• 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
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
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.
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
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
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.
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
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
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 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.
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.
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 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.
(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.
2. Chromatography
• 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.
• The component of the mixture redistributes themselves
between two phases by a process which may be adsorption,
partition, ion exchange or size exclusion.
• The stationary phase can be solid or a liquid and the
mobile Phase can be liquid, gas or a supercritical fluid.
4. ExamplesofChromatography
Thin Layer Chromatography
(TLC) is a chromatography
technique used to separate non-
volatile mixtures. Thin layer
chromatography is performed on
a sheet of glass, plastic or
aluminium foil, which is coated
with the thin layer of adsorbent
material, usually silica gel,
aluminium oxide, or cellulose.
Paper Chromatography is an
analytical method that is used to
separate coloured chemicals or
substances, especially pigments. This
can also be used in ink experiments.
Column Chromatography in Chemistry
is a method use to purify individual
chemical compounds from a mixture of
compounds. It is often used for preparative
applications on scale from micrograms up
to kilograms.
5. ExamplesofChromatography
Ion Exchange Chromatography (Ion 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 molecules including large protein,
small nucleotide and amino acids. The solution to be injected is
called Sample and individually separated components are called
analytes.
Size-Exclusion Chromatography (SEC) is a
chromatographic method in which molecules in a 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.
6. Introduction
• The Term Chromatography (chroma = a colour; graphein = to
write) is the collective term for a set of laboratory techniques
for the separation of mixtures.
• Chromatography involves a sample (or sample extract) being
dissolved in a mobile phase (which may be a gas, a liquid or a
supercritical fluid).
• The mobile phase is then forced through an immobile,
immiscible stationary phase.
• The phases are chosen such that components of the sample
have differing solubilities in each phase.
• A component which is quite soluble in the stationary phase
will take longer to travel through it than a component which is
not very soluble in the stationary phase but very soluble in the
mobile phase.
7. • As a result of these differences in mobilities, sample
components will become separated from each other as they
travel through the stationary phase.
• Techniques such as H.P.L.C. (High Performance Liquid
Chromatography) and G.C. (Gas Chromatography) use
columns - narrow tubes packed with stationary phase, through
which the mobile phase is forced.
• The sample is transported through the column by continuous
addition of mobile phase. This process is called elution.
8. History
• The subject of Chromatography was introduced into scientific
world in a very modest way by M. Tswett in 1906.
• He employed a technique to separate various pigments such
as chlorophylls and xanthophylls by passing the solution of
these compounds into the glass column which was packed
with finely divided calcium carbonate.
• Almost after three decades, in 1935 Adams and Holmes
observed the Ion Exchange characteristics in crushed
phonograph. This observation opened the field for
preparation of Ion Exchanged resins.
• The concept of Gas-Liquid Chromatography was first
introduced by Martin and Synge in 1941.
• They were also responsible for the development in Liquid-
9. Liquid chromatography.
• In 1944, from Martin laboratory, the separation of amino acid
By paper chromatography was reported.
• In 1952, the importance of the chromatography was
observed when both Synge and Martin were awarded with
Nobel Prize.
• In 1959, a technique known as Gel Filtration chromatography
was observed which is used to separate low molecular
weight substances from high molecular substances.
• In 1960, further improvement in liquid chromatography led
to the development of High Performance Liquid
Chromatography.
• The following decade of 1970’s saw an improvement in the
field of adsorption chromatography in the form of Affinity
chromatography which was mainly based on biological
interactions.
10. • A new field was originated which was supercritical fluid
chromatography.
• Supercritical fluid chromatography is a hybrid of gas and
liquid chromatography and combine advantageous feature of
the both gas and liquid chromatography.
• It will not be wrong to say that the entire twentieth century
canbe named as the century of chromatography.
11. Classification of Chromatography
On the basis of interaction of solute
to the stationaryphase
On the basis of chromatographic bed
shape
On the basis of physical state
of mobile phase
Adsorption
Chromatography
Ion Exchange
Chromatography
Two
Dimensional
Three
Dimensional
Super Critical
Fluid
Chromatography
Partition
Chromatography
Size Exclusion
Chromatography
Column
Chromatography
Gas
Chromatography
Thin Layer
Chromatography
Paper
Chromatography
Liquid
Chromatography
12. On the basis of interactionof Solute to the
stationary phase
➢ Adsorption Chromatography
➢ Partition Chromatography
➢ Ion Exchange Chromatography
➢ Size Exclusion Chromatography
13. AdsorptionChromatography
• Definition:
Adsorption chromatography is probably one of the oldest
types of chromatography around. It utilizes a mobile liquid or
gaseous phase that is adsorbed onto the surface of a
stationary solid phase. The equilibration between the mobile
and stationary phase accounts for the separation of different
solutes.
14. Principle:
• Principle of Adsorption Chromatography involves competition
of components of sample mixture for active site on adsorbent.
These active sites are formed in molecule due to Cracks, Edges
Separation occurs because of the fact that an equilibrium is
established between molecules adsorbed on stationary phase
and those which are flowing freely in mobile phase.
• The more the affinity of the molecule of particular component,
Less will be its movement.
16. PartitionChromatography
Definition:
This form of chromatography is based on a thin film formed on
the surface of a solid support by a liquid stationary phase.
Solute equilibrates between the mobile phase and the
stationary liquid.
17. Principle:
Separation of components of a sample mixture occurs because
of partition. Stationary phase is coated with a liquid which is
immiscible in mobile phase.
Partition of component of sample between sample and liquid/
gas stationary phase retard some components of sample more
as compared to others. This gives basis for separation.
The stationary phase immobilizes the liquid surface layer, which
becomes stationary phase. Mobile phase passes over the coated
adsorbent and depending upon relative solubility in the coated
liquid, separation occurs. The component of sample mixture
appears separated because of differences in their partition
coefficient.
19. IonExchangeChromatography
Definition:
Ion Exchange Chromatography (Ion 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 molecules including large protein,
small nucleotide and amino acids. The solution to be injected
is called Sample and individually separated components are
called analytes. It is often used in protein purification, water
analysis, and quality control.
20.
21. Principle:
Ion Exchange Chromatography is based on the relative retention
of the ions during their progress through an ion exchange
column which has functional group of opposite charge attached
to its surface. The stronger the charge on the ion, the greater is
the retention time in the column.
Ion chromatography is used to separate organic or inorganic
charged substances. The stationary phases used are based on
typical ion exchange resins.
22. Types:
Ion Exchange
Chromatography
Cation Exchange
Chromatography
Solute cations are attached to the
negatively charged sites covalently
bond to the stationary phase
Solute anions are attached to the
positively charged sites covalently
bond to the stationary phase
Anion Exchange
Chromatography
23. SizeExclusionChromatography
Definition:
Size-Exclusion Chromatography (SEC) is a chromatographic
method in which molecules in a 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.
25. Principle:
• A mixture of molecules dissolved in liquid (the mobile phase) is
applied to a chromatography column which contains a solid support
in the form of microscopic spheres, or “beads” (the stationary phase).
• The mass of beads within the column is often referred to as the
column bed.
• The beads act as “traps” or “sieves” and function to filter small
molecules which become temporarily trapped within the pores.
• Larger molecules are “excluded” from the beads.
• Large sample molecules cannot or can only partially penetrate the
pores, whereas smaller molecules can access most or all pores.
• Thus, large molecules elute first, smaller molecules elute later, while
molecules that can access all the pores elute last from the column.
• Particles of different sizes will elute (filter) through a stationary
phase at different rates.
27. ThinLayerChromatography
• Definition:
Thin-layer chromatography (TLC) is a chromatographic
technique that is useful for separating organic compounds.
Because of the simplicity and rapidity of TLC, it is often used to
monitor the progress of organic reactions and to check the
purity of products.
28. • Principle:
• Similar to other chromatographic methods TLC is also based
on the principle of separation. The separation depends on the
relative affinity of compounds towards stationary and mobile
phase. The compounds under the influence of mobile phase
(driven by capillary action) travel over the surface of
stationary phase. During this movement the compounds with
higher affinity to stationary phase travel slowly while the
others travel faster. Thus separation of components in the
mixture is achieved.
• Once separation occurs individual components are visualized
as spots at respective level of travel on the plate. Their nature
or character is identified by means of suitable detection
techniques.
29. PaperChromatography
• Definition:
• Paper chromatography is an analytical method that is used to
separate coloured chemicals or substances, especially
pigments. This can also be used in secondary or primary
colours in ink experiments. This method has been largely
replaced by thin layer chromatography, but is still a powerful
teaching tool.
• Double-way paper chromatography, also called two-
dimensional chromatography, involves using two solvents and
rotating the paper 90° in between. This is useful for separating
complex mixtures of compounds having similar polarity, for
example, amino acids. If a filter paper is used, it should be of a
high quality paper. The mobile phase is developing solutions
that can travel up to the stationary phase carrying the sample
along with it.
30.
31. • Principle:
• The principle involved is partition chromatography where in the
substances are distributed or partitioned between to liquid phases.
One phase is the water which is held in pores of filter paper used
and other phase is that of mobile phase which moves over the paper.
The compounds in the mixture get separated due to differences in
their affinity towards water (in stationary phase) and mobile phase
solvents during the movement of mobile phase under the capillary
action of pores in the paper.
• The principle can also be adsorption chromatography between solid
and liquid phases, where in the stationary phase is the solid surface of
paper and the liquid phase is of mobile phase. But most of the
applications of paper chromatography work on the principle of
partition chromatography i.e. partitioned between two liquid
phases.
32. On thebaseof physical state ofmobile
phase
➢ Liquid Chromatography
➢ Gas Chromatography
➢ Super Critical Fluid Chromatography
33. LiquidChromatography
• Liquid chromatography is a technique used to separate a
sample into its individual parts. This separation occurs based
on the interactions of the sample with the mobile and
stationary phases. Because there are many stationary/mobile
phase combinations that can be employed when separating a
mixture, there are several different types of chromatography
that are classified based on the physical states of those
phases. Liquid- solid column chromatography, the most
popular chromatography technique, features a liquid mobile
phase which slowly filters down through the solid stationary
phase, bringing the separated components with it.
35. Normal Phase Chromatography:
In normal phase chromatography, the stationary phase is
polar, and so the more polar solutes being separated will
adhere more to the stationary adsorbent phase. When the
solvent or gradient of solvents is passed through the column,
the less polar components will be eluted faster than the more
polar ones. The components can then be collected separately,
assuming adequate separation was achieved, in order of
increasing polarity.
36. Reverse Phase Chromatography:
In reverse phase chromatography, the polarities of the mobile
and stationary phases are opposite to what they were when
performing normal phase chromatography. Instead of
choosing a non-polar mobile phase solvent, a polar solvent
and non-polar stationary phase will be chosen.
37. Extraction Chromatography:
An important modification in terms of stationary phase is
introduced by loading the extractant used for solvent
extraction on a hydrophobized inert support and irrigating the
support with aqueous solvent. This is known as extraction
chromatography.
38. Affinity Chromatography:
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.
39. Principle:
The stationary phase is typically a gel matrix, often of agarose;
a linear sugar molecule derived from algae. Usually the
starting point is an undefined heterogeneous group of
molecules in solution, such as a cell lysate, growth medium or
blood serum. The molecule of interest will have a well known
and defined property, and can be exploited during the affinity
purification process. The process itself can be thought of as an
entrapment, with the target molecule becoming trapped on a
solid or stationary phase or medium. The other molecules in
the mobile phase will not become trapped as they do not
possess this property. The stationary phase can then be
removed from the mixture, washed and the target molecule
released from the entrapment in a process known as elution.
Possibly the most common use of affinity chromatography is
for the purification of recombinant proteins.
40. High Performance Liquid Chromatography
• High-performance liquid chromatography (HPLC; formerly
referred to as high-pressure liquid chromatography), is a
technique in analytic chemistry used to separate the
components in a mixture, to identify each component, and to
quantify each component.
• It relies on pumps to pass a pressurized liquid solvent
containing the sample mixture through a column filled with a
solid adsorbent material.
• Each component in the sample interacts slightly differently
with the adsorbent material, causing different flow rates for
the different components and leading to the separation of the
components as they flow out the column.
41.
42. GasChromatography
• Gas chromatography (GC), is a common type of
chromatography used in analytical chemistry for separating
and analyzing compounds that can be vaporized without
decomposition.
• Typical uses of GC include testing the purity of a particular
substance, or separating the different components of a
mixture (the relative amounts of such components can also be
determined).
• In some situations, GC may help in identifying a compound. In
preparative chromatography, GC can be used to prepare pure
compounds from a mixture.
• Two types of gas chromatography are encountered
a. Gas-Solid Chromatography (GSC)
b. Gas-Liquid Chromatography (GLC)
43.
44. SupercriticalFluidChromatography
• Supercritical Fluid Chromatography (SFC) is a form of
normal phase chromatography, that is used for the analysis
and purification of low to moderate molecular weight,
thermally labile molecules.
• It can also be used for the separation of chiral compounds.
• Principles are similar to those of high performance liquid
chromatography (HPLC), however SFC typically utilizes
carbon dioxide as the mobile phase; therefore the entire
chromatographic flow path must be pressurized.
• The supercritical phase represents a state in which liquid and
gas properties converge, supercritical fluid chromatography is
sometimes called "convergence chromatography."