Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
Affinity Chromatography involves the covalent attachment of an immobilized biochemical called as affinity ligand to a solid support. When a sample is passed through the column, only solute that selectively binds to the complementary ligand is retained; other sample components elute without retention. The separation exploit the “lock and key” binding that is prevalent in biological systems. The retention solutes can be eluted from the column by changing the mobile phase composition.
Affinity Chromatography involves the covalent attachment of an immobilized biochemical called as affinity ligand to a solid support. When a sample is passed through the column, only solute that selectively binds to the complementary ligand is retained; other sample components elute without retention. The separation exploit the “lock and key” binding that is prevalent in biological systems. The retention solutes can be eluted from the column by changing the mobile phase composition.
CHROMATOGRAPHY
1. INTRODUCTION
2. PRINCIPLE
3. TYPES OF CHROMATOGRAPHY
a. paper chromatography
i. principle
ii. procedure
iii. Rf value
b. affinity chromatography
c. ion exchange chromatography
d. size exclusion chromatography
e. hydrophobic interaction chromatography
Chromatography: Principle, types, application.
A complete description of Chromatography along with all the types including HPLC, GAS, COLUMN, ION EXCHANGE, AFFINITY, COLUMN, PAPER, THIN LAYER CHROMATOGRAPHY - Techniques, Steps, principles, application.
Chromatography is a widely used separation and purification technique used in laboratories to achieve high grade of purification of required molecules from a mixture. Amino acids, pigments and many other biomolecules and chemical constituents. It has high fold application in Biology and chemistry. A field of Biochemistry is heavily dependant on the technique. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate.
Chromatography is a bioanalytical technique used for separation of analytes into pure components. Biomolecules such as amino acids, proteins and carbohydrates can be purified by different chromatographic methods.
• Chromatography is a method of separation in which the components to be separated are distributed between two phases, one of these is called a stationary phase and the other is a mobile phase which moves on stationary phase in a definite direction
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Richard's entangled aventures in wonderlandRichard 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.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Chromatography by narayan sarkar and simi baruah new version
1. CHROMATOGRAPHY
Presented by –
Simi Baruah
Roll No-18
Narayan Sarkar
Roll No-6
M.Sc. 3rd semester
Guided by-
Dr. Nabanita Bhattacharyya
Assistant Professor
Dept. of Botany
3. INTRODUCTION
Chromatography is a Greek word (colour
writing), chroma =“colour” & Graphein=
“to write”
It is the collective term for a set
laboratory techniques for the separation
of mixtures.
Definition:
Chromatography is a technique for the
separation of a mixture by passing it in
solution or suspension through a
medium ,in which the components move
at different rates.
4. HISTORY OF
CHROMATOGRAPHY
To write with colors -- literally translated
from its Greek
roots chroma and graphein
Chromatography: Russian botanist
Mikhail Tswett in 1903 , first developed
this technique.
It has since developed into an invaluable
laboratory tool for the separation and
identification of compounds.
6. 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:
Molecular characteristics related to
Adsorption (liquid-solid),
Partition (liquid-solid), and
Affinity or
differences among their molecular
weights.
7. 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 the mobile
phase, and leave the system faster.
8. Basis of the chromatography technique:
Stationary phase: This phase is always
composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface solid
support”.
Mobile phase: This phase is always
composed of “liquid” or a “gaseous
component.”
Separated molecules:
The type of interaction between the
stationary phase, mobile phase, and
substances contained in the mixture is the
basic component effective on the
separation of molecules from each other.
9.
10.
11. Eluent: An eluent is a solvent used to carry
the components of a mixture through a
stationary phase. It is alternative term used
for the mobile phase.
Eluate: The mobile phase that exits the
column is termed as eluate.
Elution: The process in which solutes are
washed through a stationary phase by the
movement of a mobile phase.
13. 1)Adsorption chromatography
Separation is based on differences between the
adsorption affinities of the sample components for
the surface of an active solid stationary phase.
2)Partition chromatography
It is a type of chromatography in which the
components of the mixture get distributed into the
two phases due to differences in partition
coefficients(Kd),which is the ratio of the
concentration of solutes in two phases.
Kd= concentration of solute in phaseA
concentration of solute in phaseB
The distribution of solutes between two phases is based
on solubility differences.
14. 3)Ion exchange chromatography:
It is applicable for the separation of charged
molecules.
Stationary phase is an ion exchanger(either cation
exchanger or anion exchanger)
Solute ions of the opposite charge in the mobile liquid
phase bind reversibly to the ion exchanger.
The graeter the charge ,the stronger the interaction.
Neutral solutes show no affinity for the stationary
phase and move with the eluting buffer.
The bound solutes can be released by eluting the
column with a buffer of increased ionic strength or pH
16. 4) Size exclusion chromatography:
It separates molecules on the basis of size and
shape.
A column matrix filled with porous gel beads made
of insoluble and hydrated polymer such as
polyacrylamide or agarose acts as stationary phase.
Solution containing molecules of various sizes is
passed through the column
Molecules smaller than the pores can enter the
pores in the beads whereas larger molecules can
not.
So larger molecules move faster and elute first.
Smaller molecules have longer retention time than
the larger molecules.
17.
18. 5)Affinity Chromatography:
It involves the following steps:
Choice of an appropriate ligand
Immobilization of ligand onto a
support matrix
Binding of the molecules of interest
with the ligand
Removal of non specifically bound
molecules
Elution of the molecules of interest in
a purified form.
19. Typical biological interactions used in
affinity chromatography:
Types of ligand Molecules of interest
Enzyme Substrate analogue
Antibody Antigen
Nucleic acid Complementary base
sequence
Avidin Biotin
Calmodulin Calmodulin –binding
protein
Poly(A) RNA containing poly(U)
sequence
Proteins A and G Immunoglobulins
20.
21. PAPER CHROMATOGRAPHY
What Is Paper Chromatography?
It was discovered by Synge and Martin in the
year 1943.
It is the technique that uses paper sheets or
strips as the adsorbent being the stationary
phase through which a solution is made to pass.
Inexpensive method of separating dissolved
chemical substances by their different migration
rates.
Powerful analytical tool that uses very small
quantities of material.
22. PAPER CHROMATOGRAPHY
PRINCIPLE
It is a partition chromatography technique
Cellulose paper is a supporting medium over
which the solvents flow.
Water bound to the polar cellulose is the
stationary phase and organic solvent which flows
over it is a mobile phase. Organic solvent moves
over the hydrated cellulose fibres
As the solvent passes through an area of paper
containing a solute (mixture of components), the
solute begins to partition itself between the
aqueous and organic phases in proportion to its
relative solubility in the two phases
23. The components of the solute more
soluble in organic phase will be carried
faster along the organic phase.
Conversely, greater the affinity for
water, slower the solute will move
with respect to the solvent front.
Thus if several compounds possess
different solubility rates, each will
move across the paper at specific rate
which is generally different from that
of any other compound
24. The distance the solute moves, in
relation to the distance the solvent
moves, serves as a means of identifying
the solute and is called Rf.
25.
26. PROCEDURE OF PAPER
CHROMATOGRAPHY
Selecting a suitable type of development: It is
decided based on the complexity of the solvent, paper,
mixture, etc. Usually ascending type or radial paper
chromatography is used as they are easy to perform
Selecting a suitable filter paper: Selection of filter
paper is done based on the size of the pores and the
sample quality.
Prepare the sample: Sample preparation includes
the dissolution of the sample in a suitable solvent
(inert with the sample under analysis) used in
making the mobile phase
27.
28. Spot the sample on the paper: Samples should be
spotted at a proper position on the paper by using a
capillary tube.
Chromatogram development: Chromatogram
development is spotted by immersing the paper in the
mobile phase. Due to the capillary action of paper, the
mobile phase moves over the sample on the paper.
Paper drying and compound detection: Once the
chromatogram is developed, the paper is dried using
an air drier. Also, detecting solution can be sprayed
on the chromatogram developed paper and dried to
identify the sample chromatogram spots.
29. Paper Chromatography
Applications
Some of the uses of Paper Chromatography
in different fields are discussed below:
1)To study the process of fermentation and
ripening.
2 )To check the purity of pharmaceuticals.
3)To inspect cosmetics.
4)To detect the adulterants.
5)To detect the contaminants in drinks and
foods.
6)To examine the reaction mixtures in
biochemical laboratories.
7)To determine dopes and drugs in humans
and animals.
30. COLUMN CHROMATOGRAPHY
What Is Column Chromatography?
This method is a type of adsorption chromatography technique.
It is a technique which is used to separate a single chemical
compound from a mixture dissolved in a fluid.
It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move
through the column at different rates which allow them to get
separated in fractions.
This technique can be used on a small scale as well as large scale
to purify materials that can be used in future experiments.
31. Principle of column
chromatography
When the mobile phase along with the mixture
that needs to be separated is introduced from the
top of the column, the movement of the individual
components of the mixture is at different rates
The components with lower adsorption and
affinity to stationary phase travel faster when
compared to the greater adsorption and
affinity with the stationary phase.
The components that move fast are removed
first whereas the components that move
slowly are eluted out last.
32. The adsorption of solute molecules to
the column occurs in a reversible
manner. The rate of the movement of
the components is expressed as:
Rf = the distance travelled by solute
the distance travelled by the solvent
Rf is the retardation factor.
33.
34. Column Chromatography
Procedure
Mobile phase – This phase is made up of solvents
and it performs the following functions:
It acts as a solvent – sample mixture can be
introduced in the column.
It acts as a developing agent – helps in the separation
of components in the sample to form bands
Some examples of solvents used as mobile phase
based on their polarity are – ethanol, acetone,
water, acetic acid, pyridine, etc.
Stationary phase – It is a solid material which
should have good adsorption property.
35. The stationary phase is made wet with the
help of solvent as the upper level of the
mobile phase and the stationary phase
should match.
In the first step the compound mixture that
needs to be separated, is added from the
top of the column without disturbing the top
level.
Without disturbing the stationary phase
solvent mixture is added slowly by touching
the sides of the glass column.
36. The tap is turned on to initiate the
movement of compounds in the
mixture.
The movement is based on the polarity
of molecules in the sample.
The non-polar components move at a
greater speed when compared to the
polar components.
37. For example, a compound mixture consists of
three different compounds viz red, blue, green
then their order based on polarity will be as
follows blue>red>green
As the polarity of the green compound is less,
it will move first.
When it arrives at the end of the column it is
collected in a clean test tube.
After this, the red compound is collected and at
last blue compound is collected.
All these are collected in separate test tubes.
38. Column Chromatography Applications
Column Chromatography is used to isolate
active ingredients.
It is very helpful in Separating compound
mixtures.
It is used to determine drug estimation from
drug formulations
It is used to remove impurities.
Used to isolation metabolites from biological
fluids.
39. THIN LAYER
CHROMATOGRAPHY
What Is Thin Layer Chromatography?
It is a technique used to isolate non-volatile mixtures.
The experiment is conducted on a sheet of
aluminium foil, plastic, or glass which is coated
with a thin layer of adsorbent material.
The material usually used is aluminium oxide,
cellulose, or silica gel.
On completion of the separation, each component
appears as spots separated vertically.
40. Each spot has a retention factor (Rf)
expressed as:
Rf = dist. travelled by sample
dist. travelled by solvent
The factors affecting retardation factor
are the- solvent system,
amount of material spotted,
absorbent and temperature.
41. Thin Layer Chromatography
Principle
Like other chromatographic techniques, thin-
layer chromatography (TLC) depends on the
separation principle.
The separation relies on the relative affinity of
compounds towards both the phases.
The compounds in the mobile phase move
over the surface of the stationary phase.
42. The movement occurs in such a way
that the compounds which have a
higher affinity to the stationary phase
move slowly while the other
compounds travel fast.
On completion of the separation
process, the individual components from
the mixture appear as spots at respective
levels on the plates.
Their character and nature are identified
by suitable detection techniques.
43.
44. Thin Layer
ChromatographyExperiment
The stationary phase that is applied to the plate
is made to dry and stabilize.
To apply sample spots, thin marks are made at
the bottom of the plate with the help of a pencil.
Apply sample solutions to the marked spots.
Pour the mobile phase into the TLC chamber
and to maintain equal humidity, place a
moistened filter paper in the mobile phase.
Place the plate in the TLC chamber and close it
with a lid.
It is kept in such a way that the sample faces the
mobile phase.
45. Immerse the plate for development.
Remember to keep the sample spots
well above the level of the mobile
phase.
Do not immerse it in the solvent.
Wait till the development of spots.
Once the spots are developed, take out
the plates and dry them.
The sample spots can be observed
under a UV light chamber.
46. Thin Layer Chromatography
Applications
1)The qualitative testing of Various medicines
such as sedatives, local anaesthetics,
anticonvulsant tranquilisers, analgesics,
antihistamines, steroids, hypnotics is done by
TLC.
2)TLC is extremely useful in Biochemical
analysis such as separation or isolation of
biochemical metabolites from its blood plasma,
urine, body fluids, serum, etc.
3)Thin layer chromatography can be used to
identify natural products like essential oils or
volatile oil, fixed oil, glycosides, waxes,
alkaloids, etc.
47. 4)It is widely used in separating
multicomponent pharmaceutical formulations.
5)It is used to purify of any sample and direct
comparison is done between the sample and
the authentic sample.
6)It is used in the food industry, to separate and
identify colours, sweetening agent, and
preservatives
7)It is used in the cosmetic industry.
8)It is used to study if a reaction is complete.
48. Thin–layer chromatography offers several
benefits over the paper chromatographic
separations. Some of the benefits are:
Time Saving
The biggest advantage offered to the
chromatographer is time- saving. Paper
chromatography can take several hours to develop
the plate whereas development in thin layer
chromatography can be completed in much shorter
time (about half an hour or so)
Automation
Paper chromatography has not seen much
automation over the years but thin layer
chromatography instruments available have
automation capabilities which include
autosampler, constant volume sample dispenser,
documentation and camera for retaining pictorial
record of separations
49. Rigid Support
The cellulose paper support in paper chromatography
is flexible whereas the adsorbent in TLC is coated
onto a rigid metal, glass or plastic plate. This
contributes to reproducibility of spots and faster
development. Due to support rigidity there is less
diffusion and as a result well-defined spots are formed.
Choice of Support
TLC presents a vast choice of support adsorbent
phases including liquid coated adsorbents which can
include fluorescence inducers as well. On the other
hand choice of papers in paper chromatography is
very much limited.
Development Chamber Design
It is not necessary to suspend the plate as required in
paper chromatography from a rod on top of the
development chamber. It can be simply placed in a slanting
position with its bottom edge resting on the chamber base.
50. Sample Volume
The quantity of sample applied is small( in
microliters) and can be reproducibly applied with
the help of automated sample dispensers
Choice of Spray Reagents
Corrosive spray reagents can char the filter
paper and can even deteriorate the sample
spots. Coated plates used in thin layer
chromatography can withstand corrosive spray
reagents to a greater extent.
Heating
Thin-layer chromatography plates can be heated
if required for spot development. Paper
chromatography sheets cannot withstand heating
beyond a point.
51. CONCLUSION
Now a days, chromatography is a very
popular biophysical technique for the
separation of bimolecules from both
plants and animals.
It is used for the separation of plant
pigments such as chlorophyll ,
xanthophyll etc.
Separation of amino acids is done by
this method.
52. REFERENCES
A text book of plant physiology ,
biochemistry and biotechnology by
Verma and Verma
Biophysics and molecular biology by
Pranav Kumar
https://Byjus.com
www.biochemden.com