Spectrophotometry 1

Quantitative and Qualitative

Spectrophotometry

by
Dr. Ibrahim A. Naguib

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 Spectrophotometry 2

What is spectroscopy?
Spectroscopy is the study of interaction of spectrum of
light with the analyte, for its qualitative identification and
for quantitative determination of its concentration.

What is spectrophotometry?
Spectrophotometry is the quantitative measurement of the
reflection or transmission properties of a material as a
function of wavelength. It is more specific as it deals with
visible light, near-ultraviolet, and near-infrared.

What is the difference between Light and

electromagnetic radiations EMR?
Light is one segment of the continuous spectrum known as
EMR. EMR includes sunlight, radio waves, cosmic waves and
X-rays…etc, which have similar properties and are called
electromagnetic radiations (EMR) due to presence of both
electric and magnetic components.

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What is the Dual nature of light (Dualism)?

When it propagates; light exhibits wave property and
energy particle during its interaction with matter. The
double nature of light (being waves and particles in nature)
is known as dualism:

(1). Wave property

EMR display the property of continuous waves and can be
described by the characteristics of wave motion. Such wave
motion in conveniently classified according to the
wavelength.

So what is the Wavelength (λ)?

It is the linear distance between crest of one wave to the
next.

Units of wavelength
- Micron (µ) = 1 xl0-6 m = 1 x 10-4 cm = 1 x 10-3 mm.
- Millimicron (mµ) = nanometer (nm) = 1 x 10-9 m = 1 x
10-7 cm = 1 x 10-6mm.
- Angstrom (AO) = 1 x 10-l0 m = 1 x 10-8 cm = 1 X 10-7 mm.
- µ = 1000 mµ

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Velocity of light {C}

Light propagates at the highest known velocity= 300, 000
Km/sec. All kinds of waves propagate at the same speed
(they differ in wavelength and frequency).
C= λ . 
EMR may be visible or invisible
a) Visible radiations form only a small part of the complete
EMR, (from 400-800 nm). They are subdivided into
different colors according to their wavelength (i.e. it is
polychromatic). Each Definite color is described as
monochromatic light.
b) Invisible radiations: which are Ultraviolet (U.V.) and
Infrared (I.R)... Etc.

EMR have the property of frequency which can be

expressed by:

a) Frequency [ (nu)]: is the number of waves/ second,

cycles / second (CPS), or Hertz (Hz).
Relation between λ & υ
C= λ .  or  = C/ λ

b) Wave number (δ): is the number of waves/ cm

δ = 1/ λ

(2). Particle property

Max Planck presented light as matter (with particulate
nature) that forms of packets of energy known as
(photons). The energy (E) of photons is variable. It is
proportional to the frequency i.e related to C and λ.

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 Spectrophotometry 5

It can be expressed by max plank relation:

E = h υ (h = Max Plank constant = 3.63 x 10-27 erg. sec.)
i.e Eαυ
or E α 1/ λ α δ

Accordingly, energy of EMR increases as wavelength

decreases.
e.g. U.V range contains shorter λ, hence having more energy
than visible range, and visible range more than I.R range.
i.e the shorter the wave length, the greater the energy of
the photons and the more powerful the radiation.

The regions of I.R, visible and U.V radiations are used for
analytical purposes usually.
Day light (visible radiation) consists of colored radiations,
which are, red, orange, yellow, green, blue, indigo and violet
(ROYGBIV).
If we observe the visible radiations of all wavelengths, we
see white light (remember the color wheel!).
A beam containing several wavelengths is called
polychromatic light, while a beam single wavelength is said
to be monochromatic.

Composition of spectrum of EMR can be represented

diagramatically as follows:

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Spectrophotometry 6

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 Spectrophotometry 7

Radio waves Microwaves I.R VIS U.V x-rays

Visible range
I.R. Red Orange Yellow Green Blue Indig Violet U.V
o

800nm 400nm
decrease

increase
E

Interaction of a molecule with EMR

Q. what happens when radiant energy is absorbed
by a molecule?

* this results in gaining of energy by the molecule.

* a molecule may absorb energy in three ways:
1- by raising electrons to a higher energy level (transitional
energy), when the molecule absorbs light in visible and U.V
region.
2- by raising the vibration of the constituent nuclei
(vibrational energy), when the molecule absorb light in I.R
region.
3- by increasing rotation of the molecule around the axis
(rotational energy), when the molecule absorbs light in F.I.R
region.

The relative energies of transitional, vibrational and

rotational are roughly in the order of 10000: 100: 1.

When a molecule interacts with radiant energy in the

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 Spectrophotometry 8

visible and U.V region, it gains energy that leads to

displacement of an outer electron (valence electron) and
the molecule is said to be excited because electron
undergoes transition from original energy level (ground
state = Eg) to an excited state (Es).

The energy of transition can be represented by the

following equation:
ΔE = Es – Eg = h υ

Where h is Max Planck constant and υ is the

frequency of EMR at which the transition take
place, which is characteristic for each molecule.

Excited state Es

Ground state Eg

N.B
Absorbing large amount of energy (from far U.V region),
which is sufficient to exceed the energy of the formation
of certain bonds, may lead to bond rupture and new
compounds being formed. This phenomenon is described as
photolysis.

Emission of radiant energy.

In all cases, absorbed energy doesn't accumulate in
electronic system of a molecule. If an excited electron
returns to the ground state, it may lose absorbed energy in
the form of heat, light or molecular collision.

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 Spectrophotometry 9

If the excited electron returns directly to the ground

state heat is evolved.
When excited electron returns to the ground state via
second excited state, light is emitted as f1uorescence or
phosphorescence (see later).

The relation between spectra and chemical structure

The absorbance of EMR in the U.V-VIS regions depends
on structure of organic molecule (i.e. upon the number and
arrangement of the electrons in organic molecule).
Absorbance of EMR by organic molecule is achieved by
chromophoric groups (chromophores) assisted by
auxochromes, where the electron of absorbing molecule
gets excited, (i.e. when it undergoes transition from the
ground state to the excited state).

What is a Chromophore?
It is unsaturated organic group responsible for electronic
absorption e.g.

C C C O , N O
,

What is an Auxochrome?
It is a saturated organic group which when attached to a
chromophore, changes both the λ and intensity of
absorption maxima e.g. -OH, - NH2, - Cl.
Notice: All auxochromes have one or more non-bonding pair

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 Spectrophotometry 11

of electrons. If an auxochrome is attached to a

chromophore, it helps extending the conjugation by sharing
of non-bonding electrons.

Types of electronic transition

The outer electrons in an organic molecule may occupy one
of three different energy levels; namely:
1) Sigma (ζ) electrons: They are bonding electrons which
represent valence bonds formed due to linear overlapping
of electronic clouds of S or SP orbitals. They have the
lowest energy level (i.e. they are the most stable).
2) Pi (π) electrons: they are the bonding electrons
constituting the pi bonds (double bonds) and result from
lateral overlap of electronic clouds of P orbitals. They are
of higher energy than sigma electrons.
3) Non-bonding (n) electrons: they are of atomic ortbitals of
hetero atoms (N, O, X or S) which don't participate in
bonding. n electrons occupy the highest level of ground
state.
In excited state ζ electrons occupy an antibonding
energy level denoted as ζ* and the transition is termed ζ -
ζ * transition.
π electrons occupy the antibonding π* level, while
n electrons occupy either π * or ζ*.
The absorption spectra of organic molecules depend on
the electronic transitions occurring.
The following figure represents the different
electronic energy levels and different types of transition
which may occur:

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excited state *
*

n
ground state 


σ-Absorption:
Compounds containing only ζ-electrons are the saturated
hydrocarbons which absorb at < 170nm (i.e. in the far UV
region). They are transparent in the near UV (200-300nm),
which makes of them ideal solvents for other compounds to
be studied in this region.

n-electrons absorption in saturated compounds:

For saturated compounds containing heteroatoms (S, N, O
or halogens), the majority of these compounds show
absorption in the near UV region e.g. Methanol at 177nm,
triethylamine at 199nm and chloroform at 173nm.
Alcohols and ethers absorb at wavelength shorter than
185nm and so they are useful as common solvents at >
200nm (that will help in quantative analysis of drugs later).
However, their intense absorption usually extends to the
edge of the near UV region (Cut off wavelength) in the
200-220nm region.

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 Spectrophotometry 12
Far UV
near UV

Cut off 
max of
solvent

200 220



Absorption spectra
Absorption spectrum is the plot of Absorbance (A) as a
function of wavelength (λ). Shape of absorption spectrum
depends on electronic transitions that occurs in each
organic molecule.
Absorption spectrum characterizes each molecule. It has
characteristic shape which shows the λ of maximum
absorbance (λ max).
λ max is characteristic for each molecule, therefore it is
used for identification of a chemical substance (i.e.
qualitative analysis).
Also λmax is used for quantitative measurement, in order to
increase sensitivity and to minimize error of the analytical
method.

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 Spectrophotometry 13

N.B
Sometimes, absorption spectra may show a shoulder or
even no absorption characteristics.

Shifting of λmax
This feature usually happens due to change in configuration
of chromophore or auxochrome groups.
Types of shifting;
a- Bathochromic shift (or red shift)
is the shift of λmax to a longer wavelength due to:
- General increase in conjugation e.g. when two or more
chromophores are present in conjugation.
- Substitution with certain functional groups (e.g. -OH and -
NH2)
- Effect of the medium (solvent). For example, decreasing
polarity of solvent causes a red shift in n-π* transition of
carbonyl compounds.
b- Hypsochromic shift (or blue shift)
is the shift of λmax to a shorter wavelength due to removal
of conjugation e.g. by changing polarity of the solvent.
c- Hyperchromic effect (or shift)
is increase in the intensity of absorption. It is usually
induced by introduction of an auxochrome to the compound.

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e.g. introduction of methyl group in position 2 of pyridine

increases absorption for π-π * transition.
d- Hypochromic effect (or shift)
It involves a decrease in the intensity of absorption. This
is induced by introduction of groups which are able to
distort the geometry of the molecule.

The presented diagram represents absorption and

intensity shift.

Factors affecting absorption spectra

1- Effect of pH on absorption spectrum
The spectra of compounds containing acidic (phenolic-OH)
or basic (-NH2) groups are dependent on the pH of the
medium. Phenol and aniline are typical examples.

Phenol
The U.V spectrum of phenol in acid medium is completely
different from its spectrum in alkaline medium. In acid
medium the benzenoid form is the predominant species;
represented as phenate anion.
The spectrum in alkaline medium exhibits bathochromic

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 Spectrophotometry 15

shift (red shift) due to participation of the pair of

electrons of oxygen in resonance with the π-electrons of
aromatic ring, thus increasing the delocalization of the π -
electrons, leading to the formation of conjugated system
(quinonoid structure); i.e. electrons become more energetic
and need less energy to be excited, therefore absorb
longer λ and hyperchromic effect is observed.

.. ..
:OH :O -

-
OH +
+ H
+
H

Aniline
Aniline behaves like phenol, i.e. its spectrum exhibits
bathochromic shift and hyperchromic effect in alkaline
medium due to its conversion to the quinonoid species, while
in acid medium its spectrum exhibit hypsochromic shift and
hypochromic effect due to its conversion to the benzenoid
species.

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 Spectrophotometry 16
+ NH3 :NH2

-
OH
+
H

+NH 2

2- Effect of redox reaction on absorption spectrum.

Oxidation of diphenylamine will convert the benzenoid
spectrum of diphenylamine to the quinonoid spectrum of
the oxidised form, as shown in the following equation.

H ox.
2 N
red.

H H +
N N + 2H + 2e

red. ox.

+
N N + 2H + 2e

Note:
Generally, increase in conjugation leads to increase in
absorbance of light by a compound, which appears colored
and exhibits hyperchromic effect.

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 Spectrophotometry 17

What is the isosbestic point?

On running U.V spectrum of known concentration of phenol
as a function of pH (i.e. at different pH). The spectrum will
be shifted to different λmax by changing the pH, but all
spectra intersect at certain λ which is known as isosbestic
point.
The isosbestic point is the wavelength at which the same
absorbance is given for the same concentration at different
pH (i.e. at this point absorbance is not pH dependent but
concentration dependent).

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 Spectrophotometry 18

Laws of light absorbance

When a monochromatic light having intensity (I0) is allowed
to pass through absorbing medium, some is absorbed (Ia),
reflected (Ir), transmitted (It), refracted (If) and scattered
(Is).
i.e. I0 = Ia + It + Ir + If + Is

Is = zero for clear solution, while

If and Ir may be canceled by means of control cuvette
containing the solvent in which the substance to be anaylsed
is dissolved.
Therefore, under experimental conditions:
I0 = I a + I t
Or Ia = I 0 - It

A) Bouguert -Lambert's law

When a monochromatic light enters absorbing medium, its
intensity is decreased exponentially with the increase of
thickness of the absorbing medium (i.e. solution) (b) at
constant concentration (C)
i.e. log I0 / It α b or log I0 / It = K b
Where K is proportionality constant.

Log I0 / It It
Io
It
C C
b b
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 Spectrophotometry 19

B) Beer's Law:
When a monochromatic light enters an absorbing medium,
its intensity is decreased exponentially with the increase of
the concentration of the absorbing medium, when (b) is
constant.
i.e log I0 / It α C or log I0 / It = KC

Log I0 / It It
Io
It

b b
C C

Beer-Lambert's law:
According to lamberts' law
log I0 / It α b (thickness or pathlength)
According to Beers' law:
log I0 / It α C (Concentration)
i.e. log I0 / It α bc or log I0 / It = abc
or A = abc
where: A = log I0 / It = absorbance
a is a constant, known as absorptivity which is
the absorbance, when thickness of solution is unity (i.e. l
cm) and concentration is unity.

Molar absorptivity or epsilon (ε)

If the unit of concentration is 1M, ‘a’ is known as molar
absorptivity or epsilon (ε) or molar extinction coefficient.

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(Unit of ε is L mol1- cm1-)

A (1% - 1cm): If unit of concentration is 1%, ‘a’ is known as
A (1 %, lcm).
- Absorptivity ‘a’, can be calculated from the slope of the
curve produced on plotting (A) as function of (C) at fixed
(b).
When (b) is 1 cm.
A = a C or a = A/C

- Both ε and A (1%, 1 cm) are characteristic for each

substance and are used for qualitative purpose.

A
Tan x = = a
C
A A = slope of the curve
x
C

b
C

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Colorimetry

Principle: in colorimetry we are measuring absorbance A of

‘visible’ radiation by a colored sample under study.
To be measured colorimetrically, the substance has
to fit one of three categories:
1. The substance to be measured must be colored e.g CuSO4,
KMnO4, organic dyes,… etc.
2. If the substance to be analysed is colorless, it must be
reacted first with certain reagent (known as chromogen) to
produce equivalent colored product e.g.
orthophenanthrolene which reacts with ferrous (Fe2+) in
buffered medium (acidic pH) to produce intense red color.

2+

N N
2+
3 + Fe Fe
N N

3. If the substance to be analysed is colorless and there is

no suitable chromogen, it must be converted to a certain
derivative which has a suitable chromogen. e.g. in
determination of ester, which is first converted to
hydroxamic acid derivative through the reaction with
hydroxylamine. Hydroxamic acid derivative gives purple
color on addition of ferric (Fe3+) due to the formation of
iron chelate.

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O O
R –C– OEt + H2N – OH R –C– NH – OH + EtOH
Hydroxamic acid derivative

What are the requirements for the colored product?

1. It should be of intense color, to increase the sensitivity of
measurement.
2. The reaction of color formation have to be rapid and
quantitative.
3. It should be unaffected by pH or the pH must be specified
and maintained by suitable buffer or the measurement is
carried out at λ of isosbestic point.
4. It should be stable with time.
5. The colored product, should obey Beer-Lambert's law, i.e.
on plotting A versus C at fixed b, we obtain straight line
passing through the origin.

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What are the requirements for ideal chromogen?

1. It should be colorless or easily separated.
2. The reaction to produce colored product should be of
known mechanism and stoichiometric.
3. The full development of color must be rapid.
4. Produces only one color of specified λmax.
5. It should be selective (i.e. reacts only with the substance to
be analysed).

Instrumentation

I. Visual methods: which are applied only in case of colored

samples. It is also called ‘visual colorimetric methods’.
II. Photo-electric methods: which are applied in both colored
and colorless samples . It is also called ‘photo-electric
colorimetric methods’.

I. Visual methods
Human eye is the detector in this case and its
sensitivity varies with wavelength. Long observations
weaken the eye sensetivity.

a. Standard series method. (using polychromatic light)

Principle: This method, based on the comparison of the
colored sample with standard series of colors.
Standard colors may be freshly prepared or a permanent
colored system.
For freshly prepared standards, matched Nessler tubes

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are used.
For permanent colored system colored disc, lovibond
comparator or sealed ampoules are used

b) Variable depth or balancing method

Principle: can be represented as follows:

According to Beer-Lambert's Law:

Log Io/It = A = a b1 Cl = a b2C2
or b1 Cl = b2 C2
or Cl /C2 = b2/b1
i.e. we Compare a sample with a solution of known
concentration of the same sample under investigation

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(standard solution) till the intensity of It emerging from

sample and standard solution are the same, then we
calculate the concentration of the unknown sample from the
equation above.

To apply this method we can use:

1)Duboscq-type colorimeter:

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 Spectrophotometry 26

It depends on using a dual matched optical system made

of glass plungers (p); the depth of their immersion bring
about changes in the thickness (b) of colored solutions of
the sample and standard. The intensity of emerging light
(It) is brought into a circular field of view (E) through
prism (R). The varying depths are measured by a vernier
(V).
Intensity of emerging light (It) is, proportional to
thickness (b) and concentration of the sample (C2) is given
by:
C2 = Cl (b1/b2)

2) Hehner tube: designed to withdraw the solution from the

more concentrated solution, until the color of the sample
and that of the standard solution are matched.

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 Spectrophotometry 27

II- Photo-electric Instruments using

monochromatic light
This method depend on the photoelectric phenomenon,
where, the intensity of EMR is measured through the
intensity of electric current produced by electrons
liberated from a photosensitive metal under the influence
of incident EMR.

The instrument used consists of 5 basic components

1. Radiant energy source (light source).
2. Dispersing system (or monochromator).
3. Sample compartment (cuvette).
4. Detector.
5. Recorder (meter).
The following figure represents schematic diagram of the
instrument.

Light source Monochromator Cuvette Detector Recorder

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1. Radiant energy source (light source)

- It must be of sufficient intensity
- It must cover the desired spectral range.
- In visible range, we must use tungsten lamp, and in U.V
range we must use deuterium lamp (D2) (or hydrogen lamp.)

2. Monochromator (Dispersing system)

- It converts polychromatic light to monochromatic light,
i.e. of definite range or λ.
- Monochromatic light may be obtained by one of the
following systems:
a)- Filters
They function by selective absorption of unwanted λ and
transmit the complementary color, which is needed to be
absorbed by the sample to be analysed.
Complementary colors can be represented by colored
wheel or the following table:

Colour Wavelength Complementary

() colour
Violet 400-435 Yellowish green
Blue 435-480 Yellow
Greenish blue 480-490 Orange
Bluish green 490-500 Red
Green 500-560 Purple
Yellowish green 560-580 Violet
Yellow 580-595 Blue
Orange 595-610 Greenish blue
Red 610-750 Bluish green

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Notes:
- If a substance absorbs all visible light it will appear black,
and if a substance doesn't absorb visible light, it will
appear colorless.

- Filters may be: gelatin, liquid and tinted glass.

- These types of filter transmit a wide band of 35-50 nm
which is not exactly monochromatic.
- A narrower band can be obtained by using interfering
filters, which consist of successive multiple layers of high
and low refractive index material (e.g. MgCl2 and Agº film).
Interference results in a narrower band of 10-17 nm.

b)-Prisms
- They act by refraction of light.
- Glass prism is used in visible range, while in U.V range
we use prism made of quartz or fused silica.

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R
O
Y
G
B
I
V

c)-Grating
This one acts by diffraction and interference.
- Grating consists of a large number of parallel lines ruled
very close to each other on a highly polished surface e.g
aluminum or aluminized glass (600 line/mm).
- Each ruled groove functions as a scattering center for
light rays falling on its edge.
- Through diffraction and interference, the grating
disperses the light beam into almost single λ.

Associated optics
Associated optics, are used to control light intensity e.g
collimating lenses which act as condensers,
slit of variable width; which helps to narrow band width,
mirrors and diaphragms for proper alignment of the beam.

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Note: All optical components should be suitable for the

spectral range, i.e. glass for visible-range and quartz or
fused silica for U.V range.

3. Sample compartment (Cuvette)

Cuvette is made of glass for visible range and quartz or
fused silica for U.V range. Its standard path length is 1cm
(10 mm) and sometimes it is 1/2 cm.

Transparent surface Obaque surface

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4. Light Detector
There are two types of detectors:
A) photocells (Photovoltaic cell) e.g Barrier layer
cell

Light falling on cell

Transparent metal
layer of Ago
(Collecting electrode)
-

Photosenitive
semiconductor of selenium
+
Metal base Plate of iron

Priciple: light falls on a semiconductor surface, where

electrons are excited and produce EMF (current)
proportional to the intensity of incident light.
N.B. This type requires no external source of power.

B) Phototube (photomultiplier or photoemissive tube)

In order to obtain greater sensitivity when the light
signal is weak, multiplication of the initial photoelectrons by
secondary emission; using several anodes arranged in

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gradually increasing potential is implemented. i.e. (greater

sensitivity is obtained through magnification of EMF
produced),
N.B. this type requires external source of power).

5. Recorder {meter}
The amplified electric signal produced in detector is fed
to a sensitive galvanometer, its scale is graded in
absorbance or/and transmittance units.

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Commercial instruments
1. Filter photo-electric colorimeter

2. Compensating two-photocell/ colorimeter

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In this type, fluctuations in source intensity of EMR

source are automatically cancelled due to the two
cell set up.

3. Prism spectrophotometer

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Deviations from Beer-Lambert's law

1) Real deviations
- This happens when solute molecule in concentrated
solution doesn't absorb radiant energy in the same manner
as does the same molecule in dilute solution????? why ?????
due to charge distribution, molecular interaction, and when
absorbing species are affected by complexation or
hydration,
All of that leads to non-linear response when A is plotted
against C.
- This deviation decreases or disappears in very dilute
solution.

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 Spectrophotometry 37

2) Instrumental deviations
This type of deviation may be irregular or regular
a. Irregular deviations, may result from:
1.Use of unclean optics (lenses, mirrors or lamp)
2.The use of unmatched cuvette (due to industrial defects)
3.Use of unclean handling (e.g finger print on the cuvette)

b. Regular deviations, may result from:

1) Error in λ scale.
2) Error in slit width control (maximum opening of slit
leads to non-specific λ).

3) Error in potentiometric reading of absorbance.

4) Non-linear response of detector which gives
calibration curve that doesn't pass through the origin.
5)Stray light:
- Stray light is any radiations or λ other than that absorbed
by the sample or any light that reaches the detector
without passing through the sample.
- It may result from, incorrect choice of filters, the
presence of fluorescent impurities, aging of mirrors and
light source.

N.B. Radio & T.V waves also interfere, hence avoid their
presence close to the spectrophotometer..

3) Chemical deviations
These include:
- effect of pH (which leads to shifting of λmax),

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 Spectrophotometry 38

- temperature effect and,

- time factor (the obtained color may fade by time due
to deterioration by oxidation, reduction, hydrolysis, etc ....
).

Applications

1.Qualitative analysis.
2.Quantitative analysis.
3. Determination of some physical constants.

1- Qualitative analysis
Absorptivity (ε or A (1% - lcm), λ max., U.V and visible
absorption spectrum usually give finger print of the sample
to be analysed, hence could be used for its identification.

2- Quantitative analysis
a. Quantitative analysis of a single component.
For quantitative determination of a single component we
should consider the following:
- The substance to be analysed is well dissolved in a
suitable solvent, e.g. methanol, ether, water, etc ...
- The solvent is used as blank (to cancel its interference if
any).
- Absorbance readings are taken in the expected range
(e.g. 200-400 nm for colorless samples and 400-800 nm
for colored samples).
- Detect λmax of the substance to be analysed after

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 Spectrophotometry 39

dissolving in a suitable solvent.

- Construct a calibration curve by plotting A against C at
fixed b using standard series of the same chemical present
in the sample, at the characteristic λmax.
- The Absorbance of the sample to be analysed is
determined under the conditions adopted during
construction of the calibration curve.
- The concentration of the sample can be determined from
the calibration curve.

Or mathematically:
A = abc
i.e C = A / (a b)

b. Quantitative analysis of multicomponent

mixture
Consider a mixture of X and Y. For their quantitative
analysis, the following has to be considered:
1.The absorption spectrum of X and Y should not show
sever overlap.
2.X and Y must be chemically inert to each other.

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 Spectrophotometry 41

3.Beer-Lambert's law must be obeyed for X and Y at their

characteristic λmax.

The following 2 equations can be obtained (at b = 1cm)

At 1 A1mix = A1x + A1y

= a1x cx + a1y cy (1)

At 2 A2mix = A2x + A2y

= a2x cx + a2y cy (2)

A1mix A2mix, a1x, a1y , a2x and a2y can be determined.

By solving equations (1) and (2) we can find cx andcy

a1x A2  a2x A1 a1y A2  a2y A1

Cy  x y Cx  x y
a1 a2  a2y a2x a1 a2  a2y a2x

41
 Spectrophotometry 41

N.B

Absorbance of the mixture at i (i = isosbestic point) is

given by: Aimix = ai (Cx + Cy)

c- Determination of some physical constants

e.g. (Determination of pKa of weak acid or pkind. )
Consider the weak acid HB during its dissociation, the
following equilibrium is established:

OH-
HB + H + + B-
H
+ -
Ka = [H ][B ] / [HB]

- log Ka =- log [H+] – log [B-] / [HB]

pKa = pH – log [B-] / [HB]

To determine pKa, the absorption spectra of a fixed

concentration of HB as function of pH is constructed (as
shown in Figure A).
Then absorbance is measured as a function of pH at λ1, (λmax
of HB) and λ2 (λmax of B) (a shown in figure B).
The pH at which the two curves are intersected
represents pKa of weak acid.

41
Spectrophotometry 42

Figure A

42
 Spectrophotometry 43

Other spectrophotometric applications

1. Determination of complexation ratio (Job's

method).
Procedure: to get the complexation ratio
- prepare a series of dilutions of Metal ion (Mn+) ranging
from 0 to 10 (i.e. 0, 1, 2… 10) and ranging from 10 to 0 (i.e.
10, 9, 8 ... 0) for complexing agent (L).
- Depending on additive property of absorbance, prepare
complementary mixtures of Mn+ and L (i.e. sum up to 10).
- By measuring A of these mixtures, if there is no change in
A, this indicates that, there is no complexation reaction.
However, if A of mixtures is varied, this indicates that
complexation reaction has taken place.
- The ratio of (Mn+ : L) can be detected by their ratio at the
point of maximum or minimum absorbance (as shown in the
figure).

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 Spectrophotometry 44

2. Effect and detection of impurities

Absorbing impurities have disastrous effects in
spectrophotometric applications:
- They cause distortion of the absorption curve of the
mother substance leading to over estimation (i.e. higher
absorbance);
- They may also lead to shifting of λmax.
Accordingly it is important to detect their
presence.

Detection of impurities
a) Impurity index (I.I.)
Construct the absorption spectra of tested sample and
reference standard of the same substance contained in the
tested sample.

A' and A refer to the absorbance of the sample and

reference standard respectively.
I.I = (A'1 / A'2) – (A1/A2), it must be equal to zero if the
sample is 100% pure as the reference.

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 Spectrophotometry 45

b) Spectrophotometric Purity Index (S.P.I)

S.P.I = (A' 1/ A' 2) / (A1 / A2)
this value equals 1 in case of pure samples.

References

1- Wikipedia:http://en.wikipedia.org/wiki/Spectrophotometry
2- D. C. Harris, Quantitative Chemical Analysis, 5th ed.,
1999, W. H. Freeman and company, New York, USA.
3- P. L. Kriz, Introduction to Spectroscopy, 3rd Ed.,
Harcourt college publishers, 2001, PA, USA.

45
Spectrophotometry 46

Q. what is molecular luminescence spectroscopy?

Is a type of spectroscopy in which luminescence occur when
certain mo lecules are excited by EMR, then reemission of that
radiation occurs either of the same λ or of longer λ.
- it comprises :
√ Molecular fluorescence.
√ Molecular phosphorescence.
√ Chemiluminescence (in which emission spectrum is formed
during a chemical reaction)

- Both fluorescence & phosphorescence are referred to as

photolumines cence.
- Fluorescence & phosphorescence are alike in tha t:
(1) In both excitation occur by absorption of photons.
(2) The emission λ is longer than the excitation λ.
- Fluorescence differs from phosphorescence in that :
1- The electronic energy transi tions don't involve a change in
electron spin.
2- The lifetime of the excited state is short.
(fluorescence ceases after ≈ 10 - 7 S , while phos phorescence
after several seconds ≈ 100 S )

- Fluorescence may occur to atoms or molecules in gaseous,

liquid or solid states.
- If the radiation emitted has the same λ as the excitation
radiation, the fluorescence is called "resonance fluorescence ".
- If the radiation emitted has longer λ (of lower energy E) than
the excitation radiation, this is called stokes shift (mostly
encountered with molecular fluorescence & phosphorescence)

- To understand the difference between Fluorescence &

phosphorescence, we 1 s t should know:
a- Electron spin:
- No more than 2 electrons occupy an orbital.
- The 2 electrons must hav e opposite spin states.

46
Spectrophotometry 47

(i.e paired spins which exhibit no net magnetic field & so

called diamagnetic)
- Free radicals (which contain unpaired electrons) are attracted
to magnetic field, so called paramagnetic.

b- Singlet / Triplet Excited states :

- What is a singlet state?
Is a molecular electronic state in which all electron spins are
paired.
- What is a triplet state?
When one of a pair of electrons excited, either a singlet or a
triplet state is formed.
In the excited singlet sta te:
The spin of the excited electron is still paired with that
electron in the ground state
In the excited triplet state:
The spin of the excited electron becomes unpaired ē that
electron in the ground state & thus they become parallel.

ground singlet excited excited

state singlet state triplet state
(arrows represent the direction of the spin)

* The properties of molecules in the excited triplet state

differ from those in the excited singlet state in that :
1- Molecules are paramagnetic (while diamagnetic in the singlet
state).
2- Molecules in the excited triplet state show phosphorescence,
while those in the excited singlet state show either
fluorescence of phosphorescence (if converted into triplet
state).
3- Singlet – triplet transitions are less probable than singlet –
singlet transitions

47
Spectrophotometry 48

(so phosphorescence is less probable than fluorescence )

4- The average lifetime of an excited triplet state (range from
10 - 4 S to several seconds) while (10 - 5 – 10 - 8 S) for excited
singlet state.
(so fluorescence is short lived compared to phosphorescence ) .

* Energy level Diagrams for photoluminescent

molecules *

- Each energy level, either ground or excited, consists of

numerous vibrational energy levels.
- The lowest heavy horizontal line represents the ground state
energy level S o which is normally a singlet.

48
Spectrophotometry 49

- The 2 upper heavy line s on the left are the excited singlet
energy levels S 1 , S 2 .
- The upper heavy line on the right is the excited triplet energy
level T 1

- Notice that:
√ Excitation of the molecule may occur at λ 1 where S o → S 1
transition occur or at shorter λ 2 where S o → S 2 transition
occur.
√ The E of T 1 is less than that of S 1 and S 2 .
√ Excitation results in conversion of the molecule to any of the
several excited vibrational states.
√ No direct excitation to T 1 occur.
(i.e the electron doesn't change its spin upon d irect excitation,
but can change it during relaxation )

- The excited molecule returns to the ground state by several

mechanisms.
- This Deactivation (or relaxation) process is classified into:
a- radiative b- non radiative .

- In the graph:
√ A straight vertical arrow represent radiative relaxation
(fluorescence or phosphorescence).
√ A wavy arrow represent radiationless deactivation.

N.B the favored mechanism is that which minimizes the life time
of the excited state.

(A)- Radiationless Dea ctivation:

1- Vibrational relaxation.
2- Internal conversion.

49
Spectrophotometry 51

3- External conversion.
4- Intersystem crossing.

(1) Vibrational relaxation :

- A molecule is promoted to any of several vibrational energy
levels in the excited state.
- The excess vibra tional energy is immediately lost, Why?
due to molecular collisions between excited molecules & the
solvent molecules (where electrons pass from higher
Vibrational E levels to lower vibrational E levels in the same
excited state ).
- This relaxation p rocess is very rapid =10 - 1 2 S.
- The net result is:
√ Minute increase in solvent temp.
√ When fluorescence occur on the further step, electrons will
pass from the lowest vibrational level in the excited state to
any vibrational level in the ground state, thus the spectrum
emitted will be a band.
√ The spectrum emitted will be displaced to a longer λ (lower E)
than the absorption band (excitation spectrum) i.e stokes shift .

N.B overlap occur between the excitation spectrum & the

emission spectrum only at the resonance peak ( i.e. where
λ r = λ ŕ ) see the graph.

(2) Internal conversion : IC

- It is intermolecular process by which a molecule passes from
an excited E level (S 2 ) to a lower excited E level (S 1 ) when
the lowest vibrational E le vel of (S 2 ) coincide with one of
the vibrational levels of (S 1 ).
N.B the proof that IC is true:
√ Fluorescence is not frequent among molecules (i.e IC is
probable > fluorescence).

51
Spectrophotometry 51

√ Notice that the emission spectrum occurs at λ3 only

regardless the e xcitation spectrum wavelength was λ 1 or λ 2 (i.e
IC makes λ 1 and λ 2 excitations give the same fluorescence).

(3) External conversion : EC

Is a type of radiationless deactivation of the excited state in
which interaction (collisions) & energy transfer occur between
the excited molecules & the solvent molecules.
The evidence that EC occusr:
Conditions that tend to reduce collisions between particles (e.g
cooling) lead to increase in fluorescence (radiative deactivation
rather than radiationless).

(4) Inters ystem crossing : ISC

- Is a process in which the spin of the excited electron is
reversed (excited singlet ---- flipping-----excited triplet)
- As in IC:
The probability of this transition increases if the lowest
vibrational E level in the excited singlet state (S 1 ) overlap with
one of the vibrational E levels of the excited triplet state (T 1 ).
- The E of T 1 is less than S 1 .
- ISC is common in:
√ Molecules that contain heavy atoms e.g iodine & bromine
(called heavy – atom effect).
√ Presence of paramagnet ic species e.g molecular O2 (i.e
decrease probability of fluorescence & increase
probability of phosphorescence).
So vigorous shaking quench fluorescence because of entrapped
O2.

51
Spectrophotometry 52

(B) Radiative deactivation:

(1) Fluorescence:
- After the excited elec tron loses its vibrational E by collisions,
it will reach the lowest vibrational E level in the excited state
(S 1 or S 2 ).
- Then transition from this lowest vibrational E level to the
ground singlet state occurs with loss of E as photons [i.e
emission of EMR known as fluorescence].
e.g from S2 So
or from S 1 So (after IC of S 2 S1)

(2) phosphorescence:
- After conversion of the electron from the excited singlet
state to the excited triplet state by IS C
(S 1 T 1 ): the electron will be further deactivated either by IC
, EC or phosphorescence .
- Phosphorescence occurs when the electron in the excited
triplet state relaxes to the ground singlet state where EMR is
emitted (T 1 S o ).
- Relaxation of T 1 S o is not easy ??
because it involves reversal in the electron spin , so
T1 S o relaxation occurs after longer time.
(N.B the average lifetime of T 1 is 10 - 4 – 10 S)
- So phosphorescence will persist for some time (a fter
irradiation) longer than fluorescence.

Q. phosphorescence is observed only at low temperatures or in

highly viscous media ?
Because EC & IC compete with phosphorescence, so under
these conditions, collisions will decrease, thus the probability
of EC and IC will decrease & the probability of
phosphorescence will increase.

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Spectrophotometry 53

* Excitation & Emission spectra *

st
- 1 the compound is scanned in the UV – VIS spectrum to give
→ absorption spectrum.
- From this absorption spectrum you can determine certain
wavelengths which give high A (including λ m a x ).
- 2 n d choose one of these wavelengths (mostly λ m a x ), then in the
spectrofluorimeter, try to get the excitation spectrum &
emission spectrum.
- Excitation spectrum :
Is a plot of intensity of fluorescence at one fixed λ (λ
emission) Vs the wavelength λ used to excite the fluorescence.
(N.B it should be identical with the absorption spectrum you
obtained by the spectrophotometer)
- Emission spectrum:
Is a plot of intensity of fluorescence at one monochromatic λ
(λ excitation) Vs wavelength λ.

- If we plot the excitation & the emission spectra on the same

chart, we will notice the shift of tee emission spectrum to a
longer λ (stokes shift ) & the 2 spectra will appear as mirror
image .

53
Spectrophotometry 54

Figure 34: Emission spectrum of Metopimazine with maximum at 505 nm

(solid line) and excitation spectrum of Metopimazine with maximum at 336

nm (dotted line) for 1µg.ml-1 Metopimazine

400

300

200
Int.

100

0
270 300 400 500 610
Wavelength [nm]

Q. what is the quantum yield Φ (=quantum efficiency) ?

is the ratio of no. of molecules that fluoresce to the total of
excited molecules .
OR:
the ratio of no. of photons emitted to no. of photons
absorbed.

- for highly fluorescent substances : Φ ≈ one

- for non fluorescent substances: Φ ≈ zero .

*Quantitative fluorimetry & effect of concentratio n on

fluorescence*
.. principle : F α (I o – I)
F: power of the fluorescent radiation.
(I o – I): power of the excitation beam that is absorbed by the
system.
. . . F = K (I o – I)

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Spectrophotometry 55

K : constant dependen t on the quantum efficiency

- to relate F with concentration (C) , use Beer's law in the form

: I/I o = 10 - ε b c
ε → molar absorbitivity
εbc → A absorbance
- By substitution in the 1 s t equation;
F = K I o (1 – 10 - ε b c )
.. If A < 0.05, the exponential term will be:
F = 2.3 K ε bC I o i.e. ( F = K \ C )
Then by plotting F Vs concentration → linear relation but at low
concentration.
.. When concentration increases (so that A becomes > 0.05) →
linearity is lost.

Q. what are factors c ausing loss of linearity?

1- Increase in concentration (so that A is > 0.05).
2- Self absorption:
i.e. some of the emitted radiation is absorbed by the molecules
in solution (when emission λ overlaps with absorption λ) causing
decrease in fluorescence.
3- Self – quenching:
results from collisions of excited molecules causing decrease in
fluorescence .

* Factors affecting fluorescence *

1- Molecular structure.
2- Temperature & solvent effect.
3- pH effect.
4- Dissolved O 2 effect.

(1) Molecular structure :

A- Simple heterocyclics (e.g. pyridine, thiophene, pyrrole &
furan) don't fluoresce, why?

55
Spectrophotometry 56

Because the lowest transition n – π* occur, which is

rapidly converted to triplet & prevents fluorescence.
√ halogen substitution (specially with iodin e & bromine)
decrease fluorescence due to ISC .
√ substitution of carboxylic acid or carbonyl group on an
aromatic ring inhibits fluorescence .

B- √ generally most fluorescent compounds have aromatic

functional group.
√ compounds containing aliph atic & alicyclic carbonyl groups
or conjugated double bonds exhibit fluorescence.
√ most unsubstituted aromatic hydrocarbons fluoresce (the
quantum yield increases with increase in the no. of fused
rings) .
√ fusion of benzene ring in hetero atom results in fluorescent
compounds.
√ fluorescence is favored in molecules that possess rigid
planar structure e.g fluorene fluoresce much more than
biphenyl (due to rigidity furnished by methylene group in
fluorene ) .

.. Account: fluorescent intensity of 8 -hydroxyquinoline is

increased when it forms zinc complex??
Due to increase in rigidity of the structure.

56
 Spectrophotometry 57

(2) Temperature & solvent effect:

- fluorescence decreases by increase in temperature, Why??
because deactivation by external conversion is favored.
- decrease in solvent viscosity leads to the same result
(decrease in fluorescence).
- polar solvents may increase fluorescence .
- solvents containing heavy atoms such as carbon tetrabromide
or ethyl iodide decrease fluorescence .

(3) Effect of pH:

- The fluorescence of an aromatic compound with acidic or
basic ring substitution is pH dependent where pH affects the
emission intensity & λ of the ionized & unionized forms .

(4) Effect of dissolved O 2 :

- O 2 is paramagnetic, so it decreases fluorescence due to ISC.

* Instrumentation *
.. Principle:
- EMR from a UV – VIS source passes through a wavelength
selector then to the cell (containing the analyzed sample).
- If monochromators used → the instrume nt is called
spectrofluorimeter &
If filters used → the instrument is called fluorimeter .
- After irradiation, emission of radiation by the sample occurs
in all directions.
- The emitted radiation is measured at 90 o angle from the
path of the excit ing beam & at the centre of the cell??
to minimize the error due to scattering of light from the walls of
the cell & solution ( which occur at other angles ) & to prevent
interference from the exciting beam.

57
Spectrophotometry 58

- A second wavelength selector is used between the sample &

the detector??
to pass the most intense emitted λ (λ emission) from the
broad emission band .

* Parts of the spectrofluorimeter *

(1) Source of Energy:
- it must be highly intense ?
to allow considerable excitation.
- the two mos t commonly used sources are :
(a) Mercury – arc lamp:
- is a quartz lamp containing mercury vapor.
- upon excitation emits line spectra of several definite λ .
. . . it can't be used when scan of spectrum is required .
(b) High pressure xenon lamp:
- emits a continuum of radiation throughout the UV – VIS
regions.
. . . it is useful when spectrum scanning is needed .
- Defect: intensity of the emitted radiation (fluorescence)
varies with λ of excitation but unfortunately not due to
sample nature or change in con centration but due to
variation in source intensity leading to erroneous results.
. . . to compensate for variation in source intensity & any other
instrumental variation:
Use a double – beam instrument. (resembles blank
experiment! )
Where por tion of the excitation E is directed to a solution of
a fluorescent standard, and then compare the sample &
reference signals.

(2) Wavelength selector:

√ 2 filters (absorption or interference filters) or 2
monochromators (grating type) are used.

58
Spectrophotometry 59

√ one of them between the source & the sample & the other
between the sample & the detector .

(3) The cell:

Tetragonal or cylindrical transparent glass or quartz tubes are
used.

(4) Detectors & readout meter:

- photomultiplier tube is used ?
Since the intensity of emitted radiation is small.
- Digital or analog or nullpoint meter is used.

59
Spectrophotometry 61

* Applications of Fluorimetry *
(1) For fluorescent compounds e.g. phenobarbitone , quinine ,
emetine , adrenaline , cinchonine , reserpine , vitamin A ,
riboflavin .....
Are determine d at very low concentrations by simple
fluorimetric methods .
(2) For non fluorescent compounds
determined after a chemical reaction which transforms
them into fluorescent comps.
(3) For inorganic ions :
- determined after formation of fluorescent c helates e.g. 8 -
hydroxyquinoline (for Al) , benzoin (for Zn) , flavanol (for Zr)
OR ,
- by measuring the quenching (inhibition) of fluorescence of a
fluorescent compound by these ions.

61