. Example of such transitions are saturated alkyl halides, alcohols, ethers,
amines, aldehydes, ketones.

Such transition is brought about by radiation in the region of 150nm to 250nm. The absorption data for
some typical compounds involving n transition are give below.

Compounds max(nm) max

Water 167 1480

CH3OH 174 150
CH3OCH3 184 2520
(CH3)3N 227 900

The molar absorptivities for this type of absorption are intermediate in magnitude and usually in the
range 100-3000 mol-1.lit.cm-1.

In saturated alkyl halides, the energy required for such a transition decreases with increase in the size of
halogen atom (or decrease in the electronegativity of the atom). For example, “n” electrons on chloride
atoms are comparatively difficult to excite. The absorption for the maximum for methyl chloride is 172-
175nm whereas that for methyl iodide is 285nm as n electrons on iodide atom are loosely bound
because of its increase in size and lesser electronegativity. Max is also higher compared to methyl
chloride. Similarly amines absorb at higher wavelength as compared to alcohol and hence the excitation
co-efficient for amines will be larger. Thus, when absorption measurement are made in UV-region
compound such as aliphatic alcohol and alkyl halides are commonly used as solvent, because, they start
to absorb at 260nm. However, these solvent cannot be used when the measurement are to be made in
200-260nm. In such cases saturated hydrocarbons which only give rise to  transition must be
used. However, the drawback is that these are poor solvating agent. H-bonding shifts the UV
absorptions to shorter wavelength.
 8

c) → Transition: This type of transition occurs in the un-saturated centers of the molecule, i.e.in
compounds c containing double or triple bonds and also in aromatics.

The excitation of pi electrons requires smaller energy and hence the transition of this type occurs at
longer wavelength. A  electron of a double bond is excited to  orbital. For example, alkenes, alkynes,
carbonyl compounds, cyanides, azo compounds etc. show  transition.

Consider and alkene:

This transition requires still lesser energy as compared to n transition and therefore, absorption
occurs at longer wavelengths. Absorption usually occurs within the region of ordinary ultraviolet
spectrophotometer. In un-conjugated alkenes, absorption bands appear around 170-190 m. In
carbonyl compounds, the bond due to  transition appears around 180um and is most intense, i.e.
the value excitation co-efficient is high.

The introduction of alkyl group to olefinic linkage produces a bathochromic shift of the order of 3 to5m 
per alky group. The shift depends upon the type of the alkyl group and stereochemistry about the
double bond.

d) n→ Transition: In this type of transition, an electron of un-shared electron pair on hetro atom
gets excited to  anti-bonding orbital. This type of transition is requires least amount of energy out of
all the transitions and hence occurs. Saturated aldehydes show both the type of transitions which are
low energy n and multiple bonds double and triple bonds containing heron atom.

. Forbidden transitions: All the possible transitions are not observed, there are few restrictions called
selection rules. One important selection rule states that transitions that involve a change in the spin
quantum number of an electron during the transition are not allowed to take place; they are called
“forbidden” transitions. Additionally  and  transitions do not take place because these are
“LAPORTE FORBIDDEN”.

3: Instrumentation:
The desired parameter in spectroscopy is absorbance, but it cannot be directly measured. Thus, a UV-
visible spectrophotometer compares the intensity of the transmitted radiation with that of the incident
UV-visible radiation. Most UV-visible spectrophotometers are double-beam instruments and consist of
following main components:
1. Radiation source
2. Monochromator
3. Detector
4. Amplifier and
5. Recorder
1. Radiations Source: The hydrogen-discharge lamp is the most commonly used source of radiation in
the UV region (180-400 nm). A deuterium-discharge lamp is used in its place when more (3-5 times)
intensity is desired. A tungsten-filament lamp is used when absorption in the visible region (400-800 nm)
is to be determined.
2. Monochromator: The Monochromator spread the radiations obtained from the source into their
separate wavelengths. The most widely used dispersing element is a prism or grating made up of quartz
because quartz is transparent throughout the UV range. Glass and plastics strongly absorbs ultraviolet
radiation, hence it cannot be used in this region. Glass can be satisfactorily used in the visible region. The
dispersed radiation is divided by the beam divider into two parallel beams of equal intensity; one of
which passes through a transparent cell containing the sample solution and the other through an
identical cell containing the solvent. The former is called sample beam and the latter reference beam.
3. Detector: These have photocells or photomultiplier tubes which generate voltage proportional to
the radiation energy that strikes them. However in modern instruments photodiodes are also used.
 9

4. Amplifier:
The spectrophotometer has balancing electronic amplifier which subtracts the absorption of the solvent
from that of the solution electronically.
5. Recorder:
It automatically records the spectrum as a plot of wavelengths of absorbed radiations against absorbance
A or molar absorptivity.
A double beam UV-Visible spectrophotometer is shown below in fig.4

Fig.4

Handling Of Samples in UV-Visible Spectroscopy and Choice of Solvents:

UV-visible spectra are generally recorded either in very dilute solutions or in the vapour phase. Quartz
cells of 1 cm square are commonly used for recording the spectra. The sample is dissolved in some
suitable solvent. Generally, 1 mg of the compound with a molecular weight of 100-200 is dissolved in a
suitable solvent and made up to, e.g. 100 ml and only a portion of this is used for recording the
spectrum.
The choice of the solvent to be used in ultraviolet spectroscopy is quite important. The first criterion for
a good solvent is that it should not absorb ultraviolet radiation in the same region as the substance
whose spectrum is being determined. Usually, solvents that do not contain conjugated systems are most
suitable for this purpose, although they vary regarding the shortest wavelength at which they remain
transparent to ultraviolet radiation. Commonly used solvents are cyclohexane, 1, 4-dioxane, water, and
95% ethanol. The chosen solvent should be inert to the sample.
A second criterion for a good solvent is its effect on the fine structure of an absorption band.
A nonpolar solvent does not hydrogen bond with the solute, and the spectrum of the solute closely
approximates the spectrum that would be produced in the gaseous state, in which fine structure is often
observed. In a polar solvent, the hydrogen bonding forms a solute–solvent complex, and the fine
structure may disappear. Figure.5 shows the effects of polar and nonpolar solvents on an absorption
band.

Fig.5

PRESENTATION OF SPECTRA:
The ultraviolet–visible spectrum is generally recorded as a plot of absorbance versus wavelength. It is
customary to then re-plot the data with either e or log e plotted on the y-axis and wavelength plotted on
the x-axis. Figure 6, the spectrum of benzoic acid, is typical of the manner in which spectra are displayed.
However, very few electronic spectra are reproduced in the scientific literature; most are described by
indications of the wavelength maxima and absorptivities of the principal absorption peaks. For benzoic
acid, a typical description might be,
 10

Max = 230 nm log  =

4.2 272 3.1
282 2.9

Figure 6

Certain Terms Used in Electronic Spectroscopy Definitions:

Chromophore:
A covalently unsaturated group responsible for absorption in the UV or visible region is known as a
chromophore. For example, C =C, C =C, C =0, C =N, N =N, N02 etc. If a compound absorbs light in the
visible region (400-800 nm), only then it appears colored. Thus, a chromophore may or may not impart
color to a compound depending on whether the chromophore absorbs radiation in the visible or UV
region.
Chromophores like C =C or C =C having n electrons undergo  * transitions and those having both n
and non-bonding electrons, e.g. C =0, C=N or N=N, undergo  *, n* and n * transitions. Since
the wavelength and intensity of absorption depend on a number of factors, there are no set rules for the
identification of a chromophore.
Auxochrome:
A covalently saturated group which, when attached to a chromophore, changes both the wavelength
and the intensity of the absorption maximum is known as auxochrome, e.g. NH 2 , OH, SH, halogens etc.
Auxochromes generally increase the value of max as well as max by extending the conjugation through
resonance. These are also called color enhancing groups. An auxochrome itself does not show
absorption above 200 nm. Actually, the combination of chromophore and auxochrome behaves as a
new chromophore having different values of max and max · For example, benzene shows max 256 nm,
200, whereas aniline shows max 280 nm, max 1430 (both increased). Hence, NH2 group is an
auxochrome which extends the conjugation involving the lone pair of electrons on the nitrogen atom
resulting in the increased values of max and max ·
Absorption and Intensity Shifts:
Bathochromic Shift or Effect: The shift of an absorption maximum to a longer wavelength (Fig.7) due
to the presence of an auxochrome, or solvent effect is called a bathochromic shift or red shift. For
example, benzene shows max 256 nm and aniline shows max 280 nm. Thus, there is a bathochromic shift
of 24 nm in the max of benzene due to the presence of the auxochrome NH 2 .Similarly, a bathochromic
shift of n band is observed in carbonyl compounds on decreasing solvent polarity, e.g. max of
acetone is at 264.5 nm in water as compared to 279 nm in hexane.
 11

Figure.7

Hypsochromic Shift or Effect: The shift of an absorption maximum to a shorter wavelength is called
hypsochromic or blue shift (Fig. 7). This is caused by the removal of conjugation or change in the solvent
polarity. For example, aniline shows max 280 nm, whereas anilinium ion (acidic solution of aniline) shows
254 nm. This hypsochromic shift is due to the removal of n* conjugation t of the lone pair of
electrons of the nitrogen atom of aniline with the -bonded system of the benzene ring on protonation
because the protonated aniline (anilinium ion) has no lone pair of electrons for conjugation. Similarly,
there is a hypsochromic shift of 10-20 nm in the max of  bands of carbonyl compounds on going
from ethanol as solvent to hexane, i.e. on decreasing solvent polarity.
Hypochromic Effect: An effect which leads to an increase in absorption intensity max is called
hypochromic effect (Fig.7). The introduction of an auxochrome usually causes hypochromic shift. For
example, benzene shows B-band at 256 nm, max 200, whereas aniline shows B-band at 280 nm, max
1430. The increase of 1230 in the value max of aniline compared to that of benzene is due to the
hypochromic effect of the auxochrome NH2.
Hypochromic Effect: An effect which leads to an absorption intensity decrease in max is called
hypochromic effect (Fig.7). This is caused by the introduction of a group which distorts the
chromophore. For example, biphenyl shows max 252 nm, max 19,000, whereas 2, 2'-dimethylbiphenyl
shows max 270 nm, max 800. The decrease of 18,200 in the value of max of 2, 2'-dimethylbiphenyl is due
to the hypochromic effect of the methyl groups which distort the chromophore by forcing the rings out
of co- planarity resulting in the loss of conjugation.

Woodward-Fieser Rules for Calculating Max in Conjugated Dienes and Trienes:

Woodward (1941) formulated a set of empirical rules for calculating or predicting Max in conjugated
acyclic and six-membered ring dienes. These rules, modified by Fieser and Scott on the basis of wide
experience with dienes and trienes, are called Woodward-Fieser rules and are summarized in Table 1.

The calculated and observed values of Amax usually match within ±5 nm as shown in the following
examples illustrating the applications of WoodwardFieser rules (Table 1).
 12

Example 1. Using Woodward-Fieser rules, calculate Max for 2,3-dimethyl-1,3-butadiene.

It is an acyclic diene with two alkyl substituents. Thus, Max of this compound is

Base value 214 nm

Two alkyl substituents (2 x 5) 10 nm

Calculated Max 224 nm

Observed Max 226 nm

Example 2. Calculate the wavelength of the maximum UV absorption for Myrcene.

Since, it is an acyclic diene with one alkyl substituent, thus

Base value 214 nm
One alkyl substituent 5 nm
Calculated Max 219 nm
Observed Max 224 nm
Example 3. Predict the value of Max for -phellandrene.
This is a heteroannular diene (conjugated double bondsarenot in the same ring) with two ring residues
and one exocyclic double bond, hence
Base value 214 nm
Two alkyl substituents (2 x 5) 10 nm
One exocyclic double bond 5 nm
Predicted Max 229nm
Observed Max 232 nm

Woodward-Fieser Rules for Calculating Max in a,ß-Unsaturated Carbonyl Compounds:

Compounds containing a carbonyl group (C = 0) in conjugation with an ethylenic groups (C =C) are called
enones. UV spectra of enones are characterized by an intense absorption band (K-band) due to 
transition in the range 215-250 nm (Max usually 10,000-20,000) and a weak R-band due to 
transition in 310-330 nm region (Max usually 10-100). Similar to dienes and trienes, there are set rules
called Woodward-Fieser rules for calculating or predicting Max in a, ß-unsaturated carbonyl
compounds (enones).
These rules first framed by Woodward and modified by Fieser and by Scott are given in Table 2.
 13

Table.2.Woodward-Fieser rules for calculating Max in a,ß-unsaturated carbonyl compounds.

For calculated Max in other solvents, a solvent correction given in Table 3 must be carried out.

Table.3. Solvent corrections.

Since carbonyl compounds are polar, the positions of the K- and R-bands of
enones are dependent on the solvent. Hence, solvent corrections are required
(Table 3) to obtain the calculated vallies of Max in a particular solvent. The
Max for cisoid enones are usually <10,000, while that of transoid are >10,000.
The calculated values of Amax are usually within ±5 nm of the observed values as shown in the following
examples illustrating the applications of Woodward-Fieser rules (Table 2)

Example 1. Applying Woodward-Fieser rules, calculate the Max for the ethanolic solution of mesityl
oxide
This being an acyclic ,-unsaturated ketone with two ß-alkyl substituents, the
Max of this compound is calculated as
Base value 215 nm
Two -alkyl substituents (2 x 12) 24 nm
Calculated Max 239 nm
Observed Max 237 nm
 14

Example 2. Predict the value of Max for,

This is a six-membered cyclic ,-unsaturated ketone with one - and one -alkyl substituents. Hence,
Base value 215 nm
One -alkyl substituent 10 nm

One -alkyl substituent 12 nm

Predicted Max (EtOH) 237 nm

Observed Max (EtOH) 249nm

Calculated Max (hexane) 237 (EtOH)

Solvent correction +11

Total 248 nm

Example 3. Calculate Max (EtOH) for,

1t is a five-membered cyclic ,-unsaturated ketone with one -hydroxy and one -ring residue. Thus,
Base value 202 nm
One -hydroxy groups 35 nm
One -ring residue 12 nm
Calculated Max (EtOH) 249 nm
Observed Max (EtOH) 247 nm
,-Unsaturated Carboxylic Acidsand Esters:
The attachment of groups containing a lone pair of electrons to the carbonyl group, as in carboxylic
acids, esters and amides, shifts the n transition to shorter wavelengths (200-220 nm) region with
little effect on intensity. This hypsochromic shift is due to combined inductive and resonance effects. For
example, compared to the R-band of acetaldehyde (Max 290 nm) that of acetic acid, ethyl acetate and
acetamide appear at 204, 211 and 220 nm, respectively. , -unsaturated acids, esters and amides
display strong K-bands characteristic of the conjugated system. , --unsaturated acids and esters
follow a trend similar to that of enones. Thus, we can calculate Max of their K-bands taking the base
value 195 nm and using the data given in Table 2. As shown above.
Example 1. Calculate Max for

Base value 195 nm

One a-alkyl substituent 10 nm
Calculated Max (EtOH) 205 nm
Observed Max (EtOH) 210nm
 15

Example 2. Predict the Max for,

Base value 195 nm

Two ß-ring residues (2 x 12) 24 nm
One exocyclic C = C 5 nm
Predicted Max (EtOH) 224nm
Observed Max (EtOH) 220 nm

Terms used in Woodward-Fieser rules:

(i) Homoannular Dienes: In homoannular dienes, conjugated double bonds are present in the same ring
and having s-cis (cisoid) configuration (s = single bond joining the two doubly bonded carbon atoms).

The s-cis configuration causes strain which raises the ground state energy Ievel of the molecule leaving
the high energy excited state relatively unchanged.
Thus, the transition energy is lowered resulting in the shift of absorption position to a longer
wavelength. Acyclic dienes exist mostly in the strainless s-trans (transoid) conformation with relatively
lower ground state energy Ievel. Thus, their absorptions appear at shorter wavelengths. For example,
1,3cyclohexadiene (I) shows Max 256 nm, whereas 1,3-butadiene shows Max 217 nm. Also, due to
the shorter distance between the two ends of the chromophore, s-cis dienes give lower Max (1 0,000)
than that of the s-trans dien es (-20,000).

(ii) Heteroannular Dienes:

In heteroannular dienes, conjugated double bonds are not present in the same ring and these have s-trans
(transoid) configurations.

(iii) Exocyclic Conjugated Double Bonds: The carbon-carbon double bonds projecting outside a ring are
called exocyclic double bonds. For example,

Note that the same double bond may be exocyclic to one ring, while endocyclic to the other and
sometimes the same double bond may be exocyclic to two rings simultaneously.
 16

(iv) Alkyl Substituents and Ring Residues:Only the alkyl substituents and ring residues attached to the
carbon atoms constituting the conjugated system of the compound are taken into account. Following
examples indicate such carbon atoms by numbers and the alkyl substituents and ring residues by dotted
lines.

Instrumentation:
1. Diagram of a double-beam UV-Vis. spectrophotometer

Fig: Diagram of a double-beam UV-Vis. spectrophotometer

2. Description:

The light source

Need a light source which gives the entire visible spectrum plus the near ultra-violet
so that you are covering the range from about 200 nm to about 800 nm. (This extends
slightly into the near infra-red as well.)
It can't get this range of wavelengths from a single lamp, and so a combination of two
is used - a deuterium lamp for the UV part of the spectrum, and a tungsten / halogen
lamp for the visible part.
The combined output of these two bulbs is focused on to a diffraction grating.

The diffraction grating and the slit:

It probably familiar with the way that a prism splits light into its component colors. A
diffraction grating does the same job, but more efficiently.
 17

The blue arrows show the way the various wavelengths of the light are sent off in
different directions. The slit only allows light of a very narrow range of wavelengths
through into the rest of the spectrometer.

By gradually rotating the diffraction grating, it can allow light from the whole
spectrum (a tiny part of the range at a time) through into the rest of the instrument.

The rotating discs:

This is the clever bit! Each disc is made up of a number of different segments. Those in
the machine we are describing have three different sections - other designs may have
a different number.

The light coming from the diffraction grating and slit will hit the rotating disc and one
of three things can happen.

1. If it hits the transparent section, it will go straight through and pass through the
cell containing the sample. It is then bounced by a mirror onto a second rotating
disc.

This disc is rotating such that when the light arrives from the first disc, it meets
the mirrored section of the second disc. That bounces it onto the detector.

It is following the red path in the diagram:

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2. If the original beam of light from the slit hits the mirrored section of the first
rotating disc, it is bounced down along the green path. After the mirror, it passes
through a reference cell (more about that later).

Finally the light gets to the second disc which is rotating in such a way that it
meets the transparent section. It goes straight through to the detector.

3. If the light meets the first disc at the black section, it is blocked - and for a very
short while no light passes through the spectrometer. This just allows the
computer to make allowance for any current generated by the detector in the
absence of any light.

The sample and reference cells:

These are small rectangular glass or quartz containers. They are often designed so
that the light beam travels a distance of 1 cm through the contents.

The sample cell contains a solution of the substance you are testing - usually very
dilute. The solvent is chosen so that it doesn't absorb any significant amount of light in
the wavelength range we are interested in (200 - 800 nm).

The reference cell just contains the pure solvent.

The detector and computer:

The detector converts the incoming light into a current. The higher the current, the
greater the intensity of the light.

For each wavelength of light passing through the spectrometer, the intensity of the
light passing through the reference cell is measured. This is usually referred to as I o -
that's I for Intensity.

The intensity of the light passing through the sample cell is also measured for that
wavelength - given the symbol, I.

If I is less than Io, then obviously the sample has absorbed some of the light. A simple
bit of math’s is then done in the computer to convert this into something called the
absorbance of the sample - given the symbol, A.

For reasons which will become clearer when we do a bit of theory on another page,
the relationship between A and the two intensities is given by:
 19

On most of the diagrams you will come across, the absorbance ranges from 0 to 1, but
it can go higher than that.

An absorbance of 0 at some wavelength means that no light of that particular

wavelength has been absorbed. The intensities of the sample and reference beam are
both the same, so the ratio Io/I is 1. Log10 of 1 is zero.

An absorbance of 1 happens when 90% of the light at that wavelength has been
absorbed - which means that the intensity is 10% of what it would otherwise be.

In that case, Io/I is 100/I0 (=10) and log10 of 10 is 1.

The chart recorder

Chart recorders usually plot absorbance against wavelength. The output might look
like this:

This particular substance has what are known as absorbance peaks at 255 and 395
nm. How these arise and how they are interpreted are discussed on another page.

Operation Procedure:
1. Make sure the unit is plugged in.
2. Turn on the unit (switch is on the back of the instrument on the right side).
3. Flip up the display screen on the front of the instrument.
4. Instrument will go through a series of diagnostics. "Multi-CellOrg." diagnostic
will fail. Proceed to the next step.
5. Allow the UV source to warm up for a few minutes in order for the lamp energy
to stabilize.
6. Press the number 1 on the keypad to select "Photometric Measurement".
7. Press the GOTO l, button. A cursor will appear at the bottom of the display
screen.
8. Enter the desired wavelength using the numeric keys. Press Enter. The
wavelength will appear in the centre of the display. (If the wavelength is
entered incorrectly, repeat steps 7-8.)
9. Press the Fl button to toggle between absorbance and o/o transmittance modes.
Abs will appears on the display.
10. Fill two plastic cuvette (for aqueous solutions) or quartz cuvette (for organic
solutions) about 50% filI with solvent. One is sample and one is reference
solution
11. Place those solution in the sample and reference compartment. The cuvette will
not go down that far. Not push down very hard on the cuvette.
 20

12. Make sure the sample compartment door is closed.

13. Press the AUTO ZERO button to "zero" the spectrophotometer.
14. The reading will appear in the centre of the display.
15. Turn off the unit (switch is on the back of the instrument on the right side).
16. Flip down the display screen on the front of the instrument.

Trouble Shooting:
Possible Causes Action

Light path is blocked. Ensure that the light path is free and
clear of all obstructions

Cuvette or flow cell not installed Check and correct cell installation.
correctly.

Air bubble sticking on the quartz Use cell cleaning fluid to passivity
window of the flow cell. flow cell.

Source lens or spectrograph lenses Clean lenses.

dirty or fogged.

Shutter not functioning or partly Exchange shutter assembly.

blocking light.

Spectrophotometer data acquisition Exchange SDA board.

(SDA) board may be defective.

Spectrophotometer lamp supply Exchange SLS board.

(SLS) board may be defective.
Possible Causes Action

Main power supply (MPS) may be Exchange MPS.

defective.

Spectrograph electronics may be Exchange the optical unit.

defective.

One of the lamps may be turned off Switch on proper lamp.

when making measurements.
Measurements taken with the
deuterium lamp off exhibits excessive
noise in the UV wavelength range.
Measurements taken with the
tungsten lamp off exhibits excessive
noise in the visible wavelength range.

Noise in the UV wavelength range Exchange the lamp. Lifetime of the

may be caused by a weak or defective deuterium lamp may be influenced by
deuterium lamp. the number of ignitions.

Flow cells and cuvettes, which reduce Change cell to standard type. Glass
and/or distort the colimated light absorbs in the low UV region and
beam used in the spectrophotometer, causes high noise. Make sure that
can result in low lamp intensity at the your flow cells and cuvettes are made
 21

spectrograph. from quartz

Solvent or buffer blocks light in a If information is required in such a

certain wavelength range. wavelength range, a solvent or buffer
that is transparent in that range
should be used.

Fingerprints on cuvettes or flow cells Clean your flow cells or cuvettes with
typically absorb light in the UV range a lens cleaning tissue.
of the spectrum..

Source lens or spectrograph lenses Clean lenses.

dirty or fogged.

Spectrophotometer lamp supply Exchange SLS board.

(SLS) board may be defective.

Remedies:
1. Operators must be wear hand gloves.
2. UV- Spectrometers power plug connect properly.
3. Set λmax properly for specific compound.
4. Cuvette tube must be cleaned with distilled water.
5. High concentrated liquid must not be used, e.g.: Semisolid, suspension etc.
6. Taking absorbance from the display properly up to four digits after decimal.
7. Work properly & sincerely .

Application:
1. Determination of various chemical compound purity test of various active
pharmaceutical ingredients (API)
2. Determination of amount and concentration of API in various pharmaceutical
dosage form
3. The Determination of Food Colors in Mixtures
4. Thermal Analysis of DNA
5. Kinetics Rate Law Investigations
6. Multi-Component Analysis of a Vitamin B Mixture
7. Automation of Good Manufacturing Practice Requirements
8. Automated Measurement of the Color of Red Wines
9. Fully Automated Water Nitrate Analysis and etc.
 22

Conclusion:
Following the application we can say that UV-Spectrometer is one of the most
important analyzing and testing instrument. All pharmaceutical, chemical industry
and research institutes use it for their wide range of testing and identifying.

References:
1. Introduction to spectroscopy by Pavia, LAmpman, Kriz (Fifth edition).
2. Organic Chemistry by Dr.M.younus.