This document provides an overview of partition chromatography. It defines partition chromatography as a method of separation where components in a mixture distribute between two immiscible liquid phases due to differences in their partition coefficients. The document discusses the history of chromatography and describes partition chromatography techniques like paper chromatography and gas-liquid chromatography. It explains the basic principles, procedures, factors affecting separation, and applications of partition 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
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
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Partition Chromatography technique is defined as. the separation of components between two liquid phases viz original solvent and the film of solvent used in the column.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Partition Chromatography technique is defined as. the separation of components between two liquid phases viz original solvent and the film of solvent used in the column.
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
• Chromatography is a method of separation in which the components to be separated are distributed between two phases, one of these is called a stationary phase and the other is a mobile phase which moves on stationary phase in a definite direction
Chromatography is a bioanalytical technique used for separation of analytes into pure components. Biomolecules such as amino acids, proteins and carbohydrates can be purified by different chromatographic methods.
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.
The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment. It becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
2. SYNOPSIS
INTRODUCTION
TERMS RELATED TO CHROMATOGRAPHY
HISTORY OF CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY
PARTITION CHROMATOGRAPHY
TYPES
PRINCIPLE
PROCEDURE
APPLICATIONS
REFERENCES
3. Introduction
Chromatography is an analytical technique used for separation, identification, and analysis of
various components of a mixture. The sample components often vary in physical and/ or
chemical properties.
The compound that is separated during chromatography is called analyte.
Russian botanist - M. S. Tswett. He developed this useful technique in 1906 to separate plant
pigments under gravity using a calcium carbonate column. Tswett also coined the term
‘chromatography’, which comes from the words chroma (Greek) i.e. colour and graphein,
which means "to write". “Towrite with colors”
In fact, this technique can be used to distinguish between two
compounds that are quite similar in molecular mass or
charge; however, this requires an appropriate combination of
materials and operating conditions.
A wide range of soluble or volatile, organic or inorganic
compounds can be thus separated using chromatographic technique.
4. TERMS RELATED TO Chromatography
StationaryPhase - phasethat stays in placeinside the column
usually viscous liquid chemically bonded to the inside of acapillary
column
Mobilephase- solvent moving through the column and is either
liquid orgas.
Elution- processof passing liquid or gasthrough the column.
Eluent -fluid entering the column
Eluate-fluid leaving the column
5. History of Chromatography
• Chromatography was first employed in Russia by the Italian-born
scientist Mikhail Tswett in 1906.
• of Archer John Porter Martin and Richard Laurence Millington
Synge during the 1940s and 1950s, for which they won the 1952 Nobel
Prize in Chemistry.
• They established the principles and basic techniques of partition
chromatography, and their work encouraged the rapid development of
several chromatographic methods.
• Researchers found that the main principles of Tswett's chromatography
could be applied in many different ways.
7. PartitionChromatography
basedon athin film formed on the surfaceof a
solidsupport by aliquid stationary phase.
Solute equilibrates between the mobile phase&
the stationary liquid.
method of separation in which the components
present in the mixture get distributed more likely
into two liquid phasesbecauseof differences in partition coefficients during the
flow of mobile phasein the chromatographycolumn.
8. Partition Coefficient - the ratio of the concentrations of a solute in
two immiscible or slightly miscible liquids, or in two solids, when it is
in equilibrium acrossthe interface between them.
TYPESOFPARTITIONCHROMATOGRAPHY
Liquid –Liquid Chromatography
Gas- Liquid Chromatography
9. Liquid - LiquidChromatography
• employsliquid mobile andstationary phases
• usessmall particles with moleculesbonded to their surfaceto give a
thin filmthat hasliquid like properties
• PAPER CHROMATOGRAPHY
10. 1.PAPERADSORPTIONCHROMATOGRAPHY
Paperimpregnatedwith silicaor aluminaactsasadsorbent(stationary phase)and solvent
asmobilephase.
2. PAPER PARTITIONCHROMATOGRAPHY
Moisture / Water present in the pores of cellulose fibers present in filter paperactsas
stationary phase& another mobilephaseisused assolvent
Ingeneral,PaperChromatography=PaperPartitionChromatography
Typesof PaperChromatography
11. PaperPartitionChromatography
Instandardmethod of analysis,wherein the paperisutilized asa support with
one solvent asmobile phaseand the other is the stationaryphase
Themigration of substancesisdueto the partition coefficients
separationof similar substancesby repeated divisionsbetween two
immiscible liquids,sothat the substances,in effect, crossthe partition
betweenthe liquidsin oppositedirections;whereone of the liquidsis
boundasafilm onfilter paper.
• Ascending,
• Descending
• Circular
12.
13. Celluloselayers in filter paper contains moisture which acts as
stationary phase& organicsolvents/buffersareusedasmobile
phase
STATIONARY PHASEAND PAPERS USED
Whatman filter papers of different grades like No.1, No.2,
No.3, No.4, No.20, No.40, No.42 etc are used. In general this paper contains 98-
99% of α-cellulose, 0.3–1%β -cellulose
Principle of PaperPartitionChromatography
14. Pure solutions can be applied direct on the paper but solids are always dissolved in small
quantity of asuitablesolvent.
Biological tissues are treated with suitable solvents and their extracts obtained.
Proteins can be precipitated with alcohol and salts can be removed by treatment with
ion exchangeresin.
APPLICATIONOF SAMPLE
The sample to be applied is dissolved in the mobile phaseand applied asasmall spot on
the origin line,usingcapillarytube or micropipette.
verylow concentrationisusedto avoidlargerzone
Thespot isdried onthe filter paperandisplacedin developingchamber.
Preparation of thesolution
15.
16. • Glasstanks are preferredmost.
They are available in various
dimensional sizedepending upon
paper length and development
type.
• The chamberatmosphere
should be saturated with
solventvapor.
Chromatographic
Chamber
17. • Paper is flexible when compared to glass plate used in TLC, several types of
development are possible which increasesthe easeof operation.
• The paper is dipped in solvent in such a manner that the spots will not dip
completely into the solvent.
• The solvent will rise up and it is allowed to run 2/3rd of paper height for better
andefficient result.
Procedures
20. Temperature
Purity of the solventsused
Quality of the paper,adsorbents& impurities presentnthe
adsorbents
Chambersaturation techniques,method of drying & development
Distancetravelled bythe solute& solvent
Chemicalreaction between the substancesbeingpartitioned.
pH of thesolution
Factors affecting RfValue
21. Gas- LiquidChromatography
• mobilephaseisagasandthe stationary phaseisaliquid, usually on smallbeads
packedin along column
Pointstoremember:
Samplehasto beableto bevaporizedwithout
decomposition
Basedonboiling point/vapor pressure
• Mobilephase
o Inert carrier gaslike Helium or Nitrogen
• Stationaryphase
o Layerof liquidor polymeroninert solidsupport
o Insideaglassor metal tubing(COLUMN)
22. Procedures
• Compoundisinjectedwith syringeinto sampleinjector
• Compoundiscarriedbycarriergasandvaporized
• Vaporizedsampleinteractswith wallsof column
o Somesamplesinteract more someless
• Dueto interaction sampleseluteat differenttimes
o Retentiontimes
o Comparisonof retentionstimes iswhat isuseful
• Adetector monitors the outlet streamfrom the column
23.
24. GasPressure Regulator
• Helium - It hasan excellent thermal conductivity, low density,
inertness andit permits greater flow rates. It ishighly expensive
• Nitrogen - Itoffers reduced sensitivity and is inexpensive
• Hydrogen - It hasadistinctly better thermal conductivity and lower
density. Demerits areits reactivity with unsaturated compoundsand
hazardous explosivenature
• Air - It isemployedonly when the atmosphericO2isbeneficial to
the detectorseparation.
25. Applications
usedfor final purification natural extracts, synthetic mixtures and
biological matrices.
It is also usedfor fractionization of complex crude extracts eg. Petroleum
fractions
Determination ofwater quality
Separation of aroma moleculesof wine
Determination of pesticide residue
26. REFERENCES
• BIOPHYSICAL CHEMISTRY BY UPADHYAY & UPADHYAY NATH
• INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS BY DR.B.K.SHARMA
• A TEXTBOOK OF MICROBIOLOGY BY R.C. DUBEY & D.K. MAHESHWARI
• GOOGLE SEARCH
• SLIDESHARE