Thin layer chromatography is a technique used to separate mixtures into their components. It involves a stationary phase, such as silica gel, and a mobile phase, such as a solvent. A sample is applied to the plate and the mobile phase moves up the plate, separating the components by their interaction with the stationary phase. The separated components are then detected and quantified. TLC is useful for analyzing mixtures and is simpler and less expensive than other chromatography methods.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
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
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
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.
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
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.
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.
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.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
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.
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
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.
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.
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.
Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography.
Chromatography- Principles and application of chromatographySanchit Dhankhar
Laboratory technique for the Separation of mixtures
Chroma -"color" and graphein - "to write”.
Colour bands - separation of individual compounds
Measured or analysed.
Analytical
Determine Chemical composition of a sample
Preparative
Used to purify sufficient quantities of a substance
Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
Eluent - Fluid entering column/ solvent that carries the analyte.
Eluate - Mobile phase leaving the column.
Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner wall of the column tubing.
Examples : Silica layer - Thin Layer Chromatography
Thin layer chromatography technique - easier, cheaper.
Handling is easy. Used as an identification test also purity test. It comprises of stationary and mobile phase. There are various types of chromatography technique. TLC consists of three steps - spotting, development, and visualization. The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". CThere are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel.
ABBREVIATED NEW DRUG APPLICATION (ANDA),INVESTICATION OF MEDICINAL PRODUCTS D...GOKULAKRISHNAN S
Introduction to ANDA
Regulations applied to ANDA process
Format and content of ANDA
ANDA approval process
Exclusivity
Hatch-Waxman amendments & 180 days exclusivity
Introduction to IMPD
Contents of IMPD
Introduction to IB
Contents of IB
Introduction
Brief description of the drug and the therapeutic class to which it belongs
Chemical and pharmaceutical information
Animal Pharmacolog
Animal Toxicology
Human/Clinical Pharmacology phase I
Therapeutic exploratory trials (Phase II)
Therapeutic confirmatory trials (Phase III)
Special Studies Geriatrics, pediatrics, pregnant or nursing women
Regulatory status in other countries
Prescribing information
Samples and Testing Protocol/s
Objectives of CGMP
Layout of buildings, services, equipments & maintenance
Production organization
material management
handling and transportation
inventory management &control
Production and planning control
Sales forcasting
Budget and cost control
Industrial and personnel relationship
Total quality management
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
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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.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
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.
Chapter 3 - Islamic Banking Products and Services.pptx
Thin layer Chromatography (TLC)
1. S.GOKULAKRISHNAN
M.Pharm (Pharmaceutics) – I Year,
Department of Pharmaceutics,
College of Pharmacy,
Mother Theresa Post Graduate and Research Institute of Health Sciences,
(A Government of Puducherry Institution)
Puducherry.
THIN LAYER CHROMATOGRAPHY
GOKULAKRISHNAN TLC 1
2. GOKULAKRISHNAN TLC 2
INTRODUCTION
TLC is a form of liquid chromatography consisting
of:
A mobile phase (developing solvent).
A stationary phase (a plate or strip coated with a form
of silica gel).
Analysis is performed on a flat surface under
atmospheric pressure and room temperature.
3. GOKULAKRISHNAN TLC 3
Michael Tswett is credited as being the father
of liquid chromatography. Tswett developed
his ideas in the early 1900’s.
5. GOKULAKRISHNAN TLC 5
Normal Phase
Normal phase is the terminology used when the
stationary phase is polar; for example silica gel, and the
mobile phase is an organic solvent or a mixture of
organic solvents which is less polar than the stationary
phase.
6. GOKULAKRISHNAN TLC 6
Reversed Phase
Reversed phase is the terminology used when the
stationary phase is a silica bonded with an organic
substrate such as a long chain aliphatic acid like C-18
and the mobile phase is a mixture of water and organic
solvent which is more polar than the stationary phase.
7. GOKULAKRISHNAN TLC 7
THIN LAYER
CHROMATOGRAPHY
Thin layer chromatography is used to separate mixtures of substances into
their components.
Similar to P.C, except that a thin layer of some inert material,
i.e. Aluminium oxide, mag.oxid. , sili.oxide is used instead of
paper.
A layer of any one of these oxide is made from a slurry of power in a
suitable inert solvent.
Slurry is spread over a flat surface ( glass, metal or rigid plastic ) &
dried.
8. GOKULAKRISHNAN TLC 8
PRINCIPLE
ADSORPTION
The component with more affinity towards the S.P travels slower
The component with lesser affinity towards the S.P travels faster
ADVANTAGES OF TLC
Simple mtd. & cost of the equipment is low
Rapid technique & not time consuming like C.C
Separation of µg of the substances can be achieved
Any type of compound can be analyzed
Corrosive spray reagents can be used without damaging the plate &
needs less solvent
9. GOKULAKRISHNAN TLC 9
STEPS IN TLC ANALYSIS
The following are the important components of a typical
TLC system:
Apparatus (developing chamber)
Stationary phase layer and mobile phase
Application of sample
Development of the plate
Detection of analyte
10. GOKULAKRISHNAN TLC 10
GENERAL PROCEDURE
Decide if you are going to do Normal or Reversed
phase chromatography.
Prepare a plate or select a plate with the proper sorbent
material.
Prepare the mobile phase
Mark the plate
Apply the sample
Develop the plate
Detect the analytes
11. GOKULAKRISHNAN TLC 11
PRACTICAL REQUIREMENTS
STATIONARY PHASE
Adsorbents mixed with water or other solvents→ slurry
Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF (Silica gel + binder + fluorescent indicator)
Alumina, Cellulose powder.
13. GOKULAKRISHNAN TLC 13
2. GLASS PLATE
Specific dimensions-
20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand hightemperatures
PREPARATION & ACTIVATION OF TLC PLATES
1.Pouring ( simplest methods )
2.Dipping (used for small plates )
3.Spraying ( difficult to get uniform layers )
4.Spreading ( best technique ) TLC Spreader
15. GOKULAKRISHNAN TLC 15
ACTIVATION OF PLATES
After spreading → Air dry (5 to 10 minutes)
Activated by heating at about 100˚C for 30 min. Then plates may
be kept in desiccators
4.APPLICATION OF SAMPLE
Using capillary tube or micropipette
Spotting area should not be immersed in the mobile phase
5.DEVELOPMENT TANK
Better to develop in glass beakers, jars to avoid more wastage of
solvents
When standard method is used, use twin trough tanks
Do chamber saturation to avoid “edge effect”
16. GOKULAKRISHNAN TLC 16
6. MOBILE PHASE
M.P used depends upon various factors
Nature of the substance
Nature of the S.P
Mode of Chromatography
Separation to be achieved, Analytical/Preparative
e.g. → pyridine, pet. ether, carbon tetrachloride, acetone,
water, glycerol, ethanol, benzene….
18. GOKULAKRISHNAN TLC 18
DEVELOPMENT TECHNIQUE
One dimensional development
Two dimensional development
Horizontal development
Multiple development
19. THIN LAYER CHROMATOGRAPHY
DRYING OF CHROMATOGRAM
After the solvent has moved a certain distance for certain
time the chromatogram is taken out from the tank & position
of the solvent front is marked with a pencil.
They are dried by cold or hot air depending on volatility of
solvents. A simple hair dryer is a convenient device to dry
chromatograms.
GOKULAKRISHNAN TLC 19
20. THIN LAYER CHROMATOGRAPHY
DETECTING / VISUALISING AGENTS
If the substance are colored they are visually detected easily.
But for colorless substance, Physical and chemical methods are used
to detect the spot.
(a)Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254 or at 365nm.
GOKULAKRISHNAN TLC 20
21. (b) Specific methods (Chemical methods) or Spraying
method
EXAMPLES
Ferric chloride-Phenolic comp. & tannins
Ninhydrin in acetone-Amino acids
Dragendroff’s reagent-Alkaloids
3,5 dinitro benzoic acid-Cardiac glycosides
GOKULAKRISHNAN TLC 21
22. Following detecting tech. can also be categorized as
1) DESTRUCTIVE TECHNIQUES
Specific spray reagents, samples destroyed before detection e.g. – ninhydrin
reagent
2) NON-DESTRUCTIVE TECHNIQUES
For radio active materials - Geiger Muller counter
uv chamber, iodine chamber
QUANTITATIVE ESTIMATIONS
The method can be divided into two main groups
1.Direct techniques-
2.Indirect techniques-
GOKULAKRISHNAN TLC 22
23. THIN LAYER CHROMATOGRAPHY
Direct Measurement Method
(i) Comparison of visible spots
A rough quantitative measurements
Component in a mixture can be carried out by comparing the intensity
and size of the spot with a standard substance.
(ii) Photo densitometry
The method is used with the chromatograms of colored
compound, instrument which measures quantitatively the density of the
spots.
GOKULAKRISHNAN TLC 23
24. THIN LAYER CHROMATOGRAPHY
(iii) Fluorimetry
The compound to be determined by fluorimetry must be fluorescent or
convertible into fluorescent derivatives.
(iv) Radiotracer Method
The compound containing radioactive element is labeled and treated with
locating reagent. Using Geiger Muller counter.
(v) Polarographic & Conductometric methods
Used to measure the amount of material in the spot
INDIRECT MEASUREMENT METHOD
In this technique, the spots are cut into portions and eluted with solvents.
This solution can be analyzed by any techniques of analysis like
spectrophotometry, electrochemical methods, etc.
GOKULAKRISHNAN TLC 24
25. Rf VALUE (Retardation Factor)
In Thin layer chromatography the results are represented by Rf
value which represent the movement or migration of solute relative to
the solvent front.
Rf value=
the ratio of the distance traveled by the substance
the distance traveled by the solvent
GOKULAKRISHNAN TLC 25
26. Factors affecting Rf VALUE
The temperature
The purity of the solvents used
The quality of the paper, adsorbents & impurities
present in the adsorbents
Chamber saturation techniques, method of drying & development
The distance travelled by the solute & solvent
Chemical reaction between the substances.
pH of the solution
GOKULAKRISHNAN TLC 26
27. APPLICATIONS
Purity of sample
Examination of reaction
Identification of compounds
Biochemical analysis
In pharmaceutical industry
Separation of multicomponent pharmaceutical formulations
In food and cosmetic industry
GOKULAKRISHNAN TLC 27
28. REFERENCE
S.RAVI SHANKAR, Text book of pharmaceutical analysis,
Rx publications, 2001.Pg no : 13-4 to 13-13.
WWW.Google.com
GOKULAKRISHNAN TLC 28