Lecturer : Dr E.

Makhado
Email : [Link]@[Link]
Office No. : 1014 N-block
Ext. : 4627

1
 Module Outline

Analytical Chemistry 3 Module Code SCHA032 N0. of Credits: 16

Module Lecturers

Dr E Makhado Prof NC Hintsho-Mbita

Office No: 1014 N-block Office No: 2012 N-block

Ext. No: 4627 Ext. No: 2205
Email: [Link]@[Link] Email: [Link]-mbita@[Link]

Consultation time Important dates

Monday-Thursday Test 1 (08/08)

10:00 am-16:00 pm Test 2 (29/08)

2
 INTRODUCTION
• Analytical chemistry is the branch of chemistry dealing
with the separation, identification, and quantification of
the chemical components of natural and artificial
materials.

• The basic knowledge of analytical chemistry is applied

when detecting the presence and determining the
quantities of chemical compounds, such as:
– heavy metals during water purification or
– arsenic in tissue samples.

3
 INTRODUCTION
• It also encompasses many different spectrochemical and
electro-analytical techniques as well as separation
methods, all of which are used under various
experimental conditions.

• This branch of chemistry teaches the general theories

behind the use of each instrument as well as analysis of
experimental data.

INSTRUMENTS

4
 MODULE OUTLINE
Module Content Topics

1. Spectrochemical 2. Separation Methods 3. Electrochemical Methods

Methods

1.1. Introduction to 2.1. Introduction to 3.1. Introduction to

Spectrochemical Methods Analytical Separations Electrochemistry
1.2. Molecular Absorption 2.2. Solvent Extraction: R, 3.2. Potentiometry &
Spectrometry: UV-Vis & IR D, L-L & L-S Voltammetry
1.3. Molecular Fluorescence 2.3. Chromatography: GC, 3.3. Electrogravimetry &
Spectrometry LC & SFC Coulometry
1.4. Atomic Spectrometry:
AA, ICP & MS

5
 SPECTROCHEMICAL METHODS

After studying this topic student should be able to :

– Define each spectroscopic method
– Describe each spectroscopic method principles
– Explain major components of each spectroscopic
method and their functions
– Explain application of each spectroscopic method
– Explain factors affecting function of each
spectroscopic method
– Describe advantages & disadvantages of each
spectroscopic method
– Data analysis
6
 INTRODUCTION TO
SPECTROCHEMICAL METHODS

7
 Introduction
• Spectroscopy and spectrograph
– The measurement of radiation intensity as a function
of wavelength.
• Often used to describe experimental spectroscopic
methods

– Spectral measurement devices

• spectrometers,
• spectrophotometers,
• spectrographs or
• spectral analyzers.

8
 Introduction
– Spectroscopic studies were central to the development
of quantum mechanics.

– Spectroscopy is used in PHYSICS and CHEMISTRY

because atoms and molecules have unique spectra.

– As a result, these spectra can be used to detect,

identify and quantify information about the atoms and
molecules present in the analyte.

9
10
• Spectrochemical methods
– Absorption or emission are two fundamental processes describing
how light interacts with matter.
• Almost all analytical instrumental methods involve these processes

– Spectroscopy or spectrometry
– Photo-physical and chemical processes in solutions involve how
molecules in a solution interact with light (EM radiation) and
the subsequent changes that occur.

• Spectroscopic instrument uses

– a phototube or photomultiplier tube (PMT) as a light sensor in a
spectrophotometer.

11
 Spectrochemical analysis

An analysis of light absorbed or emitted in relation to the quantity of

analyte present in the sample is referred to as degree.
– Critical qualitative and quantitative measurements!

• Atomic spectroscopy
The elemental composition of atoms is determined by their spectral
differences.

• Molecular spectroscopy
Examines how molecules interact with EM radiation, causing transitions
between energy levels.

12
13
 Spectroscopy
• Interaction of radiation with a sample

• The study of the molecular or atomic structure of a

substance by observation of its interaction with
electromagnetic radiation.

• QUANTITATIVELY - For determining the amount of

material in a sample
• QUALITATIVELY – For identifying the chemical
structure of a sample

14
 Spectroscopy
• The basic principle shared by all spectroscopic techniques

response

response
sample detector


• The response is usually recorded as a function of radiation
wavelength.
• A plot of the response as a function of wavelength is
referred to as a spectrum.

15
 Regions of the electromagnetic spectrum
• Most of us are aware of many different ways of
transmitting energy and these phenomena come together
in one physical entity called the ELECTROMAGNETIC
SPECTRUM

• The difference between these sources of radiation is the

amount of energy they radiate.

• The radiation from these and other sources covers a

range of energies

16
 Regions of the electromagnetic spectrum

Classes of electromagnetic radiation:

– Gamma radiation
– X-ray radiation
– Ultraviolet and Visible radiations
– Infrared radiation
– Microwave radiation
– Radio waves

17
 The electromagnetic spectrum

Change of Change of Change of electron Change of nuclear

orientation configuration distribution configuration

NMR= 10 m - 100cm wavelength

ESR = 100 cm - 1cm wavelength Change of spin
18
 Radiation Energy
• The strength of the radiation energy will interact with the
molecules in different ways:

– High energy sources produce breaking of bonds

• X-Ray,
• γ Rays, …

– Medium energy sources excite electrons

• UV Spectroscopy
• VISIBLE Spectroscopy

19
 Radiation Energy
– Low energy sources produce vibrations in chemical
bonds

• Near-infrared
• Mid-infrared
• Far-infrared Energies

– Very low energy sources produce rotation of the

chemical bonds

• Microwaves
• Radio waves

20
 The particle nature of radiation energy
• Electromagnetic Spectrum: E = hv

– UV, visible, and infrared regions

• Radiation are transformed in a waveform

– Nanometer and micrometer units used for

wavelength

– Relationship
» Long wavelength = low energy
» Short wavelength = high energy

21
The particle nature of radiation energy

• Infrared region
– Have short Wavelengths or high wavenumber
– Their energy is higher than radio or television
– Harmless
»Remotes for TVs, VCRs, etc

• Dangerous radiations: UV, x-rays, and gamma rays

– Very short wavelengths
– Very high energy

22
 Effect of energy on a molecule
ELECTROMAGNETIC SPECTRUM
ENERGY
1.2 1.2 12000 31 15 0.1 0.001
( kJ/mol)
x105 x107 0 0 2 2

Electronic excitation
e-

FREQUENCY
(Hz) 1020 1018 1016 1014 1012 108

Cosmic γ x Ultra

visibl
Infrared Radio
rays violet Microwave
ray rays waves

e
s
WAVELENGTH
(m) 10-12 10-11 10-9 10-6 10-3 10-1

23
 Interaction of light with matter
LIGHT MATTER INTERACTIONS

SCATTERING ABSORPTION

Elastic, PHOTOLUMINESCENCE
Inelastic

Fluorescence Phosphorescence

24
 Interaction of light with matter
• Interaction in spectroscopy
involve transition between
energy levels of chemical
species
• Interactions: reflection,
refraction, elastic scattering,
interference and diffraction
are related to the bulk
properties of materials
• Interactions depends strongly
on the energy of the radiation
used and the mode of detection
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Absorption Methods
– It refers to spectroscopic techniques that measure the absorption of
radiation, as a function of frequency or wavelength, due to its
interaction with a sample.

– The sample absorbs energy, i.e., photons, from the radiating field.

– The intensity of the absorption varies as a function of frequency, and

this variation is the absorption spectrum.

26
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Absorption Methods
Incident light
S2

E2=hv2

Absorbance
sample

S1

E1=hv1
Transmitted light

S0
1 2
27
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Emission Spectroscopy
– It is a type of electromagnetic spectroscopy which
analyses fluorescence from a sample.

– It involves using a beam of light that excites the

electrons in molecules of certain compounds and
causes them to emit light of a lower energy.

– At low concentrations the fluorescence intensity will

generally be proportional to the concentration of the
fluorophore.

28
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Emission Spectroscopy
Incident light S2

E21=hv21

Intensity
S1
sample

E1=hv1
E2=hv2

Emitted light S0 2 1 21

29
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Photoluminescence Spectroscopy
– The emission of photons is measured after absorption.

– Light source, self-emission which means the electrons

transferred to the lowest level spontaneously

– A fluorescence excitation spectrum provides

information about the absorption spectrum of the
excited fluorescent molecule in the excited state.

30
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Substance Detection & Concentration
• Photoluminescence Spectroscopy
Incident light

sample

Transmitted & Emitted light

31
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Chemical Identification
• IR Spectroscopy
– It is a powerful analytical tool for determining the
structure of both inorganic and organic
compounds

– All substances vaporize molecules that can be

detected with their own specific spectral signature
while being exposed to an appropriate light.

– Vibrating  Energy  Frequency

32
 Spectroscopic measurements
• There are several ways to use a spectrometer:
– Chemical Identification
• Atomic Spectroscopy
– It is used for determination of elemental
composition by its electromagnetic or electron
beam.

– It is closely related to other forms of spectroscopy.

– It can be divided by atomization source or by the

type of spectroscopy used.

33
 Tutorial Questions
1. Why do analytical chemists employ spectrochemical methods? (3
marks)

2. In electromagnetic radiation, x-rays region is regarded as

dangerous radiation whereas infrared region known as a harmless
radiation. Explain, why is x-ray radiation harmful? (3 marks)

3. Explain and give a conceptual diagram to demonstrate absorption

and emission processes in spectroscopic measurements. (5
marks)

4. Define these terms: luminescence, photoluminescence,

chemiluminescence and bioluminescence. (4 marks)

34
MOLECULAR ABSORPTION
SPECTROMETRY

35
 Introduction
• Molecular spectroscopy:
– Quantitative and qualitative analyses
• UV/Vis spectroscopy • IR Spectroscopy

36
 Ultraviolet and Visible Instrument
• Single-Beam Instruments
– The light source is imaged upon the sample
– A fraction of the light is transmitted or reflected from the
sample
– The light from the sample is imaged upon the entrance slit
of the monochromator
– The monochromator separates the wavelengths of light and
focuses each of them onto the photodetector sequentially.

Fixed wavelength light source

37
38
 Ultraviolet and Visible Instrument
• Single-Beam Instruments
– two sources are used, a tungsten halogen and a
deuterium lamp
– mechanism for source selection should be available.
– The wavelength selector is a grating or prism
monochromators and
– the detector is usually a vacuum phototube or a
photomultiplier tube in higher cost instruments.
Continuous source

39
 Ultraviolet and Visible Instrument
– Double-Beam Instruments
• Schematic diagram of a double-beam UV-Vis.
Spectrophotometer
– Sources (UV and visible):
– Wavelength selector (monochromator)
– Sample containers
– Detector
– Signal processor and readout

40
 Ultraviolet and Visible Instrument
– Double-Beam Instruments

41
 Ultraviolet and Visible Instrument
– Carry 100 Double-Beam Instruments

42
[Link]
 Ultraviolet and Visible Instrument

Monochromator Detector>> PMT

– It is a device that contains an – Types of photon detectors

entrance and exit slits • Phototube and photomultiplier tube
– Wavelength selector (PMT)
– Exit slit is for isolation of a small • Photoconductive cells
band of wavelengths • Silicon photodiodes and
– Polychromator has multiple exit photo/diode arrays
slits • Charge transfer device 43
 Photomultiplier tube (PMT)
• PMT is a commonly used detector in UV-Vis spectroscopy
• It consists of a photoemissive cathode, several dynodes and an anode.
• A photon of radiation entering the tube strikes the cathode, causing
the emission of several electrons.
• The electrons strike the dynode, causing the emission of several
electrons for each incident electron.
• Each original photon has produced 106 - 107 electrons.
• The resulting current is amplified and measured.

44
Photomultiplier tube (PMT)

45
 Ultraviolet and Visible Spectroscopy
• Visible Spectroscopy
– Sunlight is white light and covers a wavelength
range of 380-750nm.
– A simple physics experiment shows that white light
is actually a composition of a range of colours i.e.,
light of different energies and hence wavelengths.

Red
Orange
WHITE
Yellow
LIGHT
Green
Blue
Indigo
Violet

46
 Ultraviolet and Visible Spectroscopy
• When a sample only absorbs light of a single
wavelength the eye sees COMPLEMENTARY colours.
Wavelength Range Colour Colour Seen By
Absorbed Absorbed Eye
380 - 430 Violet Yellow - Green
430 - 480 Blue Yellow
480 - 490 Green - Blue Orange
490 - 500 Blue - Green Red
500 - 560 Green Purple
560 - 580 Yellow - Green Violet
580 - 590 Yellow Blue
590 - 610 Orange Green - Blue
610 - 750 Red Blue - Green

LOW ENERGY 47
 UV/Vis Absorption process
• The absorption of UV or visible radiation corresponds to the
excitation of outer electrons.

– There are three types of electronic transition which can be

considered:

• Transitions involving p, s, and n electrons

• Transitions involving d and f electrons
• Transitions involving charge-transfer electrons

48
 UV/Vis Absorption process
•   * transition: high-energy, accessible in vacuum
UV (max <150 nm). Not usually observed in
molecular UV-Vis.
• n  * transition: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.

49
 UV/Vis Absorption process
• n  * and   * transitions: most common transitions observed in
organic molecular UV-Vis, observed in compounds with lone pairs
and multiple bonds with max = 200-600 nm.

• Any of these require that incoming photons match in energy gap

corresponding to a transition from ground to excited state.

• Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol

50
Absorbing species containing p, s,
and n electrons

51
 d-d or f-f electronic transitions
• Four types of transitions:
(i) Within the same atom e.g. d-d or f-f transition
(ii) To adjacent atom (charge transfer)
(iii) To a delocalized energy band (photoconductivity)
(iv) Promotion of an electron from valence band to
conduction band (band-gap in semiconductors)

52
 Charge - Transfer Absorption
• Many inorganic species show charge-transfer absorption and
are called charge-transfer complexes.
• For a complex to demonstrate charge-transfer behaviour,
one of its components must have electron donating
properties and another component must be able to accept
electrons.
• Absorption of radiation then involves the transfer of an
electron from the donor to an orbital associated with the
acceptor.
• Molar absorptivities from charge-transfer absorption are
large (greater than 10,000 L mol-1 cm-1).

53
 UV / Visible Spectroscopy - Theory
• Sample can absorb some of the radiation then the
transmitted light intensity (It ) will be less than the
incident light intensity (Io). It< Io
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I Intensity (I t

o) )

• The amount of light transmitted with respect to

the incident light is called TRANSMITTANCE (T)
ie., >>>>Fraction of incident radiation
transmitted by the solution

54
 UV / Visible Spectroscopy - Theory
• Transmittance: T=
I t

I o

I
%T = t
X 100
I o

• ABSORBANCE: A = - log T

I 2
A = - log t
B
I
A
A = log Io o 0
I 220 Wavelength(nm) 380

For of %T = 0 and 100t the corresponding absorbance

values will be 0 and 2, respectively
55
 The Laws of Spectrophotometry
• There are two very important basic laws and a third one
which is a combination of the two:

– LAMBERTS LAW – ABSORBANCE (A) is proportional

to the PATH LENGTH (l) of the absorbing medium.

– BEERS LAW - ABSORBANCE (A) is proportional to the

CONCENTRATION (c) of the sample.

– BEER- LAMBERT LAW - ABSORBANCE (A) is

proportional to c x l

56
 Beer-Lambert Law
– The Beer-Lambert law (or Beer's law) is the linear
relationship between absorbance and concentration of an
absorbing species.
– The general Beer-Lambert law is usually written as:
A = abc
where A is the measured absorbance, a is a wavelength-
dependent absorptivity coefficient, b is the path length,
and c is the analyte concentration.
– When working in concentration units of molarity, the
Beer-Lambert law is written as:
A = bc
where  is the wavelength-dependent molar absorptivity
coefficient with units of M-1 cm-1. 57
[Link]
 Beer-Lambert Law
• UNITS OF THE MOLAR EXTINCTION COEFFICIENT
– CONCENTRATION (c) - Moles litre-1
– PATHLENGTH (l) - cm

A = cl Hence  =A
cl
= 1 ˛

mole litre-1 x cm
 = mole-1 litre x cm -1
But 1 litre = 1000 cm3
 = 1000 mole -1 cm3 x cm -1
Hence Units of = 1000 cm2 mole -1 or L/mol/cm

58
 Importance of the Beer-Lambert Law
• A = cl but if  and l are constant

• ABSORBANCE  CONCENTRATION and should be linear relationship

– Prepare standards of the analyte to be quantified at known concentrations
– and measure absorbance at a specified wavelength.

– Prepare calibration curve.

• ABSORBANCE AT 300nm
• x
– From measuring absorbance of sample
• x
– Concentration of analyte in sample • x
can be obtained from the calibration curve • x
– Linear regression line (y=mx+c)
 can be obtained from the slope of the • x
• CONCENTRATION (moles litre-1 )
– calibration curve for a given wavelength ()

59
 Importance of the Beer-Lambert Law
RULES FOR QUANTITATIVE ANALYSES

ABSORBANCE AT 300nm
x
At high concentrations the calibration
curve may deviate from linearity x
– Always ensure your concentration of x
the sample falls within the linear range x
– if necessary dilute sample x
CONCENTRATION (moles litre-1 )

Absorbance not to exceed 1 to reduce

error*

60
 Importance of the Beer-Lambert Law

ABSORBANCE AT 300nm
CHOOSE CORRECT WAVELENGTH x
An analyte may give more than one absorbance x
maxima (max) value.
x

Many compounds absorb at 220-230 nm hence x

do not use A x
CONCENTRATION (moles litre-1 )

Need to choose wavelength more specific A

to compound (SELECTIVITY) and if more C max
0.6 B
than one select one with highest absorbance
as this gives less error – hence use C

0
220 Wavelength (nm) 380
61
 Example 1: Using Beers’ Law
• A 7.25 x 10-5 M solution of light emitting dye (LED) for LCD display applications has
at transmittance of 44.1% when measured in 2.10 cm cell at a 525 nm. Calculate (a)
absorbance of this solution; and (b) the molar absorptivity of LED.

– (a) A = -log T
= -log (0.441)
= 0.355

– (b) A=cl or A=bc

 = A/cl
 = 0.355/ (2.10 cm x 7.25 x 10-5 mol/L)
 =2.33 x 103 L/mol/cm

62
 Example 2: Using Beers’ Law
• At 580 nm, the wavelength of its maximum absorption in a 1.00 cm cell, the
complex FeSCN2+ has a molar absorptivity of 7.00 x 10 3 L/cm/mol.
Calculate:
(a)the absorbance of a 7.25 x 10-5 M this solution; and
(b)when the concentration is twice that in (a).
(c)the transmittance of the solutions described in (a) and (b).
(d) the absorbance of a solution that has half the transmittance of that described
in (a).
• Describe how the Beer-Lambert Law can be used to determine the
concentration of an unknown sample.

63
 Example 3: Using Beers’ Law
• Given the following set of data for a compound C: (a) Calculate
molar extinction coefficient; and (b) What is the concentration
of C when we obtain an Absorbance of 0.3321?

Conc 0.7
(M) Abs y =1.0137x +0.1378
0.6
0.1 0.2322 0.5 R2 =0.997

0.2 0.3456 0.4

Abs
0.3 0.4532 0.3
0.2
0.4 0.5331
0.1
0.5 0.6453
0
Is the fitting of the curve to
0 0.2 0.4 0.6
the equation acceptable? conc
How can you tell?
64
[Link]/2366/22/Chem_Physical_spectroscopy-_basics.pptx
 Example 3: Using Beers’ Law
Given the following set of data for a compound C: (a) What is the
concentration of C when we obtain an Absorbance of 0.3321? and
(b) Calculate molar extinction coefficient.

Method 1: The concentration is: Abs= 1.0137 Conc + 0.1378

Abs= 0.3321 – Abs blank

= 0.3321- 0.13800
= 0.1941

Conc= Abs = 0.1941 = 0.2 M

1.0137 1.0137

65
 Example 3: Using Beers’ Law

Method 2
The concentration is: Abs= 1.0137 Conc + 0.1378
Abs= 0.3321
Conc= 0.3321-0.1378 = 0.1941 = 0.2 M
1.0137 1.0137

66
 Example 3: Using Beers’ Law
Method 3 =slope=y2-y1 =1.003
x2-x1

Conc (M) Abs Corr

0.232
0.1 2 0.0944
0.345
0.2 6 0.2078
0.453
0.3 2 0.3154
0.533
0.4 1 0.3953 Therefore unknown [C]= 0.2 M
0.645
0.5 3 0.5075
unknown
C 0.3321 0.1943 67
 Application of Beer’s law to mixtures
• Each analyte present in the solution absorbs light!
• The magnitude of the absorption depends on its 
• Total absorbance of a solution at any given wavelength is
equal to the sum of the absorbances of the individual
components in the solution:
• A total = A1+A2+…+An
• A total = 1bc1+2bc2+…+nbcn
• If 1 = 2 = n then simultaneous determination is impossible
• Need ’s where ’s are different to solve the mixture

68
 Example 1: Using Beers’ Law on Mixtures
Pd(II) and Au(III) complexes can be determined simultaneously by reaction
methiomerprazine. The absorption maximum of Pd complex occurs at 480 nm, while that for
the Au complex is at 635 nm. Molar absorptivity data at theses wavelengths are as follows:

Molar absorptivity, e

A 25.0 mL sample was treated with an excess480 nm

of methiomeprazine 635 nmsubsequently diluted
and
Pd complex
to 50. 0 mL. Calculate 3. 55 xof10Pd(II),
the molar concentrations 3 c Pd, and5.64 x 102cAu, in the sample
Au(III),
if the diluted solution
Au had an absorbance of
complex 0.533
2.96 x 10at3 480 nm and 1.45
0.590x 10
at 635
4 nm when
measured in a 1.00 cm cell.

69
Example 1: Using Beers’ Law on Mixtures
ANSWER:
A480=pdbcpd + AubcAu

0.533=(3. 55 x 103 M-1cm-1)(1.00 cm)cpd + (2.96 x 103 M-1cm-1)(1.00 cm)cAu

A635=pdbcpd + AubcAu
0.590=(5.64 x 102 M-1cm-1)(1.00 cm)cpd + (1.45 x 104 M-1cm-1)(1.00 cm)cAu

70
0.533 = (3. 55 x 103) × cpd + (2.96 x 103) × cAu …………………(1)

cpd = ((0.533) - (2.96 x 103)cAu ) / 3. 55 x 103…………………..…(3)

0.590=(5.64 x 102)cpd + (1.45 x 104)cAu…………………….……….(2)

(3) into (2)
2094.5 = 300.612 – 1669 440 cAu + 51 475 000 cAu

1793.88 = 49 805 560 cAu

cAu = 3.60 × 10-5 M

cpd = (0.533) - (2.96 x 103) × 3.60 × 10-5 M / 3. 55 x 103………..(3)

cpd = 1.20 × 10-4 M 71

 STANDARD ADDITION METHOD

• Single-point method
– Two portions of the sample are taken
• One portion is measured as usual: A1=bVxcx/Vt
• A known amount of standard analyte solution is added to the second
portion: A2= bVxcx/Vt + bVscs/Vt
• Therefore cx=A1csVs/(A2Vt-A1Vx)

72
• Multiple method
– Adding several increment of a standard solution to sample aliquots of the same
size: As= bVscs/Vt+ bVxcx/Vt & k= b/Vt
= kVscs + kVxcx
– Plot of As vs Vs: straight line give As=mVs + b; m=kcs & b=kVxcx
– Ratio of m/b=(kcs )/(kVxcx)
– Therefore: cx=(bkcs )/(mkVx)
– Standard deviation of the m, b and c: (s c/cx)2=(sm/m)2+(sb/b)2
sc= cx ((sm/m)2+(sb/b)2)0.5

73
 Example 26-1 from textbook
Ten-millimeter aliquots of a natural water sample were
pipetted into 50.00 mL volumetric flasks. Exactly 0.00,
5.00, 10.00, 15.00, and 20.00 mL of a standard solution
containing 11.1 ppm of Fe were added to each, followed
by an excess of thiocyanate ion to give the red complex
Fe(SCN). After dilution to volume, absorbances for the
five solutions, measured with a photometer equipped
with a green filter, were found to be 0.240, 0.437, 0.621,
0.809, and 1.009, respectively (0.982-cm cells). (a) What
was the concentration of Fe3+ in the water sample? (b)
Calculate the standard deviation of the slope, the
intercept, and the concentration of Fe.

74
Solutions
xi yi xi2 yi2 xiyi
0 0.24 0 0.0576 0
5 0.437 25 0.190969 2.185
10 0.621 100 0.385641 6.21
15 0.809 225 0.654481 12.135
20 1.009 400 1.018081 20.18
50 3.116 750 2.306772 40.71

sxx 250
syy 0.364881
sxy 9.55
m 0.0382
b 0.2412
cx 7.01
sr 4.9E-3
sm 3.07E-4
sb 3.76E-3
sc 0.12
75
Limitations and deviations from Beer’s Law
• Real limitations
– Non-linearities due to intermolecular interactions
• Self aggregation effects and electrolyte effects
• Apparent
– Dynamic dissociation or association of analyte
• Instrumental
– Polychromatic radiation
• Different molar absorptivities at different wavelength
leads to non-linearities in Beer’s Law
– Stray radiation and Mismatched cells
• Non-zero intercept in calibration curve

76
[Link]
 Deviations from Beer’s Law

I r ( 2  1 ) 2

I 0 ( 2 1 ) 2
uccessful at low analyte concentrations (0.01M)!
gh concentrations of other species may also affect 77
UV Spectra: Spectral nomenclature of shifts

78
 Photometric/Spectrophotometric titration
• UV-visible spectrophotometric and photometric
– Useful for locating the end points of titrations.
• Spectrophotometer serves as the detector that monitors the
transmittance or absorbance or the solution at a suitable
wavelength during the addition of increments or the titrant.
• Acid/base titration can be monitored
– By adding a small amount of an indicator that is colored in
either the acidic or basic form dissociation or association of
analyte
• Titration Curves
– Plot of abs vs titrant volume, the shapes depend on the species
that absorbs radiation.

79
Photometric/Spectrophotometric titration

80
 Tutorial Questions
1. Why is a solution of metal free phthalocyanine dye blue? (2
marks)

2. Identify factors that cause the Beer’s law relationship to depart

from linearity. (3 marks)

3. A solution that was 3. 78 x 10-3 M in X had a transmittance of

0.212 when measured in a 2.00-cm cell. What concentration of X
would be required for the transmittance to be increases by a factor
of 3 when a 1.00-cm cell was used? (5 marks)

4. Describe the following terms: Photomultiplier tube and

Monochromator. (5 marks)

81
 Problems
1.1. The phosphate present in a prepared solution of the
powdered detergent is known to react with ammonium
molybdate to produce the molybdenum blue complex,
which is known to absorb strongly at a wavelength of 715
nm. Explain how can phosphate in detergent be
determined using UV-visible spectroscopy for quantitative
analysis?
(6)

82
 Problems

1.2. Determine the concentration of phosphate (w/w

%) in detergent. (6)

solution analysed absorbance @ 715 nm

5.0 mg/L PO43-(aq) standard 0.061

10 mg/L PO43-(aq) standard 0.124

15 mg/L PO43-(aq) standard 0.185

25 mg/L PO43-(aq) standard 0.310

40 mg/L PO43-(aq) standard 0.495

diluted detergent sample 0.238

83
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer

• Introduction:
– UV/vis spectrophotometer measures the intensity of light
passing through a sample (I), and compares it to the
intensity of light before it passes through the sample (Io).
– The ratio I / Io is called the transmittance, and is usually
expressed as a percentage (%T).
– The absorbance, A, is based on the transmittance:
• A = − log (%T)

84
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer

• Reagents:
– CuSO4.5H2O
– Sodium acetate
– Glacial acetic acid
– Xylenol orange
– Unknown copper solution

85
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer
Method:
o1. Preparation of the acetate buffer
o Weigh 136g NaCH3COO into 1000 ml volumetric flask. Fill
the volumetric flask to about ¾ with distilled water.
o Adjust the pH of the solution to 5.9 with glacial acetic acid
and fill up to the mark with distilled water.

o2. Preparation of the colour reagent

o Weigh 120mg of Xylenol Orange in to 1000 ml volumetric
flask and fill up to the mark with distilled water
o

86
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer
Method:
3. Preparation of the Cu2+ stock solution
o Calculate the mass of CuSO4.5H2O needed to give stock
solution of 100ppm Cu2+ in a 1000 ml volumetric flask

4. Preparation of calibration standards

oPour 400 ml of the Xylenol orange solution in to 1000 m1
beaker. Add 100 ml of the buffer and mix.
oPipette 40 m1 of this mixture in to a 100 ml volumetric flask and
fill up to the mark with distilled water. This is the base line
standard.
oThe standard copper solutions are prepared as follows:
o
87
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer
Method:
5. The standard copper solutions are prepared as follows:
o Pipette 40 ml of the xylenol/buffer mixture into sox different
100 ml volumetric flasks
o Pipette respectively 50, 40, 30, 20, 10 ml of the 5ppm Cu2+
solution into the different 100ml volumetric flasks.
o Fill up to the mark with deionized water and shake.

6. Get the unknown sample solution from your instructor.

88
 Experiment 1:
Determination of Cu2+ concentration by making
use of an UV/vis spectrophotometer
UV-Vis measurement:
o Take the highest concentration of the copper standards
and scan it on the spectrophotometer for the maximum
wavelength of absorption.
o Thereafter measure the absorbance’s for each of the
standards at the wavelength of the maximum absorbance.
o Lastly also measure the absorbance’s of the unknown
sample at the same wavelength.

89
 Infrared Spectroscopy
• Infrared absorption:

– Energy of IR photon insufficient to cause electronic

excitation but can cause vibrational or rotational excitation
– It can induce transitions in the vibrational and rotational states
– Molecules absorb specific frequencies that are characteristic
of their structure.
– These absorptions are resonant frequencies, i.e. the frequency
of the absorbed radiation matches the transition energy of the
bond or group that vibrates.

90
 Infrared Spectroscopy
• In order for a vibrational mode in a molecule to be "IR active,"
• It must be associated with changes in the dipole.
• For molecules with N atoms in them, linear molecules have 3N
– 5 degrees of vibrational modes.
• Nonlinear molecules have 3N – 6 degrees of vibrational modes
• As an example H2O, a non-linear molecule, will have 3 × 3 – 6
= 3 degrees of vibrational freedom, or modes.

91
 Infrared Spectroscopy
• Number of vibrational modes
Symmetrical Antisymmetrical
stretching stretching Scissoring

Rocking Wagging Twisting

+ + + -

Out of plane Out of plane

92
 Regions of Frequencies
Spectral Frequency Wavenumber Wavelength
Region (Hz) (cm-1) (,nm)
Near -to 3.8 x 1014 12800 to 780 to 2500
visible- IR to 1.2 x 4000
(NIR) 1014
Combinatio
n bands
Mid 1.2 x 1014 4000 to 200 2500 to 50000
Infrared to 6.0 x
Fundmental 1012
bands for
organic
molecules
Far IR 6.0 x 1012 200 to 10 50000 to
Inorganics to 3.0 x 1000000
organometal 1011
lics Rule of mutual exclusion:
If a molecule has a centre of symmetry:
IR vibration inactive
Vice versa
93
 Infrared Instruments
• Three types of spectrometers:
– Dispersive IR instruments
• Introduction
– It uses a diffraction grating in a monochromator
to disperse the different wavelengths of light.
– It separates the wavelengths of light in the
spectral range and directs each wavelength
individually through a slit to the detector.

94
 Infrared Instruments
• Three types of spectrometers:
– Fourier transform infrared spectrometer
• Introduction
– An FT-IR instrument uses a system called an
interferometer to collect a spectrum.
– It consists of a source, beam splitter, two
mirrors, a laser and a detector.
Fixed mirror

Movable Source
mirror

B
Detector

Sample
95
 Infrared Instruments
• Three types of spectrometers:
– Filter spectrometer
• Introduction
– Infrared photometers designed to monitor
the concentration of air pollutants, such
as carbon monoxide, nitrobenzene, vinyl
chloride, hydrogen cyanide, and pyridine,
are often used to ensure compliance

96
 FT-IR Instruments
• The process of collecting an infrared spectrum in an
FT-IR spectrometer

97
 A Functional Group Chart
4000 3600 3200 2800 2400 2000 1600 1200 800 group
O-H str

NH str
COO-H
=C-H
str
Csp3-H
C-H
-(C=O)-
H
CN
CC
C=O
-C=N
-C=C
phenyl
C-O
C-N
F Cl C-X I
Br
98
 FTIR absorption bands
• IR spectroscopy is often used to identify structures
because functional groups give rise to characteristic
bands both in terms of intensity and position

99
 FTIR: Isotope effects
• The different isotopes in a particular species may give
fine detail in infrared spectroscopy.
• For example, the O–O stretching frequency of
oxyhemocyanin is experimentally determined to be 832
and 788 cm−1 for ν(16O–16O) and ν(18O–18O),
respectively.
• The wavenumber of absorbance, ν can be calculated:

• where k is the force constant for the bond, c is the speed

of light, and μ is the reduced mass of the A–B system:
100
 Hydrogen stretching region in FTIR

Arom- H
H

3000cm-1

101
 FTIR Spectra
FTIR Spectra of Cellulose

- 4000 -1600 cm-1 functional group region.

- 1600- 400 cm-1  fingerprint region.

102
 Tutorial Questions
1. Give advantages of FTIR over dispersive instruments. (4 marks)

2. The infrared range of the electromagnetic spectrum is divided

into three regions. Name them and give their relation to the
visible spectrum? (3 marks)

3. Why are photomultiplier tubes unsuited for the detection of

infrared radiation? (3 marks)

4. A ketone possesses an absorption band with a peak centred

around 1710 cm-1. From this information deduce a value for the
force constant of the C=O double bond. (5 marks)

103
A ketone possesses an absorption band with a peak centred around 1710 cm -1. From this
information deduce a value for the force constant of the C=O double bond.

Reduced mass ()= (M1 x M2)/(M1+M2)

104
MOLECULAR FLUORESCENCE
SPECTROSCOPY

105
 Introduction
• Three types of Luminescence methods are:
(i) molecular fluorescence
(ii) phosphorescence
(iii) chemiluminescence
• Fluorescence:
– Photoluminescence process
• Atoms or molecules are excited by absorption of electromagnetic radiation
• The excited species then relax to the ground state, giving up their excess energy as
photons
• Absorption of photon, short-lived excited state (singlet), emission of photon.

106
 Introduction
• Phosphorescence :
– Photoluminescence process
• Absorption of photon, long-lived excited state (triplet), emission of photon.

• Chemiluminescence:
– Luminescence process involving chemical reaction
• No excitation source – chemical reaction provides energy to excite
molecule, emission of photon.

107
 Theory of fluorescence
• Types of Fluorescence:
– Resonance
• Emitted  = excitation 
– Stokes shift
• Emitted  > excitation 

108
 Theory of fluorescence
• Electron spin and excited states:
– Excited, paired = excited singlet state 
fluorescence
– Excited, unpaired = excited triplet state 
phosphorescence

109
 Theory of fluorescence
• Deactivation:
– Absorption of photon, long-lived excited state (triplet), emission of photon.
Process by which an excited molecule returns to the ground state.
– Minimizing lifetime of electronic state is preferred (i.e., the deactivation
process with the faster rate constant will predominate).

• Radiationless Deactivation:
– Without emission of a photon (i.e., without radiation).

110
Theory of fluorescence

111
 Terms from energy-level diagram
• Term: Absorption Effect: Excite
• Process: Analyte molecule absorbs photon (very fast
~ 10-14 – 10-15 s); electron is promoted to higher energy state.
Slightly different wavelength  excitation into different
vibrational energy levels.

• Term: Vibrational Relaxation Effect: Deactivate,

Radiationless
• Process: Collisions of excited state analyte
molecules with other molecules  loss of excess
vibrational energy and relaxation to lower vibrational levels
(within the excited electronic state)
[Link]/abhuiyan/Course/Chem%204414/Chapter%[Link] 112
 Terms from energy-level diagram

• Term: Internal conversion Effect: Deactivate,

Radiationless
• Process: Molecule passes to a lower energy state –
vibrational energy levels of the two electronic states
overlap (see diagram) and molecules passes from one
electronic state to the other.

• Term: Fluorescence Effect: Deactivate,

Emission of h
• Process: Emission of a photon via a singlet to
singlet transition (short – lived excited state ~10 -7 – 10-9 s).
113
[Link]/abhuiyan/Course/Chem%204414/Chapter%[Link]
 Fluorescence and Structure
• Low–energy   * (aromatic): most intense
fluorescence.
• Heterocycles do not fluoresce; heterocycles fused
to other rings fluoresce. Heteroatom increases
ISC then f decreases.
• Conjugated double bond structures exhibit
fluorescence.
• Structural rigidity (e.g., naphthalene or fluorene vs
biphenyl). Flexibility increases then f decreases.

114
[Link]/abhuiyan/Course/Chem%204414/Chapter%[Link]
 Fluorescence Quantum Yield
• The quantum yield or quantum efficiency for
fluorescence or phosphorescence is the ratio of the number
of molecules that luminesce to the total number of excited
molecule.
to tal # lu m in escin g m o lecu les
Q u an tu m Y ield ,  =
to tal # o f ex cited m o lecu les
kf
 [ k = rate co n stan t]
k f  k i  k ec  k ic  k pd  k d

•A quantum yield = 1 means that every excited molecules

deactivates by emitting a photon – such a molecule is
considered a very good fluorophore.
•Quantum yield can be express as a function of rate
constants.
115
[Link]/abhuiyan/Course/Chem%204414/Chapter%[Link]
 Fluorescence Quantum yield
• Light source, self-emission which means the electrons
transferred to the lowest level spontaneously
• A fluorescence excitation spectrum provides information
about the absorption spectrum of the excited fluorescent
molecule in the excited state.

std F . Astd .n 2
 F  F 2
Fstd . [Link]

The ratio of the number of photons emitted

to the number of photons absorbed
116
 Factors affecting Quantum Yield

•Temperature: increase fluorescence intensity with

decreasing T (reduce number of deactivating collisions).
•Solvent: increase fluorescence with increased viscosity
(decreased likelihood of external conversion –
radiationless deactivation)
•Heavy atoms such as I, Br, Th increases ISC as a
consequence f decreases
•pH: Increased resonance structures (protonation or
deprotonation)  stable excited state and greater quantum
yield
117
 Fluorescence instruments
• Sources
– Hg lamp (254 nm)
– Xe lamp (300 – 1300 nm)
• Filter/monochromator
– Isolate excitation 
– Scan excitation 
– Isolate emission  from excitation 
– Scan emission 
• Detector
– Usually PMT: very low light levels are measured.
118
Fluorescence instruments

119
Fluorescence Spectra

120
 Tutorial Questions
1. Briefly describe or define (a) resonance fluorescence, (b)
vibrational relaxation, (c) internal conversion, (d) fluorescence, (e)
stokes shift, (f) quantum yield and (g) self quenching (7 marks)

2. Why is spectrofluorometry potentially more sensitive then

spectrophotometry? (3 marks)

3. Explain why molecular fluorescence often occurs at a longer

wavelength than the exciting radiation? (2 marks)

4. Why are most fluorescence instruments double beam in design? (3

marks)

121
 MODULE OUTLINE
Module Content Topics

1. Spectrochemical 2. Separation Methods 3. Electrochemical Methods

Methods

1.1. Introduction to 2.1. Introduction to 3.1. Introduction to

Spectrochemical Methods Analytical Separations Electrochemistry
1.2. Molecular Absorption 2.2. Solvent Extraction: R, 3.2. Potentiometry &
Spectrometry: UV-Vis & IR D, L-L & L-S Voltammetry
1.3. Molecular Fluorescence 2.3. Chromatography: GC, 3.3. Electrogravimetry &
Spectrometry LC & SFC Coulometry
1.4. Atomic Spectrometry:
AA, ICP & MS

122
123
124
125
126
127
128
ATOMIC SPECTROSCOPY

129
 Introduction
• Atomic Spectroscopic Methods
– Qualitative and quantitative determination >> 70 elements
• Detect small concentration, 10 ppm to ppb
• Rapid, convenient, and high sensitivity
– Two groups
• Optical atomic spectrometry
– Uses the same principles as seen in molecular
spectrometry
– It is divided into absorption, emission, and
fluorescence
• Atomic mass spectrometry
– It consists of an ion source, a mass analyzer, and a
detector.

130
 Introduction
• Atomic Spectroscopic Methods
– Involves atomization of individual atoms
– Atomization- volatilization and decomposition of samples
into gaseous phase
– There are several methods to atomize samples
• Inductively coupled plasma-6000-8000 OC-ICPAES
• Flames-1700-3150 OC-AAS, AES & AFS
• Electrothermal-1200-3000 OC-AAS & AFS
• Direct-current plasma-5000-10000 OC-DCPS
• Electric arc-3000-8000 OC-Arc source ES
• Electric spark-Varies with time & position-Spark source
ES & MS

131
 Importance of elemental analysis
• Monitoring levels of certain elements in samples to
detect the concentrations of these elements.
– pharmaceutical products and standards

• Monitoring the levels of the toxic elements in samples to

ensure it’s safety.
– cosmetics, food supplements, entire plant or part of
it

132
 Atomic absorption spectroscopy
• Atomic absorption spectroscopy (AAS)
– Standard instruments for the determination of metal
elements
• Applications:
Food
 Pharmaceutical supplements
product

 Standards
Entire plant
or part of it

 Mixture of
 Cosmetics known & unknown
herb
133
 Atomic absorption spectroscopy
• AAS intrinsically more sensitive than AES
• Similar atomization techniques to AES
• Addition of radiation source
• High temperature for atomization necessary
– flame and electrothermal atomization
• Very high temperature for excitation not necessary
– generally no plasma/arc/spark AAS

134
 Principle of AAS
• Sample Introduction
– Flame
– Furnace
– ICP
• Sources for Atomic Absorption/Fluorescence
– Hollow Cathode Lamps
• Sources for Atomic Emission
– Flames
– Plasmas
• Wavelength Separators + Slits +Detectors
135
 Principle of AAS
• Atomized elements each absorb energy of a wavelength
that is peculiar to that element.
• The atomic absorption method uses as its light source a
hollow cathode lamp which emits light of a wavelength
that is peculiar to each element.
• Elements within a solution are heated in a flame or
electrically (2000K to 3000K) and subsequently
determined using the fact that the degree of absorption
will vary with its concentration.

136
 Sample introduction AAS systems
• Continuous sample introduction methods -
nebulizer, Flow injection, HPLC & Vapor generator

137
Flame Method With the Flame Method, the sample solution is
converted into mist-form using a nebulizer,
and then introduced into the flame. It is
atomized by the temperature of the flame. The
flames vary in temperature
Ar-H2, Air-H2, Air-acetylene & ONO-
acetylene
Measurement time: A few dozen seconds

Laminar flow burners provide a relatively

quiet flame and a long path length to enhance
sensitivity for AA and reproducibility
Region of a flame
138
 Flameless Method (Graphite Furnace)
• Electrothermal atomizer
Graphite Graphite 1400-3000 oC
cap holder
Sample
Cooling inlet
block
Aperture Graphite
plate Seal
socket tube

400-1000oC

100oC
Eject arm Spring Fixing
knob

139
 SOURCES
• Solution to line width problem: Use atomic source of same material. e.g. For
Na analysis Na vapor is used.
• Atoms are excited by electrical discharge; the excited atoms emit a
characteristic l.
• Hollow Cathode Tube : Hollow cathode made of the material needed is
vaporized and emits radiation of the characteristic wavelength.
• The ion current to the cathode controls photon intensity; Increasing the
voltage between the anode and cathode will control the current and thus total
photon flux.
• Optimum current for each lamp (»1-20ma).

140
 Hollow Cathode Lamb
• Consists of a cathode and an anode. The cathode is made of the element of
interest

• A large voltage across the anode and cathode will cause the inert gas to
ionize.
• The inert gas ions will then be accelerated into the cathode, sputtering off
atoms from the cathode.
• Both the inert gas and the sputtered cathode atoms will in turn be excited
by collisions with each other.

141
 Hollow Cathode Lamb
• When these excited atoms decay to lower energy
levels they emit a few spectral lines characteristic of
the element of interest.
• The light is emitted directionally through the lamp's
window, a window made of a glass transparent in the
UV and visible wavelengths.
• The light can then be detected and a spectrum can be
determined.
• Continuum source background correction
- Uses a deuterium lamb to obtain and estimate of the
background absorbance (AB) and
- HCL obtains total absorbance (AT)
- The corrected Abs=AT-AB 142
 Origin of Optical Atomic Spectra
Atomic Absorption Spectroscopy, AAS Excited state E1

Absorption

e
Ground state E0

e
Excited state E1
Atomic Emission Spectroscopy, AES

Emission

e
Ground state E0

143
Charnchai Suracheep, Principle of Atomic Absorption Spectrophotometry
 Origin of Optical Atomic Spectra
• Natural linewidth of an absorption spectrum is very
small (104Å) but is broadened by
– Doppler broadening: Random thermal motions of
atoms relative to the detector
– Pressure broadening: In the atomic absorption
experiment the pressure is large enough that atoms
can undergo some interatomic collisions which
cause small changes in the ground state levels.

• Normal line width of excitation lines much greater than

this line width

• Monochromator cannot be used to select l range in AA

(bandwidth  few tenths of a nm). 144
 Origin of Optical Atomic Spectra
• The most common line width broadening
effects are:
 Doppler effect- This effect arises because atoms
will have different components of velocity along
the line of observation.
 Lorentz effect- This effect occurs as a result of
the concentration of foreign atoms present in the
environment of the emitting or absorbing atoms.
The magnitude of the broadening varies with the
pressure of the foreign gases and their physical
properties.
145
 Origin of Optical Atomic Spectra
• Quenching effect
– In a low-pressure spectral source
– Quenching collision can occur in flames as the result of the
presence of foreign gas molecules with vibration levels very close
to the excited state of the resonance line.
• Self absorption or self-reversal effect
– The atoms of the same kind as that emitting radiation will absorb
maximum radiation at the center of the line than at the wings,
– Resulting in the change of shape of the line as well as its
intensity.
– This effect becomes serious if the vapor, which is absorbing
radiation is considerably cooler than that which is emitting
radiation.

146
Atomic Absorption Spectrophotometer

AA-6300

147
Atomic absorption spectra Molecular absorption spectra
1- The outer most electrons occupy The outer most electrons occupy ,
one of the atomic orbitals and have  or n electronic energy in the
its energy levels [K, L, M, .ground state
N, ......
s, s,p s,p,d s,p,d,f ]
Upon excitation electrons are -2 Upon excitation electrons
promoted to any permissible higher raised to * or * energy
atomic energy levels levels
Since there are no bonds there-3 Since there are bonds, there are
are no vibrational or rotational vibrational and rotational energy
energy levels in either the ground or levels in both the ground and
.excited state excited states
The analytical wavelength is the -4 The analytical wavelength is the
resonance wavelength of the .max
analyte
.The spectra are line form -5 The spectra are in the form
of bands due to the presence
of very close, superimposed
and unresolved vibrational and
148
rotational energy levels in the
 Atomic absorption spectroscopy: theory
• When light of a certain intensity is given to many
atom in ground state, part of this light is absorbed by
atoms.
Density C
I0 I Absorbance of

Absorbance
unknown
sample
l

Abs = -logI/I0 = k .l. C Concentration

of unknown
sample
k : proportional constant
Concentration (ppm)
l : path length
C : density (concentration) 149
Charnchai Suracheep, Principle of Atomic Absorption Spectrophotometry
 Quantitative analysis
Standard Addition Method
No.1 No.2 No.3 No.4

100 ml

30 ml
10 ml 20 ml
10 ml 10 ml 10 ml 10 ml

Mg concentration X X+0.1 X+0.2 X+0.3

after filled up Concentration of
unknown sample

10 mlUnknown sample Solvent

1.0 ppm X Standard solution (ppm : mg/1000ml)

150
 Quantitative analysis
• If the sample concentration is too high to permit
accurate analysis in linearity response range

• There are three alternatives that may help bring the

absorbance into the optimum working range:

I. sample dilution
II. using an alternative wavelength having a lower
absorptivity
[Link] the path length by rotating the burner hand.

151
 Example 1 (Problem 28.13): AAS
• A 5.00 mL sample of blood was treated with tricloroacetic acid to
precipitate proteins. After centrifugation, the resulting solution was
brought to pH 3 and extracted with two 5 mL portions of methyl
isobutyl ketone containing the Pb complex agent APCD. The extracted
was aspirated directly into an air-acetylene flame and yield an
absorbance of 0.444 at 283.3 nm. Five millilitre of aliquots standard
solutions containing 0.250 and 0.450 ppm Pd were treated in the same
manner and yielded absorbances of 0.396 and 0.599, respectively.
Calculate the concentration of Pb (ppm) in the sample assuming that
Beer’s law is obeyed.

152
 Tutorial Questions
1. What is the principle of A.A.S (4 marks)

2. What is the principle of a hollow cathode lamp and what are its
main advantages? (5 marks)

3. Why are burners with a long slot used in A.A.S? (2 marks)

4. In the atomic absorption determination of uranium, a linear

relationship is found between the absorbance at 351.5 nm and the
concentration in the range of 500 to 2000 ppm of U. At lower
concentrations, relationship becomes nonlinear unless about 2000
ppm of an alkali metal salt are use introduced. Explain. (4 marks)

153
 EXPERIMENT 2
Determination of Copper using Flame atomic
Spectrophotometer

• Introduction
– Atomic absorption spectroscopy (AA or AAS) is one of
the popular instrumental methods for analyzing
metals and some metalloids.
– It determines the presence and concentrations of
metals in liquid samples e.g Fe, Cu, Al, Pb, Ca, Zn,
Cd and many more.

154
 EXPERIMENT 2
Determination of Copper using Flame atomic
Spectrophotometer

• Reagents
– CuSO4.5H2O
• Method
– Prepare the following 250.0 ml standard solutions from
250.0ppm stock solution: 1,2,5,10,20 and 30 ppm.
– You will be issued with a solution of unknown Cu 2+
concentration.
– A demonstrator will measure the absorbance of each
solution for you.
– Measurements will be done at 324.7 and 217.9 nm
respectively.
155
 MODULE OUTLINE
Module Content Topics

1. Spectrochemical 2. Separation Methods 3. Electrochemical Methods

Methods

1.1. Introduction to 2.1. Introduction to 3.1. Introduction to

Spectrochemical Methods Analytical Separations Electrochemistry
1.2. Molecular Absorption 2.2. Solvent Extraction: R, 3.2. Potentiometry &
Spectrometry: UV-Vis & IR D, L-L & L-S Voltammetry
1.3. Molecular Fluorescence 2.3. Chromatography: GC, 3.3. Electrogravimetry &
Spectrometry LC & SFC Coulometry
1.4. Atomic Spectrometry:
AA, ICP & MS

156
 Atomic Emission Spectrometry
• Inductively coupled plasma atomic emission
spectroscopy (ICP-AES or ICP-OES)
– For elemental analysis
• Applications:

157
 ICP-Atomic Emission Spectroscopy

• It is an analytical technique used for the detection of trace

metals.
• For multi-elements determination.
• It is a type of emission spectroscopy that uses
the inductively coupled plasma.
• To produce excited atoms and ions that
emit electromagnetic radiation at wavelengths
characteristic of a particular element.
• The ICP-AES is composed of two parts: the ICP and the
optical spectrometer.

158
 ICP-Atomic Emission Spectroscopy
• Inductively coupled plasmas are at least 2X as hot as
flames or furnaces.
• The Ar plasma is the result of the flow of Ar ions in a
very strong, localized radio field.
• 6000-10000 K are common.
• Hot enough to excite most elements so they emit light.
• Hot enough to prevent the formation of most
interferences, break down oxides and eliminate most
molecular spectral interferences.

159
ICP-AES Instrumentation

160
 Major Components of ICP-AES

Sample Delivery System - pump, nebulizer,

spray chamber

Inductively Coupled Plasma - torch, RF generator

Spectrometer - Monochrometer, photomultiplier tube

161
 Sample Delivery System
Nebulization –sample is aspirated into the nebulizer,
- it is converted into an aerosol
-pushed into plasma
Laser ablation- plume of ablated material –swept to plasma
Electrothermal-hot surface to vaporize sample for plasma

162
 Inductively Coupled Plasma torch
• Radio frequency electrical current
and associated magnetic field
• Ionization of Ar gas (sparked by a
Tesla coil)
• Acceleration of ions and electrons
by magnetic field
• Energy in transferred from the
electrons to the gas by collision so
the gas is heated up.
• Production of high concentration
of both EXCITED ATOMS and
ions
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 Characteristics of the ICP
• High Temp.
• Long residence times.
• High electron number densities(few ionization
interferences)
• Free atoms formed in nearly chemically inert
environment.
• Molecular species absent or present at low levels.
• Optical thin.
• No electrodes.
• No explosive gas.

164
Direct-current plasma

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 Performance characteristics of ICP-AES
• Elements determined
– ~ 60 elements
• Line selection
– Most elements have several lines that can be selected
• Linear range
– Better than AAS
• Interferences
– Chemical interferences (lowered)
– Spectral interferences (still a problem)
• Detection limits
– Comparable or better than other atomic spectral
techniques
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Summary in Optical Atomic spectrometry

167
 Tutorial Questions
1. Describe three ways of introducing a sample into an ICP (6
marks)

2. Explain four characteristics of an ICP that renders it as an

excellent instrument? (4 marks)

3. Why is the internal standard method often used in plasma

emission spectrometry? (2 marks)

4. Why are atomic emission methods with an ICP source better

suited for multi-element analysis than are flame atomic
absorption methods (3 marks)

168
 Atomic Mass spectroscopy (MS)
• MS is the most useful technique among analytical methods due to its
unequalled sensitivity, detection limits, speed and diversity of its
applications.
• It is used to measure the masses and relative concentrations of atoms
and molecules by converting them to ions so that they can be moved
about and manipulated by external electric and magnetic fields.

169
 Atomic Mass spectroscopy (MS)
• MS has a small sample of compound is ionized, usually to cations by loss of an electron (Ion
Source).
• The ions are sorted and separated according to their mass and charge (Mass Analyzer).
• The separated ions are then detected and tallied, the results are displayed on a chart (Detector).
• The molecular ion is a radical cation with an odd number of electrons, undergoes into
fragmentations to give either a radical and an ion with an even number of electrons or a
molecule and new radical cation.

170
 The basic principle in the mass
spectrometric analysis
• The production of gas-phase ions of the compound.
• Electron ionization (M + e- M.+ + 2e-).

171
 The basic principle in the MS

Sample Count ions

introduction Separate
masses Collect results
Ionization
Minimize collisions, interferences

Sample

+
_

Ionizer Mass Analyzer Detector

172
 The Nature of MS spectra
• A mass spectrum will usually be presented as a vertical bar graph
• Each bar represents an ion having a specific mass-to-charge ratio (m/z) .
• The length of the bar indicates the relative abundance of the ion.
• The most intense ion is assigned an abundance of 100, and it is referred to as the base peak.
• The ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself.
• The highest-mass ion in a spectrum is normally considered to be the molecular ion
• Lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound.

173
 The Nature of MS spectra
• Sample is heated and energized by a beam of electrons,
usually gives a molecular ion (M+) and a lot of fragments
Example H H
H C C H
H H
H H H H
e- + H C C H H C C+ H
H H H H

H H
(M-R2)+ H C+ C H
Mass Spectrum (M-R )+
1
(M-R3)+ M+ H H

174
 Basic equations of mass spectrometry

1 2
mv  zV Ion’s kinetic E function of accelerating voltage (V) and charge (z).
2

F mv 2 / R Centrifugal force

F Bzv Applied magnetic field

mv 2 / R Bzv balance as ion goes through flight tube

Combine equations to obtain:

m / z B 2 R 2 / 2V Fundamental equation of mass spectrometry

Change ‘mass-to-charge’ (m/z) ratio by

B in gauss, r in centimeters,m in
changing V or changing B.
amu
NOTE: if B, V, z constant, then: V in volts, z in electronic charge
2 2
m 5 B r
r m 4.825 x10
z V
175
 Sample Introduction Systems
1) Gas source (lighter elements)
dual inlet - sample purified and measured with standard gas at
identical conditions precisions ~ ±0.005%
continous flow - sample volatized and purified (by EA or GC) and
injected into mass spec in He carrier gas, standards measured before
and after, precisions ~ 0.005-0.01%
2) Solid source (heavier elements)
TIMS - sample loaded onto Re filament, heated to ~1500°C,
precisions ~0.001%
laser ablation - sample surface sealed under vacuum, then sputtered
with laser precisions ~0.01%?
3) Inductively coupled plasma (all elements, Li to U)
ICPMS - sample converted to liquid form, converted to fine aerosol
in nebulizer, injected into ~5000K plasma torch
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 Mass Analyzers
Sector Analyzers
It is a magnetic sector analyzer,
separation is based on the deflection of ions in a magnetic field. The
trajectories that ions take depend on their m/z values.
Quadrupole Mass Analyzers
It consists of four cylindrical rods,
They are mass filters that only allow ions of a certain
mass-to-charge ratio to pass.
Ion motion in electric fields is the basis of separation.
Rods opposite each other are connected to dc and radio-frequency (RF)
voltages.
Time-of-Flight Mass Analyzers
The time-of-flight (TOF) mass spectrometer represents another approach
to mass analysis. In a TOF analyzer, a packet of ions with nearly
identical kinetic energies is rapidly sampled, and the ions enter a field-
free region.
177
 Inductively Coupled Plasma M.S
• ICPMS is a type of mass spectrometry which is capable
of detecting metals and several non-metals at
concentrations as low as one part in 1012 (part per
trillion).
• This is achieved by ionizing the sample
with inductively coupled plasma and then using a mass
spectrometer to separate and quantify those ions.
• ICP-MS has greater speed, precision, and sensitivity.
However, analysis by ICP-MS is also more susceptible
to trace contaminants from glassware and reagents.
• Has low detection limits with double focus than with
quadrupole MS- because of high resolution to separate
ions from background 178
 Applications of ICP-MS
• Metal speciation
- for identification of isotopic speciation of metal.
e.g. Cr (III) and Cr (VI).

• Quantification of proteins and biomolecules

- identifying and quantifying native metal cofactor
containing proteins in biofluids.

• Elemental analysis
- the ICP-MS allows determination of elements with
atomic mass ranges 7 to 250.
- this encompasses Li to U in a periodic table.
179
 Inductively Coupled Plasma M.S
detector
Shared components
of all ICPMS machines
high vacuum mass/charge
10-7 bar discriminator

sample cone

skimmer cone

“fore” vacuum
10-4 bar

instrument housing

atmospheric
pressure

180
Inductively Coupled Plasma M.S
1. Quadrupole ICPMS
- measure concentrations
as low as several ppt

- no fuss sample preparation

(dissolve in 5% HNO3)

- get beam intensity

vs. mass/charge ratio

or magnetic
sector

Faraday cup
and ion counter (electron multiplier)

181
 Tutorial Questions

1. What are the principles behind MS? (5 marks)

2. Give a short explanation of the working principle of a

quadrupole analyzer. (3 marks)

3. What are the principles behind ICP-MS? (5 marks)

4. Why are detection limits in ICP-MS generally lower with

double-focusing spectrometers than with quadrupole mass
spectrometers? (2 marks)
182