Chromatography is an analytical technique used to separate mixtures by differential partitioning between a mobile and stationary phase. There are several types including partition chromatography using paper or thin layer, and adsorption chromatography using ion exchange or affinity. Gel filtration chromatography separates biomolecules by size as they permeate gel beads. Paper and thin layer chromatography separate non-polar substances using a mobile solvent phase and cellulose or silica gel stationary phase. Ion exchange chromatography separates biomolecules based on net charge using charged resins, while affinity chromatography uses a ligand to specifically and reversibly purify a target biomolecule. These techniques are widely used in biomedical research and clinical labs.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
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Spectroscopy and it's applications as well as it's types like Infrared spectroscopy and ultraviolet spectroscopy and principle of spectroscopy why we use spectroscopy.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
Spectroscopy techniques, it's principle, types and applications NizadSultana
Spectroscopy and it's applications as well as it's types like Infrared spectroscopy and ultraviolet spectroscopy and principle of spectroscopy why we use spectroscopy.
This is helpful for understanding What is Beer Lambert's Law? What is derivation of Beer Lambert's Law?...Helpful for Pharmacy and Chemistry students....
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 is helpful for understanding What is Beer Lambert's Law? What is derivation of Beer Lambert's Law?...Helpful for Pharmacy and Chemistry students....
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
Chromatography is a laboratory technique for the separation of a mixture. 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 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.
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.
The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
The first analytical use of chromatography was described by James and Martin in 1952, for the use of gas chromatography for the analysis of fatty acid mixtures.
A wide range of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties to separate materials.
It is a powerful separation tool that is used in all branches of science and is often the only means of separating components from complex mixtures.
hromatography 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 the mobile phase, and leave the system faster.
Three components thus form the basis of the chromatography technique.
1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3. Separated molecules
Types of Chromatography
Substances can be separated on the basis of a variety of methods and the presence of characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.
This leads to different types of chromatography techniques, each with their own instrumentation and working principle.
For instance, four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion.
Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography.
Applications of Chromatography
Pharmaceutical sector
To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element composition.
Detects the unknown compounds and purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications.
In forensic pathology and crime scene testing like analyzing blood and hair samples.
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 is a widely used analytical technique in chemistry and biochemistry. It is a method for separating and identifying the individual components of a mixture based on their physical and chemical properties. The word "chromatography" itself means "color writing," and it was initially developed for separating and analyzing colored compounds, but it is now applied to a broad range of substances.
Chromatography is a powerful tool in research, quality control, and various industries because it allows for the separation and identification of complex mixtures, helping scientists and analysts understand the composition of substances and solve various analytical problems.
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June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
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The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2. INTRODUCTION
Chromatography is one of the most useful analytical technique by
which closely related molecules in a mixture of biomolecules could be
separated ,isolated and purified. The separation processes is based on
differential distribution of substances between two immiscible phase
including a mobile phase and stationary phase ,one of which moves
relatively to the other.
Based on the type of interaction between the substances and the
stationary phase, chromatographic techniques can be classified into
three major groups
1.Partition Chromatography
2.Permeation or molecular exclusion chromatography
3.Absortion chromatography
3. Based on the type of Stationary phase used, the partition and adsorption
chromatography are further classified into
Partition chromatography- Paper chromatography and Thin Layer chromatography
Adsorption chromatography-Affinity chromatography(AC)
Ion-exchange chromatography
-Cation exchange chromatography
- Anion exchange chromatography
similarly ,based on the type of mobile phase used, the chromatography could be
classified into three types: 1. Liquid chromatography(LC)
2.Gas chromatography(GC)
3. Gas-Liquid chromatography(GLC)
Paper and thin layer chromatography
In paper chromatography ,the paper (made of cellulose) acts as stationary phase and the
solvent system acts as mobile phase. When the mixture of substances moves on a stationary
phase(paper) along with the mobile phase (the solvent flowing along the paper) , the
separation is brought about by continuous partition between the mobile phase and water held
in the paper, where paper together with water acts as an adsorbent which results in
retardation of substances at certain levels of flow on the paper. Thus the paper
chromatographic separation is achieved as a resultant of propelling (mobile) and retardation
(stationary) forces.
4. Similarly ,in thin layer chromatography silica gel coated plates are being used
instead of paper. Here again the separation is achieved by the result partition
between the mobile phase and an inert stationary phase (silica).This is being
commonly used in several laboratories to separate hydrophobic non-polar soluble
substances such as lipids.
In both these techniques, one should consider certain properties of solvents as
mobile phases which include
1. Solvent should be stable in air and when mixed with small quantities acid or
alkaline vapor.
2. Components of the mobile phase should be relatively non-volatile or their
volatilability should be similar to the closed apparatus.
3. The solvent should be removed rapidly and completely from the stationary
phase after the chromatogram has been completed.
4. The solvent should remain homogenous throughout the range of temperature
experienced in laboratory.
5. The solvent should not react with any of the substances to be separated or with
stationary phase.
Identification of compounds separated
An important characteristics used in both paper and thin layer chromatography for
identification of compounds of substances that are separated is identified after using certain
5. This value (Rf) can be calculated by measuring the distances travelled by the
substance(separate) and the solvent( mobile) from their point of origin.
Permeation of Molecular exclusion or Gel filtration chromatography
Gel filtration chromatography is one of the most commonly employed tools for separating
biomolecules on the basis of their molecular weight. Thence it is other wise called as
molecular exclusion or molecular sieve chromatography . This system involves a stationary
phase (usually a preswollen gel beads) and a mobiles phase(buffer system).
Principle: In this technique, the mixture of biomolecules dissolved in a suitable buffer is
allowed to flow by gravity down a column packed with beads of an inert highly hydrated
polymeric material that has previously been washed and equilibrated with the buffer.
During this process , biomolecules of different molecular size penetrate into the internal
pores of the beads to different degrees and thus travel down the column at different rates.
Very large biomolecules cannot enter the pores of the beads,hence excluded and remain in
the exclusion volume of the column. On the otherhand ,small biomolecules which can enter
the gel pores, move more slowly through the column , sinces they spend a proportation of
the their time in stationary phase. Biomolecules of intermediate size will be excluded from
6. the beads to a degree that depends on their size. The biomolecules are therefore
eluted in a order of decreasing molecular size and thus a separation of molecules is
achieved.
The column materials that are commonly used include:
1.Sephadex G-type(G-10,G-25,G-50,G-75,G-100,& G-200)
2.Sepharose CL-series(2B,4B,6B).
3. Sephacryl(S-100,S-300).
4.Bio-gel
5.Silica gel
Applications:
The gel filtration chromatography is routinely used in several bio-medical and
research laboratories
1. To fractionate proteins,carbohydrates,nucleic acids and enzymes.
2. to fractionate cell organelles,viruses etc.,
3. To remove co-factors, inhibitors etc., from enzymes
4. To desalt the biomolecules before lyophilization or concentration or further
purification.
5. To remove free and low moleculres weight labels such as 125I,FITC from the
solution of labelled proteins.
7. Adsorption chromatography:
Ion-exchange chromatography
This techniques involves separation of biomolecules depending on their net
charges. In gel filtration techniques, a mixture of biomolecules are separated
based on molecular size, but the different biomolecules having identical or similar
molecules weight or size cannot be effectively separated. Such possibilities are
significant because many unrelated biomolecules may be identical in their
molecules weight or size. To solve this kind of problems ,other characteristics of
biomolecules such as net charge iso-electric point or their specific binding ability
are being utilized to separate the unrelated biomolecules having identical or
similar molecular weight.
APPLICATIONS:
The ion-exchange chromatography in mostly used in certain bio-medical and
research laboratories to
1. Separated many blood product such as albumin,IgG,plasma b-endorphins.
2. Separate products of recombinant DNA technology.
3.Separate T3 and T4.
4. Separate renal proteinuria in urine.
8. Affinity Chromatography
Affinity chromatography is a type of adsorption chromatography in which the
biomolecules or specific type of cells to be purified is specifically and reversibly
adsorbed by a complementary binding substances (Ligand) which is immobilized on
a insoluble support (matrix). It occupies a unique place in separation technology
since it is the only techniques which enables purification of almost any biomolecules
on the basis of its biological function or individual chemical structure .In addition
purification of a biomolecules is often in the order of several thousand fold and
recovery of active materials are generally very high and being a single step
purification it is an immense time saver over the other multi-stage purification
procedures.
Applications:
1.To purify enzyme using substrate as ligands.
2. To purify hormones using cell menbrance receptors.
3.To purify specific antibodies using antigens.
4.To purify mRNA using complementary DNA.
5.To purify interferons, viral RNA,etc.