This document provides information about high performance liquid chromatography (HPLC). It discusses the principle of HPLC, which is that mixture components travel through the column at different rates depending on their affinity for the stationary phase. The document also describes the types of HPLC based on the mode of chromatography or separation principle used. It provides details on the instrumentation of HPLC including the solvent delivery system, pumps, sample injection system, columns, detectors, and recorders/integrators. It concludes by listing some advantages of HPLC such as its ability to perform fast, efficient, and reproducible separations of complex mixtures.
Instrumentation of Thin Layer ChromatographyTanmoy Sarkar
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 system on which a material called the stationary phase is fixed.
Thin-layer chromatography is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose.
Instrumentation of Thin Layer ChromatographyTanmoy Sarkar
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 system on which a material called the stationary phase is fixed.
Thin-layer chromatography is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose.
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 types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
This presentation is all about chromatography, its types, different techniques of chromatography. Also it contains the principle, method, applications of column chromatography.
Instrumentation of HPLC, principle by kk sahuKAUSHAL SAHU
INTRODUCTION
Instrumentation of HPLC
TYPES OF HPLC
PARAMETERS
APPLICATION
CONCLUSION
REFERENCE
High-performance liquid chromatography ( HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds.
HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules.
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 types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
This presentation is all about chromatography, its types, different techniques of chromatography. Also it contains the principle, method, applications of column chromatography.
Instrumentation of HPLC, principle by kk sahuKAUSHAL SAHU
INTRODUCTION
Instrumentation of HPLC
TYPES OF HPLC
PARAMETERS
APPLICATION
CONCLUSION
REFERENCE
High-performance liquid chromatography ( HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds.
HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules.
powerpoint presentation on high performance liquid chromatography which include its definition, classification, principles of seperation, instrumentation and application.
HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High-pressure liquid chromatography).
High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.
It is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a mixture.
High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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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.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
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.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
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!
Embracing GenAI - A Strategic ImperativePeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
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Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
1. 4/30/2018
1
HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
High-performance liquid chromatography
formerly referred to as high-pressure liquid
chromatography), is a technique used to separate,
identify, and quantify components of a mixture.
It relies on pumps to pass a pressurized liquid
solvent containing the sample mixture through a
column filled with a solid adsorbent material
2. 4/30/2018
2
PRINCIPLE:
When a mixture is introduced into HPLC column,
components of mixture travel according to their relative
affinities towards the stationary phase. The component
which has more affinity towards the adsorbent, travels
slower. The component which has less affinity towards the
stationary phase travels faster.
TYPES OF HPLC:
A. Based on modes of chromatography
1. Normal phase mode
2. Reverse phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Size exclusion(or)Gel permeation chromatography
4. Affinity chromatography
C. Based on elution technique
1. Isocratic separation
2. Gradient separation
D. Based on purpose of operation
1. Analytical HPLC
2. Preparative HPLC
3. 4/30/2018
3
Instrumentation of High-Performance Liquid Chromatography
INSTRUMENTATON:
1. Solvent delivery system
2. Pumps (6k psi)
3. Sample injection system
4. Column (5-10µ particle size)
5. Detectors
6. Recorders and Integrators
4. 4/30/2018
4
Pumping systems:
– Requirement: high P (6kpsi), pulse-free, constant flow (0.1
10mL/min.),resistant to corrosion
SAMPLE INJECTION SYSTEM:
Several devices are available either for manual or auto
injection of the sample.
Different devices are: 1.Septum injectors
2. Stop flow injectors
3. Rheodyne injectors (loop valve type)
Rheodyne injector is the most popular injector. This has a
fixed volume loop like 20 μl or 50 μl or more.
5. 4/30/2018
5
COLUMNS:
– Stainless steel tubing for high pressure
– Heavy-wall glass or PEEK tubing for low P (< 600 psi)
– Analytical column: straight, L(5 ~ 25 cm), d (3 ~ 5 mm
- Micro column: L (3 ~ 7.5 cm), d (1 ~ 5 mm), high speed
and minimum solvent consumption
– Guard column: removes particulate matter and
contamination protect analytical column
-Column packing: silica, alumina, polystyrene-di vinyl benzene
synthetic or an ion-exchange resin
– Porous particle: commonly used, d: 3 ~ 10 μm. Narrow size
distribution, porous micro particle coated with thin organic
films
6. 4/30/2018
6
DETECTORS:
Detectors used depends upon the property of the compounds
to be separated. Different detectors available are:
1. Refractive index detectors
2. U.V detectors
3. Fluorescence detectors
4. Electrochemical detectors
5. IR detectors
1. Refractive index detectors:
• Nearly universal but poor detection limit
• Passes visible light through 2 compartments, sample &reference.
• When the solvent composition are the same the light passed through the compartments
the light beam that passes through is recorded as zero.
• When a solute is in the sample compartment, refractive index changes will shift the
light beam from the detector.
• Limit of detection (LOD) 10 ng of solute
7. 4/30/2018
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2. U.V detectors:
• Based on electronic transitions within
molecules.
• Most common type of detector for LC
• Fixed wavelength, Hg lamp 254 nm (π
=> π*)
•Tunable wavelength, selectable for
specific wavelengths, monochromators
or filters. Still limited to single
wavelengths.
• - 1 pg LOD
•Solvent limitations with UV-vis abs.
Detectors
•Z-shape, flow-through cell (V, 1 ~ 10
μL and b, 2 ~ 10 mm)
• Spectrophotometer: more versatile
3. Fluorescence detectors:
• Review - based on emission of excited state molecules.
• Detector 900 from excitation axis.
• LOD 10 fg
• Hg or Xe lamp
• Fluorometer and spectrofluorometer
• Fluorescing species or fluorescent derivatives
8. 4/30/2018
8
4.Electrochemical detectors:
•Based on amperometric response of
analyte to electrode usually held at
constant potential.
•If the analyte is electro active, can be
highly sensitive since response is
based on a surface phenomenon rather
than a solution bulk property (e.g. UV-
vis absorbance)
•simplicity, convenience and wide-
spreading application
• Thin-layer flow cell of Teflon :
50μm thick, 1 ~ 5 μL volume
• Indictor E: Pt, Au, C
• Multi-electrode: simultaneous
detection or sample purity indication.
5. IR detectors:
• filter instrument or FTIR
• Similar cell (V, 1.5 ~ 10 μL and b, 0.2 ~ 1.0mm)
• Limit: no suitable solvent, special optics
• FT-IR allows for spectrum records of flowing systems analogous to the diode array
system.
• Water/alcohols can be major interferences to solute detection
• LOD 100 ng
9. 4/30/2018
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RECORDERS AND INTEGRATORS:
Recorders are used to record the responses obtained from detectors after
amplification. They record the base line and all the peaks obtained, with respect to time.
Retention time for all the peaks can be found out from such recordings
Integrators are improved version of recorders with some data processing capabilities.
They can record the individual peaks with retention time, height and width of peaks, peak area,
percentage of area, etc. Integrators provide more information on peaks than recorders. Now a
days computers and printers are used for recording and processing the obtained data and for
controlling several operations.
PARAMETERS USED IN HPLC:
1.Retention time
2.Retention volume
3.Seperation factor
4. Resolution
10. 4/30/2018
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1.Retention time:
Retention time is the difference in time between the point of injection and
appearance of peak maxima. It is also defined as time required for 50% of a component to
be eluted from a column. It is measured in minutes and seconds.
2.Retention volume:
Retention volume is the volume of carrier gas required to elute 50% of the
component from the column. It is the product of retention time and flow rate.
Retention volume = Retention time × flow rate
3.Seperation factor:
Separation factor is the ratio of partition coefficient of the 2 components to be
separated.
S=Ka/Kb=(tb-to)/(ta-to)
Where to = Retention time of unretained substance
Ka, Kb = Partition coefficients of a, b
ta, tb = Retention time of substance a, b
If there is a more difference in partition coefficient between 2 compounds, the peaks
are far apart and the separation factor is more. If the partition coefficient of 2 compounds
are similar, then the peaks are closer and the separation factor is less.
4. Resolution:
Resolution is the measure of extent of separation of 2 components and the base
line separation achieved.
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6.ADVANTAGES OF HPLC:
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column separations are excellently
made.