University of Al-Ameed
College of Pharmacy
Advanced pharmaceutical analysis
lecture 2
5th class/ 2nd semester
UV/Vis spectroscopy
Dr. Abbas Abdulridha
Woodward-Fieser Rule
In 1945 Robert Burns Woodward gave certain rules for correlating λmax with molecular
structure.
In 1959 Louis Frederick Fieser modified these rules with more experimental data, and
the modified rule is known as Woodward-Feiser Rules. It is used to calculate the λmax
for a given structure by relating the type of chromophores present, the substituents on
the chromophores, and shifts due to the solvent.
According to Woodward’s rules the λmax of the molecule can be calculated using a
formula:
The Woodward-Fieser Rule provides a method for predicting the maximum wavelength of
absorption (λmax) for conjugated dienes and polyenes based on their structure. Key
points include:
1. Base Value: Each type of diene (acyclic, homoannular, heteroannular) has a fixed base
value that depends on its configuration (linear, cyclic, etc.).
2. Substituents: The presence of substituents such as alkyl groups, additional conjugated
double bonds, and polar groups (e.g., –Cl, –Br) influences λmax. Each of these
contributes specific numerical values.
3. Calculation: To obtain the λmax for a particular compound, the fixed base value is
adjusted by adding the contributions from the substituents
➢ This rule allows chemists to estimate the absorption characteristics of
conjugated systems based on their structural features.
Important Terms
1. Acyclic dienes
An acyclic diene exists mostly in s-trans (transoid) conformation as shown in the case of
butadiene.
• As acyclic diene can rotate about their single bond to give either cisoid or transoid,
• therefore they exist in trans-form due to the high stability of the later.
When this diene becomes a part of a ring system as in 1,3 cyclohexadiene, it is forced into a
cisoid configuration.
➢ The wavelength of absorption is also shifted to longer wavelength in comparison with
acyclic dienes. The absorption maxima for 1,3 cyclohexadiene is 256 nm, whereas 1,3-
butadiene shows λmax at 217 nm.
2. Cyclic dienes:
Cyclic dienes can be classified into two classes depending upon whether the double
bonds are in the same ring or in different rings.
a) Homo-annular conjugated dienes: Both conjugated double bonds are in the same ring
and are having s-cis (cisoid) configuration. The s-cis configuration causes strain which
raises the energy of ground state, while the energy of the excited state is relatively
unchanged. Thus the transition energy is lowered resulting absorption at longer
wavelength.
b) Hetero-annular dienes: Conjugated double bonds are present in different rings and
have S-trans configuration.
The homo-annular diene resembles with 1,3 cyclohexadiene, whereas hetero-annular
diene with butadiene.
3. Endocyclic double bond: Double bond/s is present inside the ring.
4. Exocyclic double bond: Double bond/s projecting outside the ring is called as
exocyclic double bonds.
➢ In example II, the double bond present within ring B is exocyclic to ring A, whereas
endocyclic to ring B.
➢ In example III, double bond present in ring A is exocyclic to ring B, similarly double
bond present in ring B is exocyclic to ring A.
➢ In example IV, both double bonds are present within ring B making them endocyclic to
ring B, while both the double bonds are exocyclic to ring A.
➢ In example V, the double bond present in the ring B is exocyclic to ring A and ring C.
Similarly, the double bond present in the ring A is exocyclic to ring B.
➢ In example VI, the double bond is exocyclic to two different rings at a time. In such
cases, the contribution would be 2 times.
Empirical rules for dienes
Base value for Butadiene system; basic value =217 nm
Base value for homoannular (cisoid) diene = 253 nm
Base value for heteroannular (transoid) diene = 214 nm
Increment for:
a) Alkyl substituent or Ring residue attached to the parent diene = 5 nm
b) Double bond extending conjugation = 30 nm
c) Exocyclic double bonds = 5 nm 1. -OCOCH3 = +0
2. -OR = +6
d) Polar substituents
3. -SR = +30
4. -Cl, -Br = +5
5. -NR2 = +60
Important Note:
1. While calculating λmax only those alkyl substituents or ring residues are counted which
are directly attached to the carbon atoms making conjugated system within the
compound.
2. If a molecule has both homoannular diene and heteroannular diene (Entry 5) then
always use the homoannular diene as the core chromophore.
➢ With the help of these rules the UV absorption maximum (λmax) can be predicted for
any alkene compound.
• Let us take some examples to explain the calculation for λmax
217
232
Base value=253 (homoannular)
4 ring residues =4X5=20
2 exoyclic bond=2X5=10
Thus λmax = 283 nm
Base value = 253 (homoannular)
6 ring residues =6X5=30
2 exoyclic bond=2X5=10
1 double bond exocyclic to two
rings= 2X5=10
1 double bond extending
conjugation=1X30=30
Thus λmax = 333 nm
For polyenes with more than 4 conjugated double bonds, Fieser-Kuhn rule is used to
calculate λmax
Fieser-Kuhn derived the following equation to calculate the wavelength of maximum
absorption
λmax and also maximum absorptivity εmax
Where,
• λmax is the wavelength of maximum absorption
• x is the number of alkyl substituents / ring residues in the conjugated system
• y is the number of conjugated double bonds
• Rendo is the number of rings with endocyclic double bonds in the conjugated system
• Rexo is the number of rings with exocyclic double bonds in the conjugated system
Where,
εmax is the maximum absorptivity
y is the number of conjugated double bonds
We can use this equation to calculate wavelength of maximum absorbance (λmax) and the
maximum absorptivity (εmax) for polyene such as β-carotene, Lycophene etc.
Let us take β-carotene as an example. The observed λmax of β-carotene is 452 nm, while
the observed εmax is 15.2 x 10
x (number of alkyl substituents) = 10
y (number of conjugated double bonds) =
11
Rendo (number of endocyclic double
bonds) = 2
Rexo (number of exocyclic double bonds) =
0
= 114 + 5(10) + 11 (48.0 – 1.7(11)) – 16.5 (2) – 10 (0)
= 114 + 50 + 11 (29.3) – 33 – 0 ε max = (1.74 x 104) y
= 114 + 50 + 322.3 – 33 = (1.74 x 104) 11
= 453.30 nm = 19.14 x 104
Woodward-Fieser Rule for α,β-unsaturated carbonyl compounds
Carbonyl compounds have two principal UV radiations, the allowed π to π* transitions
and the forbidden n to π* transitions.
➢ In amides, acids, esters or acid halides, the substituents: NR2, OH, OR, or –X on
carbonyl group show pronounced hypsochromic effect on the n to π* transitions.
➢ The hypsochromic effect is due to inductive effect of nitrogen, oxygen or halogen
atoms.
➢ The heteroatom withdraws electrons from carbonyl carbon and makes carbonyl
oxygen lone pair of electrons more stabilized due to its involvement in increasing
C=O bond order.
• As a result, the n to π* transition of these compounds is shifted to 200-215 nm range
relative to 270 nm in aldehydes and ketones.
• Conjugation of the carbonyl group with double bond shifts both n to π* and π to π*
transitions to longer wavelengths. The effect on π to π* band is more pronounced.
Base value for
a) Acyclic α, β unsaturated ketones = 215 nm Increment for
b) 6 membered cyclic α, β unsaturated ketones ii) Each double bond extending conjugation
= 215 nm = 30 nm
c) 5 membered cyclic α, β unsaturated ketones iii) Exocyclic double bonds = 5 nm
= 202 nm iv) Homoannular diene component = 39 nm
d) α, β unsaturated aldehydes = 210 nm
e) α, β unsaturated carboxylic acids and esters
= 195 nm Increment for substituents
Let us see few typical examples to elaborate the above rules:
Note: In cross conjugated system, λmax is calculated by using highly substituted conjugated
system as parent system (Entry 5).
Tips & Hints for Accurate and Precise UV/VIS Measurements
The absorption spectrum of a substance can be influenced by the solvent, the pH of the
solution, the temperature, high electrolyte concentrations, and the presence of
interfering substances.
Effects of these variables must be known and conditions for the analysis chosen so that
the absorbance will not be affected by small, uncontrolled variations in their
magnitudes. The most important effects will be described in the following paragraphs.
1. Cuvette-based UV/VIS spectroscopy
• For UV measurements, employ UV-transparent materials like quartz
• Use the same type of cuvette for both reference and sample measurements.
• Clean cuvette windows with lint-free tissues(to avoid scratches from lint on
the surface) before every use to prevent contamination (e.g., fingerprints,
grease).
2. Solvent selection
A solvent for UV/VIS spectroscopy must be transparent throughout the applied region and
should dissolve a sufficient quantity of the sample to give well-defined peaks. Every solvent
has a UV/VIS absorbance cut-off wavelength.
The solvent cut-off is the wavelength below which the solvent itself absorbs all of the light.
So, when choosing a solvent, it is important to be aware of its absorbance cut-off as well as
where the compound under investigation is expected to absorb. If they are close, a different
solvent should be chosen.
➢ Generally, non-polar solvents and non-polar molecules have the least effect on one
another.
➢ However, polar molecules exhibit quite drastic differences when they interact with a
polar solvent such as water, alcohols esters, and ketones.
➢ Interaction between solute and solvent leads to absorption band broadening and a
consequent reduction in structural resolution and maximum extinction coefficient
The next figure illustrates the effect of iso-octane and ethanol on the spectrum
of phenol.
By changing from a non-polar solvent (iso-octane) to a polar one (ethanol), the
fine structure due to the vibrations of phenol are attenuated. In general, the
spectra recorded in non-polar solvents are closer to those obtained from gas
samples.
iso-octane
Spectrum of phenol in iso-octane and ethanol. Polar solvents such as ethanol
show much lower spectral resolution
3. Sample concentration
For optimal measurement results and to comply with the Lambert-Beer Law,
the absorbance shall be determined in the linear range of the instrument. Thus,
it is better to avoid very high (>2.5 AU) and very low absorbance values (<0.3
AU) which may lead to non-linearity.
When an analyte interacts (association, dissociation) or reacts with the solvent, new
chemical species are created leading to a change of absorption behavior. It is therefore
recommended to use a non-interacting solvent and a low concentration of analyte to
avoid any side reactions.
4. wave-length
In order to obtain maximum sensitivity, spectrophotometric absorbance
measurements are performed at the wave-length located at lambda max.
The importance of measuring absorbance precisely is demonstrated in the following
figure.
➢ Errors due to either wavelength setting or instrument calibration will be at a
minimum when measurements are made at the wavelength of maximum absorption.
Figure : Left: Change in absorbance per wavelength change has significant effect in Band B,
whereas in Band A the change is very small and negligible. Right: The change effects the
calculated concentration of a sample in case B
4. Resolution in UV/VIS Spectroscopy
Importance of Resolution:
➢ The resolution of a spectrophotometer is crucial for distinguishing closely located
absorbance peaks in a sample's UV/VIS spectrum.
• High resolution allows clear separation of absorbance peaks.
• Low resolution can cause peaks to overlap, making it difficult to identify them
accurately.
In the following example, the spectrum of benzene is shown at three different resolution
settings on the spectrophotometer. This parameter is called the spectral bandwidth.
In the right-hand graph, with the lowest resolution, only a single, broad absorption band
is displayed.
• Resolution is therefore a critical factor in determining the shape of measured peaks. If
the instrumental resolution is not sufficient, the absorbance value will be lower than
the true value.
3. Calculate λ max of following compound
• Parent value for heteroannular diene : = 214 nm
• Four ring residue : 4 X5 = 20 nm
• Calculated value : = 234 nm
• Observed value : = 236 nm
4. Calculate λ max of following compound
H3C
S
• Parent value for heteroannular diene : = 214 nm
• 3 ring residue : 3 X5 = 15 nm
• Exocyclic double bond = 5 nm
• Thiomethyl substituent =30 nm
• Calculated value : = 264 nm
• Observed value : = 268 nm
7. Calculate λ max of following compound
Calculate the λmax for the following compounds :
1 2
Distinguish Isomers
43
44
Factors affecting UV/Vis absorption :
• The absorption spectrum of a chromophore is only partly
determined by its chemical structure.
• The environment also affects the observed spectrum, which
mainly can be described by three parameters :
1. Solvent polarity (dielectric constant of the solvent).
2. Substituent effects.
3. Protonation/deprotonation (pH, redox).
Solvent polarity: Affects the difference between the ground and excited states.
2- substituent effect
The non bonding pairs of electrons of the N atom are not effectively interact
with the π electrons of the ring (not completely parallel). This effect is for o >
m >>> p
➢ Resonance energy is at maximum when the rings are coplanar
