The document discusses ion exchange chromatography, which separates charged molecules by exchanging them for ions attached to an insoluble matrix. It describes the principle of reversible ion exchange between oppositely charged molecules and the matrix. The document outlines the types of ion exchange resins used, including polystyrene and cellulose. It also discusses cation and anion exchangers, preparation of ion exchangers, factors affecting separation, and applications such as water softening and analyzing nucleic acids.
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
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
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
Theoretical background
Cont’d
Ion exchangers
There are three classes of ion exchangers , these include
Resins
Gels
Inorganic exchangers
Selectivity for ion exchange
In general , ion exchangers favour the binding of ions of
Higher charge
Decreased hydrated radius
Increased polarizability
Ion exchange resins are used for the separation of small molecules.
Ion exchange gels are used for the separation of large molecules like protiens ,nucleic acids.
Separations involving harsh chemical conditions(high temperature , high radiation levels, strongly basic solutions or powerful oxidizing agents) employ inorganic ion exchangers
Advantages
Detectability: useful for the detection of many in-organic salts and organic ions with poor uv absorptivity like alkyl amines or sulfonates.
Preparative separations: usually preferred because of the availability of volatile buffers . volatile buffers makes the removal of mobile phase easier.
Useful to resolve very complex samples, i.e in the case of multi step separation
Useful for separation of mixtures of biological origin, in organic salts and some organo- metallics
Applications
Conversion from one salt to other e.g we can prepare tetra propyl ammonium hydroxide from a tetra propyl salt of some other anion.
household (laundry detergents and water filters) to produce soft water
Ion exchange is used to prepare de-ionized water
separate and purify metals
Dealkalization
analysis and purification of immunoglobulins
Separation of inorganic ions
It includes basic knowledge about affinity chromatography along with its procedure and application. and brief description of various type of affinity chromatography.
This is a type of chromatography in which similar charged ions are separated by using ion exchange resin, that exchanges ions according to their relative affinities.
Ion exchange chromatography works under the principle of reversible adsorption and this method involves the separation of ions by using different types of exchange resins based on the ions to be separated.
Ion exchange is a reversible chemical reaction wherein an ion from solution is exchanged for a similarly charged ion attached to an immobile solid particle.
Download and play it my friends it contain VIDEO
The technique of ion exchange chromatography is based upon the interaction between charged solute molecules and oppositely charged moieties covalently linked to chromatographic matrix.
The reasons for its widespread success is its applicability, high resolving power, high capacity and simplicity of the technique.
Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most experiments are performed by following : Video For Understanding Play It
Slide share on Ion-Exchange chromatography
It contains-
Introduction of ion exchange,
principle of ion exchange(cat-ion exchanger and an-ion exchanger),
mechanism of ion exchange,
types of resins,
instrumentation of IEC,
its properties,
factors affecting, and
its applications.
Uploaded by:-
Affan Ahmad
B.pharm final year
Sai Meer College of Pharmacy
Chhibramau Kannauj U.P. India
INSTRUMENTAL METHODS OF ANALYSIS, B.PHARM 7TH SEM. AND FOR BSC,MSC CHEMISTRY. This is Geeta prasad kashyap (Asst. Professor), SVITS, Bilaspur (C.G) 495001
Industrial Production of L-Lysine by FermentationKuldeep Sharma
Lysine is an essential amino acid that is used in the biosynthesis of proteins. Lysine is required for the nutrition of animals and humans. Lysine is useful as medicament, chemical agent, food material (food industry) and feed additives (animal food). It's demand has been steadily increasing in recent years. Several thousand tones of L-lysine are annually produced worldwide, almost by microbial fermentation.
±For Education Purpose Only
Patanjali Ayurved Limited is an Indian FMCG company. Manufacturing units and headquarters are located in the industrial area of Haridwar while the registered office is located at Delhi. The company manufactures mineral and herbal products.
±For Education Purpose Only
DNA Replication stress is any hindrance to progression or movement of the replication fork. Recently, its been related to genomic instability and cancer.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
(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.
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.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
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.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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.
3. Introduction
• “Ion exchange chromatography may be defined as the reversible
exchange of ions in the solution with ions electrostatically bound to
some sort of insoluble matrix or a stationary phase.”
• This technique is extremely useful in the separation of charge
compounds like proteins differing by only one charged amino acid.
• In Ion exchange chromatography technique one can choose
whether to bind the substance of interest and allow the
contamination to pass through the column and vice versa.
• This technique has been developing since 19th
century which was
firstly used for purifying the drinking water.
• Ion exchange chromatography is a distinct principle of
chromatography performed in the column.
5. Principle
• Ion exchange chromatography relies on the attraction between
oppositely charged stationary phase, known as an ion exchanger, and
analyte.
• The ion exchanger consists of an inert support medium coupled
covalently to positive (anion exchanger) or negative (cation exchanger)
functional groups.
• To these covalently bound functional groups the oppositely charged
ions are bounded (mobile counter ion), which will be exchanged with
like charge ions in the sample having charge magnitude more than the
ions bounded to the matrix.
• Thus if anion exchange chromatography is performed, negatively
charged sample components will interact more with the stationary
phase and will be exchanged for like charged ions already bounded to
the matrix.
6. Working
• Consider a column having E
-
Y+
cation exchanger in
which E
-
is negative charged exchanger and Y+
is the
mobile counter ion.
• Let X+
be the cation in the sample having charge greater
than Y+
.
• The X+
ion can exchange sites with the counter ion Y+
with satisfying the following relationship;
E
-
Y+
X+
E
-
X+
Y+
• The remaining neutral and negatively charged particles
8. Working
• Bounded interest of ion (X+
) can now be eluted by either
of the two ways;
1. By adding a component M+
having magnitude of charge
more than that of X+
so that M+
will replace X+
and X+
will
be eluting out.
2. By changing pH of the solvent (mobile phase so that X+
have no charge and is then unbounded from the matrix
and can be eluted out.
9. Regeneration
• Cation exchange resins are regenerated
by treatment with acid, then washing with
water.
• Anion exchange resins are regenrated by
treatment with NaOH, then washing with
water.
10. Factors Affecting Chromatographic
Separation
1. Ion Exchange Resin:
The swelling factor and cross linking is important for the effective
separation.
The cross linking should be controlled as its affects the exchanger’s
capacity.
Swelling helps in proper exposure of charged functional groups for
exchange of ions.
2. Sample:
The concentration and charge of ions
3. Buffer:
The pH of the buffer should impart the same charge to the sample ions as
present in the Column.
Anionic Exchange Chromatography should be carried out with cationic
buffers and vice versa because buffer ion will indulge in ion exchange,
which will be of no use.
11. Ion Exchangers
An ion exchanger consists of two basic components;
1.Ion exchange resin
2.Exchange Medium (Cation exchanger and Anion exchanger)
- Charged functional group
- Mobile counter ion
12. Ion Exchange Resin
o Two main groups of materials are used to prepare ion exchange resins;
• Polystyrene
• Cellulose
o Resin made from both of these materials differ in their flow properties, ion
accessibility, and chemical and mechanical stability.
o Selection of one or the other type of resin is done on the basis o
compounds being separated
13. 1. Polystyrene
• Polystyrene resins are prepared by polymerisation reaction of styrene and
divinylbenzene.
• Higher concentration of divinylbenzene produces higer cross linkages.
• Polystrene resin are very useful for separating small molecular weight
compounds, however, unsatisfactory for the separation of macromolecules
14. 2. Cellulose
• Cellulose is a high molecular weight compound which
can be readily obtained in a high pure state.
• Cellulose has much greater permeability to
macromolecules.
15. Exchange Medium
• The choice of ion exchangers depends upon the stability,
molecular weight, and ionic strength of the sample
components.
• The volume of exchanger used for separation is usually 2.5
fold greater than to exchange with the ion in the sample.
• The ion exchanger are packed in column having suitable
buffer.
• The ion exchangers are of two types;
1. Anion Exchangers
2. Cation Exchangers
16. 1. Anion Exchangers
• The anion exchangers have positively charged
exchanger with negatively charged mobile counter ion
available for exchange.
• If the basic functional groups are introduced, the resin
becomes anion exchanger.
• Tertiary amines Strong anion exchangers
Secondary amines Weak anion exchangers
Mobile counter ion
Positively charged exchangers
Ion exchanger resin
17. Examples: Anion Exchangers
Source: Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of
biochemistry and molecular biology. Cambridge University Press.
18. 2. Cation Exchangers
• The cation exchangers have negatively charged
exchanger with positively charged mobile counter ion
available for exchange.
• If acidic functional group are introduced, then the resin
becomes cation exchangers.
• Sulphonic acid Strong cation exchangers
Carboxylic acid Weak cation exchangers
Negatively charged exchangers
Mobile counter ion
Ion exchanger resin
19. Examples: Cation Exchangers
Source: Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of
biochemistry and molecular biology. Cambridge University Press.
20. Preparation of Ion exchangers
• Preparation of the exchange medium is essential for satisfactory
performance of ion exchange chromatography.
• Apart from removing impurities, there are three major steps that are
absolutely important in ion exchanger preparation.
1. Swelling: For anion Exchangers it is done by treating it first with an
acid (0.5 N HCl) and then with base (0.5 N NaOH). Reverse is true
for preparation of cation exchangers.
2. Removal of fine particles: Large number of such particle can
result in decreased flow rate and improper resolution. For this the
exchangers are repeatedly suspended in large volume of water.
3. Addition of counter ions: Accomplished by washing the
exchanger with suitable reagent depending upon which counter ion
to be introduced.
For e.g., NaOH (Na+
), HCl (H+
), etc,.
22. Applications
• Softening of hard water
• Demineralization of water
• To analyze base composition of nucleic acid
• To concentrate the metal ions in the sample
• To measure the additives in food and drug sample
• To separate protein mixtures