The document describes various analytical techniques used in biochemistry to separate, identify, and quantify biomolecules. It discusses chromatography techniques including paper chromatography, ion-exchange chromatography, affinity chromatography, and high-performance liquid chromatography. It also covers electrophoresis methods like paper electrophoresis, gel electrophoresis, and isoelectric focusing. Other techniques mentioned are spectrophotometry, centrifugation, and enzyme-linked immunosorbent assay. The document provides details on the principles and procedures of these analytical methods.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
Centrifugation is the separation technique commonly used in clinical and research laboratories.
It is based on the behavior of particles in an applied centrifugal field.
More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
Centrifugation is the separation technique commonly used in clinical and research laboratories.
It is based on the behavior of particles in an applied centrifugal field.
More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
INTRODUCTION, DEFINATION OF ELECTROPHORESIS, ELECTROPHORESIS PRINCIPLE, TYPES OF ELECTROPHORESIS, FREE ELECTROPHORESIS, ZONE ELECTROPHORESIS,PAPER ELECTROPHORESIS, WORKING OF PAPER ELECTROPHORESIS, PROCEDURE FOR PAPER ELECTROPHORESIS, VISUALISATION, FACTORS AFFECTING SEPARATION OF MOLECULES, APPLICATIONS, working of paper electrophoresis ,procedure for paper electrophoresis ,visualisation ,factors affecting separation of molecules ,applications ,forensics ,dna fingerprinting ,molecular biology ,microbiology information about the organisms ,biochemistry mapping of cellular components ,paper electrophoresis is also used in study of sic ,hemoglobin abnormalities ,separation of blood clotting factors ,serum plasma proteins from blood sample ,used in separation and identification of alkaloids ,used for testing water samples ,toxicity of water ,drug industry to determine presence of illelgal drUGS
Electrophoresis along with its history, application and types are discussed here to give a brief yet understanding outlook to the topic explained which is an important topic for the biotechnology as well as all biological research field.
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase.
The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights
Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster.
This is based on protein-ligand interaction physical method, which gives us knowledge about how our body protein interacts with other molecule and protein function.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
DERIVATION OF MODIFIED BERNOULLI EQUATION WITH VISCOUS EFFECTS AND TERMINAL V...Wasswaderrick3
In this book, we use conservation of energy techniques on a fluid element to derive the Modified Bernoulli equation of flow with viscous or friction effects. We derive the general equation of flow/ velocity and then from this we derive the Pouiselle flow equation, the transition flow equation and the turbulent flow equation. In the situations where there are no viscous effects , the equation reduces to the Bernoulli equation. From experimental results, we are able to include other terms in the Bernoulli equation. We also look at cases where pressure gradients exist. We use the Modified Bernoulli equation to derive equations of flow rate for pipes of different cross sectional areas connected together. We also extend our techniques of energy conservation to a sphere falling in a viscous medium under the effect of gravity. We demonstrate Stokes equation of terminal velocity and turbulent flow equation. We look at a way of calculating the time taken for a body to fall in a viscous medium. We also look at the general equation of terminal velocity.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...
Analytical techniques ppt
1. A
SEMINAR
ON
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
PROF. J.P. SHARMA (DIRECTOR)
DR. R.K. RAO (PRINCIPAL)
GUIDED BY - HUMA NAZZ
SIDDIQUI
PRESENTED BY..
NIKITA DEWANGAN
M.Sc.1st SEM
BIOTECHNOLOGY
G.D. RUNGTA COLLEGE OF SCIENCE & TECHNOLOGY
KOHKA-KURUD,BHILAI DURG (C.G.)
2. INTRODUCTION
VARIOUS ANALYTICAL TECHNIQUES
PAPER CHROMATOGRAPHY
ION-EXCHANGE CHROMATOGRAPHY
GEL FILTRATION CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
PAPER ELECTROPHORESIS
GEL ELECTROPHORESIS
ISOELECTRIC FOCCUSING
SPECTROPHOTOMETER
CENTRIFUGE
ENZYME LINKED IMMUNO SORBANT ASSAY
CONCLUSION
SUMMARY
REFERENCES
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3. Introduction
In analytical biochemistry uses instruments and used to
separate, identify and qualify biomolecules.
Definition
An analytical techniques is a method that is used to
determine the concentration of a chemical compound.
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5. INTRODUCTION
Chromatography is an analytical technique dealing with the separation of
closely related compound from a mixture. These include protein, peptides,
amino acid, lipids, carbohydrates, vitamins and drugs.
PRINCIPLE
Chromatography (Greek ; chroma – colour, graphein – to write) usually
consists of a mobile phase and a stationary phase.
The mobile phase refers to the mixture of substances( to be separate),
dissolved in a liquid or a gas. The stationary phase is a porous solid matrix
through which the sample contained in the mobile phase percolates.
The interaction between the mobile and stationary phases results in the
separation of the compounds from the mixture.
TYPES OF CHROMATOGRAPHY - PAPER CHROMATOGRAPHY, ION
EXCHANGE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY.
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6. INTRODUCTION
Paper chromatography is useful for separating the mixture of amino acid,
sugar, chemicals, lipid , urea and some drugs.
PRINCIPLE
The filter paper are used to support a stationary water phase while mobile
organic phase moves down the suspended paper strip in a cylinder.
Separation is based on a liquid-liquid partition of the compounds.
Thus, this is essentially a form of partition chromatography between 2 liquid
phases, although adsorption to the paper may also take place.
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7. PROCEDURE –
The sample mixture is applied to a piece of
filter paper the edge of the paper is
immersed in a solvent.
The aqueous component of the solvent
system binds to the paper & forms a
stationary phase.
The organic component that migrates on the
paper is the mobile phase.
When the migration of the solvent is
upwards, it is referred to as ascending
chromatography.
In descending chromatography, the solvent
moves downwards.
As the solvent flows, it takes along with it
the unknown substances.
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ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
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Fig. 1; paper chromatography
8. The rate of migration of the molecules depends on the relative solubility in the
stationary phase (aqueous) & mobile phase (organic).
The paper (chromatography) is then removed, dried & developed
for the identification of the specific spots.
Identification of the compound :-
The distance travelled by a particular component of a mixture (or solute)is used
to identify it.
Resolution Factor(RF) = Distance from origin run by the solute
Distance from origin run by the solvent 7
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
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9. INTRODUCTION
Ion exchange chromatography is used for separation of amino acids, proteins,
nucleotides and charged molecule.
PRINCIPLE
Ion exchange chromatography involves the separation of molecules on the basis of
their electric charges. Ion exchange resins- cat ion exchanges & anion exchanges are
used for this purpose.
PROCEDURE- An anion exchanges (R⁺ A⁻) exchanges its anion (A⁻) with
another anion (B⁻) in solution.
R⁺ A⁻ + B⁻ = R⁺ B⁻ + A⁻
A cation exchanges ( H⁺R⁻) exchanges its cat ion (H⁺) with another cat ion ( C⁺) in
solution.
H⁺ R⁻ + C⁺ = C⁺ R⁻ + H⁺
Thus, in ion exchange chromatography, ions in solutions are reversibly replaced by
ion-exchange resins.
The binding abilities of ions bearing positive or negative charges are highly pH
dependent, since the net charge varies with pH .
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10. FIG.:- 2 Ion exchange chromatography
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11. INTRODUCTION
Affinity chromatography is a method of separating biochemical mixtures based on a
highly specific interaction such as that between antigen and antibody, enzyme and
substrate, Or receptor and ligand.
PRINCIPLE
The principle of affinity chromatography is based on the property of specific &
noncovalent binding of proteins to other molecules, referred to as ligands. For instance
enzymes bind specifically to ligands such as substrates or cofactors.
PROCEDURE
This technique involves the use of ligands covalently attached to an inert & porus
in a column.
The immobilized ligands acts as molecular fish hooks to selectively pick up the
desired protein while the remaining proteins pass through the column.
The desired protein captured by the ligands, can be eluted by using free ligand
molecules.
Alternatively, some reagents that can break protein- ligand interaction can also be
employed for the separation.
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
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INTRODUCTION
High Performance Liquid Chromatography (HPLC) is used to separate
components of a mixture by using a variety of chemical interactions between the
substances being analyzed & the chromatography column.
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
12Fig; 4. HPLC
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PRINCIPLE
It may employ the principles of adsorption, partition, ion-exchange, exclusion
& affinity chromatography. This makes it an extremely versatile technique.
PROCEDURE
The sample to be analyzed is introduced in a small volume to the stream of
mobile phase.
It is retorted to specific chemical or physical interactions with the stationary
phase. It travels the length of the column.
The amount of retardation depends on the nature of the analyte, stationary phase
& mobile phase composition.
At the time at which a specific analyte elutes is called the retention time.
The use of pressure increases the linear velocity giving the components less
time to diffuse with in the column.
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INTRODUCTION
The movement of charged particles(ions) in an electric field resulting in
their migration towards the oppositely charged electrode is known as
electrophoresis.
TYPES OF ELECTROPHORESIS – PAPER ELECTROPHORESIS,
GEL ELECTROPHORESIS, ISOELECTRIC FOCUSSING.
PAPER ELECTROPHORESIS - Separation of charged particles is determined by
differences in their migration rate which varies with electrical charge, size of
particles & shape of particles.
PROCEDURE -
In this the sample is applied on a strip of filter paper wetted with dried buffer
solution.
The end of the strip are dipped into the buffer reservoirs in which electrodes are
placed. Then electric current is supplied allowing the molecules to migrate for
sufficient time.
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The paper is removed, dried & stained with a dye that specifically colors the
substance to be detected which can be identified by comparing with a set of
standards run simultaneously.
For the separation of serum proteins whatman No.1 filter paper, veronal or tris
buffer at pH 8.6 & the stains amido black or bromophenol blue are employed.
Fig 5, PROCESS OF PAPER ELECTROPHORESIS
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
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INTRODUCTION
It is used for the separation of proteins & nucleic acid.
This technique involves the separation of molecules based on their size, in
addition to electric charges.
PROCEDURE
In this technique gel is used as a stabilizing media.
Gel contains wells made with the help of comb.
Buffer is added in the apparatus.
In the well the sample is loaded.
The electric current is switched on.
The separated components can be identify either by labeling with a radio
isotope or by UV- visible spectrophotometer.
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INTRODUCTION
It is used for purification of proteins.
This technique is primarily based on the
immobilization of the molecules at isoelectric
pH during electrophoresis.
PROCEDURE
Stable pH gradients are set up (usually in gel)
converting the pH range to include the
isoelectric points of the components in a
mixture.
As the electrophoresis occurs, the molecules
migrate to positions corresponding to their
isoelectric points, gets immobilized & form
sharp stationary bonds.
The gel blocks can be stained & identified.
Fig -7 Isoelectric focusing
20. SPECTROPHOTOMETER – Spectrophotometer is a method to measure how much a
chemical absorbs light, by measuring the intensity of light as a beam of light passes
through sample solution.
PRINCIPLE OF SPECTROPHOTOMETER - The principle of spectrophotometer is
based on Lambert’s Beer’s law. :-
1. Lambert’s law (Bouguer’s law) - this law states that the amount of light absorbed is
directly propotional to the length or thickness of the solution under analysis.Thus,
I / I0 = e -kb
I = the intensity of the transmitted light, I0 = the intensity of the incident light, b = the
absorbing thickness, better known by the term path-length, k = the linear absorption
coefficient of the absorbing material,
the power term in the above relationship can be removed by converting to the logarithmic
form, thus,
In I /I0 = - kb or In I0 /I = kb,
Changing the common logarithms we get2.
2.303 Log10 I0/I = kb
2. Beer’s law - this law states that the amount of light absorbed is directly proportional
to the concentration of the solute in solution. Thus,
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
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21. 2.303log10 I0/I = kʹC
Where kʹ = absorptivity constant , C = the concentration of the
absorbing material.
The beer- lambert’s law then becomes:
Log10 I0/ I = abC
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20Fig - 8 spectrophotometer
22. CENTRIFUGE is device for separating particles from a solution according
to there size, shape, density, viscosity of the medium.
PRINCIPLE : - An object moving in a circle at a steady angular velocity will
experience a force F directed outwards. This is the basis of centrifugation. Angular
velocity ω and the radius of rotation ‘r’ in cm collectively determine the magnitude
of the force F,
F = ω2r
If F is expressed in the form of gravitational force it is divided by 980.
RCF = ω2r / 980
RCF = relative centrifugal force
But ω = 2π radian = 2π/60 r/min
RCF = 4π2( r/m)2r/ 3600× 980
RCF = 1.118 × 15-5 rpm2r
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23. ENZYME LINKED
IMMUNOSORBANT
ASSAY
ELISA is based on affinity.
An ELISA test can
detect both current and
past infections. As well as
antibodies, an ELISA test
can also be used to detect
antigens (substances that
provoke an immune
response, such as bacteria
or proteins) or enzymes.
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Fig ; 9ELISA
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Analytical techniques in biochemistry provides an opportunity for
researchers and scientists to explore the advanced and latest research
developments in the field of biochemistry .
Biochemistry deals with the structures, functions and interactions of
biological macromolecules such as proteins, nucleic acids, carbohydrates and
lipids which provide the structure of cells and perform many functions
associated with life.
It can be used both for separation & identification of macromolecules
& micro molecules.
In biochemistry, the term macromolecule is applied to the four
conventional biopolymers ( nucleic acids, proteins , carbohydrates & lipids )
as well as non polymeric molecules with large molecular mass.
The constituent molecules from which macromolecules are assembled are
called monomers, dimers or tetramers.
23
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
25. An analytical technique is a method that is used to determine the concentration of
a chemical compound.
CHROMATOGRAPHY is used to separation of closely related compound from a
mixture.
Chromatography types – PAPER CHROMATOGRAPHY, ION- EXCHANGE
CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY, HPLC.
ELECTROPHORESIS – The movement of charged particle an electric field
resulting in their migration towards the oppositely charged electrode .
Electrophoresis types – PAPER ELECTROPHORESIS, GEL
ELECTROPHORESIS, ISOELECTRIC FOCUSING.
Spectrophotometer is the quantitative measurement of the reflection or
transmission properties of a material as a function of wavelength.
CENTRIFUGE - it is a device for separating particles from a solution according to
their size, shape, density and velocity of medium.
ELISA - An enzyme-linked immunoassay (ELISA) is a test that detects
antibodies in the blood.
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ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
26. U. SATYANARAYANA 2010 BIOTECHNOLOGY
J.L JAIN SIXTH EDITION 2010 FUNDAMENTAL
OF
BIOCHEMISTRY
UPADHAYAY FOURTH EDITION2007 BIOPHYSICAL
AND
BIOCHEMISTRY
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