BP701T INSTRUMENTAL METHODS OF ANALYSIS (Theory) 3L + 1T / week

Lesson Plan

Lecture Contents
Number
1  Types of energy and energy levels in a molecule
 Electronic energy levels, bonding, non-boding and anti-bonding molecular
orbitals
 Quantized energy levels and Energy gaps
 Excitation and relaxation
 Electronic transitions
 EMR and EMS – Energy, wavelength, frequency, wavenumber, intensity
 UV – Visible radiation and its interaction with molecules, energy of UV –
Visible radiation and energy gaps associated with π to π* and n to π*
2 Terms-
 Absorption of EMR
 Chromophore
 Transmittance
 Absorbance
 Absorption spectrum (why a band spectrum?)
 Absorptivity and Wavelength maxima
Factors affecting absorbance and absorptivity
Beer Lambert’s law - derivation
3 Beer Lambert’s law – Limitations
Conditions to be ensured for Beer Lambert’s law to hold true
 Choice of solvent
 Narrow bandwidth of wavelengths
 Concentration range of analyte
Effect of conjugation on energy gap of chromophore
Effect of solvent on energy gap of chromophore
Bathochromic, Hypsochromic, Hyperchromic and Hypochromic shift
4 Instruments used for measurement of absorption of UV – Visible radiation by
molecules
 Components-
 Sources
 Wavelength selectors
 Filters and monochromators
5  Sample cells
 Detectors
Types of instruments
 Photometers and Spectrophotometers
 Double beam and single beam
Comparison between different types of instruments
6 Applications –
Titrations
Single component analysis
Numericals
7 Applications –
Multicomponent analysis
Numericals
8 Fluorescence spectroscopy –
 Jablonski diagram
 Excitation
 Relaxation processes
 Quantum yield
9 Quenching
Factors affecting fluorescence intensity
Instruments
 Photofluorimeter
 Spectrofluorimeter
10 Components of instruments, Applications of fluorescence spectroscopy
Derivatization to add fluorophore
11 IR Spectroscopy –
 IR radiation – Near, Mid and Far IR
 Molecular bond vibrations and types
 Energy of molecular bond vibrations
 Vibrational and rotational energy levels
 Energy gap and energy of IR radiation
 Necessary and sufficient condition for absorption of IR radiation
12  Fundamental absorptions and overtones
 Factors affecting bond vibration frequency
 IR absorption spectrum
 Finger print region and group absorption frequencies
13 Dispersive IR spectrophotometer Components –
 Source, sample cell, monochromator, detectors
 Block diagram
 FTIR spectrophotometer
 Components
 Source, sample cell, interferometer, detectors
 Comparison of dispersive and FTIR spectrophotometer
14 Applications of IR spectroscopy –
 Correlation chart and identification of molecules
 Structural elucidation
 Reaction monitoring
 IR spectra of 3 - 4 simple molecules and correlation with structure (ketone,
amide, acid, alcohol – 2 aliphatic, two aromatic)
15 Atomic spectroscopy
 Atomic energy levels (absence of vibrational and rotational levels)
 Partial energy level diagram of Na or K
 Excitation and relaxation in atoms of Na or K
 Line spectrum
 Need for gaseous state of sample
 Atomic emission and conditions under which emitted intensity is directly
proportional to concentration
 Wavelength and intensity of emission (importance of Boltzmann equation)
16 Flame photometer Components –
 Excitation source – types of flames and burners
 Wavelength selectors
 Detector
 Interferences
 Applications
17 Atomic absorption spectroscopy
 Proportion of excited atoms to ground state atoms in vapor state as per
Boltzmann equation and Absorption of monochromatic radiation by
ground state atoms
 Proportionality of absorbed radiation to concentration
 Comparison of AES and AAS
 Conditions under which one is preferred over the other
18 Atomic absorption spectrophotometer
 Source – HCL
 Wavelength selector
 Excitation source - flame
 Detector
 Interferences
 Applications
19 Nepheloturbidimetry
 Scattering of light and factors affecting the same
 Nephelometry – Principle
 Instrument – components, block diagram and working
20 Turbidimetry –
 Principle
 Instrument – components, block diagram and working
 Applications of nepheloturbidimetry
21 Introduction to chromatography –
 Definition of chromatography
 Classification
 Theory of chromatography
 Plate theory – Only concept of theoretical plate and effect of number and
height of theoretical plate on separation
 Rate theory – Only concept of difference in migration rates being
responsible for separation
22 Van Deemter equation and how separation can be improved
Terms
 Chromatogram
 Retention time
 Adjusted retention time
 Dead time
 Capacity factor
 Selectivity factor
 Resolution
23 Conventional column chromatography
Adsorption chromatography –
 Principle involved in separation
 Columns
 Stationary phase and packing material
 Mobile phases
 Procedure
24 Partition chromatography
 Principle involved in separation
 Types of partition chromatography – Normal and reverse phase
 Columns
 Stationary phase and packing material
 Mobile phases
 Procedure
 Isocratic and gradient elution in column chromatography
25 Planar chromatography
Thin layer chromatography
Plates
Stationary phases
Solvent systems
Steps involved –
 Preparation of plates
 Sample application
 Preparation of solvent system
 Chamber saturation
 Development of plates
 Visualization of spots
 Rf values
 Results and conclusion
26 Selection of solvent system – steps involved
 Trial and error approach
 Single solvents from different classes
 Mixtures
 pH adjustment
Advantages and disadvantages
Applications
27 Paper chromatography
 Type of paper used
 Mechanism involved
 Steps involved –
 Sample application
 Preparation of solvent system
 Chamber saturation
 Development of chromatogram – ascending, descending, radial
 Visualization of spots
 Rf values
28  Selection of solvent system – steps involved
 Advantages and disadvantages
 Applications
29 Electrophoresis –
 Definition
 Principle
 Factors affecting electrophoretic mobility
 Types of electrophoresis – Zone and Moving boundary
30 Apparatus used in and procedure of
 Paper electrophoresis
 Gel electrophoresis
 Capillary electrophoresis
31 Gas chromatography
 Principle involved in separation
 Solubility of gas in liquid, effect of vapour pressure, temperature and
pressure
 Carrier gas as mobile phase
 Instrument components
 Carrier gas supply and regulator
 Column oven
32  Sample injector – split and splitless
 GC columns – Packed and open tubular
 Packing materials
 Detectors – FID
33  Detectors – TCD, ECD
 Isothermal and temperature programming in gas chromatography
 Advantages and disadvantages of GC
34  Applications of GC
 Headspace analysis and Residual solvents
 Derivatization in GC
 Retention indices
35 HPLC
Transition from conventional column chromatography to HPLC
 Reduction in particle size of stationary phase
 Uniform particle size
 Dense packing
 Increased number of plates
 Increased back pressure
 Use of pump
 Closed system
 Stainless steel column
 Smaller internal diameter and length of column
 In-line detector
 Sample introduction in closed system without interrupting mobile phase
flow
  Instrument components
 Solvent reservoir(s)
 Pump
 Manual and automated sample injection
36 Guard columns
Analytical columns
Packing material – Silica, types of silica
Stationary phases – reverse and normal phase
Most popular stationary phases
Detectors
 Solute property – UV, DAD, fluorescence, electrochemical
 Bulk property – RI
 ELSD
37 Mobile phase selection for reverse phase HPLC with UV detection
 Solvent strength selectivity
 Solvent type selectivity
 pH dependent selectivity
 Need for buffered mobile phases
 Influence of mobile phase flow rate
 Choice of detector wavelength
 UV cutoff of mobile phase solvents
38 Applications of HPLC
Quantitation methods - Area normalization, external standard method, internal
standard method
Numericals for calculation of capacity factor, selectivity factor, resolution,
number of plates, HETP
39 Ion exchange chromatography
Ion exchange reaction and mechanism of separation
40 Role of pH of buffer in mobile phase and pKa/ pI value of components
Stationary phase – Ion exchange resins, types of ion exchange resins
Mobile phase
41 Applications
Separation of proteins and amino acids
Methodology
42 Gel chromatography
Definition
Mechanism involved
43 Stationary phase and mobile phase
Other instrument components
Applications
44 Affinity chromatography
Definition
Mechanism
45 Stationary phase and mobile phase
Applications
 Blueprint
MCQs

Unit Number of MCQs

1 5
1.1 2
1.2 1
1.3 1
1.4 1
2 5
2.1 1
2.2 1
2.3 1
2.4 1
2.5 1
3 3
3.1 1
3.2 or 3.3 1
3.4 1
4 4
4.1 2
4.2 2
5 3
5.1 1
5.2 1
5.3 1

Total MCQs 20

LAQ: 3 questions of 10 marks each

Unit Questions
1 and 2 1
3 and 5 1
4 1

SAQ: 9 questions of 5 marks each

Unit Questions
1 2
2 2
3 2
4 2
5 1