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BIO F244
Instrumental Methods of Analysis (IMA) 1 Spectroscopy Characteristic of atomic and molecular spectroscopy
(Pre-Mid Sem)

2 Atomic Spectroscopy Infrared Spectroscopy

(Pre-Mid Sem) Atomic Absorption Spectroscopy

3 Molecular Visible and Ultraviolet Spectroscopy, Fluorescence Spectroscopy

Spectroscopy
(Pre-Mid Sem)

4 Optical Spectroscopy Polarimetry and Circular Dichroism

(Pre-Mid Sem)
5 Electrophoresis SDS-PAGE
(Pre-Mid Sem)

6 Chromatography High-Performance Liquid Chromatography

(Post-Mid Sem)

7 Molecular Biology ELISA, PCR

Techniques
(Pre-Mid Sem)

8 Mass Spectrometry Basic principles and applications

(Post-Mid Sem)
Definition: Spectroscopy is the study of the interaction between matter and electromagnetic radiation.

Significance: Provides critical information about the structural, electronic, and vibrational properties of atoms and molecules.
 Basic and Universal Elements of Spectroscopic Setups

Light Source
1. Provides electromagnetic radiation for interaction with the sample.
2. Common examples:
1. Continuous sources: Tungsten lamps (visible), deuterium lamps (UV).
2. Line sources: Lasers, hollow cathode lamps.
3. Broadband sources: Xenon arc lamps, synchrotrons.

Monochromator
1. Isolates a specific wavelength or a narrow band of wavelengths.
2. Key components:
1. Prisms or gratings: Separate wavelengths based on dispersion.
2. Slits: Control the bandwidth and resolution.

Sample Holder:
1. Holds the sample for interaction with the light source.
2. Types:
Gas cells: For gaseous samples.
Cuvettes: For liquids, typically made of quartz (UV) or glass (visible).
Solid-phase supports: For thin films, powders, or solid materials.
Optical Path
IR Spectrophotometer
 Interaction of Light with Matter:
Absorption: Matter absorbs specific wavelengths, causing transitions to higher energy levels.
Emission: Matter releases energy as light when transitioning to lower energy levels.
Scattering: Light is redirected by particles or molecules in a non-uniform medium.
Quantization of Energy Levels:
Atoms and molecules have discrete energy levels for electronic, vibrational, and rotational states.
Transitions occur between these levels when the energy of light matches the energy gap.
Selection Rules:
Govern allowed transitions based on the conservation of angular momentum and symmetry.
Resolution and Bandwidth:
• Resolution: The ability to distinguish between closely spaced spectral features.
• Bandwidth: The range of wavelengths passing through the monochromator.
Signal-to-Noise Ratio (SNR):
Spectral Calibration:
Ensures wavelength accuracy using known standards (e.g., mercury or neon lamps).
Reference and Baseline Correction:
Corrects for instrumental or environmental factors to isolate sample-specific spectral features.
 Selection Rules
Electronic Transitions (Atomic Spectroscopy):
Spin Rule: ΔS=0 (no spin change allowed).
Orbital Rule: Δl=±1 (change in orbital angular momentum).
Parity Rule: Transitions between states of the same parity are forbidden.
Vibrational Transitions (Molecular Spectroscopy):
Dipole Moment: IR-active if dipole moment changes.
Polarizability: Raman-active if polarizability changes.
Quantum Number Rule: Δv=±1 (fundamental transitions).
Rotational Transitions (Molecular Spectroscopy):
Rotational Rule: ΔJ=±1 (adjacent rotational levels).
Dipole Requirement: Requires a permanent dipole moment.
Relaxed/Forbidden Transitions:
Occur due to spin-orbit coupling, overtones (Δv>1), or external fields.
Lower intensity but can still appear in spectra.
Applications:
Predict spectral lines and intensities.
Analyze molecular symmetry and electronic configurations.
Atomic Spectroscopy
Involves transitions between electronic energy levels in isolated atoms.

Key Features:
Discrete Lines: Spectra consist of sharp, well-defined lines due to quantized electronic transitions.
Types:
Emission Spectroscopy: Atoms emit radiation when excited electrons return to lower energy states.
Absorption Spectroscopy: Atoms absorb specific wavelengths of light, leading to transitions to higher energy levels.
Fluorescence Spectroscopy: Atoms re-emit absorbed light at longer wavelengths.

Selection Rules: Govern allowed transitions based on quantum mechanical principles.

Applications:
• Identifying elements in a sample (e.g., flame tests, atomic absorption spectrometry).
• Determining isotopic compositions.
Molecular Spectroscopy
Involves transitions between electronic, vibrational, and rotational energy levels in molecules.
Key Features:
Broader Spectra: Due to additional vibrational and rotational degrees of freedom.
Types:
Rotational Spectroscopy: Transitions between rotational energy levels (microwave region).
Vibrational Spectroscopy: Transitions between vibrational levels (infrared region).
Electronic Spectroscopy: Transitions between electronic energy levels (UV-visible region).
Selection Rules: Depend on dipole moment changes and molecular symmetry.

Applications:
Identifying functional groups in molecules.
Studying molecular structure and bonding.
Analyzing reaction dynamics.
UV-Vis Spectroscopy
Principle:
Measures the absorption of ultraviolet (UV) or visible (Vis) light by a substance.
Absorption occurs when photons excite electrons to higher energy levels.
Key Features:
Wavelength Range: UV (200–400 nm), Vis (400–700 nm).
Transitions: Primarily electronic transitions (e.g., π→π∗ , n→π∗).
Beer-Lambert Law:
A=εcl
A: Absorbance.
ε: Molar absorptivity.
c: Concentration of the sample.
l: Path length of the light through the sample.
Instrumentation:
Light Source: Deuterium (UV) or tungsten (Vis) lamp.
Detector: Photodiode or CCD.
Monochromator: Isolates specific wavelengths.
Applications:
Identifying compounds and functional groups.
Quantifying concentrations.
Studying reaction kinetics.
Atomic Absorption Spectroscopy
 30%
70%
100% PMT

Cathode Metal
 KBr pellet method and Attenuated Total Reflectance (ATR)
KBr Pellet: Best for solid samples that can be ground into a fine powder. It is not suitable for liquids and gases.
ATR (Attenuated Total Reflectance): Versatile for solids, liquids, and even gases. It is especially useful for samples that are
difficult to prepare in other forms, such as viscous or sticky substances.

Non-destructive

KBr Pellet: The sample is dispersed in a matrix of potassium bromide (a transparent salt in the IR), and IR light passes
through the pellet. The IR light is absorbed according to the molecular vibrations of the sample.
ATR: Involves an IR crystal with a high refractive index. IR light enters the crystal and reflects internally, creating an
evanescent wave that extends beyond the surface of the crystal into the sample. The absorption occurs at the surface where
the sample interacts with the evanescent wave.
Amide I Segment
1600-1700 cm-1
Protein Secondary Structures
Protein-Conformational/Structural fingerprints in FTIR Spectrum

 Amide I secondary structure assignments

Amide I band 80% C=O stretch, near 1650cm-1

Amide I fingerprints (in Water) Secondary Structures Assigned

1616 Aggregates

1630 Beta Sheets

1644 Disorder

1651 Alpha Helix

1665 Turns

1678 Beta sheets (weak)

Principles of UV-Vis Spectroscopy
Ultraviolet-Visible (UV-Vis) spectroscopy:
i. Analytical technique used to measure the absorption (or sometimes
transmission) of ultraviolet (UV) and visible (Vis) light by a sample.
ii. Provides information about the electronic transitions in molecules or
atoms, which are related to their molecular structure, concentration,
and various other properties.

1. Basic Concept
• Electromagnetic Spectrum Range: Focuses on the UV region
(approximately 200–400 nm) and the visible region (approximately
400–800 nm) of the electromagnetic spectrum.

• Electron Transitions: UV-Vis absorption arises from the promotion of

electrons from lower-energy states (e.g., bonding orbitals) to higher-
energy states (e.g., anti-bonding orbitals). Common transitions
include:
o π→π∗
o n→π∗
o σ→σ∗
Beer–Lambert Law
UV-Vis spectroscopy often utilizes the Beer-Lambert Law to
relate absorbance (A) to concentration (c) of the analyte:
A=ε l c
where:
A is the measured absorbance (no units).
• ε (epsilon) is the molar absorptivity or extinction coefficient
(L⋅mol−1⋅cm-1)
• l is the path length of the cell (typically 1 cm).
• c is the concentration of the absorbing species (in mol⋅L−1).
 Types of UV-Vis Measurements
Single Beam vs. Double Beam Instruments
In a single-beam spectrophotometer, the intensity of light is measured sequentially for reference (blank) and
sample.
In a double-beam spectrophotometer, the beam is split so that the intensities of the reference and the sample are
measured simultaneously, improving stability and accuracy.
Absorbance Spectra
Plot of absorbance vs. wavelength.
Characteristic absorption bands help identify functional groups or electronic transitions.
Kinetic Measurements
Monitoring how absorbance changes over time to study reaction rates.
Quantitative Analysis
Using the Beer–Lambert Law to determine the concentration of absorbing species.
A more realistic model: partial unfolding due to damage to Trp residues

protein
aggregation

UV light can catalyze side chain oxidation (so can some heavy metals).
This type of damage can cause a protein to “open up” or misfold.
Damage to W42 or W130 of HγD leads to aggregation at 37 °C and pH7

Note: all the proteins fold up at low temperature despite the destabilizing mutations.

Chowdhury, Serebryani et.al. Elife 2023, Serebryany et al., Protein Sci. 2016
Principles of Fluorescence
1.Absorption and Emission
1. A molecule (fluorophore) absorbs a photon of a specific wavelength (excitation), promoting an electron from the
ground state (S₀) to an excited state (S₁, S₂, etc.).
2. After brief relaxation processes within the excited state (internal conversion, vibrational relaxation), the electron
returns to the ground state and emits a photon of lower energy (longer wavelength) compared to the absorbed
photon.
3. This phenomenon is illustrated by the Jablonski diagram, showing absorption (upward arrows) and emission
(downward arrows).
2.Stokes Shift
1. The Stokes shift is the difference in wavelength (or frequency) between the absorbed light and the emitted light.
2. Typically, the emission wavelength is longer (lower energy) than the excitation wavelength due to the energy lost
through non-radiative processes in the excited state.
Fluorescence Lifetime
1. The lifetime of fluorescence is the average time a molecule spends in the excited state before emitting a photon.
2. Typical fluorescence lifetimes range from picoseconds to nanoseconds, though longer lifetimes (microseconds)
can be observed for phosphorescence or certain rare fluorophores.

Quantum Yield
1. The quantum yield (Φ) is the ratio of the number of photons emitted to the number of photons absorbed.
2. A higher quantum yield indicates a more efficient fluorescence process. Values can range from near 0 (weakly
fluorescent compounds) to nearly 1 (highly fluorescent compounds).
Jablonski Diagram
Optical Path- Fluorescence Spectrophotometer
Fluorescence Spectrophotometer Readouts (Trp Fluorescence) to determine the folded-unfolded states of a protein (N
terminal domain of Crystallin Protein)

Fraction Unfolded (N.ter+Inositol)

6 Fraction Unfolded (N.ter)

5
Fraction Unfolded
4

0
0 1 2 3 4 5 6
Gdn.HCl
Circular Dichroism
Most biological molecules are asymmetrical or chiral

Therefore, molecules have a “handedness”

Higher order structures have a “hand” associated with it

Most DNA and protein helices are right-handed

Plane-polarized light propagated through a chiral sample will emerge plane
polarized but at different rotational angle.

Ellipticity is defined as the angle θ

given in radians as the value of its tangent of minor axis/major axis
optical rotation is defined by the angle α

Circular dichroism (CD) is the differential absorption of right- and

left-circularly polarized light

Ap = log10 (I0p/Ip)= εpCl

Expressed as ΔA=AL-AR=εCl
AL and AR is the absorbances for left and right circular polarized
light

CD is defined in terms of the molar ellipticity [θ]

Standard Secondary Assignments [negative ellipticity peaks] in a CD spectrum
CD spectrum from a thermal melt experiment-
With the rise in temperature CD spectral features are lost indicating loss of secondary structures , structural instability and
high degrees of conformational freedom [chain-entropy]
Electrophoresis-

Technique to separate charged molecules under an electric field

Migration depends on molecule’s charge, size, and shape
Commonly used for proteins (SDS-PAGE) and nucleic acids (agarose gels)

Electrophoretic Velocity (v) ~ (Charge / Friction) × Electric Field

Symbolically: v=μ×Ev (μ is mobility, E is field strength)

Horizontal Gel Vertical Gel

Sample Preparation Gel Preparation Electrophoresis Run Staining &
Visualization
Mix protein with SDS + Stacking gel (lower Apply electric field;
reducing agent (e.g., β- acrylamide) aligns proteins migrate
Use Coomassie, silver
mercaptoethanol) proteins toward the positive
stain, or fluorescent dyes
Boil to ensure denaturation Resolving gel (higher electrode
Compare bands with a
acrylamide) separates
molecular weight marker
proteins by size

Native PAGE
Maintains protein’s native fold

Separates by charge, size, and shape

No SDS or reducing agents

Allows study of protein complexes and

quaternary structures
PAGE (polyacrylamide gel) acts as a sieve to separate by size

SDS denatures proteins and imparts a uniform negative

charge

Simplifies analysis by focusing on molecular weight

differences only

APS (ammonium persulfate) + TEMED: Initiate polymerization

of acrylamide
Acrylamide + Bis-acrylamide: Form crosslinked gel

SDS (detergent): Denatures proteins, imparts uniform negative

charge
Tris + Glycine Buffers: Maintain pH and provide ions for current
 Stacking Gel
Lower acrylamide concentration (large pore size)
Aligns and concentrates proteins into a narrow band

Resolving Gel
Higher acrylamide concentration (smaller pore size)

Separates proteins by molecular weight

 Factors Controlling Migration in SDS-PAGE
Molecular Weight of the Protein:
SDS coats proteins with a uniform negative charge, making migration size-dependent.
Smaller proteins migrate faster, while larger proteins move slower through the gel matrix.
Gel Concentration (Polyacrylamide %):
Higher % gel (e.g., 12-15%): Better for small proteins (tight separation).
Lower % gel (e.g., 6-8%): Better for large proteins (faster migration).
Voltage and Electrophoretic Conditions:
Higher voltage speeds up migration but may cause overheating and distortion.
Lower voltage provides better resolution but takes longer.
Buffer Composition & pH:
Tris-Glycine-SDS buffer maintains a stable negative charge on proteins.
Ensures consistent ion flow and pH stability, affecting migration efficiency.
Protein Folding & Sample Preparation:
Incomplete denaturation (without SDS, DTT, or heating) may cause aggregation or abnormal migration.
Proper treatment with SDS (to provide uniform charge) and DTT (to break disulfide bonds) is crucial.
Gel Polymerization Quality:
Uneven polymerization or poor stacking gel formation can disrupt migration.
Proper TEMED and APS concentrations ensure uniform gel polymerization.
Presence of Glycosylation or Post-Translational Modifications:
Glycosylated or heavily modified proteins may migrate abnormally, appearing at higher molecular weights than expected.
Loading Volume & Sample Overloading:
Excess protein loading can cause smearing or distortion.
Consistent and appropriate sample amounts improve band sharpness.
2D- Gel Electrophoresis

Isoelectric focusing (pI) in the first

dimension

SDS-PAGE in the second dimension

Separates proteins by both charge
(pI) and molecular weight

Produces a 2D map of protein

spots for complex mixture analysis
 PCR (Polymerase Chain Reaction)

PCR is a technique used to amplify small segments of DNA.

It generates millions of copies of a specific DNA sequence,
making it easier to study and analyze.

Steps in PCR
Denaturation (94-98°C):
The double-stranded DNA is heated to separate it into two
single strands.
Annealing (50-65°C):
Short primers (single-stranded sequences) bind to their
complementary sequences on the single-stranded DNA.
Extension (72°C):
The enzyme Taq polymerase synthesizes new DNA strands by
extending the primers, creating two copies of the target
sequence.
Components-
DNA Template: The sample DNA to be amplified.
Primers: Short, single-stranded sequences that flank the target region.
Taq Polymerase: Heat-stable enzyme that synthesizes new DNA.
Nucleotide Mix (dNTPs): Provide the building blocks for DNA synthesis.
Buffer Solution: Maintains the optimal environment for the reaction.

PCR Applications:
Genetic Testing: Detection of mutations, genetic disorders, or disease-causing pathogens.
Forensic Analysis: DNA fingerprinting.
Cloning and Sequencing: Amplification of DNA for cloning or sequencing purposes.
Pathogen Detection: Detection of bacteria, viruses, and fungi in clinical samples.
RT-PCR
ELISA - Enzyme-Linked Immunosorbent Assay

To detect and quantify soluble substances such as

proteins, peptides, antibodies, and hormones.

The test involves the use of antibody-antigen interactions,

where the antigen is captured by the antibody, and the signal is
detected via an enzyme reaction.

Procedure:
1.Coating: The antigen or antibody is immobilized on a solid
surface (typically a well in a microplate).
2.Blocking: Unoccupied sites are blocked to prevent non-
specific binding.
3.Detection: The antibody (or antigen) specific to the target
analyte is added, followed by the enzyme-labeled secondary
antibody.
4.Substrate Addition: A substrate is added that reacts with
the enzyme to produce a detectable signal (color change or
fluorescence).
Types of ELISA
Types of ELISA:
Direct ELISA: Antigen (Ag) is bound to the plate, and a labeled primary antibody directly detects it.
Indirect ELISA: Uses an unlabeled primary antibody followed by a labeled secondary antibody, improving sensitivity.
Sandwich ELISA: A capture antibody immobilizes the antigen, and a detection antibody binds it, offering high specificity.
Competitive ELISA: An inhibitor antigen competes with the sample antigen for binding, used for small molecules and hormone detection.
Detection Mechanism:
Enzyme-linked antibodies react with a substrate, producing a color change proportional to antigen concentration.
Key Applications:
Disease diagnosis (e.g., HIV, COVID-19, cancer markers).
Drug and toxin detection (e.g., doping tests, pesticide screening).
Food safety analysis (e.g., allergen detection).
Antibody/antigen quantification in research and diagnostics.
Advantages of Different ELISA Types:
Direct ELISA: Fast and simple but less sensitive.
Indirect ELISA: More sensitive due to signal amplification.
Sandwich ELISA: High specificity, ideal for complex samples.
Competitive ELISA: Suitable for small molecules with low antigen availability.

Critical Considerations:
Blocking steps prevent non-specific binding.
Washing steps are crucial to reduce background noise.
Standard curves help quantify unknown samples.