L T P C

2 1 0 3
Course Code: CHY206
Semester: III
ANALYTCAL TECHNIQUES FOR BIOTECHNOLOGY

Course Objective:
1. To provide an insight, into the fundamentals of the interaction of electromagnetic radiation
with matter with special emphasis on spectroscopy
2. To deliver the need for UV-vis, NMR spectroscopy and mass spectrometry in bio-
molecular characterisation
3. To provide an insight, into the commonly used chromatographic and electrophoretic
techniques used in biomolecules separation
4. To rationalize the use of radioactive tracers in biology and medicine

UNIT - I 12 Periods
Electromagnetic Spectrum
Interaction of electromagnetic radiation with matter; Frequency; Wave number; Wave length;
Regions and wavelengths.
Absorption (UV-Visible) & Emission (Fluorescence) Spectroscopy
Absorption and Emission spectroscopy; Atomic and Molecular difference; Principle; Beer-
Lambert law; Deviation from Beer-Lambert law; Types of electronic transitions; Metallic (d-d)
and Non-metallic (σ, π, n); Instrumentation – Double Beam Spectrophotometer; Applications
(Lowry’s method for protein estimation (metallic); Enzyme Assays (non-metallic); Calibration
curves for estimation using UV-Vis); Real time Case studies - Drunken Driving, Blood sugar
estimation, Ca2+ levels in Blood); Principle of Fluorescence phenomenon; Jablonski diagram;
need for fluorophore; Fluorescence quenching and solvent effects. Initiation into Förster
resonance energy transfer (FRET); Fluorimetry.

UNIT - II 11 Periods
NMR Spectroscopy
1
H NMR phenomenon; Chemical shift; Spin-Spin splitting (Prediction of types of 1H and
splitting patterns in simple and substituted aliphatic/aromatic molecules and biomolecules;
first order effects only); spin decoupling; Introduction to 13C NMR (Predictions of types of 13C
signals); Relaxation T1 and T2 states; Introduction to Magnetic Resonance in Imaging (MRI)

Mass Spectrometry
Principle; Molecular Ion Peak; Base Peak; Ionization – Electrospray (ESI) and MALDI;
Analysis – Quadrupole and Time of Flight (TOF); Instrumentation – ESI-TOF and MALDI-TOF;
Examples of MATRIX; nitrogen rule; Fragmentation ions of peptides; Application (Molecular
weight determination of proteins).

UNIT - III 11 Periods

Chromatography
Principle; Retardation Factor; Retention times; Resolution; Techniques – Thin Layer; Liquid
chromatography- Normal Phase (Adsorption); Reverse phase (Partition); Gel Permeation
(Size Exclusion); Ion Exchange; Affinity; High Performance Liquid Chromatography (HPLC);
Instrumentation – HPLC; Applications (Molecular weight determination of a protein using Gel
Permeation; Separation of proteins using Ion Exchange; Purification of mRNA from Total RNA
and Isolation of DNA binding proteins using Affinity).
UNIT - IV 11 Periods
Electrophoresis
Principle; Expressions for Electrophoretic Mobility and Zeta Potential; Types of
electrophoresis – native (agarose, PAGE); denatured (SDS-PAGE), discontinuous; Isoelectric
focusing; PFGE; 2D electrophoresis; Examples.

Tracers in Biology and Medicine

Radioactivity; Principles of tracer techniques- advantages, limitations; Applications (Radio
Immunoassay (RIA); Clinical applications).

TEXTBOOKS

1. Upadhyay A., K. Upadhyay and N Nath, Biophysical Chemistry, 4/e, Himalaya Publishing
House, Mumbai, 2016.
2. Klostermeier, D. and M.G. Rudolph, Biophysical Chemistry, 1/e, CRC Press, 2017.

REFERENCES

1. Lundblad, R.L. and F.M. Macdonald, Handbook of Biochemistry and Molecular biology,
5/e, CRC Press, 2017.
2. Willard, H.H., L.L. Merritt, J.A. Dean and F.A. Settle, Instrumental Methods of Analysis,
7/e,CBS Publishers, Delhi, 2004.
3. Berg, J.M., J.L. Tymoczko, G.J Gatto and L. Stryer, Biochemistry, 8/e, W.H. Freeman
Publishers, 2015.
4. Ghatak, K.L., Techniques and methods in Biology, 1/e, PHI Learning Pvt. Ltd., 2010.

ONLINE MATERIAL

http://nptel.ac.in/courses/102103044/

LEARNING OUTCOMES

Upon completion of each unit, the learner will be able to:

Unit - I Use the spectroscopic methods to study the biomolecules.

Unit - II Utilise NMR and mass spectrometry for characterisation of biomolecules

Apply different chromatographic methods for the separation and purification
Unit - III
of biomolecules
Unit - IV Use the different electrophoresis and tracer techniques in biotechnology.
Course Code: CHY206
Semester: III
ANALYTICAL TECHNIQUES FOR BIOTECHNOLOGY

UNIT – I 12 Periods
Electromagnetic Spectrum
Interaction of electromagnetic radiation with matter; Frequency; Wave number; Wave
length; Regions and wavelengths.

Absorption (UV-Visible) & Emission (Fluorescence) Spectroscopy

Absorption and Emission spectroscopy; Atomic and Molecular difference; Principle;
Beer-Lambert law; Deviation from Beer-Lambert law; Types of electronic transitions;
Metallic (d-d) and Non-metallic (σ, π, n); Instrumentation – Double Beam
Spectrophotometer; Applications (Lowry’s method for protein estimation (metallic);
Enzyme Assays (non-metallic); Calibration curves for estimation using UV-Vis); Real
time Case studies - Drunken Driving, Blood sugar estimation, Ca2+ levels in
Blood); Principle of Fluorescence phenomenon; Jablonski diagram; need for
fluorophore; Fluorescence quenching and solvent effects. Initiation into Förster
resonance energy transfer (FRET); Fluorimetry.

Course plan for the Unit – I:

Module No of Topics to be Learning Outcomes

No periods covered
1 1 Electromagnetic The learners will be able to
Spectrum - (i) define and demonstrate the
Interaction of electromagnetic spectrum
electromagnetic (ii) analyze the interaction of
radiation with electromagnetic radiation with matter
matter; Frequency; (iii) explain the properties of
Wave number; electromagnetic radiation
Wave length; (iv) identify the regions of
Regions and electromagnetic spectrum
wavelengths.
2 2 Absorption (UV- The learners will be able to
Visible) (i) differentiate the atomic and
spectroscopy- molecular absorption spectroscopy
Atomic and (ii) explain the principle of absorption
Molecular spectroscopy
difference; (iii) derive the Beer-Lambert law to
Principle; Beer- explain the theory of light absorption
Lambert law; (iv) analyse the deviations from the Beer-
Deviation from Lambert law
Beer-Lambert law (v) analyse the factors affecting
absorption
3 1 Types of electronic The learners will be able to
transitions; Metallic (i) list various electronic transitions
(d-d) and Non- occurs during the absorption of light
metallic (, , n) in the UV-Visible region
(ii) differentiate the metallic and non-
metallic transitions
4 1 Instrumentation – The learners will be able to
Double Beam (i) depict the double beam
Spectrophotometer spectrophotometer
 (ii) demonstrate the parts involved in the
double-beam spectrophotometer and
explain its advantages and
applications
5 2 Applications The learners will be able to
(Lowry’s method (i) apply the principle of UV-Visible
for protein spectroscopy for protein estimation
estimation by Lowry’s method
(metallic); Enzyme (ii) study the enzyme assay for the
Assays (non- estimation of protein concentration
metallic); with the help of UV-Visible
Calibration curves spectroscopy
for estimation (iii) analyze the unknown concentration
using UV-Vis) of analytes by calibration curves
6 1 Real time Case The learners will be able to apply the
studies - Drunken principle of UV-Visible spectroscopy to
Driving, Blood (i) identify the drunken drivers
sugar estimation, (ii) estimate the blood sugar
Ca2+ levels in (iii) estimate the amount of Ca+2 in blood
Blood) samples
7 1 Emission The learners will be able to
spectroscopy- (i) differentiate the atomic and
Principle of molecular emission spectroscopy
Fluorescence (ii) explain the principle of emission
phenomenon spectroscopy (fluorescence
phenomenon)
8 2 Jablonski diagram; The learners will be able to
need for (i) demonstrate the Jablonski diagram
fluorophore; and explain the processes involved
Fluorescence in it
quenching and (ii) explain need for fluorophore
solvent effects. (iii) identify the reasons of fluorescence
Initiation quenching
into Förster (iv) analyze the effects of solvents on
resonance energy fluorescence
transfer (FRET) (v) explain the importance of Förster
resonance energy transfer (FRET)
9 1 Fluorimetry The learners will be able to
(i) explain the instrumentation of
spectroflurimetry

UNIT- II 11 Periods
NMR Spectroscopy
1
H NMR phenomenon; Chemical shift; Spin-Spin splitting (Prediction of types of 1H
and splitting patterns in simple and substituted aliphatic/aromatic molecules and
biomolecules; first order effects only); spin decoupling; Introduction to 13C NMR
(Predictions of types of 13C signals); Relaxation T1 and T2 states; Introduction to
Magnetic Resonance in Imaging (MRI)

Mass Spectrometry
Principle; Molecular Ion Peak; Base Peak; Ionization – Electrospray (ESI) and
MALDI; Analysis – Quadrupole and Time of Flight (TOF); Instrumentation – ESI-TOF
and MALDI-TOF; Examples of MATRIX; nitrogen rule; Fragmentation ions of
peptides; Application (Molecular weight determination of proteins).

Course plan for the Unit – II:

Module No of Topics to be Learning Outcomes

No periods covered
1
1 1 H NMR The learners will be able to
phenomenon (i) demonstrate the phenomenon,
principle and selection rules of NMR
spectroscopy for the characterization
of organic and biomolecules
2 2 Chemical shift, The learners will be able to
Spin-Spin splitting (i) predict the chemical shift of different
(Prediction of kinds of protons
types of 1H and (ii) identify the splitting pattern of peaks
splitting patterns in for simple aliphatic/aromatic organic
simple and compounds and biomolecules
substituted
aliphatic/aromatic
molecules and
biomolecules; first
order effects only)
3 1 Spin decoupling; The learners will be able to
Introduction to 13C (i) explain the spin decoupling in NMR
NMR (Predictions (ii) explain the principle of 13C NMR and
13
of types of C difference between 1H NMR and 13C
signals) NMR
(iii) predict the types of 13 C NMR signals
4 1 Relaxation T1 and The learners will be able to
T2 states; (i) differentiate the relaxation states T1
Introduction to and T2
Magnetic (ii) apply the principle of NMR to MRI
Resonance in and study its applications
Imaging (MRI)
5 1 Mass The learners will be able to
Spectrometry- (i) differentiate between spectroscopy
Principle; and spectrometry
Molecular Ion (ii) demonstrate the principle of mass
Peak; Base Peak spectrometry for the identification of
molecules by mass to charge ratio
(iii) identify molecular ion peak and base
peak in a mass spectrum
6 1 Ionization – The learners will be able to
Electrospray (ESI) (i) identify different ionization
and MALDI; techniques useful for the analysis of
biomolecules
(ii) explain the principle of Electrospray
(ESI) and MALDI ionization
techniques for the ionization of
biomolecules
7 1 Analysis – The learners will be able to
Quadrupole and (i) identify various analyzation
Time of Flight techniques useful for the analysis of
 (TOF); biomolecules
(ii) explain the principle of quadrupole
and TOF techniques
8 2 Instrumentation – The learners will be able to
ESI-TOF and (i) explain the instrumentation of ESI-
MALDI-TOF; TOF and MALDI-TOF
Examples of (ii) significance of MATRIX used in
MATRIX; nitrogen MALDI
rule; (iii) significance of nitrogen rule in mass
spectrometry
9 1 Fragmentation The learners will be able to
ions of peptides; (i) apply the fragmentation methods for
Application the analysis of peptides by ESI and
(Molecular weight MALDI techniques
determination of (ii) determine the molecular weight of
proteins) proteins using fragmentation of
peptides

UNIT - III 11 Periods

Chromatography
Principle; Retardation Factor; Retention times; Resolution; Techniques – Thin Layer;
Liquid chromatography- Normal Phase (Adsorption); Reverse phase (Partition); Gel
Permeation (Size Exclusion); Ion Exchange; Affinity; High Performance Liquid
Chromatography (HPLC); Instrumentation – HPLC; Applications (Molecular weight
determination of a protein using Gel Permeation; Separation of proteins using Ion
Exchange; Purification of mRNA from Total RNA and Isolation of DNA binding
proteins using Affinity).

Course plan for the Unit – III:

Module No of Topics to be Learning Outcomes

No periods covered
1 1 Chromatography The learners will be able to
Principle; (i) explain the principle of
Retardation chromatography and learn the
Factor; Retention terminologies involved in
times; Resolution chromatography
(ii) demonstrate the terms-retardation
factor, retention time, resolution, etc.
2 3 Techniques – Thin The learners will be able to
Layer; Liquid (i) learn various techniques involved in
chromatography- chromatography
Normal Phase (ii) differentiate between thin layer,
(Adsorption); liquid chromatography
Reverse phase (iii) differentiate the normal phase and
(Partition) reverse phase liquid chromatography

3 2 Gel Permeation The learners will be able to

(Size Exclusion)- (i) explain the principle of gel
Molecular weight permeation chromatography, various
determination of a gels, column preparation, elution, etc
protein using Gel (ii) determine the molecular weight of a
Permeation protein using gel permeation
 chromatography
4 2 Ion Exchange- The learners will be able to
Separation of (i) explain the principle of ion-exchange
proteins using Ion chromatography, types of ion-
Exchange; exchange resins, choice of buffer,
column preparation, elution, etc
(ii) analyze the separation of proteins
using ion-exchange chromatography
5 2 Affinity- The learners will be able to
Purification of (i) explain the principle of affinity
mRNA from Total chromatography, supporting matrix,
RNA and Isolation immobilized ligand, spacer arm,
of DNA binding column preparation, elution, etc
proteins using (ii) illustrate the purification of mRNA
Affinity from Total RNA
(iii) demonstrate the isolation of DNA
binding proteins using affinity
chromatography
6 1 High Performance The learners will be able to
Liquid (i) explain the principle and
Chromatography instrumentation of High Performance
(HPLC); Liquid Chromatography for the
Instrumentation – separation of chemical and
HPLC biomolecules in ultra low level
quantities.

UNIT - IV 11 Periods
Electrophoresis
Principle; Expressions for Electrophoretic Mobility and Zeta Potential; Types of
electrophoresis – native (agarose, PAGE); denatured (SDS-PAGE), discontinuous;
Isoelectric focusing; PFGE; 2D electrophoresis; Examples.

Tracers in Biology and Medicine

Radioactivity; Principles of tracer techniques- advantages, limitations; Applications
(Radio Immunoassay (RIA); Clinical applications)

Course plan for the Unit – IV:

Module No of Topics to be Learning Outcomes

No periods covered
1 3 Principle; The learners will be able to
Expressions for (i) explain the principle of
Electrophoretic electrophoresis for the separation of
Mobility and Zeta biomolecules
Potential (ii) determine the electrophoretic
mobility and zeta potential of a
biomaterial
2 4 Types of The learners will be able to
electrophoresis – (i) identify various techniques involved
native (agarose, in electrophoresis for the separation
PAGE); of biomolecules based on charge
denatured (SDS- (ii) differentiate between native PAGE,
PAGE), SDS-PAGE, and discontinuous
 discontinuous; electrophoresis techniques
Isoelectric (iii) explain the principle of isoelectric
focusing; PFGE; focusing, PFGE and 2D
2D electrophoresis with suitable
electrophoresis; examples
Examples.
3 2 Tracers in Biology The learners will be able to
and Medicine- (i) explain the principle of radioactivity
Radioactivity; (ii) demonstrate the principle and
Principles of tracer significance of tracer techniques in
techniques- biology and medicine, their
advantages, advantages and limitations
limitations
4 2 Applications The learners will be able to
(Radio (i) demonstrate the principle of Radio
Immunoassay Immunoassay to analyze any
(RIA); Clinical antigen or anti-body in the patient’s
applications) serum to diagnose the disease
(ii) explain the clinical applications of
tracer techniques in diagnosis and
therapy