Minutes of Meeting for PCI Syllabus Orientation

Sem: VII Final Year B. Pharm Date: 11/07/2022

Course Code: BP701T
Course name: 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

 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
● Bathochromic, Hypsochromic, Hyperchromic and Hypochromic shift

4 ● Instruments used for measurement of absorption of UV – Visible

radiation by molecules (Only Block Diagrams)

Components:
i. Sources
ii. Wavelength selectors
iii. Filters and monochromators
5 iv. Sample cells
v. Detectors

● Types of instruments
Photometers and Spectrophotometers
Double beam and single beam
● Comparison between different types of instruments

6 ● Applications –
Spectrophotometric titrations
Single component analysis
Numerical based on Beer-Lambert’s Law
7 ● Applications –
Multicomponent analysis (Solvent extraction, Simultaneous
equation, Absorbance ratio, Derivative)
8 ● Fluorescence spectroscopy –
Jablonski diagram
Excitation
Relaxation processes
Quantum yield
9 ● Quenching

● Factors affecting fluorescence intensity

● Instruments

Photofluorimeter
Spectrofluorimeter
10 ● Components of instruments
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
● 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
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
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
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
● 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

● Numericals for calculation of capacity factor, selectivity factor,

resolution, number of plates, HETP
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
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
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 –
i. Sample application
ii. Preparation of solvent system
iii. Chamber saturation
iv. Development of chromatogram – ascending, descending, radial
v. Visualization of spots
vi. Rf values
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
28 ● Selection of solvent system – steps involved
● Advantages and disadvantages
● Applications
● Numerical based on planar chromatography
29 ● Electrophoresis –
Definition
Principle
Factors affecting electrophoretic mobility

● Types of electrophoresis – Zone and Moving boundary

30 ● Principle, Instrumentation & Applications 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
i. Carrier gas supply and regulator
ii. Column oven

32 iii. Sample injector – split and splitless

iv. GC columns – Packed and open tubular
v. Packing materials
vi. Detectors – FID
33 vii. Detectors – TCD, ECD
viii. Isothermal and temperature programming in gas
chromatography

● Advantages and disadvantages of GC

34 ● Applications of GC

Headspace analysis and Residual solvents

Derivatization in GC
35 ● HPLC
● Transition from conventional column chromatography to HPLC
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
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-visible, Diode Array, fluorescence,
electrochemical
- Bulk property – Refractive Index, Evaporative Light
Scattering

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 cut-off of mobile phase solvents
38 ● Applications of HPLC
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022

● Quantitation methods - Area normalization, external standard

method, internal standard method
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

Instrumentation
Applications
44 ● Affinity chromatography

Definition
Mechanism
45 Stationary phase and mobile phase
Instrumentation
Applications

Blueprint
MCQs
Unit Number of MCQs
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
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 (5 + 5)

Unit Questions
1 and 2 1
3 and 5 1
4 1
 Minutes of Meeting for PCI Syllabus Orientation
Sem: VII Final Year B. Pharm Date: 11/07/2022
SAQ: 9 questions of 5 marks each
Unit Questions
1 2
2 2
3 2
4 2
5 1

Compiled by: Reviewed & Approved by:

Dr. Paraag Gide Dr. Pournima Hamrapurkar