Instrumental Analysis

ً‫التحلٌل اآلل‬
Instrumental Analysis )CHM 351(

Part one: Spectrophotometric Methods

1
 ‫الهدف من المقرر‬

‫دراسة الطرق التحلٌلٌة باستخدام االجهزة الحدٌثة االلٌة المختلفة‬ ‫‪‬‬

‫والتدرٌب على تطبٌقات هذه الطرق فً المعمل والصناعة والمعامل‬

‫الصحٌة وغٌرها وٌشتمل علً جزٌئٌن رئٌسٌن ‪.‬‬

‫‪ -1‬دراسة الطرق التحلٌلٌة الطٌفٌة المختلفة والتدرٌب على تطبٌقات‬ ‫‪‬‬

‫هذه الطرق فً المعمل والصناعة والمعامل الصحٌة وغٌرها‪.‬‬

‫‪ -2‬دراسة الطرق التحلٌلٌة الكهربائٌة المختلفة والتدرٌب على تطبٌقات‬ ‫‪‬‬

‫هذه الطرق فً المعمل والصناعة والمعامل الصحٌة وغٌرها‪.‬‬ ‫‪2‬‬

 : ‫طرق تقٌٌم الطالب‬
5. Schedule of Assessment Tasks for Students During the Semester

Assessment task (i.e., essay, test, quizzes, group project, Proportion of Total
Week Due
examination, speech, oral presentation, etc.) Assessment

First semester exam. 7 20 %

1
Duties 1-15 5%
2
Quiz 1 6 5%
3
Quiz 2 13 5%
4
Oral and discussions 1-15 5%
5
Practical 16 20 %
6
Final 17 40 %
7
3
 ‫طرق تقٌٌم الطالب ‪:‬‬

‫مجموع الدرجات‬
‫‪ 100‬درجة‬

‫االختبار النظري النهائً‬ ‫اعمال السنة‬

‫‪ 40‬درجة‬ ‫‪60‬درجة‬

‫شفوى و‬
‫مناقشات‬
‫اسئلة قصٌرة‬ ‫اختبار فصلى واجبات‬ ‫عملً‬
‫‪ 5‬درجات‬ ‫‪ 20‬درجات ‪ 5‬درجات‬ ‫‪ 20‬درجة‬
‫‪ 10‬درجات‬ ‫‪4‬‬
 Analytical Methods
The majority of industrial analyses are performed using one of the following techniques:
‫معظم عملٌات التحلٌل تشمل الطرق التالٌة‬
Simple methods: ‫الطرق البسٌطة‬
Gravimetric ‫الطرق الوزنٌة‬
Titrimetric ‫الطرق الحجمٌة‬
Spectroscopic Methods: ‫الطرق الطٌفٌة‬
Atomic Absorption Spectrophotometry (AAS) (GFAAS)
Inductively Coupled Argon Plasma Spectrophotometry (ICAP)
Ultraviolet/Visible Spectrophotometry
X-ray Diffraction (XRD, XRF….)
Electroanalytical Methods ‫الطرق الكهربٌه‬
Conductometric methods
Voltametric methods
Amperometric methods
Chromatographic Methods: ً‫طرق الفصل الكروماتغراف‬
Gas Chromatography (GC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography (IC)
Microscopic Methods
Electron Microscopy (Transmission and Scanning microscopes) AFM, SEM, TEM
Optical Microscopy (Phase-contrast and polarized-light microscopes) 5
 Spectrophotometric Methods of
Analysis
Spectroscopy

It is the branch of science that deals with the study of

interaction of matter with light.
OR
It is the branch of science that deals with the study of
interaction of electromagnetic radiation with matter.

6
 Light
 Light is electromagnetic radiation

 Electromagnetic radiation is a form of energy that

has both Wave and Particle Properties (Dual nature
of light).

7
 Wave Properties of Electromagnetic Radiation

Electromagnetic waves are consists of an

oscillating electric field (E) & an oscillating
magnetic field (M) which are perpendicular to
each other.

8
 Some properties of electromagnetic waves

wavelength () ً‫الطول الموج‬

 It is the distance between any two consecutive
maxima or minima of an electromagnetic wave
 The distance between two consecutive peaks

9
 Wavelength units
ً‫وحدات الطول الموج‬

Units: m, cm, m, nm or Å

micrometer Angstrom
Meter ‫انجستروم‬
Centimeter ‫مٌكرومتر‬
‫متر‬
‫سنتمتر‬ Nanometer
‫نانومتر‬
1m=100cm=106µm=109nm=1010Å

Å=10-10m=10-8 cm=10-4 µm=10-1nm

10
 Frequency () ‫التردد‬
 Number of complete oscillations (complete
cycle) that the waves makes each second
 the number of cycles which pass a fixed point in
space per second

11
Frequency () units ‫وحدات التردد‬

Hertz (Hz) ‫هٌرتز‬

‫او‬
 second-1 (s-1) 1- ‫ثانٌة‬

1 megahertz (MHz) = 106s-1 = 106Hz

Relation between wavelength and frequency

Wavelength is inversely proportional to frequency

‫الطول الموجً ٌتناسب عكسً مع التردد‬
α
1

• The Higher the Frequency the Shorter the Wavelength .
• The Longer the Wavelength the Lower the Frequency. 12
 Relation between velocity (speed) of light,
wavelength and frequency
‫العالقة بين سرعة الضوء و الطول الموجي و التردد‬

The velocity of light (c), is given by the equation:

C=  
Velocity of light (c) is constant
1. At wavelength  2. At wavelength  unit
unit is cm and is m and frequency
frequency  unit is s-1  unit is s-1 or Hz or
or Hz or cycles/sec cycles/sec
Velocity of light Velocity of light
c = 3.00x1010 cm/s c = 3.00x108 m/s
13
 Amplitude
 Maximum height of a wave in the electric field

Wave number
ً‫الرقم الموج‬
 Is the number of waves per cm
 Is the reciprocal of wavelength
unit
=1/ = /c cm-1 14
 Example
 Calculate the wave length of a wave of frequency
1011Hz travelling with a speed of light

 = C/ 

 unit, m = 3.00x108/1011=3.00x10-3m

 unit, cm = 3.00x1010/1011=3.00x10-1cm

15
 Example
 Calculate the wave number of a wave of
frequency 1011Hz travelling with a speed of light

=1/ = /c
= 1011/3.00x1010=3.33cm-1

16
 Particle Properties of Electromagnetic Radiation

Electromagnetic radiation consists of a beam of

energetic particles called photons
Photon: A particle of light carrying an amount of energy
equal to h
 The energy of a single photon, in joules, is related
to its frequency, wavelength, or wavenumber by
the following equations
E =h  - frequency
= h c/   - wavelength
= hc -wavenumber
 h – Planck‘s constant =6.63x10-34 J s photon-1 17
Therefore: Energy is directly proportional to frequency
is inversely proportional to wavelength

the energy of 1 mol of photons is Planck‘s equation

multiplied by Avogadro‘s number to its wavelength,
frequency and wavenumber by
E = NAh
h – Planck‘s constant =6.63x10-34 J s photon-1
NA - Avogadro‘s number = 6.022x1023 photon mol-1

18
 Example

Calculate the energy, in joules, of a photon of violet light

that has a frequency of 6.15x1014 s-1 (Planck‘s constant =
6.63x10-34 J s photon-1)

E =hυ
E= 6.63x10-34 J s photon-1 x6.15x1014s-1
E = 4.07x10-19 J photon-1

19
 Example
A laser products red light of wavelength 632.8 nm .
Calculate the energy, in kilojoules, of 1 mol of photons
of this red light (Planck‘s constant =6.63x10-34 J s photon-1
and Avogadro‘s number = 6.022x1023 photon mol-1 )

 = 632.8x10-9= 6.328x10-7m
= c/  = 3x108 /6.328x10-7 = 4.738x1014s-1
E = NAh
E= 6.022x1023 x 6.63x10-34 x4.738x1014 =1.891x105 J/mol
= 189.1 KJ/mol

20
 Questions

a. Complete the following

1. Electromagnetic radiation has both ………..

and ……….Properties .

2. Light waves are consists of an ……… field

and a ………………. field.

3. Å= ……. nm
21
4. Wavelength is inversely proportional to….
5. Energy is directly proportional to …… …and
inversely proportional to………

b. Define the following

1. Wavelength
2. Frequency

c. The wavelength of a certain wave is 6m and

30 waves pass a fixed point per minute, what is
the speed of the wave.
d. calculate the frequency of an X ray that has
a wavelength of 8.21 nm. 22
e. calculate the frequency, in hertz, of a
microwave that has a wavelength of 1.07 mm
f. calculate the wavelength, in nanometers,
of infrared radiation that has a frequency of
9.67x1013Hz
g. express the wave length 2500 Å in
micrometers and nanometers.
h. convert the wavelength 4000 Å into
frequency (Hz) and into wave numbers
(cm-1)

23
 k. The lower wavelength limit of visible light
is about 400nm. What is the energy of this
radiation, expressed in kilojoules per mole of
photon ((Planck‘s constant =6.63x10-34 J s
photon-1 and Avogadro‘s number =
6.022x1023 photon mol-1 )

24
 Electromagnetic radiation

Electromagnetic radiation is classified into:

1. Gamma rays
2. X-rays
3. Ultraviolet radiation
4. Visible light
5. Infrared radiation
6. Microwaves
7. Radio waves
25
 The Electromagnetic Spectrum
The division of electromagnetic radiation on the basis of a
photon‘s energy

26
 Wavelength range
Region (approx.)
Gamma radiation (< 0.01 nm)
X-ray radiation (0.01 nm -10 nm)
Vacuum ultraviolet radiation 10 nm - 200 nm

Ultraviolet radiation (185 nm – 380 nm)

Visible radiation (380 nm - 780 nm )

Infrared radiation (780 nm – 1mm)

Microwave radiation (1 mm – 10cm)
Radio waves ( 10cm - 10m)

27
l. Arrange the following in order of
increasing wave length and frequency?
Uv, IR and Visible
If you know the energy increase in the
following sequence : UV > Vis > IR

28
Division of visible radiation region

29
Color absorbed determines the color observed

For example
dichromate ions
absorbs blue light, so
their solutions appear
orange.

Permanganate ions
absorb green light, so
permanganate
solutions are purple.

30
 Principles of Spectroscopy

 The principle is based on the measurement of spectrum

of a sample containing atoms / molecules.

 Spectrum is a graph of intensity of absorbed or emitted

radiation by sample verses frequency (ν) or wavelength (λ)
or wavenumber .

 Spectrometer is an instrument design to measure the

spectrum of a compound.

31
 Spectroscopy
 It is used to study atomic and molecular structure
So spectroscopy is conveniently divided into:

Atomic spectroscopy Molecular spectroscopy

- Used for elemental analysis is the study of absorption
- Deals with the absorption and emission of light by
and emission of radiation by molecules
atoms Examples
- Deals with free atoms - Molecular absorption spectroscopy
- Molecular emission spectroscopy
Examples - Molecular Fluorescence Spectroscopy
Atomic absorption spectroscopy - Nuclear Magnetic Resonance (NMR)
Atomic emission spectroscopy - UV-VIS
Atomic fluorescence spectroscopy - IR
- MS 32
 Spectroscopy is conveniently divided into two broad
classes:

In one class of techniques:

energy is transferred between a photon of electromagnetic
radiation and the analyte.

In the second broad class of spectroscopy:

the electromagnetic radiation undergoes a change in amplitude,
phase angle, polarization, or direction of propagation as a result
of its refraction, reflection, scattering, diffraction, or dispersion
by the sample.

33
 Spectroscopies Involving an Exchange of Energy

Luminescence Emission Absorption

spectroscopy spectroscopy spectroscopy

34
 a. Absorption Spectroscopy

An analytical technique which concerns with the measurement

of absorption of electromagnetic radiation.
Note that: At room temperature the molecules are in the
lowest energy levels E0.

In absorption spectroscopy

 The energy carried by a photon is

absorbed by the analyte,
promoting the analyte from a
lower-energy state to a higher-
energy, or excited, state

35
 The intensity of photons passing through a sample containing
the analyte is attenuated because of absorption.

 The measurement of this attenuation, which we call

absorbance, serves as our signal.

Absorbance (A)
 The attenuation of photons as they pass through a sample

 Is based on the amount of light absorbed by the sample

 Absorption only occurs when the photon‘s energy matches

the difference in energy, DE, between two energy levels.

36
 A plot of absorbance as a function of the photon‘s energy is
called an absorbance spectrum

Ultraviolet/visible absorption
spectrum for bromothymol blue

Absorbance or absorption spectrum

A graph of a sample‘s absorbance of electromagnetic
radiation versus wavelength (or frequency or wavenumber).
Or is a plot of the amount of light absorbed by a sample as a
function of wavelength

37
hv

I0 (radiant intensity) I (transmitted intensity)

Higher energy state
Excited state or

hv

Ground state or lower energy state

38
 Molecular Energy Levels
Molecules have three types of energy levels:
I. Electronic energy levels
II. Vibrational energy levels
III. Rotational energy levels
I) electronic energy levels
 At room temperature the molecules are in the lowest
energy levels E0.

 When the molecules absorb UV-visible light from

electromagnetic radiation, one of the outermost bond /
lone pair electron is promoted to higher energy state such
as E1, E2, …En, etc is called as electronic transition and
the difference is as:
∆E = hν = En - E0 where (n = 1, 2, 3, … etc)
39
 II) Vibrational energy levels

 These are less energy level than electronic energy

levels.

 The spacing between energy levels are relatively small

i.e. 0.01 to 10 kcal/mole.

 e.g. when IR radiation is absorbed, molecules are

excited from one vibrational level to another or it vibrates
with higher amplitude.

40
 III) Rotational energy levels

 These energy levels are quantized & discrete.

 The spacing between energy levels are even smaller than
vibrational energy levels.

Note that
 Total excitation energy of molecules:
Etotal=Eelectronic+Evibrational+Erotational

 Energy sequence

∆Erotational < ∆Evibrational < ∆Eelectronic

41
Molecular Energy Levels

42
 Molecular Energy Levels

Excited
Electronic
state

Vibrational
Rotational Energy Levels
Energy Levels

Ground
Electronic
state

Rotational Vibrational Electronic

Transition Transition Transition

1-20 cm-1 2000-4000 cm-1 10000-50000 cm-1

Microwave Infrared radiation UV-Visible radiation

43
 Final Conclusion

What happened to molecule in:

 The visible and ultraviolet regions, the transitions occur
between electronic states accompanied by vibrational and
rotational changes
 The infrared region, transitions between vibrational states
take place with and without transitions between rotational
states and to promote bond stretching

Symmetrical Asymmetrical
Scissoring (Bending)
stretching stretching

Rocking Wagging Twisting

44
 The microwave region, transitions in molecules with unpaired
electrons and transitions between rotational states take place

The far ultraviolet and X-ray regions, transitions occur that

can ionize or dissociate molecules

45
 b) Emission spectroscopy

Emission of a photon occurs when an analyte in a higher-

energy state returns to a lower-energy state
Higher energy state

hv
Excited state or

hv

hv hv hv

Ground state and lower energy state

Emission
The release of a photon when an analyte returns to a lower-
energy state (or ground state) from a higher-energy state (or
excited state).
46
 The higher-energy state (Excitation) can be achieved in
several ways, including thermal energy, radiant energy from
a photon, or by a chemical reaction.
 Emission following the absorption of a photon is also called
photoluminescence.
 Emission following a chemical reaction is called
chemiluminescence.
 Radiating particles produced
radiations of a definite wavelength
producing discontinuous or line
spectra
 A graph of emission intensity
versus wavelength (or frequency
or wavenumber) is called
emission spectrum A typical photoluminescence emission
spectrum of the dye coumarin 343
47
48
 c) Scattering spectroscopy:

if the incoming radiation strikes with the solid particles

suspended in the solution, the light transmitted at an angle
other than 1800 from the incident light. This spectroscopy is
called scattering spectroscopy.

49
 Principles of
Ultraviolet and Visible (UV-Vis)
Absorption Spectroscopy

50
Ultraviolet and visible (UV-Vis) absorption spectroscopy is
the measurement of the attenuation of a beam of light after
it passes through a sample.
 Ultraviolet and visible (UV-Vis) spectroscopy is also known
as electronic spectroscopy because it involves the
transition of electrons from a lower (ground state) to
higher (exited state) energy level or vice versa by
absorption or emission of radiations falling in the UV-
Visible range.
 Absorption measurements can be at a single wavelength
or over an extended spectral range
 A molecule absorb ultraviolet or visible radiation of
frequency (), the electron in that molecule undergo
transition from lower to higher energy level.
The energy can be calculated by the equation,
E= h
51
 The UV radiation region extends from 10 nm to 380 nm and
the visible radiation region extends from 380nm to 780 nm.
Near UV Region: 180 nm to 380 nm
Far UV Region: below 180 nm
 Far UV spectroscopy is studied under vacuum condition

Ultraviolet and visible absorption spectra arise from

transition of electron with in a molecule from a lower (or
ground state) level to a higher (or excited state) level.

52
Electronic
Transitions
53
 Electronic spectra and molecular structure

The electronic transitions that take place in the visible and

ultraviolet regions of the spectrum are due to the absorption
of radiation by specific types of groups, bonds, and functional
groups within the molecule.

Electrons in a molecule can be classified into four different

types:
1. Closed-shell electrons
2. Covalent single-bond electrons
3. Paired nonbonding outer-shell electrons
4. Electrons in π (pi) orbitals
54
1- Closed-shell electrons that are not involved in bonding.
These have very high excitation energies and do not
contribute to absorption in the visible or UV regions.

2- Covalent single-bond electrons (σ, or sigma electrons).

These have high an excitation energy to contribute to
absorption in the visible or UV radiation (e.g., single valence
bonds in saturated hydrocarbon, —CH—CH2—).
55
3- Paired nonbonding outer-shell electrons (n electrons)
such as those on N,O,S and halogens.
These are can be excited by visible and UV radiation
1s22s22p63s23p5

4- Electrons in π (pi) orbitals (π bonds),

for example, in double or triple bonds
These are the most readily excited and are responsible for a
majority of electronic spectra in the visible and UV regions.

56
 Molecular orbitals:
1- Bonding orbitals :σ , π 2- Non-bonding orbitals: n
3- Anti-bonding orbitals: σ* , π*
The possible electronic transitions can graphically shown as:

57
The possible electronic transitions are

1 • σ → σ* transition
2 • π → π* transition

3 • n → σ* transition

4 • n → π* transition

5 • σ → π* transition

6 • π → σ* transition
58
 1 • σ → σ* transition

σ electron from orbital is excited to

corresponding anti-bonding orbital σ*.

 The energy required is large for this transition.

 e.g. Methane (CH4)

 has C-H bond only

 can undergo σ → σ* transition

 shows absorbance maxima at 125 nm.

59
 2 • π → π* transition

 π electron in a bonding orbital is excited to

corresponding anti-bonding orbital π*.

 Compounds containing multiple bonds like

alkenes, alkynes, carbonyl, nitriles, aromatic
compounds, etc undergo π → π* transitions.

 e.g. Alkenes generally absorb in the region 170

to 205 nm.

60
 3 • n → σ* transition

 Saturated compounds containing atoms with

lone pair of electrons like O, N, S and halogens
are capable of n → σ* transition.
 These transitions usually requires less energy
than σ → σ* transitions.
 The number of organic functional groups with n → σ*
peaks in UV region is small (150 – 250 nm).

 =190  =201  =237

61
 4 • n → π* transition
 An electron from non-bonding orbital is promoted
to anti-bonding π* orbital.
 Compounds containing double bond involving
hetero atoms (C=O, C≡N, N=O) undergo such
transitions.
 n → π* transitions require minimum energy and
show absorption at longer wavelength around 300
nm.

62
 5 • σ → π* transition
& • π → σ* transition 6
• These electronic transitions are forbidden transitions &
are only theoretically possible.

• Thus, n → π* & π → π* electronic

transitions show absorption in
region above 200 nm which is
accessible to UV-visible
spectrophotometer.
• The UV spectrum is of only a few
broad of absorption.
63
64
CHROMOPHORE CONCEPT

It is a Greek word.

Chroma = ―color‖ & phoros = ―bearer‖

65
 Chromophore

 Any Functional group which is responsible for imparting

colour to the compound is called as chromophore.
Ex: NO2
 Covalently unsaturated groups responsible for the
imparting of the colours.
Ex: N=O, C=O, C=N, C≡N, C=C, C=S

 The functional groups containing multiple bonds capable of

absorbing radiations above 200 nm due to n → π* & π → π*
transitions
A molecule containing a chromophore is called a chromogen
66
 Types of Chromophores

a) Independent chromophore
One chromophore is sufficient to import color to the compound
e.g. Azo group
4-hydroxyazobenzene

b) dependent chromophore
When more than one chromophore is required to produce color

e.g. acetone having one ketone group is

colorless
where as Diacetyl having two ketone group is
yellow.
67
 Auxochrome
 It is the group which itself does not act as a chromophore
but when attached to chromophore it shifts the absorption
maximum towards longer wavelength along with an
increase in intensity of adsorption.
 It is a saturated group containing unshared electrons
e.g. OH, NH2, Cl etc.
The functional groups attached to a chromophore which
modifies the ability of the chromophore to absorb light,
altering the wavelength or intensity of absorption.
OH NH2

Benzene λmax = 255 nm Phenol λmax = 270 nm Aniline λmax = 280 nm

68
 Types of auxochrome

a. Bathochromic groups

 Those groups which deepen the colour of chromogen are

called bathochromic groups.
 Deepening of colour means displacement to longer
wavelength
b. Hypsochromic group

 Those groups which diminish or lighten the colour of

the chromogen are called hypsochromic groups.

 They cause displacement to shorter wavelength

69
70
 1 • Bathochromic Shift (Red Shift)

 When absorption maxima (λmax) of a compound shifts to

longer wavelength, it is known as bathochromic shift or
red shift.

 The effect is due to presence of an auxochrome or by the

change of solvent

 e.g. An auxochrome group like –OH, -OCH3 causes

absorption of compound at longer wavelength.

71
 In alkaline medium, p-nitrophenol shows red shift.
Because negatively charged oxygen delocalizes more
effectively than the unshared pair of electron.

- -
O + O O + O
N N

-
OH

Alkaline
medium -
OH O

λmax = 255 nm λmax = 265 nm

72
 • Hypsochromic Shift (Blue Shift)

 When absorption maxima (λmax) of a compound shifts to

shorter wavelength, it is known as hypsochromic shift
or blue shift.
 The effect is due to presence of an group causes
removal of conjugation or by the change of solvent.
 Aniline shows blue shift in acidic medium, it loses
conjugation. + -
NH2 + NH 3 Cl
H
Acidic
medium

λmax = 280 nm λmax = 265 nm

73
 3 • Hyperchromic Effect

 When absorption intensity (ε) of a compound is

increased, it is known as hyperchromic shift.

 If auxochrome introduces to the compound, the

intensity of absorption increases.

N N CH3
Pyridine 2-methyl pyridine
λmax = 257 nm λmax = 260 nm
ε = 2750 ε = 3560
74
4 • Hypochromic Effect

 When absorption intensity (ε) of a compound is

decreased, it is known as hypochromic shift.

CH3

Naphthalene 2-methyl naphthalene

ε = 19000 ε = 10250

75
 Shifts and Effects
Hyperchromic shift

Blue shift Red shift

Absorbance ( A )

Hypsochromic Shift Bathochromic Shift

Hypochromic shift

λmax
Wavelength ( λ )
 Factors affecting the position of the spectrum (max)
and intensity of absorption of radiation

a. Conjugation

 Conjugation occurs when you have systems containing

alternate single and double bonds
The conjugation of double or triple bonds lead to
increased absorptivity and determine the shift of λmax to
higher wavelengths (bathochromic shift):

77
Conjugation = most efficient means of bringing about a
bathochromic and hyperchromic shift of an unsaturated
chromophore:

78
79
 b. The effects of substitution

 Placing a substituent (auxochrome) on a Chromophore may

produce change in λmax by two mechanisms: introduction of
an entirely new transition and/or shifting the wavelength of
existing transitions.
 Because auxochrome inters into resonance interaction with
the chromophore , thus increase the extent of conjugation,
shift the absorption maximum to longer wave length

Examples are hydroxyl groups, amino groups and halogens.

These possess unshared (n) electrons that can interact with
(π) electrons in the chromophore (n π conjugation).
80
 Examples

Benzene Chlorobenzene Phenol Aniline Thiophenol

81
Another example

82
 c. Effect of solvent

Solvent is the important factor for u.v./visible spectroscopy.

 The solvents used in any spectroscopic method should

ideally be transparent (non-absorbing) to the
electromagnetic radiation being used

 Usually solvents which do not contain conjugated systems

are most suitable for this purpose, although they vary as to
the shortest wavelength at which they remain transparent
to ultraviolet radiation.

83
  Ideal solvent :
 It should be cheaper.
 It should be easily available.
 It should be transparent & less polar
 Should not possess any kind of absorption when it is
exposed to radiation. nm

 Most commonly used

solvent is 95% ethanol
 Commercial ethanol is not
used because it is having
benzene which absorbs
strongly in u.v.region.

84
 The position as well as the intensity of absorption
maximum get shifted for particular chromophore by
change in the polarity of solvent.

 The λmax for the non-polar compounds is usually shifted

by change in polarity of solvent.

 π π* transitions leads to more polar excited state that is

more easily stabilized by polar solvent associations (H-
bonds). The π* state is more polar and stabilized more in
polar solvent relative to nonpolar one, thus in going from
nonpolar to polar solvent there is a red shift or
bathochromic shift (increase in λmax, decrease in ΔE).

85
 For n π* transition, the n state is much more easily
stabilized by polar solvent effects (H-bonds and
association), so in going from nonpolar to polar solvent
there is a blue shift or hypsochromic shift (decrease in λmax,
increase in ΔE).

86
a, b-Un saturated carbonyl compounds - For increased
solvent polarity

In general,
a) If the group (carbonyl) is more polar in the ground state
than in the excited state, then increasing polarity of the
solvent stabilizes the non-bonding electron in the ground
state due to hydrogen bonding. Thus, absorption is
shifted to shorter wave length.
b) If the group (carbonyl) is more polar in the excited state,
the absorption is shifted to longer wavelength with increase
in polarity of the solvent which helps in stabilizing the non-
bonding electrons in the excited state.

87
Absorption characteristics of 2-methylpyridine

Solvent max max

Hexane 260 2000
Chloroform 263 4500
Ethanol 260 4000
Water 260 4000
Ethanol - HCl (1:1) 262 5200

88
 d. Effect of geometry

Trans-stilbene absorbs at a longer wavelength than cis-

stilbene due to steric effects. Co-planarity is needed for the
most effective overlap of the π-orbitals. The cis-isomer is
forced in to a non planar conformation due to steric effects.
The cis isomer are twisted slightly out of plane by steric
interactions so that the degree of conjugation in the π system
is slightly less than the trans isomers, resulting in greater
energy for the transitions.
89
 e. Effect of pH
 Protonation/deprotonation can alter the degree to which non-
bonding electrons are involved in π systems
 The spectra of compounds containing acidic (phenolic-OH) or
basic (-NH2) groups are dependent on the pH of the medium.
Example: Phenol
.. ..
 The U.V spectrum of phenol in acid :OH :O -
medium, benzenoid form while in alkaline -
medium is the phenate anion ,quinonoid OH +
+ H
form +
H
 The free pair of electrons of oxygen
increasing the electron localization of the
-electrons, leading to the formation of
conjugated system. O
 So , electrons become more energetic and
need less energy to be excited, therefore
absorb longer  bathochromic shift ; red shift)
-
with hyperchromic effect )
90
 Example: Aniline
 Its spectrum exhibits bathochromic shift and hyperchromic
effect in alkaline medium due to its conversion to the
quinonoid species
+ N H :N H 2
3

 While in acid medium O H

-
(anilinium) lost the free pair H
+
electrons of nitrogen Aniline
decrease the conjugation

+ N H 2

Its spectrum in acid medium

exhibit hypsochromic shift
-
and hypochromic effect
due to its conversion to the benzenoid species.

91
 Example: Benzoic acid

f. Effect of temperature:

Temperature provides more energy to ground state.

As a result energy required for excitation will be less, so there
is bathochromic shift

92
Fundamental Laws of Spectroscopy
 Transmittance and Absorbance
When a beam of light with the intensity I0 passes through a
sample
A portion of the incident light (I0 )
is absorbed as (Ia ), a negligible
portion of it is reflected (Ir) and
the reminder is transmitted (IT )
with net effect of intensities:
I0 = Ia + IT + Ir
I0 = intensity of incident radiation Ir= intensity of the reflected light

Ia= intensity of absorbed light IT= intensity of transmitted light

94
 However, in actual practice it has been observed that the
amount of reflected light (Ir ) is so small
I0 = Ia + IT

Transmittance (T)
Transmittance is defined as the ratio of the electromagnetic
radiation’s power exiting the sample, It , to that incident on the
sample from the source, I0
IT
T=
I0
I0 = Intensity of the incident light

IT = Intensity of the light transmitted through the sample

95
Percent transmittance
IT
%T = X 100
I0
Another definition of transmittance

 The fraction of original light not absorbed by the sample

 The ratio of the radiant power passing through a sample to

that from the radiation’s source (T).

96
 Absorbance (A)

Absorbance (A) is the relative amount of light absorbed

by the sample and is related to transmittance (T).
I0 IT
A = log = - log = - log T
IT I0

Example
A sample has a percent transmittance of 50.0%. What is its
absorbance?

With a percent transmittance of 50.0%, the transmittance of

the sample is 0.500
A = –log T = –log(0.500) = 0.301
97
 Lambert‘s Law
 When a monochromatic radiation is passed through a
solution, the decrease in the intensity of radiation with
thickness of the solution is directly proportional to the
intensity of the incident light.
Let Io be the intensity of incident radiation.
x be the thickness of the solution.
Then

dI
- aI So, - = KI
dI
dx dx
 Integrate equation between limit

I = Io at x = 0 and I = I at x=b
We get,
I I
ln = - Kb 2.303 log = -Kb
I0 I0
I K
log =- b
I0 2.303
I0
Where, log =A Absorbance
I
K
= Absorption coefficient A=  b
2.303
Lambert’s Law 99
 Beer‘s Law
When a monochromatic radiation is passed through a solution,
the decrease in the intensity of radiation with thickness of the
solution is directly proportional to the intensity of the incident
light as well as concentration of the solution.

Let Io be the intensity of incident radiation.

x be the thickness of the solution.
C be the concentration of the solution.
Then

- dI αC I So - dI = K C I
dx dx
100
 Integrate equation between limit

I = Io at x = 0 and I = I at x=b,
We get,
I I0
ln = -k b c 2.303 log = k b c
I0 I
I0 k
log = bc
I 2.303
I0
Where, log =A Absorbance
I
K Molar extinction
=ε coefficient
2.303
101
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 also called the molar
absorption coefficient or molar extinction coefficient

Therefore

A=ε b c Beer‘s Law

Absorbance is directly related to the analyte concentration,

cell pathlength and molar absorptivity

102
 Quiz
If the molar absorptivity of a colored complex is 3.20x103 at
240 nm, calculate the absorbance of a 5.0x10-5 M solution in
a 50mm cell when measured at a wavelength at 242 nm
where:

A = bc

A= 3.20x103x5x 5.0x10-5=0.800

103
The molar absorption coefficient of tyrosine in water is 1280
M-1cm-1 at 280 nm. Calculate the concentration of a tyrosine
solution in water if the absorbance of the solution is 0.34 in a
1 cm path length cell.

Ans: Given:
λ = 280 nm ε = 1280 M-1cm-1 b = 1 cm A = 0.34

From Beer-Lambert Law: A = εbc

A 0.34
c= c=
 b 1280x1
= 0.000265 M = 265 μM
104
A 5.00 x 10-4 M solution of an analyte is placed in a sample
cell that has a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the absorbance of the solution is
found to be 0.338. What is the analyte‘s molar absorptivity
at this wavelength?
A
 =
A=bc bc
0.338
 = = 676 M-1 cm-1
5.00 x 10- 4 x 1

105
 Quantitative analysis
a. Determination of Concentration When  is Known:

In this case,  = known, b= known

C= unknown
A = measured by spectrophotometer

For example, the cuvette is 1 cm,  = 3600 M-1cm-1, and

the absorption of solution measures 0.650, then you can
solve for c:

A = bc
0.650 = (3600 M-1cm-1)(1 cm)c
c = 1.80 x 10-4 M
106
b- one standard method (of the same substance)
Single-point calibration
Beer‘s law A = εbC

A1 = εbC1 (1) A1 and C1 are absorption and concentration of

standard

A2 = εbC2 (2) A2 and C2 are absorption and concentration of

unknown sample
A1 C1
From (1) and (2) =
A2 C2
Astan d ard Cstan d ard
=
Asamp le Csamp le
Cstan d ard
C sa mp le = Asamp le
Astan d ard
107
Question

A standard solution of Cu ions (0.045M) has an

absorbance of 0.67 What is the concentration
of Cu ions solution at absorbance of 0.98

108
c- Calibration curve

1- Measure the absorbance of several known

concentrations of the solution

109
3- Find the absorbance of unknown sample
4- From the calibration curve find the
concentration of unknown sample
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14 16 18 110
 Beers Law and Multicomponent Samples
‫قانون بٌٌر و عٌنات متعددة المكونات‬
Beer‘s law can be extended to samples containing several
absorbing components provided that there are no
interactions between the components.
Individual absorbances, Ai, are additive.
For a two-component mixture of X and Y, the total
absorbance, Atot, is
Atot = AX + AY = XbCX + YbCY

‫قانون بٌٌر ٌمكن ان ٌمتد لشمل عٌنات تحتوى العدٌد من المكونات التً لها قدرة‬
.‫على امتصاص الضوء بشرط اال ٌحدث تداخل بٌن المكونات‬
‫االمتصاص الخاص بكل مكون ٌمكن جمعه فمثال‬

111
Generalizing, the absorbance for a mixture of n
components, ATotal, is given as:

ATotal=A1+A2+A3……………+ An

ATotal=1b1c1+2b2c2+3b3c3……+ nbncn

b the same in all experiments

ATotal=1bc1+2bc2+3bc3……+ nbcn

112
 Validity of Beer Lambert Law

The law is valid under the following conditions:

1. The light used must be monochromatic , or
otherwise for two different wavelength (1 , 2 ) two
values of absorbance will be obtained.
‫الضوء المستخدم ٌجب أن تكون أحادٌة اللون‬

2. The law is not applicable for highly concentrated

solutions.

‫القانون غٌر مطبق فً حالة المحالٌل عالٌة التركٌز‬

113
3. If a dilute solution during measurement
undergoes chemical reaction such as
hydrolysis, association, dissociation or
polymerisation then the law is not valid.

‫إذا حدث للمحلول المخفف خالل عملٌة القٌاس تفاعل كٌمٌائً مثل التحلل‬
‫اواالتحاد اوالتفكك أو البلمرة فان القانون غٌر صالح‬

4. Solutions exhibiting stray fluorescence or

suspensions may not strictly adhere to Beer law

‫المحالٌل التً تظهر فلوره طائشة او تكون معلقات فأنها ال تلتزم بقانون بٌٌر‬

114
 Deviation from Beer Lambert Law
According to Beer‘s law, a relationship between absorbance
and the concentration of analyte in a series of standard
solutions should be a straight line passing through the
origin with a slope of b. In many cases, however,
calibration curves are found to be nonlinear
‫طٌقا لقانون بٌٌر فان العالقة بٌن االمتصاص و تركٌز‬
‫المادة المراد تحلٌلها فً سلسلة من التحالٌل القٌاسٌة‬
‫البد ان تكون خط مستقٌم ٌمر بنقطة االصل وله مٌل‬
‫ٌساوى معامل االمتصاص الموالرى و مسار االشعة‬
(‫داخل المحلول )عرض الخلٌة‬

‫فً بعض االحٌان منحنى المعاٌرة ٌكون غٌر خطى‬

115
These deviations from the Beer Lambert law can be
classified into three categories:

1. Fundamental (Real) deviations ‫االنحرافات االساسٌة‬

2. Chemical Deviations ‫االنحرافات الكٌمٌائٌة‬

3. Instrument Deviations ‫االنحرافات اآللٌة‬

116