PHARMACEUTICAL

INSTRUMENTAL ANALYSIS
Dr Aref Zayed
COURSE OUTLINE
1. Introduction to Instrumental Methods of Analysis.
2. Spectrometry:
 Introduction to Electromagnetic Radiation
 Quantum-Mechanical Properties of Radiation.
3. Ultra-Violet (UV) & Visible Spectrometry
4. Infrared Spectroscopy (IR).
5. Nuclear Magnetic Resonance (NMR) Spectroscopy :
Uses in Identification of Organic Compounds
6. Mass Spectrometry (MS): Uses in Quantitative and
Qualitative
7. Chromatography:
1. Liquid Chromatography (LC)
2. Gas Chromatography (GC).
PHARMACEUTICAL
INSTRUMENTAL ANALYSIS
Selecting an Analytical Method
 INTRODUCTION

 Types of Analyses:
1. Quantitative
2. Qualitative

 Classification of Analytical Methods

(Historical):
1. Classical
2. Instrumental
CLASSIFICATION OF ANALYTICAL METHODS
1. Classical Methods:
 Analytes separated by precipitation, extraction or
distillation.
 If Qualitative: Analytes recognised by Colour, B.P,
M.P , Solubility, Odour, Refractive Index.
 If Quantitative: Amount determined by
Gravimetric or Titrimetric measurement.

2. Instrumental Methods:
 Reliable and modern instruments were introduced.
 Measurement of physical properties like conductivity,
electrode potential, mass to charge ratio, light
absorption/ emission.
 Highly efficient separation techniques:
Chromatography, Electrophoresis.
INSTRUMENTS FOR ANALYSIS
An instrument for chemical analysis converts
information stored in the physical or chemical
characteristics of the analyte to information that
can be manipulated and interpreted by a human.

Stimulus Response

Energy Analytical
Source Information
Sample
TYPES OF INSTRUMENTAL METHODS
 According to characteristic properties employed
to generate analytical signal:

1. Properties involving Electromagnetic Radiation:

 Absorption, emission, scattering…
e.g. UV, IR, NMR, fluorescence, phosphorescence

2. Electrical Properties:
 Electrical potential, charge, current or resistance.

3. Miscellaneous:
 Mass-to-charge ratio, mass, radioactivity.
e.g. Mass Spectrometry (MS)
ANALYTICAL METHOD SELECTION

Sample

Instrument
ANALYTICAL METHOD SELECTION

 Define the problem:

1. Accuracy (e.g need 2-figure or 5-figure balance?)

2. Amount / concentration of sample e.g. measure drug

concentration in blood
3. Concentration Range

4. Interferences (e.g. Melamine contamination)

5. Matrix physical and chemical properties

6. Number of samples (cost, time, automation)

ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics:
1. Precision:
 Agreement among data obtained in same way (replicate
measurements)
 Measure of random errors
e.g. Measure glucose levels in a blood sample (3
replicates) by two methods:

Method A: 70, 80 and 90 mg/dL

Method B: 78, 80, and 82 mg/dl

Which method is more precise?

ANALYTICAL METHOD SELECTION
 Precision can be expressed by calculating
standard deviation

 For method A in the previous example :

1. 𝑥ҧ = (70+80+90)/3= 80
2. (x-𝑥)=
ҧ 70-80, 80-80, 90-80= -10, 0, 10
3. (x-𝑥)ҧ 2= 100, 0 , 100
4. 𝛴(x-𝑥)ҧ 2 = 100+0+100= 200
200
5. s= = 10
3−1
ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics
2. Bias: a measure of systematic error

bias= µ - xt

Mean value True value

USEFUL TERMS
 Blank : is a sample that contains all of the
components of the original sample except for the
analyte.

 Calibration curve: is used by introducing

several standards containing exactly known
concentrations of the analyte into the
instrument. y = mx + b
Instrument
Concentration Instrument
Response
Response
0 10
50 45
100 90 50 100

Concentration
ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics
3. Sensitivity
 Ability of method to distinguish between small
differences in analyte concentration.
 Depends on :
 Slope of calibration curve
 Precision (reproducibility)

Which method is more sensitive?

y = mx + b
Instrument
Instrument

Response
Response

50 100 50 100

Concentration Concentration
ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics
4. Detection limit (DL): The minimum concentration or mass
of the analyte the can be detected at a known confidence
level.

Lower Limit of Quantification (LLOQ): The minimum concentration

or mass of the analyte the can be quantified at a known confidence
level.
Standard
3 𝑠𝐵 deviation of
DL = the blank
𝑚
Calibration
10 𝑠𝐵 curve slope
LLOQ =
𝑚

Sensitivity and DL?

ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics
5. Dynamic Range: It is the range that extends from the
lowest concentration at which quantitative measurements
can be made (LLOQ) to the concentration at which the
calibration curve departs from linearity.
Instrument Response

LLOQ

Dynamic Range

Concentration
ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics

6. Selectivity: The degree to which the analytical method is

free from interference by other species contained in the
sample matrix.
ANALYTICAL METHOD SELECTION
 Instrument Performance Characteristics

 Other characteristics for selecting analytical

methods:
6. Speed
7. Ease and convenience.
8. Skill required of operator.
9. Cost and availability of equipment.
10. Per-sample cost.
NOTES

 Calibration of Instrumental Methods:

(self study from the book)

 Calibration curves
 Standard addition methods
 Internal standard method
ANALYTICAL PROCESS
•Obtain a representative bulk sample from the lot.
Sampling •Obtain a homogenous laboratory sample from the bulk sample

•Convert sample into a form suitable for analysis.

Sample •Remove species that interfere with chemical analysis
Preparation •e.g: Sample pre-concentration, protein precipitation,
liquid-liquid extraction, filtration, solid phase
extraction

Analysis •Run standards and samples.

•Record and evaluate

Results results of the analysis

Report and
Evaluation
INTRODUCTION TO
SPECTROMETRIC METHODS
OUTLINE
 Spectroscopy: Science the deals with interaction
of various types of radiation with matter.
 Originally, only electromagnetic radiation with matter
 Now include interaction of matter with other forms of
energy e.g. beams of electrons and ions

 Spectrometry: Measurement of the intensity of

radiation with an electronic device (photoelectric
transducer).
 Spectrometric methods: A group of analytical
methods that are based on atomic and molecular
spectroscopy. (e.g. AAS , IR)
PROPERTIES OF ELECTROMAGNETIC
RADIATION ( WAVE OR PARTICLE?)
 Two models:
1. Wave model: wavelength, frequency, velocity
and amplitude
 Requires no supporting medium (vs sound).
 What about absorption and emission of radiant
energy?
2. Particle model: stream of discrete particles of
energy (wave packets) called photons.
 Energy 𝛂 frequency

 Wave-Particle duality applies to electrons, protons

and other elementary particles
 WAVE PROPERTIES OF E-MAG RADIATION
 Electric and magnetic fields which oscillate at
right angles to each other and to the direction of
propagation.

Amplitude (A)

Y: Electric field
X: Time or distance.
WAVE PARAMETERS
 Amplitude (A): Length of electric vector at a maximum
in the wave

 Period (p): Time in seconds required for the passage of

successive maxima or minima through a fixed point in
space.

 Frequency (v): Number of oscillations of the field that

occur per second (1/p)

 Wavelength (𝛌): Linear distance between any 2

equivalent points on successive waves e.g. (maxima or
minima).
ELECTROMAGNETIC SPECTRUM
ELECTROMAGNETIC SPECTRUM
WAVE PARAMETERS

Frequency

c= v 𝛌

3.00 x 1010 cm/s Wavelength

(vacuum)
WAVE PARAMETERS
 Wavenumber (𝑣):
ҧ Reciprocal of the wavelength in cm,
units (cm-1).
 Mainly used in IR.
 𝑣ҧ = kv

Proportionality
constant
(medium dependent)
QUANTUM MECHANICAL
PROPERTIES OF RADIATION
(Chapter 6)
COMMON SPECTROSCOPIC METHODS
Type Spectroscopy Usual Wavelength Range
NMR 0.6-10 m
IR absorption and Raman 0.78-300 µm
Scattering
UV visible absorption, emission 180-780 nm
and fluorescence

Questions:
 Which color has a longer wavelength, red or
blue?
 Which color has a higher frequency, yellow or
violet?
 Which radiation has a higher energy, UV or IR?
PHOTOELECTRIC EFFECT
 When electromagnetic radiation is emitted or
absorbed , a permanent transfer of energy occurs.
 This cannot be explained by describing radiation
as waves but rather as a stream of discrete
particles called photons.
 Discovery of photoelectric effect in the 19th
century made it necessary to adapt the particle
model.
PHOTOELECTRIC EFFECT
 Hertz (1887): Spark jumped more readily
between 2 charged spheres when
illuminated with light.
 Einstein (1905): Theoretical explanation of
photoelectric effect.
 Millikan (1916): Systematic studies
confirmed Einstein’s theory.
hv

-
- - -
A

V
PHOTOELECTRIC EFFECT
 Alkali metal-coated photocathode is irradiated
with monochromatic radiation (𝛌)
 Electrons emitted from its surface with a range of
kinetic energies.
 Current produced.

 When voltage is adjusted so that anode is slightly

–ve, electrons are repelled and current decrease.
 Some electrons have sufficient kinetic energy to
overcome –ve potential and current is still
observed.
 The negative voltage at which current is zero is
called V𝛐 (stoppage voltage).
PHOTOELECTRIC EFFECT
 Stoppage voltage corresponds to the potential at
which the most energetic electrons are just repelled
from the anode.
 V𝛐 x e (-1.60 x 10-19 coulombs) = Maximum kinetic
energy (joules) : stopping energy eV𝛐

 Main Results:
1. Light with constant frequency : intensity of
radiation 𝛼 current
2. Stoppage voltage V𝛐 depends on frequency (v)
3. Stoppage voltage V𝛐 depends on coating metal.
4. Stoppage voltage V𝛐 is independent of the intensity of
radiation.
PHOTOELECTRIC EFFECT
 Frequency and maximum kinetic energy
Max kinetic energy,

Planck constant Work function

6.6254 x 10-34 J.s
eV𝛐

Slope = h eV𝛐 = hv + ω

ω E = hv
Frequency v
𝒄
E = h = eV𝛐 - ω
𝝀
EXAMPLE
 Calculate the energy of
 (a) 5.3- Aሶ X-ray photon
 (b) 530-nm photon of visible radiation

Solution:
𝒄
E = hv=h
𝝀

−
6.63 x 10 34 J.s x 3.00 𝑥 108 m/s
(a) E= − = 3.75 x 10-16 J
5.3 A x (10 10 m/A)
−
6.63 x 10 34 J.s x 3.00 𝑥 108 m/s =
(b) E= − 3.75 x 10-19 J
530 nm x (10 9 m/nm)
ENERGY STATES OF CHEMICAL SPECIES
 Quantum theory:
1. Atoms, ions and molecules can exist only in
certain discrete states, characterized by definite
amount of energy. When a species changes its
state, it absorbs or emits an amount of energy
EXACTLY equal to the energy difference
between the states.

2. When atoms, ions or molecules absorb or emit

radiation
𝒄
E1 - E0 = hv = h
𝝀
ENERGY STATES OF CHEMICAL
SPECIES
ENERGY STATES OF CHEMICAL SPECIES
 Quantum theory:
1. Atoms, ions and molecules can exist only in certain
discrete states, characterized by definite amount of
energy.
 E0 = ground state
E1, E2, E3... = excited states
 Excitation can be electronic, vibrational or rotational
 Measuring energy levels gives means of identification –
spectroscopy.

2. When a species changes its state, it absorbs or emits

an amount of energy EXACTLY equal to the energy
difference between the states.
𝛥E = E1 - E0

When atoms, ions or molecules absorb or emit radiation

𝒄
E1 - E0 = hv = h Einstein-Planck’s relation
𝝀
EXAMPLE
 Find the wavelength of a photon emitted/ absorbed when an
electron jumps from the n = 1 energy level to the n = 2 energy
level. Where is this photon in the electromagnetic spectrum?
(1 eV = 1.6 x 10-19 Joules).

𝒄
Energy Level Energy
E1 - E0 = hv = h 1 -13.6 eV
𝝀
E2-E1= (-3.4) - (-13.6) = 10.2 eV 2 -3.4 eV
3 -1.51 eV
→ 𝛌 =1.21 x 10-7 m 4 -.85 eV
5 -.54 eV

 A photon with an energy of 10.2 eV has a wavelength of 1.21 x

10-7 m, in the ultraviolet part of the spectrum. So when an
electron wants to jump from n = 1 to n = 2, it must absorb a
photon of ultraviolet light. When an electron drops from n = 2
to n = 1, it emits a photon of ultraviolet light.
ENERGY STATES OF CHEMICAL SPECIES
 For ions and atoms (elemental): Energy of any
state arises from motion of electrons around the
+ve charged nucleus → Electronic States.

 For molecules : In addition to electronic states,

they have quantized vibrational states
(associated with energy of interatomic
vibrations), and quantized rotational states that
arises from the rotation of the molecules around
their centre of gravity.

 The ground state of an atom or molecule is its

lowest energy state.
 Excited states are higher energy states.
ENERGY STATES OF CHEMICAL SPECIES
1. Emission of Radiation
2. Absorption of Radiation
3. Relaxation Processes
EMISSION OF RADIATION
 Radiation is produced when excited particles
relax to lower energy levels giving up energy as
PHOTONS
 To excite particles:

1. Bombardment with 𝑒ҧ (emission of X-radiation)

2. Exposure to AC spark or heat of flame (UV,

visible and IR radiation)
3. Irradiation with electromagnetic radiation
(fluorescent radiation)
EMISSION OF RADIATION
EMISSION OF RADIATION
 Plot of emission intensity vs. v or 𝛌 is called
emission spectrum

1. Line Spectra: Sharp well defined peaks caused

by excitation of atoms
2. Band Spectra: Groups of lines which are not
completely resolved caused by small molecules
or radicals.
3. Continuum Spectra: Responsible for the
increase in the background
FIG.1 EMISSION SPECTRUM OF BRINE
 LINE SPECTRA
Excitation by
thermal,
4p
electrical or
radiant 3p
energy

3s

Fig. : Energy level diagram for Na atom the source of line spectrum

 Produced from atoms.

 In UV-Vis region: line spectra produced from
individual atoms that are well separated in a gas
phase. Electronic transition in outer shell electrons.
 X-Ray line spectra are also electronic transition
involving inner shell electrons. Emission spectrum is
the same regardless of the environment.
 Lifetime= 10-8 seconds
SODIUM LINE SPECTRA
SODIUM STREET LIGHTS
BAND SPECTRA
 Encountered when gaseous radicals or small
molecules are present.
 e.g. OH, MgOH and MgO bands in Fig1.
 Bands: Lines are not fully resolved.
 Arise from vibrational levels superimposed on the
ground-state level of a molecule.
 Radiation results from transition from the lowest
vibrational level of an excited electronic state to
any of the vibrational levels of the ground state.

Rotational

Vibrational
CONTINUUM SPECTRUM
 Very broad spectrum in emission from solids.
 Produced by blackbody radiation- Thermal
excitation and relaxation of many vibrational and
rotational levels.

Fig2. Blackbody spectrum

ABSORPTION OF RADIATION &
RELAXATION PROCESSES
ABSORPTION OF RADIATION
 Certain frequencies are selectively removed when
radiation passes through solids, liquid or gas by
absorption, a process in which electromagnetic
radiation is transferred to particles (atoms,
molecules).

 Absorption promotes atoms/ molecules from

ground state to excited to state.

 Since energy differences are unique for each

species, a study of absorbed frequencies can be
used to characterise the constituents of a sample.
ABSORPTION OF RADIATION
 Absorption spectrum : Plot of absorbance vs v or 𝛌
 Just as in emission spectra an atom, ion or molecule
can only absorb radiation if energy matches
difference between two energy states.
Atoms:
 No vibrational energy levels- sharp well defined
line spectra with few features
e.g. Na 3s→ 3p 589.0, 589.6 nm (yellow)
Na 3s→ 5p 285.0, 285.1 nm (UV)
 UV and Vis enough energy for outer shell
(bonding) electrons
 X-ray enough energy for inner most electrons.
 ABSORPTION OF RADIATION

Molecules:
Electronic, vibrational and rotational energy levels –broad
band spectra with many features

𝛥E= 𝛥E electronic + 𝛥 E vibrational + 𝛥 E rotational

For each electronic state-many vibrational states,

For each vibrational state- numerous rotational states
→Molecular Spectra has many features (compared to atomic)
ABSORPTION SPECTRA
 Absorption spectra influenced by
 Complexity, physical state and environment of the
absorbing species.
 Whether the spectra for atoms or molecules.
PARTIAL ENERGY LEVEL DIAGRAMS
PURE VIBRATIONAL AND ROTATIONAL
 Pure vibrational absorption is observed in IR
region - energy is not enough to cause electronic
transition.

 Pure rotational absorption can be observed in

the microwave region.
ABSORPTION INDUCED BY A MAGNETIC FIELD
 Additional energy levels can be observed when
electrons of certain elements are subjected to strong
magnetic field.

 Differences in energy between induced states are

small→ only absorption of long-wavelength (low
frequency) radiation can produce transition between
the states.

 For nuclei , radio waves 30-500 MHz are involved

(nuclear magnetic resonance NMR)

 For electrons, microwaves of 9500 MHz (electron spin

resonance ESR)
MRI
RELAXATION PROCESSES
1. Non-radiative Relaxation:
 Loss of energy in a series of small steps
 Excitation energy converts to kinetic energy by
collision with other molecules→ increase in
temperature.
2. Fluorescence and Phosphorescence:
 Atoms or molecules are excited by absorption of
electromagnetic radiation
 Excited species return to ground state→ radiant
emission.
 Fluorescence (10-5 s) occurs more rapidly than
phosphorescence which may continue for min or hours.
 Fluorescence and phosphorescence are easily observed
at a 90-deg angle to excitation beam.
 FLUORESCENCE
 Resonance fluorescence:
 Frequency of emitted light = frequency of
excitation light
 Commonly produced by atoms in gaseous
state.

 Non-resonance Fluorescence:
 Produced by irradiation molecules in
solution or gaseous state.
1. Molecules are excited into a vibrational
state in an excited electronic level.
2. Lifetime vibrational (10-15 s)<< excited
electronic state (10-8 s).
3. Non-radiative relaxation occur before
emission.
4. Energy (frequency) of emitted light <
energy of absorbed light.
5. The shift to lower frequency is called
stokes shift.
PHOSPHORESCENCE
 Produced when excited molecule relaxes to
metastable excited electronic state (triplet state),
which has an average lifetime > 10-5 s.
 Examples of drugs that have phosphorescence
property : Aspirin, benzoic acid and morphine,
QUANTITATIVE ASPECTS OF
SPECTROCHEMICAL
MEASUREMENTS
SPECTROCHEMICAL METHODS
Class Radiation Measured Type of Methods
Emission Emitted Radiation • Atomic Emission
Luminescence Luminescent Radiation • Atomic and Molecular
Fluorescence
• Phosphorescence
Scattering Scattered Radiation • Raman Scattering
• Turbidmetry
Absorption Incident and transmitted • Atomic and Molecular
Radiation (Absorbed) Absorption.

• Emission and Luminescence:

S=kc
Electrical Signal Concentration
Constant
ABSORPTION METHODS
 Quantitative absorption methods require two
power measurements:
1. Before the beam pass through the sample (P0)
2. After the beam pass through the sample (P).

 Transmittance: The fraction of incident radiation

transmitted by the medium (sample).
T = P / P0
%T = P / P0 x 100%

 Absorbance : A = - log T = log P0 / P

BEER’S LAW
 For monochromatic radiation, absorbance (A) is
proportional to the path length (b) through the
medium (sample) and the concentration (c) of the
absorbing species: b

P0 c P

Absorbing solution of
concentration c

A = abc

Absorptivity Path length Concentration

(L. g-1 cm-1 ) (cm) (g. L-1)

A = 𝜖 bc

Molar Absorptivity Path length Concentration

(L. mol-1 cm-1 ) (cm) (mol. L-1)
MEASUREMENT OF ABSORBANCE AND
TRANSMITTANCE

P0 P

Single beam photometer for absorption measurement in the visible region

QUANTITATIVE ASPECTS OF
SPECTROCHEMICAL
MEASUREMENTS
SPECTROCHEMICAL METHODS
Class Radiation Measured Type of Methods
Emission Emitted Radiation • Atomic Emission
Luminescence Luminescent Radiation • Atomic and Molecular
Fluorescence
• Phosphorescence
Scattering Scattered Radiation • Raman Scattering
• Turbidmetry
Absorption Incident and transmitted • Atomic and Molecular
Radiation (Absorbed) Absorption.

• Emission and Luminescence:

S=kc
Electrical Signal Concentration
Constant
ABSORPTION METHODS
 Quantitative absorption methods require two
power measurements:
1. Before the beam pass through the sample (P0)
2. After the beam pass through the sample (P).

 Transmittance: The fraction of incident radiation

transmitted by the medium (sample).
T = P / P0
%T = P / P0 x 100%

 Absorbance : A = - log T = log P0 / P

QUANTITATIVE ANALYSIS
Beer’s Law
BEER’S LAW
 For monochromatic radiation, absorbance (A)
is proportional to the path length (b) through the
medium (sample) and the concentration (c) of the
absorbing species: b

P0 c P

Absorbing solution of
concentration c

A = abc

Absorptivity Path length Concentration

(L. g-1 cm-1 ) (cm) (g. L-1)

A = 𝜖 bc

Molar Absorptivity Path length Concentration

(L. mol-1 cm-1 ) (cm) (mol. L-1)
MEASUREMENT OF ABSORBANCE AND
TRANSMITTANCE

P0 P

Single beam photometer for absorption measurement in the visible region

BEER’S LAW

A = abc

Absorptivity Path length Concentration

(L. g-1 cm-1 ) (cm) (g. L-1)

A = 𝜖 bc

Molar Absorptivity Path length Concentration

(L. mol-1 cm-1 ) (cm) (mol. L-1) or
(M)
MWT of Analyte 𝜖 bc
M=
(g. mol-1 ) 𝑨
Concentration
Molar Absorptivity (g. L-1)
(L. mol-1 cm-1 )
BEER’S LAW

A = 𝑨𝟏%
𝟏𝒄𝒎 bc

Concentration
Specific absorbance
(g. 100 ml-1)
(absorbance of 1% w/v
or (w/v%)
solution in 1 cm cell)

MWT of Analyte
10𝜖
M=
(g. mol-1 ) 𝑨𝟏%
𝟏𝒄𝒎
PRACTICE EXAMPLES
 Q1 : The molar absorptivity of a particular
chemical is 1.5 M-1 cm-1. What is the
concentration of a solution made from this
chemical that has an absorbance of 0.72 with a
cell path length of 1.1cm? (www.chem.ucla.edu)

A = 𝜖 bc
PRACTICE EXAMPLES
 Q2 : A solution with a concentration of 0.14M is
measured to have an absorbance of 0.43. Another
solution of the same chemical is measured under
the same conditions and has an absorbance of
0.37. What is its concentration?
(www.chem.ucla.edu)

𝑨 𝟏 𝒄𝟏
= Same analyte, same conditions
𝑨 𝟐 𝒄𝟐

C1 = 0.12M
PRACTICE EXAMPLES
 Q3: A compound had a molar absorptivity of
2.17 x 103 L cm-1 mol-1 . What concentration of the
compound would be required to produce a solution that
has a transmittance of 8.42% in 2.50-cm cell?
(Problem 6-19 Skoog 5th & 6th Ed, but different Numbers!)

A = 𝜖bc
A= -log T
c = 1.98 x 10-4 M
(c = 1.35 x 10-4 M in 6th Ed)
 PRACTICE EXAMPLES
 Q4: Chloroaniline (MWT =127.6) in a sample is
determined as the amine picrate. A 0.0265 g sample is
reacted with picric acid and diluted to 1L. The
absorbance of this solution in 1 cm cell is equal to 0.368.
What is the % chloroaniline in the sample? (Molar
absorptivity for chloroaniline = 1.25 x 104 L mol-1. cm-1)

A=𝜖bc
c chloroaniline= 2.94 x 10-5 mole/L
So there is 2.94 x 10-5 mole/L x 127.6 g/mole= 3.75 x 10-3 g in 1 L
solution.
3.75 x 10−3
x 100%= 15.0 % choloraniline in the sample
2.65 x10−2
PRACTICE EXAMPLES
 Q5: A compound of formula weight 280 absorbed
65% of the radiation at a certain 𝛌 in a 2-cm cell at
concentration of 15.0 g/ml. Calculate its molar
absorptivity at the same 𝛌. (Past exam paper )

%T= 35%
T=0.35
A=-log 0.35= 0.456
𝜖 bc
M=
𝑨
Concentration
𝑴𝑨 (g. L-1)
𝜖=
bc
PRACTICE EXAMPLES
 Q6: The absorbance of pure compounds X and Y each in 5x10-5 M
concentration are as follows:
Compound A 264 A 378
X 0.510 0.192
Y 0.335 0.150

 A solution of one of these pure compounds of unknown concentration

yielded absorbance equal to 0.395 at 264 nm and 0.147 at 378 nm.
Which compound is this sample? What is the concentration of the
solution? (Past exam paper)

𝑨 𝟏 𝒄𝟏
= Same analyte, same conditions
𝑨 𝟐 𝒄𝟐
c2 should be the same for the same compound at any wavelength
So calculate c2 for each compound at the 2 wavelengths
𝟎.𝟓𝟏𝟎 𝟓𝐱𝟏𝟎−𝟓 𝟎.𝟏𝟗𝟐 𝟓𝐱𝟏𝟎−𝟓
For X at 264 nm = . At 378 nm =
𝟎.𝟑𝟗𝟓 𝒄𝟐 𝟎.𝟏𝟒𝟕 𝒄𝟐
𝟎.𝟑𝟑𝟓 𝟓𝐱𝟏𝟎−𝟓 𝟎.𝟏𝟓𝟎 𝟓𝐱𝟏𝟎−𝟓
For Y at 378 nm = . At 378 nm =
𝟎.𝟑𝟗𝟓 𝒄𝟐 𝟎.𝟏𝟒𝟕 𝒄𝟐
The unknown is compound X (c2 calculated at 264 & 378 nm is the same)
QUANTITATIVE ANALYSIS OF MIXTURES
 Beer’s law also applies to samples containing
more than one kind of absorbing species. As long
as there is no interaction between the species.

 For a mixture solution of M and N

 A total = AM + AN

= 𝜖M b cM + 𝜖N b cN
QUANTITATIVE ANALYSIS OF MIXTURES
 PRACTICE EXAMPLE
 A 0.0450 M solution of para-aminobenzoic acid (PABA) had an
absorbance of 0.844 at 267 nm in a 1.00 cm cuvette, and an
absorbance of 0.034 at 240 nm. A 0.0366 M solution of nicotinic acid
had absorbances of 0.010 and 0.755 at 267 and 240 nm, respectively.
A MIXTURE of PABA and nicotinic acid had absorbances of 0.552
and 0.403 at 267 and 240 nm, respectively. Calculate the
concentration of PABA and nicotinic acid in the mixture.

b = 1.00 cm, 𝜖 = A/bc, so 𝜖 = A/c A = 𝜖 b cPABA + 𝜖 b cNICT at 240 nm

A = 𝜖 b cPABA + 𝜖 b cNICT at 267 nm
𝜖 at 240 nm: 0.267 = 18.76 cPABA + 0.2732 cNICT
PABA: 𝜖 = 0.844 / 0.045 = 18.76 0.403 = 0.7556 cPABA + 20.63 cNICT
NICT: 𝜖 = 0.010 / 0.0366 = 0.2732

𝜖 at 267 nm: cPABA = 0.0140 M and cNICT = 0.0190 M

PABA: 𝜖 = 0.034 / 0.045 = 0.7556
NICT: 𝜖 = 0.755 / 0.0366 = 20.63
NOTES
 Practice:
 Lectures
 Examples in the book
 Problems at the end of the chapter
 Past exam papers
 Internet
 Any reference in Instrumental Analysis.
PRACTICE EXAMPLES
 Q5: A compound of formula weight 280 absorbed
65% of the radiation at a certain 𝛌 in a 2-cm cell at
concentration of 15.0 g/ml. Calculate its molar
absorptivity at the same 𝛌. (Past exam paper )

%T= 35%
T=0.35
A=-log 0.35= 0.456
𝜖 bc
M=
𝑨
Concentration
𝑴𝑨 (g. L-1)
𝜖=
bc
PRACTICE EXAMPLES
 Q6: The absorbance of pure compounds X and Y each in 5x10-5 M
concentration are as follows:
Compound A 264 A 378
X 0.510 0.192
Y 0.335 0.150

 A solution of one of these pure compounds of unknown concentration

yielded absorbance equal to 0.395 at 264 nm and 0.147 at 378 nm.
Which compound is this sample? What is the concentration of the
solution? (Past exam paper)

𝑨 𝟏 𝒄𝟏
= Same analyte, same conditions
𝑨 𝟐 𝒄𝟐
c2 should be the same for the same compound at any wavelength
So calculate c2 for each compound at the 2 wavelengths
𝟎.𝟓𝟏𝟎 𝟓𝐱𝟏𝟎−𝟓 𝟎.𝟏𝟗𝟐 𝟓𝐱𝟏𝟎−𝟓
For X at 264 nm = . At 378 nm =
𝟎.𝟑𝟗𝟓 𝒄𝟐 𝟎.𝟏𝟒𝟕 𝒄𝟐
𝟎.𝟑𝟑𝟓 𝟓𝐱𝟏𝟎−𝟓 𝟎.𝟏𝟓𝟎 𝟓𝐱𝟏𝟎−𝟓
For Y at 378 nm = . At 378 nm =
𝟎.𝟑𝟗𝟓 𝒄𝟐 𝟎.𝟏𝟒𝟕 𝒄𝟐
The unknown is compound X (c2 calculated at 264 & 378 nm is the same)
BEER’S LAW
Quantitative analysis of mixtures and deviations
from Beer’s law.
QUANTITATIVE ANALYSIS OF MIXTURES
 Beer’s law also applies to samples containing
more than one kind of absorbing species. As long
as there is no interaction between the species.

 For a mixture solution of M and N

 A total = AM + AN

= 𝜖M b cM + 𝜖N b cN
QUANTITATIVE ANALYSIS OF MIXTURES
 PRACTICE EXAMPLE
 A 0.0450 M solution of para-aminobenzoic acid (PABA) had an
absorbance of 0.844 at 267 nm in a 1.00 cm cuvette, and an
absorbance of 0.034 at 240 nm. A 0.0366 M solution of nicotinic acid
had absorbances of 0.010 and 0.755 at 267 and 240 nm, respectively.
A MIXTURE of PABA and nicotinic acid had absorbances of 0.552
and 0.403 at 267 and 240 nm, respectively. Calculate the
concentration of PABA and nicotinic acid in the mixture.

b = 1.00 cm, 𝜖 = A/bc, so 𝜖 = A/c A = 𝜖 b cPABA + 𝜖 b cNICT at 240 nm

A = 𝜖 b cPABA + 𝜖 b cNICT at 267 nm
𝜖 at 240 nm: 0.267 = 18.76 cPABA + 0.2732 cNICT
PABA: 𝜖 = 0.844 / 0.045 = 18.76 0.403 = 0.7556 cPABA + 20.63 cNICT
NICT: 𝜖 = 0.010 / 0.0366 = 0.2732

𝜖 at 267 nm: cPABA = 0.0140 M and cNICT = 0.0190 M

PABA: 𝜖 = 0.034 / 0.045 = 0.7556
NICT: 𝜖 = 0.755 / 0.0366 = 20.63
 PRACTICE EXAMPLE
 A 0.0450 M solution of para-aminobenzoic acid (PABA) had an
absorbance of 0.844 at 267 nm in a 1.00 cm cuvette, and an
absorbance of 0.034 at 240 nm. A 0.0366 M solution of nicotinic acid
had absorbances of 0.010 and 0.755 at 267 and 240 nm, respectively.
A MIXTURE of PABA and nicotinic acid had absorbances of 0.552
and 0.403 at 267 and 240 nm, respectively. Calculate the
concentration of PABA and nicotinic acid in the mixture.

b = 1.00 cm, 𝜖 = A/bc, so 𝜖 = A/c ATotal = APABA + ANICT

A = 𝜖PABA b cPABA + 𝜖NICT b cNICT at 240 nm
A = 𝜖PABA b cPABA + 𝜖NICT b cNICT at 267 nm
𝜖 at 267 nm:
PABA: 𝜖 = 0.844 / 0.045 = 18.76
NICT: 𝜖 = 0.010 / 0.0366 = 0.2732 0.267 = 18.76 cPABA + 0.2732 cNICT
0.403 = 0.7556 cPABA + 20.63 cNICT
𝜖 at 240 nm:
PABA: 𝜖 = 0.034 / 0.045 = 0.7556
NICT: 𝜖 = 0.755 / 0.0366 = 20.63
cPABA = 0.0140 M and cNICT = 0.0190 M
PRACTICE EXAMPLE
What is the concentration of a and b compounds in a mixture solution
which has an absorbance of 0.870 at λ 465, and 0.362 at λ 540
(http://fshn.ifas.ufl.edu/)

Sample 𝜖 at λ 465 𝜖 at λ 540

Compound a 11636 26579
Compound b 17949 2667
 Set up equations:
(1) 0.870 = 11636ca + 17949cb
(2) 0.362 = 26579ca + 2667cb
 Multiply equation (2) by 6.73, giving equation (4):
(3) 0.870 = 11,636ca + 17,949cb
(4) 2.436 = 178,877ca + 17,949cb
 Subtract (3) from (4), giving equation (5):
(5) 1.566 = 167,240ca
 Solve for ca:
(6) ca = 9.36 x 10-6 moles/L = concentration of compound a
 Substitute value from (6) into equation (1) and solve for cb:
0.870 = 11636(9.36 x 10-6) + 17949cb
0.870 = 0.109 + 17949cb
0.760 = 17949cb
 cb = 4.24 x 10-5 moles/L = concentration of compound b
NOTES
 Practice:
 Lectures
 Examples in the book
 Problems at the end of the chapter
 Past exam papers
 Internet
 Examples Dr Adnan’s notes.
 Any reference in Instrumental Analysis.
LIMITATIONS TO BEER’S LAW
1. Real limitations:
 Deviations from linearity at:
 High concentrations of the analyte (usually >0.01M).
 High concentrations of others species in the sample
(even at low analyte concentrations).
2. Apparent chemical deviations:
 When analyte dissociate, associate or reacts with
a solvent to produce a product with different
absorption spectrum than analyte.
 e.g. Aqueous solutions of acid/base indicators.
HIn ⇄ H+ + In-
LIMITATIONS TO BEER’S LAW
3. Apparent instrumental deviation with
polychromatic radiation:
 Instruments produce a band of λ’s (not a single λ)
around the desired one, thus a range of 𝜖 is obtained.
INSTRUMENTAL DEVIATION
 The bigger the difference between 𝜖’s the greater
the departure from linearity.
 Deviation from Beer’s law, resulting from
polychromatic beam, is small when changes in
absorption rate with 𝛌 is small.
 Therefore, measurement of absorption is better
at peak maximum (or minimum) in absorption
spectrum. i.e. 𝛌max (or 𝛌min).
 However, 𝛌max (and not 𝛌min) is used since the
absorptivity and thus sensitivity will be higher.
INSTRUMENTAL DEVIATION
 PRACTICAL SCENARIOS OF BEER’S LAW
 Diagnosis of jaundice
 Normal range of bilirubin : 5mg/ dl in infants, 2mg/ dl adults
 Determination by in plasma by spectrophotometry and
applying Beer’s law.
 Phototherapy
EXPERIMENTAL ASPECTS

 You received a sample aspirin of unknown

concentration. How do you measure its
concentration using a spectrophotometer and
applying Beer’s law.

 Think in practical way!

 UV-VIS MOLECULAR
ABSORPTION SPECTROMETRY
113
UV-VIS MOLECULAR ABSORPTION
 Determination and identification of organic and
inorganic species.
 Molecular UV-Vis are the most widely used
methods for quantitative analysis in chemical
and clinical labs.
 Molar absorptivities (𝜖) in the UV-Vis molecular
absorption range from 0-105.

114
ABSORBING SPECIES

 Atomic or molecular UV-Vis absorption is a 2-step

process
 First step involves electronic excitation:
M+ hv → M*

Atomic or molecular species Photon Excited species

 Life time of M* is 10-9-10-8 s. (its concentration at any

instant is negligible).
 M* is terminated by relaxation processes.
 The most common type of relaxation is conversion of
excitation energy to heat :
M*→ M + heat
 Amount of thermal energy is not detectable ( does not 115
disturb the system under study)
ABSORBING SPECIES
 Relaxation may also occur by photochemical reaction:
decomposition of M* to form a new species.
 Relaxation may involve reemission of fluorescence or
phosphorescence.
 UV-Vis absorption result from excitation of bonding
electrons → wavelength of absorption peaks could be
correlated to types of bonds and identification of
functional groups.
 More important, is the application of UV-Vis
absorption to quantitative analysis.

116
TYPES OF ELECTRONIC TRANSITIONS

 Three types:
1. 𝝅, 𝜹 and n electrons. (mainly organic species)

2. d and f electrons (inorganic species)

3. Charge transfer electrons.

117
ABSORPTION BY ORGANIC SPECIES (𝝅, 𝜹 and
n electrons)
 All organic species absorbs electromagnetic radiation
because they contain valence electrons that can be
excited.
 Single bonds: electrons have high excitation energy
so absorption is restricted to 𝛌 < 185 (vacuum UV
region)- experimentally difficult region.
 Spectrophotometric investigation of organic
compounds is thus done at 𝛌 > 185 (long UV and
visible).
 Absorption of long UV and visible is restricted to a
limited number of functional groups called
chromophores which contains electrons with low
excitation energy. 118
TYPES OF ABSORBING ELECTRONS
1. Electrons that participate directly in bonding
between atoms.
2. Nonbonding (unshared) outer electrons (O, S, N,
halogen)
 Overlap of atomic orbitals results in molecular
orbitals which are occupied by bonding electrons.
 This overlap results in low-energy bonding
orbital and high-energy antibonding orbital.
 In the ground state, electrons of a molecule
occupy low-energy bonding orbital.
119
TYPES OF ABSORBING ELECTRONS

 Single bonds in organic molecules have molecular

orbitals which are called sigma (𝜹) orbitals.
 The double bond in an organic molecule contains
a sigma (𝜹) orbital corresponding to one pair of
electron, and a pi (𝝅) molecular orbital associated
with the other pair. (overlap of 2 p orbitals)
 Antibonding sigma and pi orbitals are designated
by 𝜹* and 𝝅* .
 Nonbonding electrons (unshared electrons in O, S,
N, halogen) are designated by n.
120
ELECTRONIC TRANSITIONS
𝜹*
𝝅*

n
𝝅

𝜹
4 possible electronic transitions :
𝜹 → 𝜹*

𝛥 Energy
n → 𝜹*
121
𝝅 → 𝝅*

n → 𝝅*
TYPES OF MOLECULAR ORBITALS IN
FORMALDEHYDE

=𝜹
H =𝝅
C O =n

122
ELECTRONIC TRANSITIONS
𝜹 → 𝜹*
 Requires large energy. (vacuum UV region).
 Single bonds in organic molecules e.g C-C, C-H
 e.g. CH4 exhibits 𝛌max 125 nm, C2H6 exhibits 𝛌max 135 nm
 Absorption maxima due to 𝜹 → 𝜹 * are never observed in
ordinarily accessible region.

n → 𝜹*
 Saturated compounds containing atoms with unshared
electron pairs (non-bonding electrons). E.g. CH3OH
 Requires less energy than 𝜹 → 𝜹 * .
 Absorption due to n → 𝜹 * is in the region 150-250 nm
with most peaks <200 nm.
 𝛜 are low to intermediate and range between 100-3000 L cm-1
mol-1
 Number of organic functional groups with n → 𝜹 * is small.
123
 Absorption maxima shift to shorter 𝛌 in polar solvents.
EXAMPLES OF ABSORPTION DUE TO n → 𝜹 *
TRANSITIONS

124
ELECTRONIC TRANSITIONS

n → 𝝅 * and 𝝅 → 𝝅 *
 Most absorption spectroscopy applications are based
upon these transitions.
 Absorption peaks are in the region 200- 700 nm
(experimentally convenient spectral region).
 Both require the presence of an unsaturated functional
group (double bond) to provide 𝝅 orbitals.
 Unsaturated absorbing centres are called
chromosphores.
 𝛜 for n → 𝝅 * 10 -100 L cm-1 mol-1, 𝛜 for 𝝅 → 𝝅 * 1000-
10000.
125
EXAMPLE OF ABSORPTION SPECTRUM

126
SOLVENT EFFECTS

Solvent effects on absorption 𝛌:

 n → 𝝅 * peaks are shifted to shorter 𝛌 (hypsochromic
or blue shift) with increasing polarity of the solvent.
 Increased solvation of the unbonded electron pair lowers
the energy of n orbital, but not the 𝝅 * excited state.
 Higher blue (hypsochromic) shift (30 nm or more) is
observed with water or alcohols (extensive H-bonding
between solvent protons and unbonded electrons).

127
SOLVENT EFFECTS
 A small (5 nm) bathochromic (red) shift with
increasing solvent polarity affects both n → 𝝅 *
and 𝝅 → 𝝅 * transition.
 Polarisation forces between the solvent and the
absorber lower energy level of both unexcited
and excited states, but effect on excited is
greater. Thus small effect is observed.
 Effect is overshadowed in n → 𝝅 * due to the larger
hypsochromic effect.
 In 𝝅 → 𝝅 * small bathochromic or red shift is observed
with increasing solvent polarity.

128
ORGANIC CHROMOPHORES

129
Absorption maxima depends on chromophore and is affected by
solvent and structural details of the molecule.
EFFECTS OF CONJUGATION OF CHROMOPHORES
 In molecular orbital, electrons are considered to
be delocalised by conjugation (at least 4 atomic
centres involved)
 Conjugation lowers 𝝅 * energy and thus
absorption maxima are shifted to longer 𝛌
(bathochromic effect).
e.g. 1,3-butadiene CH2=CHCH=CH2 has longer 𝛌 than
unconjugated diene.
Bathochormic effect is even larger when 3 double bonds
are conjugated and so on.
130
EFFECT OF MULTICHROMOPHORE ON
ABSORPTION

131
EFFECT ON MOLAR ABSORPTIVITY
 Conjugation also results in higher absorption signal
(higher 𝛜) . This is called hyperchromic effect.
e.g. At 185 nm,1-hexene has a molar absorptivity of
about 10,000 L mol-1 cm-1 but hexa-1,4-diene has a
molar absorptivity of twice as much as 1-hexene.
However, when the double bonds are conjugated as
in hexa-1,3-diene the molar absorptivity is about
21,000 L mol-1 cm-1

132
EFFECTS OF CONJUGATION OF CHROMOPHORES

 If greater than one single bond apart:

 𝛜 are relatively additive (hyperchromic shift)
 l constant

CH3CH2CH2CH2CH=CH2 lmax= 184 emax = ~10,000

CH2=CHCH2CH2CH=CH2 lmax=185 emax = ~20,000

 If conjugated
 - shifts to higher l’s (red shift)
H2C=CHCH=CH2 lmax=217 emax = ~21,000 133
SPECTRAL NOMENCLATURE OF SHIFTS

134
SUMMARY
s → s* transition in vacuum UV (single bonds)

n → s* saturated compounds with non-bonding

electrons
 l ~ 150-250 nm
 𝛜 ~ 100-3000 ( not strong)

n → p*, p → p* requires unsaturated functional

groups (eq. double bonds) most commonly used,
energy good range for UV/Vis
 l ~ 200 - 700 nm
 n → p* : 𝛜~ 10-100
 p → p*: 𝛜~ 1000 – 10,000 135
136
CONJUGATION
CONJUGATION
 Conjugation between doubly bonded Oxygen of
aldehydes, ketones and carboxylic acids results in
bathochromic shift.


O=
e.g. CH2=CHCH2CH2CCH3 𝛌max = 282 nm (n→𝝅*)
O=

CH2=CHCCH3 𝛌max = 324 nm (n→𝝅*)

𝛌max = 219 nm (𝝅 →𝝅*)
The weak absorption peak of unsaturated aldehydes and
ketones (n→𝝅*) is shifted by 40 nm or more. The strong
absorption peak (𝝅 →𝝅*) is shifted from vacuum UV to long
UV region.
CONJUGATION

 Do you expect difference in 𝛌 max ? Why? If different

which compound has longer 𝛌 max ?

𝛌 max = 295 nm 𝛌 max = 340 nm (𝝅 →𝝅*)

𝛌 max = 440 nm (n→𝝅*)
CONJUGATION
 Bathochromic effect is also observed when 2
carbonyl or carboxylate group are conjugated.

 Wavelengths of absorption peaks for conjugated

systems are sensitive to the types of groups
attached to the doubly bonded atoms.
ABSORPTION BY AROMATIC SYSTEMS
 UV spectra of aromatic hydrocarbons are characterised
by 3 sets of bands that originate from 𝝅 →𝝅*
transitions.

 Benzene has a strong absorption peak at 184 nm (𝛜max ~

60,000); a weaker band, called the E2 band at 204 nm (𝛜max
~ 7900); and still a weaker band, termed the B band, at 265
nm (𝛜max ~ 200).

 The 3 characteristic bands are strongly affected by ring

substitution.

 E2 and B bands are of interest (long UV-Vis region)

ABSORPTION CHARACTERISTICS OF
AROMATIC COMPOUNDS

142
AUXOCHROMES
 Auxochrome is a functional group that does not
itself absorb on the UV-Vis region but has the
effect of shifting chromophore peaks to longer 𝛌
as well as increasing their intensities.
 Auxochromic substituents have at least one pair of n
pair of electron capable of interacting with 𝝅
electrons of the ring.
e.g. –OH –NH2 have auxochromic effects , particularly
with respect to the B band.
 Interaction stabilises the 𝝅* state lowering its energy
thus resulting in bathochromic shift.
AUXOCHROMIC EFFECT
SOLVENTS
 In choosing a solvent to perform absorption
measurement, solvent should be transparent in
the UV-Vis region.
 Solvent transparency minimum: The wavelength
below which solvent cannot be used because of
absorption.
 The type of solvent affects the absorption
maxima of the analyte.
 Common solvents:
 UV spectrophotometry : water, 95% ethanol,
cyclohexane and 1,4-dioxane.
 Visible region: any colourless solvent.
UV-VIS ABSORPTION
INSTRUMENTATION
INSTRUMENT COMPONENTS
1. Sources
2. Wavelength selector
3. Sample container
4. Radiation transducer
5. Signal processors and readout devices
SOURCES
 A continuum source is required whose power does
not change sharply over a considerable range of
wavelengths.

Deuterium and Hydrogen Lamps

 A continuum spectrum in the UV region is
produced by electrical excitation of deuterium or
hydrogen at low pressure.
 Spectrum range 160- 375 nm.
 > 400 nm : emission lines→ nuisance
 Quartz windows should be employed. Glass
absorbs strongly < 350 nm.
SOURCES
Tungsten Filament Lamps
 Most common source of visible and near-IR
radiation.
 Wavelength range: 350- 2500 nm. The glass
envelope of the filament imposes the lower limit.
 Tungsten/ halogen lamps
 Contain a small quantity of iodine.
 Lifetime double that of the ordinary lamp.
 Tungsten sublimes and reacts with iodine→ volatile WI2
 WI2 hits strike the filament→ decomposition→ W redeposit

Xenon Arc Lamps

 Wavelength range: 200-1000 nm.
 Peak intensity at 500 nm.
SAMPLE CONTAINERS
 Sample containers are called “cells” or “cuvettes”
 Material should be transparent to radiation in
the spectral region of interest.
 Quartz or fused silica:
 Suitable for UV region (<350 nm):
 Also transparent in the visible region
 Silicate glass can be employed in the 350- 2000
nm region.
 Plastic can be used in some visible region
application.
SAMPLE CONTAINERS
SAMPLE CONTAINERS
 Most common cell length is 1 cm.
 0.1- 10 cm can be also used.

 Quality of spectra is critically dependent upon

cell handling and maintenance.
 Use matched cells
 Clean cells before and after use
 Surfaces of cell windows should not be touched
during the handling. Fingerprints and grease alter
transmission.
 Never dry by heat, potential physical damage.
TYPES OF INSTRUMENTS
1. Single-beam
2. Double-beam in space
3. Double-beam in time.
4. Multichannel.
SINGLE-BEAM INSTRUMENTS

 Cells interposed alternately in the radiation beam.

DOUBLE-BEAM IN SPACE

 Many modern spectrophotometers are based on this

design.
 Two detectors.
DOUBLE-BEAM IN TIME

 Beam separated in time by a rotating sector

mirror.
 Entire beam directed first through the reference
cell and then through the sample cell.
DOUBLE-BEAM INSTRUMENTS
 Compensate for fluctuations in the source radiant
output as well as drift in detector and amplifier.
 Compensate for wide variations in source
intensity with wavelength.
 Suitable for continuous recording of absorbance
spectra
MULTICHANNEL INSTRUMENTS
 Most recent type of spectrophotometers.
 Single beam instrument based on diode array
transducer.
 Light (polychromatic) passes through the sample and
then passes into grating (acts like a prism).
 Dispersed radiation falls onto diode array transducer.
 Diode array consists of array of several hundreds of
photodiods (15-50 µm each) on a silicon chip (1-6 cm).
 Radiation on any diode surface results in a current
proportional to the radiant power.
 Each diode corresponds to a different wavelength.
 Entire spectrum is obtained in < 1s.
DIODE ARRAYS OF VARIOUS SIZE
PHOTODIODE ARRAY

 Entire spectrum is obtained in < 1s.

 Powerful tool for:
 Transient intermediates in fast reactions.
 Kinetic studies
 Quantitative and qualitative determination in liquid
chromatography.