Analytical Chemistry‐2
Level II Students (2nd Semester)
Spectrophotometry
(Lecture 9)
Dr. Mohamed El-Awady
15-Apr-2015
Lec. 9
Objectives
After this lecture, you should know:

What are the cases in which Beer’s law is not obeyed?

What are the basic steps for any spectrophotometric
determination?

What are the factors affecting the absorption spectrum
(i.e. the factors that can change max or absorbance in the
absorption spectrum)?
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Deviations from Beer’s law OR Limitations of Beer’s law
{Types & Causes}
These are deviations from the direct proportionality or
from the linear relationship between absorbance and
concentration (i.e. they are cases in which Beer’s law is
not obeyed).The deviation may be positive or negative.
There are 3 types of deviations: (Real - Chemical – Instrumental)
1- Real Deviation:
- At very high concentrations more than about 0.01 M,
absorbance is not directly proportional to concentration.
- This because at very high concentration, the distances between the absorbing
particles decrease, so each particle will affects the charge distribution and thus
the extent of absorption of the surrounding particles.
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2- Chemical Deviation:
It occurs when the absorbing compound undergoes a chemical reaction with the
solvent that gives a product which absorbs differently from the original compound.
Ex.: Unbuffered solution of dichromate (Cr2O72-) changes its color from orange to
yellow upon dilution with water due to its conversion into chromate (CrO42-) as
given by the following equilibrium:
Cr2O72-
+
H2O
⇌
(Orange)
max = 440 nm
2 H+ +
2 CrO42(Yellow)
max = 390 nm
In strong acidic pH (e.g. in presence of H2SO4), Cr2O72- is predominant while
in strong alkaline pH [e.g. in presence of KOH], CrO42- is predominant.
3- Instrumental Deviation:
a) Unmatched or Unclean cells (cuvettes).
b) Use of polychromatic radiation instead of monochromatic.
c) Stray light  ‫( الضوء الشارد‬any light reaching the detector outside the specified
wavelength band due to scattering, reflection, instrumental problem ….. etc).
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Steps for a spectrophotometric determination
1-
Determination
of
max
by
drawing the Absorption Spectrum:
2-
Drawing
the
Standard
Calibration Curve:
Relation between A & c
using constant c
using constant  (max)
Absorbance
Absorbance
Relation between A & 
λmax
concentration
Wavelength
3- Determination of unknown sample concentration from the standard
calibration curve: by measuring its absorbance and find the corresponding
concentration from the calibration curve.
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Notes:
The calibration curve is constructed by plotting the measured absorbance of
a series of standard solutions of the analyte (of known concentration) at λmax
giving a straight line.


Avoid very high or low absorbance when
drawing a standard calibration curve. The
best results are obtained with 0.1 < A < 1.
This because after this value deviation of
Beer law will occur (i.e the relation
between concentration and absorbance

The unknown concentration of the
sample can be determined by measuring its
absorbance
and
then
find
corresponding concentration from
the
Absorbanc
e
become non-linear).
the
calibration curve by extrapolation or by the
equation of the calibration line [y = b + ax].
concentration
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Factors affecting absorption spectrum
They are factors that can change the max or the absorption
intensity (absorbance) of the absorption spectrum.
These factors include:
1- Effect of pH.
2- Effect of dilution.
3- Effect of solvent.
4- Effect of temperature.
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Types of Spectral Shifts in Spectrophotometry
The change in the absorption spectrum can be one of the followings:
Hyperchromic effect
= Increase in absorbance
Hypsochromic
shift
= shift of the
absorption
spectrum to
shorter
wavelength
(Blue Shift)
(Red Shift)
Hypochromic effect
= Decrease in absorbance
Bathochromic
shift
= shift of the
absorption
spectrum to
longer
wavelength
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1- Effect of pH on the absorption spectrum:
The spectra of compounds containing acidic or basic groups are dependent on the pH
of the solution.
Example 1: Phenol and phenolic compounds
hyper-
The spectrum of phenol or other phenolic compounds exhibits (shows) bathochromic
shift (→) and hyperchromic effect (↑) in alkaline medium compared to acidic
medium… Why??
batho-
UV spectrum
of phenol
in acidic and
in alkaline
media
phenol in alkaline pH
phenol in acidic pH
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Explanation:
The spectrum of phenol exhibits (shows) bathochromic shift (→) and hyperchromic
effect (↑) in alkaline medium compared to acidic medium because:
(benzenoid form)
ACIDIC MEDIUM
(quinonoid form)
ALKALINE MEDIUM
In acidic medium, phenol is in the benzenoid form, while in alkaline medium it is in the quinonoid
form (phenate anion) in which the lone pair of electrons on the oxygen atom increases the
delocalization of electrons (i.e. additional conjugation to the aromatic ring) leading to:
- Increase in the absorbance {hyperchromic effect}.. Remember: conjugated double bond is a chromophore.
- Increase in the wavelength absorbed by the compound {bathochromic shift} because the
compound becomes more energetic and so the energy needed for excitation of electrons is low
(↓ energy ≡ ↑ wavelength).
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Example 2: Aniline and aromatic primary amines
The spectrum of aniline or other aromatic primary amines exhibits (shows)
hypsochromic shift (←) and hypochromic effect (↓) in acidic medium compared to
alkaline medium…………………….. Why??
Aniline in
alkaline pH
UV spectrum
of aniline
in acidic and
in alkaline
media
hypo-
hypso-
Aniline in
acidic pH
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Explanation:
The spectrum of aniline or other aromatic primary amines exhibits (shows)
hypsochromic shift (←) and hypochromic effect (↓) in acidic medium compared to
alkaline medium because:
(quinonoid form)
ALKALINE MEDIUM
(benzenoid form)
ACIDIC MEDIUM
In alkaline medium, aniline is in the quinonoid form, while in acidic medium it is in the benzenoid
form (anilinium cation) in which the nitrogen atom loses the lone pair of electrons and so the
delocalization of electrons is decreased (i.e. less conjugation with the aromatic ring) leading to:
- Decrease in the absorbance {hypochromic effect} ………… Remember: ↓ conjugation ≡ ↓ absorbance.
- Decrease in the wavelength absorbed by the compound {hypsochromic shift} because the
compound becomes less energetic and so the energy needed for excitation of electrons is high
(↑ energy ≡ ↓ wavelength).
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2- Effect of Dilution on the absorption spectrum:
An example of this effect is the change of color of dichromate solution, upon dilution
with water as given by the following equilibrium:
Cr2O72(Orange)
max = 440 nm
+
H2O
⇌
2 H+ +
2 CrO42(Yellow)
max = 390 nm
3- Effect of Solvent on the absorption spectrum:
The same substance usually has different spectra in different solvents; the spectrum in
polar solvent varies from that in non-polar solvent. One must specify the solvent used
in preparation of solutions of a compound to be quantitatively measured.
4- Effect of Temperature on the absorption spectrum:
Changes in temperature can shift the ionic equilibrium of some compounds and so
affect their absorption spectrum. e.g: Increasing the temperature causes
bathochromic shift in case of FeCl3 solution in HCl (on heating its color changes
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from yellow to red). So the temperature must be the same for all measurement.
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Summary
Spectrophotometry
 Deviations (Limitations) of Beer’s Law:
(1) Real deviation.
(2) Chemical deviation.
(3) Instrumental deviation.
 Steps for any spectrophotometric determination:
1. Absorption Spectrum.
2. Standard Calibration Curve.
3. Determination of Unknown Sample Concentration.
 Factors affecting absorption spectrum:
(Hyperchromic & Hypochromic effects) , (Bathochromic & Hypsochromic shifts)
1. pH…. e.g. Phenol & Aniline
2. Dilution…. e.g. Dichromate/Chromate
3. Solvent.
4. Temperature.
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Questions
I- Complete the following sentences:
1- Examples of factors affecting absorption spectrum are ………, …………, ……. and .…….
2- Hyperchromic effect is ………………………………………………………………………………
while hypsochromic shift is ……………………………………………………………………
3- …............. is the shift of the absorption spectrum to longer wavelength.
4- The spectrum of phenol exhibits a ……… shift and a …….. shift in alkaline pH compared to
acidic pH.
5- Deviations of Beer’s law is classified into ……… , ……….. and ……………….
II- Give reason for the following sentences:
1- Beer’s law is not obeyed at concentrations more than 0.01 M.
2- Beer’s law can be deviated due to instrumental factors.
3- Phenol exhibits a bathochromic shift and a hyperchromic effect in alkaline medium.
4- Aniline exhibits a hypsochromic shift and a hypochromic effect in acidic medium.
5- Color change of potassium dichromate solution upon dilution.
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III- Write () or ():
1- FeCl3 solution in HCl exhibits a hypsochromic shift upon increasing the temperature (
2- The calibration curve in spectrophotometry is a plot of absorbance against wavelength (
).
).
IV- Choose the correct answer:
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Thank You
15-Apr-2015