Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
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
Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
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
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
chromatography, principle, adsorbent of TLC, mobile phase of TLC, techniques in TLC, preparation of TLC plate, standards for TLC, advantages, disadvantages of TLC, Application of TLC.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
It is instrumental analytical technique. it is one of the major type of chromatography technique. its basic principle is adsorption. it has many applications in various fields
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
chromatography, principle, adsorbent of TLC, mobile phase of TLC, techniques in TLC, preparation of TLC plate, standards for TLC, advantages, disadvantages of TLC, Application of TLC.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
It is instrumental analytical technique. it is one of the major type of chromatography technique. its basic principle is adsorption. it has many applications in various fields
Chromatography is a powerful and advanced techniques for separating mixtures. Many types of chromatographic techniques are known, such as paper, thin layer, column chromatography, each with its own strength and weakness.
Chromatography system in general have a stationary phase and a mobile phase.
In column chromatography both phases are placed in a column container, i.e. all the chromatographic operations are carried out using column.
Column chromatography in chemistry is a method using for the Identification, separation and purify individual chemical compounds from mixtures of compounds in the large amount.
Column Chromatography is a separation technique in which components of mixture is separated by using a glass column packed with stationary phase and liquid mobile phase flowing continuously through the column.
It is suitable for the physical separation of gram quantities of material. A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase.
Usually a glass tube with a diameter from 1cm to 10cm and a height of 20 cm to 50cm with a tap at the bottom, is used for this purpose.
Depending upon the flow of solvent down, column chromatography may be separated into two categories.
Gravity column chromatography
If the solvent is allowed to flow down the column by Gravity, or downward process, it is known as gravity column chromatography .
Flash chromatography
If the solvent is forced down the column by positive air pressure , it is called flash chromatography.
The present slide gives us an insight into the different aspects of application of columnn chromatrography the principle behind it and at the same time its recent advances.
Cell signaling is part of any communication process that governs basic activities of cells and coordinates all cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity, as well as normal tissue homeostasis
Both Alzheimer’s disease and Parkinson’s disease are diseases of the brain. Both may cause forgetfulness. However, the similarities end there. In fact, researchers believe that even the memory disorder that results from Parkinson’s is distinct from the memory disorder that Alzheimer’s causes.
One main difference between the diseases is how they affect the body:
Alzheimer’s disease primarily affects memory. In advanced stages, the disease also impairs motor functions.
Parkinson’s disease primarily affects movement and coordination. In advanced stages, it may impair memory and other cognitive functions.
Rotavirus is a contagious virus that can cause gastroenteritis (inflammation of the stomach and intestines). Symptoms include severe watery diarrhea, often with vomiting, fever, and abdominal pain. Infants and young children are most likely to get rotavirus disease.
Glaucoma is an eye disease that is often associated with elevated intraocular pressure, in which damage to the eye (optic) nerve can lead to loss of vision and even blindness. Glaucoma is the leading cause of irreversible blindness in the world.
Human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) is a spectrum of conditions caused by infection with the human immunodeficiency virus (HIV).
Endocarditis usually occurs when germs from elsewhere in the body travel through the blood and attach to damaged areas of the heart. People with damaged or artificial heart valves or other heart conditions are most at risk.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
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Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
2. CONTENT
History and introduction to chromatography
Principle of column chromatography
Requirements for column chromatography
Factors affecting column chromatography
Applications
3. History & introduction to
chromatography
M. Tswett (1906) defined chromatography as the method
in which the components of a mixture are separated on an
adsorbent column in a flowing system.
The international union of pure & applied
chemistry(IUPAC) definition of chromatography:
“Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary, while the
other(mobile phase)moves in a definite direction”
4. Column chromatography
Introduction:
Column chromatography is a separation technique in
which components of mixture is separated by using a glass
column packed with stationary phase and the liquid
mobile phase flowing continuously through the column.
Principle:
Column adsorption chromatography
5. Principle
A solid stationary phase and a liquid mobile phase is used and the
principle of separation is adsorption.
When a mixture of component dissolved in the mobile phase, & is
introduced in to the column, the individual component move with
different rate depending upon the relative affinities.
The compound with less affinity toward the stationary phase
(adsorbent) moves faster and hence eluted out of the column first.
The one with greater affinity towards the stationary phase moves
slower down the column & hence it is eluted later. Thus the
compounds are separated.
The type of interaction between the stationary phase and the solute is
reversible in nature.
The rate of movement of a component(R) is given by:
R = rate of movement of component rate of movement of mobile
phase
6. Practical requirements
1. Column characteristics & selection
2. Stationary phases
3. Mobile phases
4. Preparation of column
5. Introduction of sample
6. Development of column
7. Detection of components
8. Recovery of components
7. COLUMN CHARACTERISTICS
The material of the column is mostly good quality neutral glass since it should not
be affected by solvent, acid or alkali.
The column dimension are important for effective column separations.
The length : diameter ratio ranges from 10:1 to 30:1, for more efficiency the
length : diameter ratio ranges from 100:1
The length of the column depends upon:
Number of compounds to be separated
Type of adsorbent used
Quantity of the sample
Affinity of compounds towards the adsorbent used
Better separation will be obtained with a long narrow column than short thick
column because number of theoretical plates will be more.
10. MOBILE PHASE OR ELUENTS OR SOLVENTS
USED
Mobile phase act as a solvent to introduce the mixture into the column as
developer to develop the zones for separation and as an eluent to remove the
pure component out to the column.
Strain(1942) has arranged the solvents in order of eluting power. A grouping
of solvents in order of polarity index is known as eluotrophic series.
Increasing eluting power: Light petroleum ether (petroleum ether, hexane,
heptane), Carbon tetra chloride, Cyclohexane, Carbon disulphide ,Benzene
Toluene, Chloroform, Ether Ethyl acetate, Acetone, Alcohols ,Water, Pyridine
Organic acids, Inorganic acids & bases.
11. Preparation (packing) of column:
The bottom portion of the column is packed with cotton wool or glass
wool, above which the column of adsorbent is packed.
After packing the column with the adsorbent, a similar paper dics is
kept on the top, so that the adsorbent layer is not disturbed, during
the introduction of the sample or mobile phase.
There are two types of packing techniques:
1. Dry packing technique
2. wet packing technique
12. Dry packing technique
In this technique, the required quantity of adsorbent is
packed in the column in dry form and the solvent allowed
to flow through the column till equilibrium is reached.
Disadvantage:
1. air bubbles are entrapped in the column
2. cracks appear in the adsorbent present in the column
3. hence the uniformity flow character & clear band of
the separated component may not be obtained.
13.
14.
15. Wet packing technique:
This is the ideal technique. The required amount of adsorbent is
mixed with the mobile phase in a beaker and poured in to the column.
The stationary phase settles uniformly in the column.
Advantages:
1. there is no entrapment of air bubbles.
2. there will not be any crack in the column of the adsorbent.
3. the band eluted from the column will be uniform and ideal for
separation.
16. Introduction of sample
The sample which is usually a mixture of component is
dissolved in minimum quantity of the mobile phase.
The entire sample is introduced into the column at once
and get adsorbed on the top portion of the column.
From this zone, the individual samples can be separated
by a process of elution.
17. Development technique (elution)
Elution:
It is a common method used in column chromatography.
In this method a small volume of mixture to be separated
is added on the top of column & mobile phase is allowed
to flow through the column.
There are two elution technique:
1. Isocratic elution technique
2. Gradient elution technique
18.
19. Isocratic & Gradient elution technique:
Isocratic elution technique :
In this elution technique, the same solvent composition or solvent of same
polarity is used through out the process of separation.
Ex. Chloroform only
Gradient elution technique :
In this elution technique, solvents of gradually increasing polarity or
increasing elution strength are used during the process of separation.
Initially low polar solvent is used followed by gradually increasing the polarity
of the solvent.
Ex. Initially benzene, then chloroform, then Ethyl acetate etc.
20. Detection of component
If the compounds separated in a column chromatography procedure are
colored, the progress of the separation can simply be monitored visually.
If the compounds to be isolated from column chromatography are colorless.
In this case, different techniques are used;
Using UV/visible detector.
Using fluoresence detector.
Refractive index detector.
By monitoring the fraction by thin layer chromatography.
21. Recovery of components:
The best technique to recover the components by process of elution.
The components are called as eluate, the solvent are called as eleunt.
Recovery is done by collecting different fraction of mobile phase of equal
volume like 10ml, 20ml, with fraction of 10 or 20 min.
Eluting the sample: Components a, b, and c separate as column progresses,
Fractions can be collected in test tubes, vials, beakers.
Analyze the fractions by thin-layer chromatography or by detectors.
22. Factor affecting column efficiency:
1. Dimension of the column: column efficiency has been improved by
increasing length/width ratio of the column.
2. Particle size of column packing: separation to be improved by decreasing
the particle size of the adsorbent.
3. Activity of the adsorbent
4. Temperature of the column: The speed of the elution increases at higher
temperatures.
5. Packing of the column: Dry or wet packing
6. Quality of solvents: solvents having low viscosities is giving better results.
23. Application of column chromatography
►Separation of mixture of compounds
►Purification process
►Isolation of active constituents
►Estimation of drugs in formulation
►Determination of primary and secondary glycosides in
digitalis leaf.
► separation of diastereomers & separation of
geometrical isomers.
24. Advantages & disadvantages:
Advantages of C.C:
Any type of mixture can be separated & Any quantity of mixture can be
separated
Used as qualitative as well as quantitative analysis
Wider choice of Mobile Phase
Automation is possible
Disadvantages of C.C:
Time consuming
more amount of Mobile Phase are required
To over come this, HPLC is developed with a pump.