Mr. Darshan N U is studying for his M Pharmacy first semester in the Department of Pharmaceutical Chemistry. The document provides an introduction to paper chromatography, covering its history, principles, requirements, factors affecting it, applications, and advantages over other methods. Paper chromatography is defined as a technique where unknown substances are analyzed mainly by the flow of solvents on filter paper. It has been used to separate mixtures like amino acids, food colors, and biological components.
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.
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.
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.
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
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
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
In this slide contains principle of IR spectroscopy and sampling techniques.
Presented by: R.Banuteja (Department of pharmaceutical analysis).
RIPER, anantpur.
Paper chromatography is an analytical method used to separate coloured chemicals or substances.It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (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.
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
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
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
In this slide contains principle of IR spectroscopy and sampling techniques.
Presented by: R.Banuteja (Department of pharmaceutical analysis).
RIPER, anantpur.
Paper chromatography is an analytical method used to separate coloured chemicals or substances.It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).
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 : A seperation techniqueSHIVANEE VYAS
Chromatography is a method of seperating mixture of components into individual components through equlibrium distribution between two phases.
Each chromatographic method essentially consists of 2 phases a staionary phase and a mobile phase.
Stationary phase : solid or liquid
Mobile phase : liquid or gas
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
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.
How to Make a Field invisible in Odoo 17Celine George
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.
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
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
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.
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.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
Cambridge International AS A Level Biology Coursebook - EBook (MaryFosbery J...
paper chromatography
1. Mr. Darshan N U
M Pharmacy first semester
Dept. of Pharmaceutical Chemistry
1
2. INTRODUCTION
Paper Chromatography (PC) was introduced by Schonbein (1865) under the name
capillary analysis.
PC became popular only when a team of Gorden, Martin and Syngle in England in 1944
done a outstanding work.
Paper chromatography is defined as the technique in which the analysis of unknown
substances is carried out mainly by the flow of solvents on specially designed filter paper.
2
3. PRINCIPLE OF SEPARATION
This technique is a type of partition chromatography in which substance are
distributed between two liquids , i.e., one is the stationary liquid ( usually water)
which is held in the fibers of the paper & called the stationary phase; the other is the
moving liquid or developing solvent and called the moving phase. The components
of the mixture to be separated migrate at different rates and appear as spots at
different points of paper.
3
5. Rf value
Rf (Retardation factor) is the fraction of an analyte in the mobile phase of a chromatographic system.
Rf = Distance travelled by solute / Distance travelled by solvent front
The Rf value ranges from 0 to l . But the ideal values are from 0.3 to 0.8
R is a function of the partition coefficient .
5
6. Rx values
It is nothing but the ratio of distance travelled by the sample and the distance travelled by standard.
Rx value is always closer to l. In some cases, the solvent front runs off the end of filter paper, the moment of a
substance in such cases is expressed as Rx .
RM values
RM value is used in qualitative analysis to find out whether the compounds belong to a homologous series. If
they belong to a homologous series, the RM values are constant. According to Bate-smith , RM is defined as
follows
RM = log (1/Rf -1)
6
8. 1. STATIONARY PHASE AND PAPERS USED
Paper of chromatographic grade consists of -cellulose 98 to 99%, ß-cellulose 0.3 to 1% , pentosans 0.4 to
0.8%, ether soluble matter 0.015 to 0.02%, Ash- 0.01 to 0.07% . Whatman filter papers of different grade like
No. l , No.2, No.3, No.3MM, No.4. No. 17, No.20 etc are used. These papers differ in sizes, shapes, porosities
and thickness.
choice of filter paper depends upon thickness, flow rate, purity, technique, etc.
Modified papers- Acid or base Washed filter paper, glass fibre type paper.
Hydrophilic papers - Papers modified with methanol, formamide. glycol, glycerol etc.
Hydrophobic papers - Acetylation of OH leads to hydrophobic nature, hence can be used for reverse phase
chromatography. silicone pre treatment and organic non-polar polymer can also can be impregnated to give
reverse phase chromatographic mode.
8
9. Preparation of paper
• Cut the paper into desired shape and size
depending upon work to be carried out.
• The most common shape of the filter paper
is rectangular, although square paper can
also be used.
• 15 – 30 cm in length and 5 to several cm in
width.
• The starting line is marked on the paper with
an ordinary pencil 2cm from the bottom
edge.
• On the staring line marks are made 2cm
apart from each other.
9
10. 2. APPLICATION OF SAMPLE
The sample to be applied is dissolved in the suitable solvent and applied using capillary tube or using
micropipette. Very low concentration is used to avoid larger zone.
10
11. 3. Mobile Phase
Pure solvents, buffer solutions, or mixture of solvents are used.
Some of the examples of
Hydrophilic mobile phases:
Isopropanol : Ammonia : Water -9 : 2 : 1
n-Butanol : glacial acetic acid : water - 4:1:5
Methanol : water - 7 : 3
t-Butanol : water : Formic acid - 40:30:30
11
12. 4. Development technique
Since paper is flexible when compared to glass plate used in TLC. Several types of development are possible
which increases the ease and efficiency of operation . They are
Ascending development: The solvent flows against gravity. The spots are kept at the bottom portion of paper
and kept in a chamber with mobile phase solvent at the bottom.
12
13. • Descending development: This is carried out in a special chamber where the solvent holder is at the top.
The spot is kept at the top and the solvent flows down the paper. The advantage is that the flow of solvent is
assisted by gravity and hence the development is faster.
13
14. Ascending-Descending development:
This is a combination of ascending and descending type. Only the length of separation is increased by
using a combination of techniques. In this technique, the upper part of the ascending chromatography can
be folded over a glass rod allowing the ascending development to change over into the descending after
crossing the glass rod
14
15. Circular/ radial development :
In this technique a circular filter paper is employed. Then the various material to be analysed are placed at the
centre. After drying the spot the paper is fixed horizontally on the petri-dish possessing the solvent so that the
wick of the paper dips into the solvent. Cover the paper by means of petri-dish cover. The solvent rises through
the wick. When solvent front has moved through a sufficient large distance, the components get separated in
the form of concentric circular zones.
15
16. • Two dimensional development:
This technique is similar to 2 Dimensional TLC. The paper is developed in one direction and after
development, the paper is developed in the second direction allowing more compounds or complex mixtures
to be separated into individual spots . In the second direction, either the same solvent system or different
solvent system can be used for development
16
17. Drying the chromatogram
After the solvent has moved a certain distance for a certain time the
chromatogram is taken out from the tank .
They are dried by cold or hot air depending on volatility of solvents.
A simple hair dryer is a convenient device to dry chromatograms.
17
18. 5. DETECTING OR VISUALISING AGENTS
• After the development of chromatogram the spots should be visualised. Detecting coloured spots can be
done visually. But for detecting colourless spots, any one of the following techniques can be used.
A. Non specific methods: Where the number of spots can be detected, but not the exact nature or type of
compound.
• Examples
• Iodine chamber method: where brown or amber spots are observed when the plates are kept in a tank with
few iodine crystals at the bottom.
• UV chamber for flourescent compounds: When compounds are viewed under UV chamber, at 254nm
(short ) or at 365nm (long ), flourescent compounds can be detected. Bright spots are seen against a
dark background.
18
19. B. Specific methods:
Specific spray reagents or detecting agents or visualising agents are used to find out the nature of
compounds or for identification purposes.
Example:
• Ferric chloride - for Phenolic compounds and tannins
• Ninhydrin in acetone - for amino acids.
• Dragendroff”s reagent - for alkaloids
• 3,5 - Dinitro benzoic acid - for cardiac glycoside
• 2,4 - Dinitrophenyl hydrazine - for aldehydes and ketones
19
20. o For radioactive materials, detection is by using autoradiography or Geiger muller counter.
o For antibiotics, the chromatogram is layed on nutrient agar inoculated with appropriate
strain and the zone of inhibition is compared.
20
21. Factors affecting Paper Chromatography
• Quality of paper used: Some papers are better absorbed and retained than the other , so the
different types and brands of papers are used.
• Length of the paper: Usually, separation will be better on long paper.
• Type of Solvent used: The solubility of each compound have different, so for different
effects of separation occur in the different solvent.
• The thickness of the paper: Paper thickness is difficult to travel the spot upwards.
• The concentration of the sample or spot: the concentrated spot cannot separate properly
and if the very diluted so it cannot be seen properly.
• Effect of Temperature: Temperature can affect the separation of analytes.
21
22. Precautions in Paper Chromatography
1. Minimum volume of the concentrated solution of the sample should be applied on the
chromatographic paper so as to avoid diffusion through the paper.
2. Complexing agents should not be unnecessarily used as they form a complex with the solute
having different properties resulting in multiple spots.
3. Solvent for elution should be properly selected.
4. Vapor – solvent equilibrium should be properly established.
5. Natural salts should be avoided because they disturb the cellulose water which results in
separation of water on to the paper.
22
23. Advantages over other methods
i. The equipment is very simple and is easily available.
ii. It has high efficiency of separation.
iii. Separation can be effected on macro, micro and semi-micro scale.
iv. Closely related homologues, isotopes, isomers and very labile and reactive
substances can be separated readily and satisfactorily.
23
25. Paper chromatography has been applied to the separation of many organic
and biochemical products.
For example, it has been utilized in the determination of indoles in urine and
in the study of barbiturates, antibiotics, carbamyl phosphates, hormones and
amino acids,
It also been used in the study of inorganic metal salts and complex ions
25
26. Separating mixture amino acids
Paper chromatography is especially useful in characterizing amino acids. The different
amino acids move at differing rates on the paper because of differences in their R groups.
The rate of movement of a biomolecule during paper chromatography is reported as its
relative mobility .
alanine 0.38
Methionine 0.55
arginine 0.20
leucine 0.73
26
27. FOODS
• Both natural and synthetic food colours are added to foods to improve their acceptability
and to make them more popular. Paper chromatography has been primarily used for
analysis of food colors in ice creams, sweets, drinks and beverages, jams and jellies. To
ensure that no non-permitted coloring agents are added to the foods, only edible colors are
permitted for use.
27
28. Pathology And Forensic Science
• For investigation of crimes, paper chromatography is useful in the field of
forensic science, as this process can be successfully carried out with even
very small quantities of material. Using this technique, samples from
crime scenes are collected to be analyzed and identified.
• Used in DNA and RNA fingerprinting. Moreover, to detect the presence
of alcohol or chemicals in blood, pathological laboratories use paper
chromatography.
28
29. Separation of Biological components
a. Several compounds of biological origin, such as carbohydrates, phosphorylated
sugars, lipids, steroids, bile acids, hormones, nucleosides and nucleotides, alkaloids
etc., have been studied by PC.
b. Pesticides, halogenated insecticides and organo-phosphorous insecticides can be
identified by PC.
29
30. • Nowadays, microfluidic paper-based devices (μPAD) technology has become widely used in
almost every field of science due to several advantages of paper such as low-cost, mechanical
flexibility and strength, easy to store. Doing paper chromatography on μPADs has been reported
by Murphy et al. for separation of ascorbic acid and dopamine .
30
31. • Separation and Purification of Dolichol and Dolichyl Phosphate by
Anion-Exchange Paper Chromatography
Dolichyl phosphate
31
32. REFERENCES
• Text book of pharmaceutical analysis by Dr .S. Ravishankar
• Instrumental Method of Chemical Analysis by Gurdeep R. Chatwal
• Journal by Fereshte Mohamadi Gharaghani, Morteza Akhond, Bahram,
Hemmateenejad, A. Kupferberg, M. L. Tomassoni, and M. Mersel*
32