HPLC, or high-performance liquid chromatography, is an analytical technique used to separate, identify, and quantify components in a mixture. It works by pumping a pressurized mobile liquid phase through a column containing a stationary phase, which causes the components in a sample to separate as they migrate through the column at different rates. Common components of an HPLC system include the pump, injector, column, detector, and recorder or computer system. HPLC has advantages of speed, efficiency, accuracy, and versatility in chemical analysis. It is used in various applications such as drug analysis, environmental analysis, and industrial quality control.
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
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
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
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
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
HPLC- Introduction, Theory, Instrumentation, Advantage, Applications
High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.
The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable glass columns was further developed to the HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, Solvent is forced through under high pressures of up to 400 atmospheres.
Principle
The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase.
Instrumentation
1.Solvent reservoir and degassing system
2. Pumping System (Screw- driven syringe pump, Reciprocating pump, Pneumatic or constant- pressure pump)
3. Sample injection system(Septum injectors, Stop flow septum- less injection, Micro- volume sampling valves)
4. Columns- (1. Guard columns 2.separating column)
5. Detectors( The commonly used detectors in HPLC are
Bulk property detectors- examples
1. Refractive-index detectors
2. Conductivity detectors
Solute property detectors- Examples
1. UV detectors
2. Fluorescence detectors
Multipurpose detectors- Example-
1. Perkin-Elmer 3D system (UV absorption+ fluorescence + conductometric detection altogethers)
Electrochemical Detectors- Examples
1.Amperometric, 2. Coulometric detectors)
6. Recorder( There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for an LC system which not only data acquisition but features, like peak-fitting baseline correction, automatic concentration calculation, molecular weight determination, etc.) Type of HPLC- Normal phase, Reverse Phase, ion exchange, size exclusion, Applications-Stability study- eg Atropin
Bioassays- HPLC is commonly used for the bioassay and analysis of peptide harmones and some antibiotics- cotrimoxazole, penicillins, sulphates and chloramphenicol
In cosmetic industries- used for analyzing the quality of various cosmetic products such as lipsticks, gels, creams etc
Isolation of Natural pharmaceutically Active Compounds– use in the isolation of different type of Alkaloids and Glycosides ( analysis of cinchona, liquorice, ergot extracts and digitalis.)
Control of microbiological processes- HPLC is used in analyse antibiotics (eg. Tetracyclines, chloramphenicol, strptomycin and penicillins )
Assay of cephalosporins
Advantage, Limitation
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It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
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The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
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Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
How to Create Map Views in the Odoo 17 ERPCeline George
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Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Ethnobotany and Ethnopharmacology:
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Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
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2. INDRODUCTION
High performance liquid chromatography or commonly
known as HPLC is an analytical technique used to separate,
identify or quantify each component in a mixture.
The mixture is separated using the basic principle of
column chromatography and then identified and quantified
by spectroscopy.
In the 1960s the column chromatography LC with its low-
pressure suitable glass columns was further developed to the
HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column
liquid chromatography. Instead of a solvent being allowed to
drip through a column under gravity, it is forced through
under high pressures of up to 400 atmospheres.
3.
4. PRINCIPLE OF HPLC
The purification takes place in a separation column
between a stationary and a mobile phase.
The stationary phase is a granular material with very small
porous particles in a separation column.
The mobile phase, on the other hand, is a solvent or
solvent mixture which is forced at high pressure through the
separation column.
Via a valve with a connected sample loop, i.e. a small
tube or a capillary made of stainless steel, the sample is
injected into the mobile phase flow from the pump to the
separation column using a syringe.
5. Subsequently, the individual components of the sample
migrate through the column at different rates because they
are retained to a varying degree by interactions with the
stationary phase.
After leaving the column, the individual substances are
detected by a suitable detector and passed on as a signal to
the HPLC software on the computer.
At the end of this operation/run, a chromatogram in the
HPLC software on the computer is obtained.
The chromatogram allows the identification and
quantification of the different substances.
7. INSTRUMENTATION OF HPLC
The Pump
The development of HPLC led to the development of the
pump system.
The pump is positioned in the most upper stream of
the liquid chromatography system and generates a flow of
eluent from the solvent reservoir into the system.
High-pressure generation is a “standard” requirement of
pumps besides which, it should also to be able to provide a
consistent pressure at any condition and a controllable and
reproducible flow rate.
Most pumps used in current LC systems generate the
flow by back-and-forth motion of a motor-driven piston
(reciprocating pumps). Because of this piston motion, it
produces “pulses”.
8. Injector
An injector is placed next to the pump.
The simplest method is to use a syringe, and the
sample is introduced to the flow of eluent.
The most widely used injection method is based on
sampling loops.
The use of the autosampler (auto-injector) system
is also widely used that allows repeated injections in a set
scheduled-timing.
9. Column
The separation is performed inside the column.
The recent columns are often prepared in a
stainless steel housing, instead of glass columns.
The packing material generally used is silica or
polymer gels compared to calcium carbonate.
The eluent used for LC varies from acidic to basic
solvents.
Most column housing is made of stainless steel
since stainless is tolerant towards a large variety of solvents.
10. Detector
Separation of analytes is performed inside the column,
whereas a detector is used to observe the obtained separation.
The composition of the eluent is consistent when no analyte is
present.
While the presence of analyte changes the composition of the
eluent.
What detector does is to measure these differences.
This difference is monitored as a form of an electronic signal.
There are different types of detectors available.
11. Recorder
The change in eluent detected by a detector is in
the form of an electronic signal, and thus it is still not visible
to our eyes.
In older days, the pen (paper)-chart recorder was
popularly used. Nowadays, a computer-based data processor
(integrator) is more common.
There are various types of data processors; from a
simple system consisting of the in-built printer and word
processor while those with software that are specifically
designed for an LC system which not only data acquisition but
features like peak-fitting, baseline correction, automatic
concentration calculation, molecular weight determination,
etc.
12. Degasser
The eluent used for LC analysis may contain gases
such as oxygen that are non-visible to our eyes.
When gas is present in the eluent, this is detected
as noise and causes an unstable baseline.
Degasser uses special polymer membrane tubing to
remove gases.
The numerous very small pores on the surface of
the polymer tube allow the air to go through while preventing
any liquid to go through the pore.
13. Column Heater
The LC separation is often largely influenced by the
column temperature.
In order to obtain repeatable results, it is important to
keep consistent temperature conditions.
Also for some analysis, such as sugar and organic acid,
better resolutions can be obtained at elevated temperatures
(50 to 80°C).
Thus columns are generally kept inside the column
oven (column heater).
14. TYPES OF HPLC
Normal phase:
Column packing is polar (e.g silica) and the mobile phase is
non-polar. It is used for water-sensitive compounds,
geometric isomers, cis-trans isomers, and chiral compounds.
Reverse phase:
The column packing is non-polar (e.g C18), the mobile phase
is water+ miscible solvent (e.g methanol). It can be used for
polar, non-polar, ionizable and ionic samples.
Ion exchange:
Column packing contains ionic groups and the mobile phase is
buffer. It is used to separate anions and cations.
Size exclusion:
Molecules diffuse into pores of a porous medium and are
separated according to their relative size to the pore size.
Large molecules elute first and smaller molecules elute later.
15. APPLICATION OF HPLC
The HPLC has developed into a universally applicable method
so that it finds its use in almost all areas of chemistry,
biochemistry, and pharmacy.
Analysis of drugs
Analysis of synthetic polymers
Analysis of pollutants in environmental analytics
Determination of drugs in biological matrices
Isolation of valuable products
Product purity and quality control of industrial products and
fine chemicals
Separation and purification of biopolymers such as enzymes
or nucleic acids
Water purification
Pre-concentration of trace components
Ligand-exchange chromatography
16. Speed
Efficiency
Accuracy
Versatile and extremely precise when it comes to
identifying and quantifying chemical components.
LIMITATION OF HPLC
Cost: Despite its advantages, HPLC can be costly, requiring
large quantities of expensive organics.
Complexity
HPLC does have low sensitivity for certain compounds, and
some cannot be detected as they are irreversibly adsorbed.
Volatile substances are better separated by gas
chromatography.
Ion-exchange chromatography of proteins
ADVANTAGE OF HPLC