INSTRUMENTAL ANALYSIS

Collected by
Dr. Abdul-Aziz Al-Attar
Lecturer of Pharmaceutical Analytical
Chemistry
 Anions

Qualitative
Cations

Classical
e.g.: titration

Instrumental
Analytical 1. Spectro-chemical methods
chemistry 2. Electrochemical methods
3. Separation methods
a. HPLC
Quantitative b. GC
c. Electrophoresis
d. Hybrid techniques
LC-MS=liquid chromatography mass spectrometry
GC-MS=gas chromatography mass spectrometry
GC-IR=gas chromatography infrared spectroscopy
 Classical Instrumental
Accurate and precise at gm% level Highly sensitive analysis
or higher ppm (µg/ml)
Or
ppb (ng/ml)
Disadvantages • Requires skills • Cost
• Time consuming • Complexity
• Destructive technique • Maintenance
(not suitable for forensic analysis) • Requires reference
standard materials

Advantages • Low cost • Highly sensitive

• No complexity (ppm & ppb)
• No maintenance • Time saving
• Automated
• Most are mutli-channel
technique (give more
information)
 Spectro-chemical methods
Measurement of light absorbed Measurement of light emitted

UV-Visible spectroscopy = Atomic emission spectroscopy

Spectrophtometry

IR spectroscopy Fluorescence spectroscopy

PMR = proton magnetic resonance

 Spectrophtometry

The study of interaction of spectrum of light

with a substance to be analyzed for its
identification (qualitative analysis) and/or
determination (quantitative analysis)
• EM radiations were found to behave as waves i.e. they
are refracted, reflected and diffracted. They travel in
space in straight line and have two fields represented
by an electric field and magnetic field. Both fields are
perpendicular on each other, which in turn are
perpendicular on the direction of propagation of the
radiation.
Light (Electromagnetic radiation= EMR)
has two properties:

•Wave properties
1

2
•Particle properties
• Dual nature of light

Light exhibits wave property during its

propagation and particle properties
during its interaction with matter. The
double nature of light (Waves and
particles) is known as dualism.
 Properties of EMR
1-Wave Properties:
EMR is characterized by:
Velocity • C: in vacuum is 3*108 m/sec or 3*1010 cm/sec

• λ: distance between two successive maxima(crests&

Wavelength tops) or minima (troughs & bottoms)

• ῡ: No. of waves in 1 cm
Wavenumber • ῡ=1/λ i.e. reciprocal of wavelength

Frequency
the number of cycles (waves) per second
(S-1) [Hertz (Hz)] ʋ=C/λ
Relation between C, ῡ & λ

ʋ=C/ λ=C*ῡ
 2. Particle properties
Light is considered as a stream of energy
packets called photons or quanta and can
expressed by Max Plank relation

Energy of photon “E” =hʋ=hc/λ

=hc ῡ

h:Plank’s constant=6.63*10-34 J.sec

 Max Plank Equation
(E=hʋ=hc/λ=hc*ῡ)

The energy of photons is directly

proportional to frequency and wave
number and inversely proportional to
wavelength.
Electro Magnetic Radiation “EMR”
The spectrum of light is known as
electromagnetic radiation (EMR) which
involve the following:
• Ƴ- rays
• x- rays
• UV (Ultra violet)
• VL (Visible light)
• IR (Infra red)
• Micro waves
• Radio waves
 The UV- Visible Radiation
• The UV region of spectrum extends from about 200 - 400
nm.
• The visible region of spectrum extends from about 380
nm to about 780 nm. The eye can normally detect only
the colors within this wavelength range, that is why it is
called visible.
• The visible spectrum consists of seven colors which are;
violet, indigo, blue, green, yellow, orange and red, each
color has characteristic wave length region.
• Polychromatic light is a beam of light
containing several wavelengths.

• Monochromatic light is a beam of light

containing one wavelength.
• When all the wavelengths or colors of the visible
light are transmitted or reflected together, the light
appears as white light.

• While if all wavelengths or colors of visible light are

absorbed, it appears black.

• Colored substances appear colored because they

selectively absorb some of wavelengths of visible light
and transmit or reflect other wavelengths or colors.
The Visible Spectrum and their complementary colors
Complementary Wavelength region
Color (apparent) Color (nm)

Violet Yellow-green 400-435

Blue Yellow 435-480

Blue-green Orange 480-490
Green-blue Red 490-500
Green Purple 500-560
Yellow-green Violet 560-580

Yellow Blue 580-595

Orange Green-blue 595-650
Red Blue-green 650-750
 Interaction of substance with EMR
• When a substance (atom, molecule or ion) interacts
with EMR, it will absorb energy and the molecule is
said to be excited because electrons undergo
transition from original energy level (ground state =
Eg) to an excited state (Es).
• Transition energy is given by:
Et or ∆E = Es – Eg = h ʋ
M + E → M* (excitation or activation)
• After a brief period (10-6 - 10-9 S) M* relaxes to its
ground state.
M* → M + E (relaxation)
• Relaxation process (or deactivation) takes place
through collision process in which energy is usually
lost as heat. In some cases, relaxation occurs by
emission of light usually of longer wavelength a
process known a photoluminescence.

Absorption
M + h M* M + heat
Ground UV-Visb. Excited or
state radiation state
M + h
 Ways of absorption of Energy
• A molecule may absorb light energy in three ways:
1- By raising an electron to a higher energy level
(electronic E).
2- By increasing the vibration of constituent of nuclei
(vibrational E)
3- By increasing the rotation of molecule around its axis
(rotational E)
• The total energy of a molecule is given by:
• Etotal = Eelectronic + Evibrational + Erotational .
• The relative energies is about 10000:100:1
NB:
• The UV-Vis radiation causes all types of
.

energy.
• IR radiation cause vibrational and rotational
energy, while,
• Microwaves cause only rotational energy.
• If large amount of energy (from far UV) is
absorbed by the molecule, bonds may be
ruptured and new compounds are formed. This
phenomenon is described as photolysis.
Blue color is weakly absorbed by sea water that is
why the sea is blue.
Red color is strongly absorbed by sea water that’s
why the divers cannot see their blood when they
injured under the sea water
Types of Electronic Transitions
Theoretically

1. δ → δ*
2. δ → π*
3. π → π*
4. π → δ*
5. n → δ*
6. n → π*
 Actually
1.δ → δ (X)
*

2.δ → π* (X)
3.π → π*
4.π → δ* (X)
5.n → δ* (X)
6.n → π*
 δ* anti-bonding

Excited
state π* anti-bonding

n Non-bonding

Ground π bonding
state

δ bonding
 Absorption Spectrum
• Absorption Spectrum: the relation between absorbance
and wavelength.
• It is characteristic for each compound in specific solvent
(considered as finger print for each substance).
• The absorption spectrum is characterized by the presence
of λ max
• λmax is the wavelength of maximum absorbance, it is used
in quantitative measurements.
Absorption Spectrum
1.0
 max
UV Visible
Absorbance

0.0
200 400 800

Wavelength, , generally in nanometers (nm)

 Some Important
Terms in Spectrophotometry
• Chromophores:
• Unsaturated groups imparts color to the molecules
(responsible for absorption in UV – Visible region) e.g.
C=C, C=O, N=N, N=O…. the absorption of the molecule is
the sum of absorption of all its chromophores.
• Increasing the number of chromophores leading to
increase the absorption intensity.
• Auxochromes: .

Saturated groups possess unshared lone pair of

electrons; when attached to chromophores they
alter both the wavelength (λmax) and the intensity of
absorption maxima e.g. –OH, –NH2, ……etc.

• Auxochromes function by entering into resonance

interaction with nearby chromophore, thus
increase the extent of conjugation
• Bathochromic shift: the shift of λmax to
.

longer wavelength; known by red shift, due

to substitution and / or solvent effect.
• Hypsochromic shift: the shift of λmax to
shorter wavelength; known by blue shift,
due to substitution and / or solvent effect.
• Hyperchromic effect:
An increase in absorption intensity.
• Hypochromic effect:
A decrease in absorption intensity.
 Abbreviations
Symbol Meaning

C Velocity of light in vacuum (m/sec) &

Concentration

λ Wavelength (nanometer=nm)

ʋ Frequency (Hertz =Hz=S-1)

ῡ Wavenumber (cm-1)

h Plank’s constant (J.sec)

EMR electromagnetic radiation

UV Ultra violet
Lower Case Greek Letter Lower Case Greek Letter Name
Letter Name Letter
α Alpha ξ Xi
β Beta ο Omicron
γ Gamma π Pi
δ Delta ρ Rho
ε Epsilon σ,ς * Sigma
ζ Zeta τ Tau
η Eta υ Upsilon
θ Theta φ Phi
ι Iota χ Chi
κ Kappa ψ Psi
λ Lambda ω Omega
μ Mu Mu
ν Nu Nu
 Questions
Define the following
1. Wavelength
2. Wavenumber
3. Frequency
4. Bathochromic shift= red shift
5. Hypsochromic shift= blue shift
6. Hyperchromic effect
7. Hypochromic effect
8. Auxochromes
9. Chromophores
10. Polychromatic light
11. Monochromatic light
12. Absorption spectrum
 Write short notes on:
1. Dualism of light

2. Max Plank Equation

3. Interaction of substance with EMR

4. Ways of energy absorption

5. Types of electronic transitions

Mark (T) for the true statement or (F) for
the true statement
1. Wavelength is inversely proportional to frequency ( )
2. Wavelength is directly proportional to frequency ( )
3. Visible region wavelength is shorter than UV region ( )
4. 1/λ =wavenumber ( )
5. The longer the wavelength, the lower the frequency ( )
6. Polychromatic light is composed of single wavelength ( )
7. Absorption spectrum is a relationship between absorption and concentration ( )
8. Blue color is weakly absorbed by the sea water ( )
9. Blue color is strongly absorbed by the sea water ( )
10. Wave number is the number of waves per meter ( )
11. UV is important to prevent rickets in children ( )
12. If a driver was injured in the sea, he would not see his red blood ( )
13. In UV/visible region the wavelength is expressed in angstrom unit ( )
14. Absorption spectrum is useful for qualitative analysis ( )
15. The color of a solution is the complement of the color of the light that is absorbs ( )
16. In EMR, the electric field is parallel to the magnetic field ( )
17. The part of a molecule responsible for light absorption is called a chromophore ( )
18. Shifting λmax to longer λ is called bathochromic effect ( )
 Complete the following statements
1. The ………is the distance between two successive crests or troughs.
2. Relationship between the energy of a photon to its wavenumber can
be given by ………..
3. A beam of light carrying radiation of only one λ is called ……………..,
while that carrying several λs is called …………………
4. When you double the wavenumber, you …………….the energy.
5. When you double the wavelength, you …………….the energy.
6. When you double the frequency, you …………….the energy.
7. The ……………is the number of complete oscillations that the wave
makes each second.
8. ………………is a plot of absorbance versus λ.
9. Unsaturated organic functional groups that absorb EMR in the
UV/visible region are called…………….
10. ………………..is a functional group that does not absorb EMR in the
UV/visible region but increasing the amplitude.