Unit - 1: Principle: Microscopy- Principle, mechanism, and application of photo-optical instruments

(different types of Microscopes), Phase contrast microscope, Bright Field Microscope, Dark Field
Microscope, Fluorescence microscopy, Confocal microscopy, Scanning and Transmission Electron
Microscopy, Expansion microscopy, Micrometry.
Practical: Ray diagrams of phase contrast microscopy and Electron microscopy. Measurement of
a microscopic object using an occular micrometer and stage micrometer.

Microscopes:
● Compound Microscope: The workhorse of biological research, using multiple lenses
(objective and eyepiece) to achieve high magnification and resolution for studying cells and
tissues.
● Simple Microscope: A single lens magnifies the specimen, offering a lower magnification
suitable for basic observations.
● Stereo Microscope: Provides a three-dimensional view of a specimen, ideal for examining
the surface features of insects, plants, or small objects.
● Electron Microscope: Utilizes a beam of electrons instead of light to achieve much higher
magnification and resolution, allowing visualization of structures like viruses and individual
molecules.
While the core principle remains the same, microscopy offers a diverse toolbox of techniques, each
with its strengths:
● Bright-field microscopy: The most common technique, using transmitted light to illuminate
the specimen. This is suitable for visualizing cells, tissues, and other stained samples.
● Dark-field microscopy: Ideal for observing transparent or unstained specimens. Here, light
scatters off the edges of the object, creating a bright image against a dark background.
● Phase-contrast microscopy: Offers superior contrast for observing transparent or low-
contrast biological structures. It utilizes slight phase shifts in light waves to enhance details.
● Fluorescence microscopy: Employs fluorescent dyes that bind to specific structures within
the specimen. When exposed to light of a particular wavelength, these dyes emit light,
highlighting the target structures with vibrant colors.

The Core Principles: Magnification and Resolution

At the heart of microscopy lies the ability to magnify and resolve tiny objects.
● Magnification increases the apparent size of a specimen, allowing us to see details
invisible to the naked eye.
● Resolution determines the level of detail we can distinguish. A higher resolution
microscope can differentiate finer structures within a specimen.
Photo-optical instruments utilize the principle of optical microscopy, which involves the interaction
of visible light with specimens to produce an image. The key components of a photo-optical
microscope include:

1. Light Source: Provides illumination, typically a halogen lamp or LED, which produces light
in the visible spectrum.
2. Condenser: Focuses and directs light onto the specimen, ensuring uniform illumination.
3. Specimen: The object or sample being observed, placed on a glass slide or other
transparent substrate.
4. Objective Lens: Magnifies the specimen and collects light from it. The quality of the
objective lens determines the resolution and magnification of the microscope.
5. Ocular Lens (Eyepiece): Further magnifies the image formed by the objective lens and
allows the viewer to observe the specimen.
 6. Stage: Supports and holds the specimen in place, allowing for precise positioning and
manipulation.
7. Mechanical Stage Controls: Enable movement of the stage along both X and Y axes,
facilitating scanning of the specimen.
8. Focus Adjustment: Allows for precise focusing of the specimen by adjusting the distance
between the objective lens and the specimen.
Mechanism:
1. Illumination: The light source emits visible light, which passes through the condenser and
is focused onto the specimen.
2. Interaction with Specimen: The light interacts with the specimen, undergoing various
processes such as absorption, transmission, reflection, and scattering, depending on the
properties of the specimen.
3. Image Formation: Light transmitted or scattered by the specimen is collected by the
objective lens, which magnifies and forms an image of the specimen.
4. Magnification and Observation: The magnified image formed by the objective lens is
further magnified by the ocular lens, allowing the viewer to observe the specimen.
Applications:
Photo-optical instruments have a wide range of applications in various fields, including:

1. Biological Sciences: Used for observing cellular structures, tissues, microorganisms, and
biological processes in fields such as microbiology, histology, and cell biology.
2. Material Sciences: Used for examining the microstructure and properties of materials, such
as metals, polymers, ceramics, and composites.
3. Medical Diagnostics: Used in clinical laboratories for analyzing blood smears, urine
samples, and tissue biopsies for diagnostic purposes.
4. Forensic Science: Used for examining trace evidence, such as fibers, hair, and biological
fluids, in criminal investigations.
5. Quality Control and Inspection: Used in manufacturing industries for quality control,
inspection, and failure analysis of products and components.
6. Education and Research: Widely used in educational institutions and research laboratories
for teaching, training, and conducting scientific research.

1. Brightfield Microscope:
Principle: The brightfield microscope is the simplest and most commonly used type of optical
microscope. It uses white light to illuminate a specimen, and the specimen appears darker against
a bright background due to absorption of light. Light passes through the sample, and structures
that absorb light appear darker, while those that allow light to pass through appear lighter.

Mechanism:

● Light from the source (typically a halogen lamp) passes through the condenser lens and is
focused onto the specimen.

● The objective lens collects the light transmitted through the specimen and forms an image.

● The magnified image is observed through the ocular lens.

Applications:
 ● Observation of stained biological specimens in histology, cytology, and microbiology.

● Material science applications for examining the microstructure of materials.

● Limitations: Transparent or unstained specimens offer low contrast and limited detail.
2. Phase Contrast Microscope:
Principle: Phase contrast microscopy enhances the contrast of transparent and unstained
specimens by exploiting differences in refractive index. It converts phase differences in light
passing through the specimen into differences in brightness or contrast. Utilizes slight phase
shifts in light waves as they pass through different parts of the specimen. These phase shifts are
converted into variations in brightness, creating an image with enhanced contrast for transparent
or low-contrast samples.

Mechanism:

● Light from the source passes through the condenser and a phase plate, creating phase
differences in the light waves.
● The phase differences cause a shift in the phase of light passing through different parts
of the specimen.
● The objective lens collects the phase-shifted light and forms an image where the phase
differences are translated into differences in brightness or contrast.
● The magnified image is observed through the ocular lens.

Applications:

● Observation of live cells and tissues without the need for staining, making it useful in cell
culture and medical diagnostics.
● Studies of cell morphology and dynamics.
● Limitations: Requires specialized condenser and objective lenses.
3. Dark Field Microscope:
Principle: Dark field microscopy illuminates the specimen with oblique or off-axis lighting, causing
only scattered light to reach the objective lens. Employs a specialized condenser that scatters light
at a high angle, preventing direct illumination of the specimen. Only light scattered by the edges of
the object or refracted within the specimen reaches the objective lens, This results in a bright
image of the specimen against a dark background.

Mechanism:

● Light from the source is directed toward the specimen at an oblique angle, usually by
using a specialized condenser with an opaque stop.
● Only light scattered by the specimen reaches the objective lens, while direct light is blocked.
● The scattered light forms a bright image of the specimen against a dark background.
● The magnified image is observed through the ocular lens.

Applications:

● Observation of unstained or thinly stained specimens with low contrast, such as living
microorganisms or nanoparticles.
● Examination of transparent or refractive specimens.
● Limitations: Offers lower resolution compared to bright-field microscopy and may require
 specialized objectives
4. Fluorescence Microscopy:
Principle: Fluorescence microscopy involves excitation of fluorophores within the specimen by
specific wavelengths of light, resulting in emission of fluorescence at longer wavelengths. Employs
fluorescent dyes that bind specifically to target structures within the specimen. When exposed to
light of a particular wavelength (excitation light), these dyes emit light of a different wavelength
(emission light), making the target structures appear fluorescent against a dark background.

Mechanism:

● Light from the source passes through a filter to select the excitation wavelength, which is
focused onto the specimen.
● Fluorophores within the specimen absorb the excitation light and emit fluorescence at a
longer wavelength.
● A barrier filter blocks the excitation light and allows only the emitted fluorescence to pass
through to the objective lens.
● The objective lens collects the emitted fluorescence and forms an image of the specimen.
● The magnified fluorescent image is observed through the ocular lens.

Applications:

● Visualization and localization of specific molecules and structures within cells and tissues.
● Immunofluorescence staining for identifying and studying proteins, antibodies, and
cellular components, visualizing specific proteins, DNA, RNA, etc.

● Limitations: Requires fluorescent dyes specific to the target molecule and specialized
filters for excitation and emission light.
5. Confocal Microscopy:
Principle: Confocal microscopy uses point illumination and a pinhole aperture to eliminate out-of-
focus light, resulting in high-resolution, three-dimensional images. This creates high-resolution,
optical sections of a thick specimen, allowing visualization of structures at different depths.

Mechanism:

● Laser light is focused onto a single point on the specimen, illuminating a small area.
● Light from the illuminated point passes through a pinhole aperture, which blocks out-of-
focus light from reaching the detector.
● The objective lens collects the emitted fluorescence from the illuminated point and forms
an image slice at a specific depth within the specimen.
● By scanning the laser and pinhole aperture across the specimen, multiple image slices are
acquired to construct a three-dimensional image.

Applications:

● High-resolution imaging of biological samples with optical sectioning capability.

● Study of cell morphology, structure, and dynamics in three dimensions.
● Limitations: Imaging can be slower compared to conventional fluorescence microscopy,
and samples may require special preparation.
6. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy
(TEM):
 ● Principle: SEM and TEM are electron microscopy techniques that use electron beams
instead of light to image specimens at higher resolution.
● Electron Microscopes: These powerful tools utilize a beam of electrons instead of light to
achieve much higher magnification and resolution. Electrons interact with the specimen,
and the resulting signals are used to create an image.
● Scanning Electron Microscopy (SEM): Provides detailed information about the surface
features of a specimen. Electrons interact with the specimen's surface, generating
secondary electrons that reveal the topography and texture.
● Transmission Electron Microscopy (TEM): Allows visualization of the internal structure of
a specimen at the atomic level. Electrons are transmitted through a very thin slice of the
specimen, revealing details of organelles, molecules, and even individual atoms.
● Applications: SEM is used in material science, biology (cell surface features), and
forensics (analysis of fracture surfaces). TEM is used in biology (organelles, viruses),
material science (nanoparticles), and chemistry (molecular structure).
● Limitations: Electron microscopes are expensive, require specialized training for operation,
and often necessitate complex sample preparation techniques.

Mechanism:

● In SEM, a focused electron beam scans the surface of the specimen, and secondary
electrons emitted from the specimen are detected to form an image.
● In TEM, electrons are transmitted through a thin specimen, and the resulting electron
transmission pattern is used to form an image.

Applications:

● High-resolution imaging of subcellular structures and nanomaterials.

● Study of surface morphology, topography, and composition.
7. Expansion Microscopy:
Principle: Expansion microscopy is a recent technique that physically expands biological
specimens, uses a swellable hydrogel to physically expand a biological sample enabling imaging
at higher resolution using conventional microscopes.

Mechanism:

● Specimens are embedded in a swellable hydrogel matrix and then expanded by adding
water.
● As the hydrogel expands, biological structures within the specimen also expand
proportionally.
● The expanded specimen is then imaged using conventional microscopy techniques.

Applications:

● Imaging of large biological specimens, such as intact tissues or organs, at nanoscale

resolution.
● Study of cellular structures and protein distributions with enhanced spatial resolution.
● Limitations: Still under development, and the expansion process can introduce artifacts
and distort the sample's native structure
8. Micrometry:
Principle: Micrometry involves the measurement and quantification of microscopic features and
dimensions using calibrated scales or software tools.

Mechanism:

● Images of specimens are captured using microscopy techniques such as brightfield,

fluorescence, or electron microscopy.
● The dimensions of features within the images are measured using calibrated scales,
rulers, or software tools.
● Measurements may include lengths, areas, diameters, and angles of structures within the
specimen.

Applications:

● Quantification of cell sizes, organelle dimensions, and other microscopic features.

● Analysis of morphological changes, growth rates, and spatial distributions in biological
samples.

Unit - 2: Principle: Purification and separation techniques: Principle and techniques with
applications (Partition, adsorption, ion exchange, size exclusion, 2-D, HPLC, GLC, and affinity
chromatography). Electrophoretic technique (agarose and polyacrylamide gel) its Components,
working, and applications. Principles of Centrifugation and Ultracentrifugation Techniques and
their Applications. The basic idea of salting out, Dialysis.
Practical: Separation of mixtures by paper/ thin layer chromatography - Amino acid, Sugar;
Separation of protein mixtures by Polyacrylamide Gel Electrophoresis (PAGE); Separation of
components of a given mixture using a laboratory scale centrifuge; Understanding density
gradient centrifugation with the help of pictures.

Based on Physical Properties:

● Filtration: Separates particles based on size. A filter medium with pores of a specific size
allows the liquid to pass through while retaining larger particles. (Applications: Separating
sand from water, isolating cells from a broth)
● Distillation: Separates components based on their boiling points. The mixture is heated,
and the lower boiling point component vaporizes first and is condensed back into a liquid
form. (Applications: Purifying water, separating alcohol from a mixture)
● Crystallization: Relies on the controlled formation of crystals from a saturated solution.
The desired component crystallizes out of the solution, leaving impurities behind.
(Applications: Purifying table salt, growing crystals for electronics)
Based on Chemical Properties:
● Precipitation: Involves adding a reagent to a solution to induce the formation of an
insoluble product (precipitate) that can be separated from the remaining liquid
(supernatant). (Applications: Removing heavy metals from wastewater, isolating specific
ions)
● Neutralization: Reacting an acid with a base to form a neutral salt and water. This can be
used to separate acidic or basic components from a mixture. (Applications: Treating
heartburn, controlling acidity in soil)
● Chromatography:
 o Principle: Chromatography separates components of a mixture based on differences
in their affinity for a stationary phase and a mobile phase. Components with higher
affinity for the stationary phase elute more slowly.
o Applications: Widely used in analytical chemistry, biochemistry, and pharmaceuticals
for separation and analysis of complex mixtures, purification of biomolecules, and
quality control of drugs and chemicals. Common types include:
● Gas Chromatography (GC)
● Liquid Chromatography (LC)
● High-Performance Liquid Chromatography (HPLC)
● Thin-Layer Chromatography (TLC)
● Extraction:
o Principle: Extraction separates components based on differences in their solubility
between two immiscible phases. The desired compound is transferred from one
phase to another using a suitable solvent.
o Applications: Used in organic chemistry, pharmaceuticals, and natural product
isolation for extracting and purifying compounds from solid, liquid, or gaseous
mixtures.
● Electrophoresis:
o Principle: Electrophoresis separates charged molecules such as proteins, nucleic acids,
and carbohydrates based on their size, shape, and charge under an electric field.
o Applications: Widely used in molecular biology, genetics, and biochemistry for
analyzing DNA fragments, protein complexes, and other biomolecules.
● Centrifugation:
o Principle: Centrifugation separates particles based on their size, density, and shape by
subjecting them to high-speed rotation. Heavier particles sediment more rapidly,
forming a pellet at the bottom of the centrifuge tube.
o Applications: Utilized in laboratory and clinical settings for cell isolation, separation of
subcellular organelles, and purification of biological samples such as blood and
urine.
● Adsorption:
o Principle: Adsorption separates components based on their affinity for an adsorbent
material. The mixture is passed through the adsorbent, where certain components
are retained while others pass through.
o Applications: Used in purification of gases, removal of impurities from liquids, and
separation of components in chromatographic techniques such as column
chromatography and solid-phase extraction.

Purification and Separation Techniques: Principles and Applications

1. Partition Chromatography:
● Principle: Partition chromatography separates components based on their differential
partitioning between a stationary phase and a mobile phase. Components with
higher affinity for the stationary phase elute later, while those with higher affinity for
the mobile phase elute earlier.
● Technique: Common techniques include thin-layer chromatography (TLC) and paper
chromatography.
● Applications: Used for separating and analyzing mixtures of organic compounds,
amino acids, and pharmaceuticals.
2. Adsorption Chromatography:
● Principle: Adsorption chromatography separates components based on their
differential adsorption onto a solid stationary phase. Components with stronger
interactions with the stationary phase elute later.
● Technique: Common techniques include column chromatography, flash
 chromatography, and high-performance liquid chromatography (HPLC).
● Applications: Widely used in pharmaceutical, environmental, and biochemical analysis
to separate and purify compounds such as drugs, proteins, and environmental
pollutants.
3. Ion Exchange Chromatography:
● Principle: Ion exchange chromatography separates components based on their
interactions with charged functional groups on a stationary phase. Components with
similar charge to the functional groups are retained longer.
● Technique: Common techniques include strong and weak ion exchange
chromatography.
● Applications: Used for purification and separation of charged molecules such as
proteins, peptides, nucleic acids, and amino acids.
4. Size Exclusion Chromatography (SEC):
● Principle: Size exclusion chromatography separates components based on their size
and molecular weight. Larger molecules elute faster as they are excluded from the
pores of the stationary phase.
● Technique: Also known as gel filtration chromatography.
● Applications: Used for molecular weight determination, purification of proteins,
polysaccharides, and nucleic acids, and analysis of polymer molecular weight
distributions.
5. Two-Dimensional Chromatography (2-D Chromatography):
● Principle: Two-dimensional chromatography combines two separation techniques to
achieve higher resolution and peak capacity.
● Technique: Common techniques include 2-D liquid chromatography (LCxLC) and 2-D
gas chromatography (GCxGC).
● Applications: Used for complex sample analysis, separation of closely related
compounds, and characterization of complex mixtures such as natural products,
petroleum, and environmental samples.
6. High-Performance Liquid Chromatography (HPLC):
● Principle: HPLC separates components based on their interactions with a stationary
phase packed inside a column under high pressure. Components are eluted using a
liquid mobile phase.
● Technique: Widely used in analytical and preparative chromatography.
● Applications: Used in pharmaceuticals, food and beverage analysis, environmental
monitoring, forensic sciences, and biochemistry for separation and quantification of
compounds.
7. Gas-Liquid Chromatography (GLC):
● Principle: GLC separates components based on their partitioning between a gaseous
mobile phase and a liquid stationary phase coated on a solid support.
● Technique: Components are vaporized and passed through a heated column.
● Applications: Used for analysis of volatile organic compounds, fatty acids, and
environmental pollutants in various industries such as petrochemical,
pharmaceutical, and environmental monitoring.
8. Affinity Chromatography:
● Principle: Affinity chromatography separates components based on specific
interactions between a target molecule and a ligand immobilized on the stationary
phase.
● Technique: Used for purification of proteins, antibodies, enzymes, and other
biomolecules.
● Applications: Widely used in biotechnology, biochemistry, and pharmaceutical
industries for protein purification, drug screening, and protein-protein interaction
studies.
Electrophoretic Techniques: Agarose and Polyacrylamide Gel Electrophoresis

Components:

1. Gel Matrix:
● Agarose Gel: Agarose, derived from seaweed, forms a porous matrix when mixed with
buffer and heated. It is suitable for separating large DNA fragments, typically ranging
from several hundred base pairs to several kilobases.
● Polyacrylamide Gel: Polyacrylamide, a synthetic polymer, forms a dense and uniform
matrix when polymerized with a cross-linking agent (e.g., bisacrylamide). It is used
for separating smaller DNA fragments, RNA molecules, proteins, and peptides with
higher resolution.
2. Buffer System:
● A suitable buffer system is essential for maintaining the pH and conductivity required
for efficient electrophoresis. Common buffers include Tris-acetate-EDTA (TAE) and
Tris-borate-EDTA (TBE) for DNA electrophoresis and Tris-glycine for protein
electrophoresis.
3. Electrophoresis Apparatus:
● An electrophoresis chamber with electrodes and a power supply is used to apply an
electric field across the gel. The samples are loaded into wells at one end of the gel,
and the electric current drives the molecules through the gel matrix.
4. Loading Dye:
● Loading dyes containing tracking dyes and density agents are added to the samples
to aid in sample loading and visualization during electrophoresis.

Working:

1. Agarose Gel Electrophoresis:

● Agarose powder is mixed with buffer and heated to form a gel matrix.
● The gel is poured into a casting tray with a comb inserted to create sample wells.
● Once the gel solidifies, the comb is removed, leaving wells for sample loading.
● Samples mixed with loading dye are loaded into the wells, and an electric current is
applied across the gel.
● Negatively charged molecules migrate through the gel towards the positive electrode
(anode), with smaller molecules migrating faster and traveling farther than larger
molecules.
● After electrophoresis, the gel is stained with a dye (e.g., ethidium bromide for DNA) and
visualized under UV light.
2. Polyacrylamide Gel Electrophoresis:
● Polyacrylamide gel is prepared by polymerizing acrylamide and bisacrylamide in the
presence of a catalyst.
● The gel is cast and assembled similar to agarose gel electrophoresis.
● Samples mixed with loading dye are loaded into the wells, and electrophoresis is
conducted as described above.
● Polyacrylamide gels offer higher resolution and are suitable for separating smaller
molecules such as oligonucleotides, proteins, and peptides.

Applications:

1. DNA Electrophoresis:
● Agarose Gel Electrophoresis: Used for separating and analyzing DNA fragments in
 molecular biology applications such as DNA fingerprinting, restriction enzyme digestion
analysis, PCR product analysis, and DNA size estimation.
● Polyacrylamide Gel Electrophoresis: Suitable for resolving smaller DNA fragments with
higher resolution, such as single-stranded DNA, PCR products, and sequencing
fragments.
2. RNA Electrophoresis:
● Both agarose and polyacrylamide gel electrophoresis can be used for separating and
analyzing RNA molecules, including mRNA, rRNA, tRNA, and small RNA species.
3. Protein Electrophoresis:
● Polyacrylamide gel electrophoresis (PAGE) is commonly used for separating proteins
based on their size, charge, and conformation. Techniques such as SDS-PAGE
(sodium dodecyl sulfate polyacrylamide gel electrophoresis) and native PAGE are
utilized for protein analysis, quantification, and purification.
4. Western Blotting and Southern/Northern Blotting:
● Gel electrophoresis is often combined with blotting techniques such as Western
blotting (protein), Southern blotting (DNA), and Northern blotting (RNA) for
transferring and immobilizing separated molecules onto membranes for further
analysis with specific probes or antibodies.
5. Clinical Diagnostics:
● Gel electrophoresis is used in clinical laboratories for analyzing serum proteins,
hemoglobin variants, and genetic markers associated with diseases such as sickle
cell anemia and cystic fibrosis.
Choosing the Right Gel: The choice between agarose and polyacrylamide depends on the size of
the biomolecules being separated:
● Large Molecules (DNA fragments): Agarose gel with its larger pores is ideal for separating
DNA fragments ranging from a few hundred base pairs to several kilobases.
● Small Molecules (Proteins): Polyacrylamide gel with its finer pores offers higher resolution
for separating proteins based on both size and charge.

Principles of Centrifugation and Ultracentrifugation Techniques:

1. Centrifugation:
● Principle: Centrifugation exploits the principle of sedimentation, where particles in a
suspension settle under the influence of gravity. In a centrifuge, the force of gravity is
augmented by spinning samples at high speeds, leading to the separation of
components based on their density, size, and shape.
● Working: When a sample is spun in a centrifuge, centrifugal force causes denser
particles to sediment towards the bottom of the tube, forming a pellet, while lighter
particles remain suspended in the supernatant. The separation achieved depends on
factors such as centrifugal force, duration of centrifugation, and properties of the
sample (e.g., density, viscosity).
● Applications: Centrifugation is used for various purposes in research, clinical
diagnostics, and industrial processes, including:
● Separation of cellular components such as organelles, proteins, and nucleic acids
in cell biology and molecular biology.
● Purification of biomolecules such as DNA, RNA, proteins, and viruses.
● Clarification of suspensions, removal of particulate matter, and concentration of
samples in biotechnology and environmental science.
● Density gradient centrifugation for isolating subcellular fractions, purification of
viruses, and separation of macromolecules based on buoyant density.
2. Ultracentrifugation:
● Principle: Ultracentrifugation is an advanced form of centrifugation that operates at
 much higher speeds and generates greater centrifugal forces, enabling finer separation of
particles and macromolecules.
● Working: Ultracentrifuges are equipped with rotors capable of spinning at speeds
exceeding 100,000 revolutions per minute (rpm), generating centrifugal forces in the
order of hundreds of thousands of times gravity (g). This high centrifugal force
enables the separation of particles based on subtle differences in size, shape, and
density.
● Applications: Ultracentrifugation is employed in specialized fields requiring high-
resolution separation and analysis, including:
● Density gradient ultracentrifugation for isolating and purifying subcellular
organelles, membrane vesicles, and macromolecular complexes.
● Analytical ultracentrifugation for studying the size, shape, mass, and interactions
of biomolecules such as proteins, nucleic acids, lipoproteins, and viruses.
● Isolation of lipoprotein fractions and determination of lipoprotein profiles in
clinical lipidology and cardiovascular research.
● Sedimentation velocity and sedimentation equilibrium analysis for determining
molecular weights, stoichiometry, and binding affinities of macromolecules in
biochemistry and biophysics.

Applications of Centrifugation and Ultracentrifugation:

1. Separation and purification of cellular components such as organelles, membranes, and

cytoplasmic fractions in cell biology and molecular biology research.
2. Isolation and purification of biomolecules including DNA, RNA, proteins, lipids,
carbohydrates, and viruses for biochemical and biotechnological applications.
3. Concentration and clarification of suspensions, cell cultures, fermentation broths, and
biological fluids in bioprocessing, pharmaceutical manufacturing, and environmental
monitoring.
4. Fractionation and analysis of lipoproteins, lipids, and other macromolecules in clinical
diagnostics, lipid metabolism research, and cardiovascular disease studies.
5. Characterization of macromolecular structures, dynamics, interactions, and properties
using analytical ultracentrifugation, sedimentation velocity, and sedimentation equilibrium
analysis in biophysics and structural biology.
6. Separation and analysis of nanoparticles, colloids, polymers, and nanoparticles in
materials science, nanotechnology, and industrial chemistry.

Salting Out:

Salting out is a technique used to separate and purify biomolecules, particularly proteins and
nucleic acids, based on their solubility in aqueous solutions. The principle behind salting out relies
on the addition of high concentrations of salts to a solution containing the biomolecules of
interest, causing the biomolecules to precipitate out of solution due to reduced solubility.

● Idea: This technique utilizes the addition of salt to a protein solution to decrease the
solubility of the protein, causing it to precipitate out of solution.
● Mechanism: Salting out works by disrupting the interactions between water molecules and
the protein's surface. Proteins have both hydrophilic (water-loving) and hydrophobic (water-
hating) regions. At high salt concentrations, the salt ions compete with water molecules for
interaction with the protein's surface. This weakens the stabilizing hydrogen bonds between
water and the protein, making the protein less soluble and more likely to clump together and
precipitate.
● Specificity: Salting out has limited specificity, meaning it can precipitate various proteins
from a solution, not just the target protein. However, by using specific salts at controlled
 concentrations and temperatures, scientists can partially purify a protein of interest.

Basic Idea:

1. Protein Solubility: Proteins are typically more soluble in water due to the presence of
hydrophilic amino acid residues on their surface. However, their solubility can be influenced
by factors such as pH, temperature, and the presence of salts.
2. Effect of Salts: Addition of certain salts, such as ammonium sulfate ((NH4)2SO4), sodium
chloride (NaCl), or magnesium sulfate (MgSO4), to a protein solution can disrupt the
interactions between water molecules and the protein surface. This disrupts the hydration
shell around the protein molecules, leading to decreased solubility.
3. Salting Out Process: When salts are added to the protein solution at high concentrations,
they compete with the protein for water molecules, effectively reducing the availability of
water molecules to solvate the protein molecules. As a result, the proteins become less
soluble and tend to aggregate and precipitate out of solution.
4. Precipitation and Separation: The precipitated proteins can be separated from the solution
by centrifugation, filtration, or decantation. The choice of method depends on the nature of
the proteins and the experimental requirements.

Applications:

1. Protein Purification: Salting out is commonly used as an initial step in protein purification
procedures to concentrate and partially purify proteins from crude biological samples. It
can help remove contaminants such as lipids, nucleic acids, and small molecules.
2. Fractionation: Salting out can also be used to fractionate proteins based on their solubility
characteristics. By adjusting the salt concentration or using different salts, proteins with
different solubility properties can be selectively precipitated and separated.
3. Enzyme Stabilization: Salting out can be employed to stabilize enzymes by reducing their
solubility and preventing denaturation under certain conditions. This can be useful in
enzyme storage or formulation of enzyme-based products.

Dialysis:

Dialysis is a technique used to separate solutes based on differences in their diffusion rates
across a semipermeable membrane. It is commonly employed in biochemistry and molecular
biology for buffer exchange, desalting, and removal of small molecules from biomolecule
solutions.

● Idea: This technique removes salt and other small molecules from a protein solution by
utilizing a semipermeable membrane.
● Mechanism: A dialysis membrane acts as a barrier that allows water and small molecules
like salts to pass through but restricts the passage of larger molecules like proteins. The
protein solution is placed inside a dialysis bag made of this semipermeable membrane and
submerged in a bath of clean water. Over time, the salt and other small molecules diffuse
out of the bag through the membrane into the surrounding water, while the protein remains
trapped inside. This process reduces the salt concentration in the protein solution,
essentially "desalting" it.

Basic Idea:

1. Semipermeable Membrane: Dialysis involves placing the sample containing the solutes of
interest into a container that is separated from the surrounding solution by a
 semipermeable membrane. The membrane allows the passage of solvent molecules (e.g.,
water) but restricts the passage of solutes based on their size and charge.
2. Diffusion: Due to the concentration gradient across the membrane, solutes in the sample
solution diffuse through the membrane into the surrounding solution, while solvent
molecules move in the opposite direction.
3. Equilibrium: Dialysis continues until an equilibrium is reached, where the concentrations of
solutes inside and outside the dialysis membrane are equal. At this point, further net
movement of solutes ceases, and the sample is effectively desalted or buffer exchanged.
4. Buffer Exchange: Dialysis can be used to exchange the buffer solution surrounding the
sample with a different buffer solution. This is achieved by placing the sample in a dialysis
membrane and immersing it in the desired buffer solution. Through diffusion, the buffer
components from the surrounding solution replace the original buffer components in the
sample.

Applications:

1. Desalting: Dialysis is commonly used to remove salts and small molecules from
biomolecule solutions, such as proteins, nucleic acids, and enzymes. This is particularly
useful for downstream applications where high salt concentrations may interfere with
assays or experiments.
2. Buffer Exchange: Dialysis is employed for exchanging the buffer surrounding biomolecule
samples to optimize conditions for downstream applications, such as protein purification,
enzyme assays, and nucleic acid manipulations.
3. Concentration Adjustment: Dialysis can be used to concentrate biomolecule samples by
allowing solvent molecules to pass through the semipermeable membrane while retaining
the solutes within the sample compartment. This is useful for concentrating dilute samples
prior to analysis or storage.
4. Removal of Small Molecules: Dialysis can selectively remove small molecules, such as
detergents, dyes, or inhibitors, from biomolecule samples while retaining the biomolecules
of interest. This is advantageous for removing interfering substances or contaminants from
samples.
Working Together:
Salting out and dialysis are often used sequentially for protein purification:
1. Salting Out: Salt is added to the protein solution to precipitate the protein of interest (along
with other proteins).
2. Centrifugation: The precipitated protein pellet is separated from the supernatant (liquid) by
centrifugation.
3. Dialysis: The protein pellet is resuspended in a buffer and placed in a dialysis bag. The
bag is submerged in clean water to remove the salt used in the salting out step.
4. Result: The final product is a partially purified protein solution with a lower salt
concentration.

Unit - 3: Principle: Biophysical Principles: Osmosis, osmotic pressure, Donan equilibrium, diffusion
potential, diffusion coefficient, endocytosis & exocytosis, the gradient of chemical potential as
driving force in transport, membrane potential & ionophores. Practical: Demonstration of the
protoplast formation using lysozyme.
gemini
Biophysical principles bridge the gap between the physical sciences (physics, chemistry) and the
biological world. These principles explain how the laws of physics and chemistry govern the
structure, function, and behavior of living organisms at all levels, from molecules to cells, tissues,
organs, and whole organisms. Here's an exploration of some key biophysical principles:
1. Thermodynamics:
● This branch of physics deals with energy transfer and transformation. In living systems, it
helps us understand:
o Energy flow: How organisms acquire energy (through sunlight or food) and utilize it for
various functions like growth, movement, and reproduction.
o Entropy: The tendency of systems to become more disordered over time. Living
systems are highly ordered, but they maintain this order by constantly using energy
to counteract the natural increase in entropy.
o Equilibrium: The tendency of systems to reach a state of minimal free energy. Many
biological reactions reach equilibrium states, ensuring a balance between reactants
and products.
2. Intermolecular Forces:
● These are the attractive forces between atoms or molecules. They play a crucial role in:
o Protein structure: Hydrogen bonding, ionic interactions, and hydrophobic interactions
determine how proteins fold into specific three-dimensional shapes, which are
essential for their function.
o Nucleic acid structure: Hydrogen bonding between base pairs holds the two strands of
DNA together in a double helix shape, critical for storing genetic information.
o Membrane structure: Phospholipid molecules in cell membranes self-assemble due to
hydrophobic and hydrophilic interactions, creating a barrier that separates the
internal environment of the cell from the external environment.
3. Kinetics:
● This branch of chemistry focuses on the rate of chemical reactions. Biophysical principles
are applied to understand:
o Enzyme activity: How enzymes act as catalysts, accelerating the rate of essential
biochemical reactions in cells.
o Transport across membranes: How molecules move across cell membranes,
influenced by factors like concentration gradients and the properties of the
molecules themselves.
o Signal transduction: How cells receive and transmit signals, often involving a series of
biochemical reactions that occur at specific rates.
4. Macromolecules:
● These are large, complex molecules essential for life. Biophysical principles help us
understand:
o Protein function: How the structure of proteins dictates their function, such as
enzymes that catalyze reactions or antibodies that recognize specific antigens.
o Nucleic acid function: How DNA stores and transmits genetic information, and how
RNA plays a role in protein synthesis.
o Carbohydrates: How carbohydrates provide energy and structural support in cells.
o Lipids: How lipids form cell membranes and store energy.
5. Biomechanics:
 ● This field applies the principles of mechanics to living systems. It helps us understand:
o Movement: How muscles generate force and contraction to enable movement.
o Skeletal structure: How bones and cartilage provide support and structure for the body.
o Fluid dynamics: How blood flows through the circulatory system and how air moves
through the respiratory system.

Molecular Interactions:
● Non-covalent Interactions: The intricate structures and functions of biological molecules
like proteins, nucleic acids, and carbohydrates depend on various non-covalent interactions.
These include:
o Hydrogen bonding: Plays a crucial role in maintaining protein structure and DNA base
pairing.
o Ionic interactions: Important for protein folding and stability, and neuron signaling.
o Hydrophobic interactions: Drive protein folding and assembly of membranes.
o Van der Waals forces: Weak interactions that contribute to protein structure and
molecular recognition.
● Electrostatics: The distribution of electrical charges on biomolecules influences their
interactions with other molecules and their behavior in solutions.
Transport Processes:
● Diffusion and Osmosis: These principles govern the movement of molecules across
membranes. Diffusion is the passive movement of molecules from an area of high
concentration to low concentration. Osmosis is the diffusion of water across a
semipermeable membrane driven by differences in solute concentration. These processes
are essential for transporting nutrients and waste products across cell membranes.

Gpt

Biophysical principles encompass the study of physical principles and mechanisms underlying
biological processes and phenomena at the molecular, cellular, and organismal levels. These
principles integrate concepts from physics, chemistry, and biology to understand the structure,
function, dynamics, and interactions of biological systems. Some key biophysical principles
include:

1. Structural Biology:
● Principle: Understanding the three-dimensional structures of biological
macromolecules (proteins, nucleic acids, lipids) and their complexes is crucial for
elucidating their functions and interactions.
● Techniques: X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy,
cryo-electron microscopy (cryo-EM), and computational modeling are employed to
determine and analyze molecular structures.
2. Biomechanics:
● Principle: Investigating the mechanical properties and forces involved in biological
processes, such as cell movement, muscle contraction, and tissue mechanics,
provides insights into physiological functions and disease mechanisms.
● Techniques: Atomic force microscopy (AFM), optical tweezers, microrheology, and
computational modeling are used to study mechanical properties and forces at the
molecular and cellular levels.
3. Electrophysiology:
● Principle: Understanding the electrical properties of biological membranes, ion
 channels, and neuronal signaling is essential for studying nerve function, muscle
contraction, and sensory perception.
● Techniques: Patch-clamp electrophysiology, voltage-clamp recordings, and
fluorescence imaging of membrane potential are employed to study ion currents and
membrane dynamics.
4. Molecular Biophysics:
● Principle: Investigating the physical principles governing molecular interactions,
dynamics, and conformational changes is essential for understanding biochemical
processes and regulatory mechanisms.
● Techniques: Fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Förster
resonance energy transfer (FRET), and single-molecule techniques are used to study
molecular structure, dynamics, and interactions.
5. Biophysical Chemistry:
● Principle: Exploring the thermodynamic and kinetic principles underlying biological
reactions, enzyme catalysis, and ligand binding provides insights into metabolic
pathways, signal transduction, and drug design.
● Techniques: Isothermal titration calorimetry (ITC), surface plasmon resonance (SPR),
fluorescence resonance energy transfer (FRET), and enzyme kinetics are employed to
study reaction kinetics, binding affinities, and thermodynamic parameters.
6. Bioenergetics:
● Principle: Investigating the conversion of energy in biological systems, such as ATP
synthesis, electron transport, and metabolic pathways, is crucial for understanding
cellular functions and organismal physiology.
● Techniques: Oxygen consumption measurements, spectroscopic analysis of electron
carriers, and metabolic flux analysis are used to study energy conversion and
metabolic regulation.
7. Systems Biology:
● Principle: Integrating quantitative data from multiple omics technologies (genomics,
transcriptomics, proteomics, metabolomics) with computational modeling enables
the holistic understanding of biological systems and networks.
● Techniques: Mathematical modeling, network analysis, and bioinformatics tools are
employed to analyze complex biological data and predict system behavior.

Osmosis:

● Principle: Osmosis is the movement of solvent molecules (usually water) across a semi-
permeable membrane from a region of lower solute concentration to a region of higher
solute concentration, in order to equalize the solute concentration on both sides of the
membrane.
● Mechanism: Water molecules pass through the semi-permeable membrane via passive
diffusion, driven by the concentration gradient of solute particles.
● Significance: Osmosis is vital for biological processes such as cellular hydration, nutrient
uptake, and waste removal in organisms.
● Driving Force: The difference in solute concentration across the membrane creates a
pressure gradient. Water moves to equalize the concentration on both sides, hence the term
"osmotic pressure."
● Examples:
Shriveled plant cells in salty water lose water due to osmosis.
Red blood cells burst in hypotonic solutions (lower solute concentration outside the cell).

Osmotic Pressure:
 ● Definition: Osmotic pressure is the pressure exerted by the movement of solvent molecules
across a semi-permeable membrane to equalize the concentration of solute particles on
both sides of the membrane.
● Importance: Osmotic pressure is crucial for maintaining cell structure and volume in living
organisms, and it influences processes such as water transport in plants, kidney function in
animals, and osmoregulation in cells.

Donnan Equilibrium:

● Principle: The Donnan equilibrium describes the distribution of ions across a semi-
permeable membrane that separates two solutions of different concentrations of
electrolytes. It states that at equilibrium, there is an unequal distribution of ions due to the
presence of impermeable solutes, resulting in the establishment of an electrical potential
across the membrane.
● Significance: The Donnan equilibrium is important in biological systems, such as cell
membranes, where it influences ion distribution, cell volume regulation, and membrane
potential.

Diffusion Potential:

● Definition: Diffusion potential, also known as concentration potential or concentration

gradient, is the electrical potential difference that arises across a membrane due to the
uneven distribution of ions or molecules.
● Mechanism: The movement of ions along a concentration gradient generates a separation
of charge across the membrane, creating a voltage difference.
● Role: Diffusion potential is a key factor in processes such as nerve conduction, ion
transport across membranes, and the establishment of resting membrane potential in cells.

Diffusion Coefficient:

● Definition: The diffusion coefficient is a measure of how quickly a substance diffuses

through a medium, such as a gas, liquid, or solid.
● Factors Affecting Diffusion: Factors influencing diffusion coefficient include temperature,
size and shape of the diffusing particles, viscosity of the medium, and presence of
obstacles or barriers.
● Applications: Diffusion coefficients are used in various fields, including chemistry, physics,
biology, and engineering, to describe the rate of diffusion and predict the movement of
molecules in different environments.

Endocytosis & Exocytosis:

● Endocytosis: Endocytosis is a cellular process by which cells internalize substances from

the external environment by engulfing them in vesicles formed from the cell membrane.
There are different types of endocytosis, including phagocytosis (engulfment of solid
particles) and pinocytosis (uptake of liquid particles), Receptor-mediated endocytosis
( Selective uptake of specific molecules through specific receptor proteins on the cell
surface).
●
● Exocytosis: Exocytosis is the opposite process of endocytosis, in which cells release
substances from intracellular vesicles into the extracellular environment. It involves the
fusion of vesicles with the cell membrane, leading to the expulsion of their contents.
● Functions: Endocytosis is involved in nutrient uptake, receptor-mediated signaling, and
immune response, while exocytosis is essential for secretion of hormones,
 neurotransmitters, and digestive enzymes, as well as for membrane recycling and cell
communication.

Chemical Potential (μ):

● This concept combines the concentration and inherent energy of a molecule or ion in a
specific environment. It reflects the "tendency" of a molecule to move from one location to
another.

The gradient of chemical potential serves as a driving force for various transport processes across
biological membranes, including the establishment of membrane potential and the action of
ionophores.

Gradient of Chemical Potential: The chemical potential gradient represents the difference in the
free energy per unit of substance between two regions, typically separated by a membrane. It is
determined by the concentration gradient, electrical potential gradient, and pressure gradient of the
substance.

● Transport Across Membranes :Cell membranes are selectively permeable, allowing the
passage of specific molecules and ions. The chemical potential gradient across the
membrane is the primary driving force for passive transport processes (occurring without
energy input from the cell).
1. Passive Transport: In passive transport, substances move across the membrane along
their chemical gradient, from an area of higher concentration to an area of lower
concentration. This process does not require energy input.
● Example: Simple diffusion of oxygen and carbon dioxide across the lipid bilayer of the
cell membrane.
2. Facilitated Diffusion: Facilitated diffusion involves the movement of substances across
the membrane through specific transport proteins, which facilitate the passage of
molecules that cannot freely diffuse across the membrane.
● Example: Glucose transporters facilitate the movement of glucose into cells down its
concentration gradient.
3. Active Transport: Active transport involves the movement of substances against their
concentration gradient, requiring energy input (e.g., ATP hydrolysis) to pump molecules
across the membrane.
● Example: The sodium-potassium pump actively transports sodium ions out of the cell
and potassium ions into the cell, against their respective concentration gradients.

Membrane Potential: The membrane potential refers to the electrical potential difference across a
cell membrane, typically resulting from the uneven distribution of ions (e.g., sodium, potassium,
chloride) across the membrane. The gradient of chemical potential of ions contributes to the
establishment of membrane potential, which plays crucial roles in various cellular processes,
including:

● Nerve impulse transmission

● Muscle contraction
● Ion transport
● Cell signaling

Ionophores: Ionophores are molecules that facilitate the transport of ions across biological
membranes by altering the permeability of the membrane to specific ions. They can enhance ion
transport by:
 1. Forming complexes with ions and transporting them across the membrane.
2. Creating ion channels or pores in the membrane through which ions can pass.
3. Facilitating ion exchange between ions inside and outside the cell. Ionophores can be
natural (e.g., ionophoric antibiotics) or synthetic (e.g., valinomycin, gramicidin), and they
find applications in research, medicine, and biotechnology for studying ion transport
mechanisms, modulating cellular processes, and designing drug delivery systems.

Unit - 4: Principle: Principle, mechanism, and application of instruments used in

Spectrophotometric techniques (UV, visible, IR, Fluorescence, NMR, ESR). The basic concept of CD,
ORD. Radioactivity: Laws of Radioactivity, Half-life & Average life, types of radiation (α, β, γ
radiations) application of radioactive isotopes in biology. Radioisotope dilution technique and
Autoradiography.
Practical: Spectrophotometric determination of DNA/RNA concentration and its purity checking
without and chromogenic reaction.

Spectrophotometric techniques involve the measurement of the interaction between

electromagnetic radiation and matter to obtain qualitative and quantitative information about the
sample's properties. Here's an overview of the principle, mechanism, and applications of
instruments used in various spectrophotometric techniques:

1. UV-Visible Spectroscopy:
● Principle: UV-Visible spectroscopy measures the absorption of ultraviolet (UV) and
visible light by a sample. It relies on the Beer-Lambert law, which states that
absorbance is directly proportional to the concentration of the absorbing species and
the path length of the sample.
● Mechanism: A UV-Visible spectrophotometer emits a beam of UV or visible light
through the sample. The intensity of the transmitted light is measured by a detector,
and the absorbance is calculated based on the difference between incident and
transmitted light intensities.
● Applications: UV-Visible spectroscopy is widely used in chemistry, biochemistry, and
environmental science for quantifying concentrations of chromophores, determining
reaction kinetics, analyzing electronic transitions in molecules, and characterizing
complex mixtures.
2. Infrared (IR) Spectroscopy:
● Principle: IR spectroscopy measures the absorption, transmission, and reflection of
infrared light by a sample. It detects the vibrations of chemical bonds in molecules,
providing information about their functional groups and structures.
● Mechanism: An IR spectrophotometer passes infrared radiation through the sample.
The absorption of specific wavelengths of light by molecular vibrations is detected
by an infrared detector, and a spectrum is generated based on the absorbance at
different frequencies.
● Applications: IR spectroscopy is used in organic chemistry, pharmaceuticals, materials
science, and forensics for identifying functional groups, analyzing chemical
composition, determining molecular structure, and monitoring chemical reactions.
3. Fluorescence Spectroscopy:
● Principle: Fluorescence spectroscopy measures the emission of light by a sample after
it absorbs light at a specific wavelength. It exploits the phenomenon of fluorescence,
where molecules absorb photons and then emit light at longer wavelengths.
● Mechanism: A fluorescence spectrophotometer irradiates the sample with excitation
light, typically UV or visible light. The emitted fluorescence is measured
perpendicular to the excitation beam, and the fluorescence intensity is recorded as a
function of wavelength.
● Applications: Fluorescence spectroscopy is utilized in biochemistry, molecular biology,
 environmental science, and medical diagnostics for studying biomolecular interactions,
detecting fluorescent probes, analyzing protein structure, and monitoring cellular
processes.
4. Nuclear Magnetic Resonance (NMR) Spectroscopy:
● Principle: NMR spectroscopy measures the absorption and emission of radiofrequency
radiation by atomic nuclei in a magnetic field. It exploits the magnetic properties of
atomic nuclei and their interaction with external magnetic fields.
● Mechanism: In an NMR spectrometer, a sample is exposed to a strong magnetic field
and radiofrequency pulses. The resonance frequencies of atomic nuclei in the
sample are detected by a receiver coil, and a spectrum is generated based on the
resonance signals.
● Applications: NMR spectroscopy is employed in chemistry, biochemistry, and structural
biology for determining molecular structures, elucidating chemical composition,
studying molecular dynamics, and analyzing biomolecular interactions.
5. Electron Spin Resonance (ESR) Spectroscopy:
● Principle: ESR spectroscopy, also known as electron paramagnetic resonance (EPR)
spectroscopy, measures the absorption of microwave radiation by paramagnetic
species with unpaired electrons. It exploits the magnetic properties of unpaired
electrons in a magnetic field.
● Mechanism: In an ESR spectrometer, a sample containing paramagnetic species is
exposed to microwave radiation and a magnetic field. The absorption of microwave
energy by unpaired electrons is detected by a receiver, and a spectrum is generated
based on the resonance signals.
● Applications: ESR spectroscopy is used in chemistry, biochemistry, and materials
science for studying free radicals, paramagnetic ions, transition metal complexes,
and magnetic properties of materials.
Choosing the Right Technique :The choice of spectrophotometric technique depends on the type
of information you're seeking:
● UV-Vis: Useful for studying electronic transitions and quantifying colored solutions.
● IR: Identifies functional groups and provides structural information.
● Fluorescence: Detects specific molecules and studies protein interactions.
● NMR: The most powerful tool for detailed structural analysis of organic molecules.
● ESR: Identifies the presence and type of free radicals.

Chirality:
● Chiral molecules are non-superimposable mirror images, like a right hand and a left hand.
They lack symmetry and exhibit a unique property called optical activity.
Optical Activity:
● When plane-polarized light interacts with a chiral molecule, the plane of polarization can
be rotated. This rotation can be clockwise (positive) or counterclockwise (negative).

Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) are spectroscopic techniques used
to study the chiral properties of molecules, particularly in the field of structural biology and
chemistry. Here's a basic concept of CD and ORD:

Circular Dichroism (CD):

● Principle: Circular dichroism measures the differential absorption of left- and right-handed
circularly polarized light by chiral molecules. It arises from the differential absorption of
light by chiral chromophores due to their interaction with circularly polarized light, resulting
 in a CD spectrum.
● Mechanism: When circularly polarized light passes through a sample containing chiral
molecules, the intensity of the transmitted light is measured for both left-handed (L) and
right-handed (R) circular polarization. The difference in absorbance between L and R
circularly polarized light gives rise to the CD signal.
● Applications: CD spectroscopy is used to study the secondary structure of proteins,
conformational changes, protein-ligand interactions, nucleic acid structures, and chiral
compounds in solution. It is particularly valuable in structural biology and drug discovery.

Optical Rotatory Dispersion (ORD):

● Principle: Optical rotatory dispersion measures the wavelength-dependent rotation of

linearly polarized light upon passing through a chiral medium. It provides information about
the specific rotation of a molecule as a function of wavelength, yielding an ORD spectrum.
● Mechanism: When linearly polarized light passes through a chiral medium, the plane of
polarization rotates due to the interaction of the light with chiral molecules. The amount of
rotation depends on the wavelength of light and the concentration of chiral molecules in the
sample.
● Applications: ORD spectroscopy is used to determine the absolute configuration of chiral
molecules, quantify enantiomeric excess in mixtures, study the stereochemistry of organic
compounds, and investigate the chiroptical properties of natural products and synthetic
compounds.

Key Differences:

1. Measurement Principle: CD measures the differential absorption of circularly polarized

light, while ORD measures the rotation of linearly polarized light.
2. Information Obtained: CD provides information about the chiral properties of molecules,
including their secondary structure and conformational changes, whereas ORD yields
information about the specific rotation of chiral molecules as a function of wavelength.
3. Applications: CD is commonly used in structural biology and chemistry to study
biomolecular structure and interactions, while ORD is often employed in organic chemistry
and stereochemistry to determine the absolute configuration of chiral compounds.
Radioactivity:

Radioactivity is the spontaneous emission of radiation from the nucleus of an unstable atom,
resulting in the transformation of the atom into a different nucleus or a different energy state. This
phenomenon was first discovered by Henri Becquerel in 1896 and further elucidated by Marie Curie
and Pierre Curie. Radioactivity plays a crucial role in various fields, including physics, chemistry,
medicine, and environmental science.

Laws of Radioactivity:

1. Law of Radioactive Decay: The rate of radioactive decay of a substance is directly

proportional to the number of radioactive atoms present. Mathematically, it is expressed as:
dt / dN = − λN where dt / dN is the rate of decay, N is the number of radioactive
atoms, and λ is the decay constant.
2. Law of Exponential Decay: The number of radioactive atoms present in a sample
decreases exponentially with time. The decay of a radioactive substance can be described
by the equation: N(t) = N0 e − λt where N(t) is the number of radioactive atoms at time
t , N0 is the initial number of radioactive atoms, λ is the decay constant, and e is the base
of the natural logarithm.
Half-life & Average Life:

● Half-life (T½): The half-life of a radioactive substance is the time it takes for half of the
radioactive atoms in a sample to undergo decay. It is a characteristic property of each
radioactive isotope and is used to quantify the rate of radioactive decay.
● Average Life (Tav): The average life of a radioactive substance is the average time it takes
for a radioactive atom in a sample to decay. It is related to the half-life by the equation:

Tav=T½ / ln2 Tav=ln2T½

Types of Radiation:

1. Alpha (α) Radiation: Alpha radiation consists of alpha particles, which are helium-4 nuclei
(
4
He
2 ). Alpha particles are positively charged and relatively heavy, consisting of two

protons and two neutrons. They have low penetrating power and can be stopped by a sheet
of paper or a few centimeters of air.
2. Beta (β) Radiation: Beta radiation consists of beta particles, which are high-energy
electrons (β−) or positrons (β+). Beta particles are negatively or positively charged,
depending on their type. They have higher penetrating power than alpha particles and can
penetrate several millimeters of material, such as aluminum or plastic.
3. Gamma (γ) Radiation: Gamma radiation consists of gamma rays, which are high-energy
photons emitted from the nucleus during radioactive decay. Gamma rays have the highest
penetrating power among the three types of radiation and can penetrate several centimeters
of material, including lead and concrete.

Applications of Radioactive Isotopes in Biology:

40
● Radiometric Dating: Radioactive isotopes, such as carbon-14 (
14
C ), potassium-40 ( K
6 19 ),
238
and uranium-238 ( U92 ), are used for radiometric dating of archaeological artifacts,
fossils, and geological samples.
● Medical Imaging: Radioactive isotopes, such as technetium-99m (99mTc43 ) and iodine-131

( I 53 ), are used as tracers for thyroid function test in diagnostic imaging techniques,
131

including positron emission tomography (PET) and single-photon emission computed

tomography (SPECT).
● Radiation Therapy: Radioactive isotopes, such as cobalt-60 (60Co27 ) and cesium-137
( Cs55 ), are used in radiation therapy for the treatment of cancer by targeting and
137

destroying cancerous cells.

● Genetic Research: Radioactive isotopes, such as phosphorus-32 (32P15 ) and sulfur-35
35
( S16 ), are used as radioactive tracers in molecular biology and genetic research to label
and track DNA, RNA, and proteins.

Radioisotope Dilution Technique:

The radioisotope dilution technique is a method used to accurately measure the concentration of a
substance in a sample by introducing a known quantity of a radioactive isotope (the tracer) into
the sample and measuring its distribution. This technique is widely used in analytical chemistry,
biochemistry, and environmental science for quantifying the concentration of various substances,
such as metals, proteins, and hormones.

● Principle: This technique utilizes a known amount of a radioisotope-labeled compound

(radioactive standard) to determine the quantity of the same unlabeled compound (analyte)
present in a sample.
● Mechanism:
1. A known amount of the radioisotope-labeled compound is added to the sample
containing the unlabeled compound (analyte).
2. The mixture is allowed to equilibrate, ensuring both labeled and unlabeled
compounds are present in the same chemical form throughout the sample.
3. The mixture is then isolated and purified to separate it from interfering substances.
4. The total radioactivity of the isolated and purified mixture is measured.
● Calculation: By knowing the specific activity (radioactivity per unit mass) of the labeled
compound and the measured total radioactivity, the amount of the unlabeled compound
(analyte) in the original sample can be calculated.
● Advantages
● High accuracy and sensitivity, especially for measuring small amounts of analyte.
● Applicable to a wide range of analytes, including proteins, hormones, and small molecules.
● Less dependent on matrix effects compared to some other quantification methods.

Applications:

1. Metal Analysis: The radioisotope dilution technique is used to determine the concentration
of metals in environmental samples, such as soil, water, and biological tissues. It is
valuable for assessing pollution levels and monitoring environmental contaminants.
2. Protein Quantification: In biochemistry, the radioisotope dilution technique is employed to
measure the concentration of proteins and enzymes in biological samples. It provides
accurate quantification of protein concentrations for research and diagnostic purposes.
3. Hormone Assays: Radioisotope dilution assays are utilized in endocrinology to measure
the concentration of hormones, such as insulin, thyroid hormones, and steroids, in
biological fluids. These assays are important for diagnosing hormonal disorders and
monitoring hormone levels in patients.

Autoradiography:

Autoradiography is a technique used to visualize and localize radioactive substances in a sample

by exposing it to a photographic film or emulsion that detects the emitted radiation. It is commonly
employed in molecular biology, neuroscience, and histology for studying the distribution and
localization of radioactively labeled molecules, such as DNA, RNA, proteins, and metabolites.

● Principle: This technique uses radioactively labeled molecules to visualize their location
within a sample, typically on a two-dimensional surface like a tissue section, cell culture, or
chromatography plate.
● Mechanism:
1. The sample is incubated with a radiolabeled compound (probe) that can bind to
the molecule of interest.
2. The unbound radiolabeled probe is removed through washing steps.
 3. The sample is then placed in contact with an X-ray film or a special phosphor
screen.
4. The emitted radiation from the radiolabeled compound interacts with the film or
screen, creating a latent image over time.
5. The film or screen is then processed to reveal the image, showing dark spots
where the radiolabeled probe has bound to the molecule of interest in the sample.

Applications:

1. DNA and RNA Analysis: Autoradiography is used to visualize the distribution of

radioactively labeled nucleic acids in gel electrophoresis experiments, such as DNA
sequencing, Southern blotting, Northern blotting, and RNA transcription assays.
2. Protein Localization: In protein research, autoradiography is employed to study the
subcellular localization of radioactively labeled proteins within cells and tissues. It is
valuable for elucidating protein expression patterns, protein-protein interactions, and cellular
signaling pathways.
3. Neuroscience Studies: Autoradiography is widely used in neuroscience to investigate the
distribution and binding sites of neurotransmitters, receptors, and ligands in brain tissue. It
provides insights into brain function, neuronal connectivity, and neuropharmacology.
Radioisotope dilution and autoradiography can be used together. For instance, radioisotope
dilution can be used to quantify the total amount of a molecule in a sample, while
autoradiography can reveal its spatial distribution within the sample.