الطرق الطرق تصنيف تصنيفالكروماتوجرافيةالكروماتوجرافية

CHROMATOGRAPHYCHROMATOGRAPHY

مدكور / لطفي مدكور / د لطفي د

ChromatographyChromatography

HistoryHistory

Mikhail Tswett, Russian, 1872-Mikhail Tswett, Russian, 1872-19191919

BotanistBotanistIn 1906 Tswett used to chromatography to In 1906 Tswett used to chromatography to separate plant pigmentsseparate plant pigments

He called the new technique He called the new technique chromatography because the result of the chromatography because the result of the analysis was 'written in color' along the analysis was 'written in color' along the length of the adsorbent columnlength of the adsorbent columnChroma means Chroma means ““colorcolor”” and graphein means to and graphein means to ““writewrite””

ImportanceImportance

Chromatography has application in every branch of the Chromatography has application in every branch of the physical and biological sciencesphysical and biological sciences

1212 Nobel prizes were awarded between 1937 and 1972 alone Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital rolefor work in which chromatography played a vital role

Chromatography is a physical method of Chromatography is a physical method of separation in which the components to be separation in which the components to be separated are distributed between two separated are distributed between two phasesphases

one of which is stationary (one of which is stationary (stationary phasestationary phase) ) while the other (the while the other (the mobile phasemobile phase) moves ) moves through it in a definite directionthrough it in a definite direction..

The chromatographic process occurs due to The chromatographic process occurs due to differences in the differences in the distribution constantdistribution constant of of the individual sample componentsthe individual sample components..

ChChromromatoatogragraphyphy

Is a technique used to separate and identify the components of a

mixture.

Works by allowing the molecules present in the mixture to distribute themselves between a

stationary and a mobile medium.

Molecules that spend most of their time in the mobile phase are carried

along faster.

التحليل: كروماتوجرافياكروماتوجرافيا لطرق العام األسم التحليل: هو لطرق العام األسم هويمكن أكثر،حيث أو مادتين علي يحتوي يمكن لمخلوط أكثر،حيث أو مادتين علي يحتوي لمخلوط

لميلهما إستنادآ مخلوطهما من فيزيائيآ لميلهما فصلهما إستنادآ مخلوطهما من فيزيائيآ فصلهماصلب ) ساكن طور طورين بين توزيعهما صلب ) ومعامل ساكن طور طورين بين توزيعهما ومعامل

) يمكن( ) وبالتالي ، أوغاز سائل متحرك وطور ( أوسائل يمكن( ) وبالتالي ، أوغاز سائل متحرك وطور أوسائلخالل من وذلك وكميآ نوعيآ عليهما والتعرف خالل فصلهما من وذلك وكميآ نوعيآ عليهما والتعرف فصلهما

المختلفة المعامالت المختلفة تعيين المعامالت ::تعيين

اإلستبقاء اإلستبقاء زمن t= trr = زمن

التوزيع التوزيع معامل K=Kxx=معامل

السعة السعة معامل k’=k’xx=معامل

==NN النظرية الطبقات النظرية عدد الطبقات عدد

==HH النظرية للطبقات مكافئ النظرية أعلي للطبقات مكافئ أعلي

= = αα اإلختيارية اإلنتقاء اإلختيارية معامل اإلنتقاء معامل

==RR والوضوح الفصل والوضوح درجة الفصل درجة

Classification of chromatography according Classification of chromatography according to mobile phaseto mobile phase::

11 - -Liquid chromatography: mobile phase is a Liquid chromatography: mobile phase is a liquid. (LLC, LSC)liquid. (LLC, LSC)..

22 - -Gas chromatography : mobile phase is a Gas chromatography : mobile phase is a gas. (GSC, GLC)gas. (GSC, GLC)..

Classification according to the packing of the Classification according to the packing of the stationary phasestationary phase::

11 - -Thin layer chromatography (TLC): the stationary Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or phase is a thin layer supported on glass, plastic or aluminium platesaluminium plates..

22 - -Paper chromatography (PC): the stationary phase Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert is a thin film of liquid supported on an inert supportsupport..

33 - -Column chromatography (CC): stationary phase Column chromatography (CC): stationary phase is packed in a glass columnis packed in a glass column..

Classification according to the force of Classification according to the force of separationseparation::

11 - -Adsorption chromatography (LSC)Adsorption chromatography (LSC)..

22 - -Partition chromatography (LLC)Partition chromatography (LLC)..

33 - -Ion exchange chromatographyIon exchange chromatography..

44 - -Gel filtration chromatographyGel filtration chromatography..

55 - -Affinity chromatographyAffinity chromatography..

Thin layer chromatography (TLC)Thin layer chromatography (TLC)

is a method for is a method for identifyingidentifying substances and substances and testing the puritytesting the purity of compounds of compounds..

TLC is a useful technique because it is TLC is a useful technique because it is relatively relatively quickquick and requires and requires small small quantitiesquantities of material of material..

Separations in TLC involve distributing a mixture of two Separations in TLC involve distributing a mixture of two or more substances between a or more substances between a stationary phasestationary phase and a and a

mobile phasemobile phase . .

The stationary phaseThe stationary phase:: is a thin layer of adsorbent (usually silica gel or alumina) is a thin layer of adsorbent (usually silica gel or alumina)

coated on a platecoated on a plate . .

The mobile phaseThe mobile phase:: is a developing liquid which travels up the stationary is a developing liquid which travels up the stationary

phase, carrying the samples with itphase, carrying the samples with it . .Components of the samples will separate on the Components of the samples will separate on the stationary phase according tostationary phase according to

how much they adsorb on the stationary phase versus how much they adsorb on the stationary phase versus how much they dissolve in the mobile phasehow much they dissolve in the mobile phase..

Thin Layer Chromatography (TLC)Thin Layer Chromatography (TLC)

TLCTLC

Preparing the ChamberPreparing the Chamber

To a jar with a tight-fitting lid add enough of To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it the appropriate developing liquid so that it

is 0.5 to 1 cm deep in the bottom of the jaris 0.5 to 1 cm deep in the bottom of the jar . .

Close the jar tightly, and let it stand for about Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the jar 30 minutes so that the atmosphere in the jar becomes saturated with solventbecomes saturated with solvent..

Preparing the Plates for Preparing the Plates for DevelopmentDevelopment

With a pencil, etch two small notches into the adsorbent With a pencil, etch two small notches into the adsorbent about 2 cm from the bottom of the plateabout 2 cm from the bottom of the plate..

The notches should be on the edges of the plate, and each The notches should be on the edges of the plate, and each notch should be the same distance up from the bottom of notch should be the same distance up from the bottom of the platethe plate..

The notches must be farther from the bottom of the plate The notches must be farther from the bottom of the plate than the depth of the solvent in the jarthan the depth of the solvent in the jar..

Using a drawn-out capillary tube, spot the samples on the Using a drawn-out capillary tube, spot the samples on the plate so that they line up with the notches you etchedplate so that they line up with the notches you etched..

Question: What is wrong with the plate shown Question: What is wrong with the plate shown belowbelow??

Developing the PlatesDeveloping the Plates

After preparing the development chamber and spotting After preparing the development chamber and spotting the samples, the plates are ready for developmentthe samples, the plates are ready for development . .

Be careful to handle the plates only by their edges, and Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as try to leave the development chamber uncovered for as little time as possiblelittle time as possible..

When the plates are removed from the chamber, quickly When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the trace the solvent front (the highest solvent level on the plate) with a pencilplate) with a pencil..

Identifying the Spots (visualization)Identifying the Spots (visualization)

If the spots can be seen, outline If the spots can be seen, outline them with a pencilthem with a pencil..

If no spots are obvious, the most If no spots are obvious, the most common visualization technique common visualization technique is to hold the plate under a UV is to hold the plate under a UV lamplamp..

Many organic compounds can be Many organic compounds can be seen using this technique, and seen using this technique, and many many commercially made plates often contain a substance which often contain a substance which aids in the visualization of aids in the visualization of compoundscompounds..

Visualizing AgentsVisualizing Agents

Alkaloids: DragendorffAlkaloids: Dragendorff’’s reagents reagent

Cardiac glycosides: Antimony trichlorideCardiac glycosides: Antimony trichloride

Sugar: Aniline phthalateSugar: Aniline phthalate

Amino acids: NinhydrinAmino acids: Ninhydrin

Interpreting the DataInterpreting the Data

The RThe Rff (retention factor) value for each spot (retention factor) value for each spot should be calculatedshould be calculated . .

It is characteristic for any given compound It is characteristic for any given compound on the same stationary phase using the same on the same stationary phase using the same

mobile phase for development of the platesmobile phase for development of the plates . .

Hence, known RHence, known Rff values can be compared to values can be compared to those of unknown substances to aid in their those of unknown substances to aid in their

identificationsidentifications..

((Note: RNote: Rff values often depend on the temperature and values often depend on the temperature and

the solvent used in the TLC experimentthe solvent used in the TLC experiment..

the most effective way to identify a compound is to spot the most effective way to identify a compound is to spot known substances known substances –– authentic - next to unknown authentic - next to unknown

substances on the same platesubstances on the same plate).).

In addition, the purity of a sample may be estimated In addition, the purity of a sample may be estimated from the chromatogramfrom the chromatogram..

An impure sample will often develop as two or more An impure sample will often develop as two or more spots, while aspots, while a pure sample will show only one spot

SummarySummaryA TLC plate is a sheet of glass, metal, or plastic which is coated with A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina)a thin layer of a solid adsorbent (usually silica or alumina)..

A small amount of the mixture to be analyzed is spotted near the A small amount of the mixture to be analyzed is spotted near the bottom of this platebottom of this plate . .

The TLC plate is then placed in a shallow pool of a solvent in a The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in developing chamber so that only the very bottom of the plate is in the liquidthe liquid..

This liquid, or the eluent, is the mobile phase, and it slowly rises up This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary actionthe TLC plate by capillary action..

As the solvent moves past the spot that was applied, an equilibrium is As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the established for each component of the mixture between the molecules of that component which are adsorbed on the solid and molecules of that component which are adsorbed on the solid and the molecules which are in solutionthe molecules which are in solution..

In principle, the components will differ in solubility and in In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than components will be carried farther up the plate than

othersothers . .

When the solvent has reached the top of the plate, the plate is When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the removed from the developing chamber, dried, and the separated components of the mixture are visualizedseparated components of the mixture are visualized..

If the compounds are colored, visualization is If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the platesso a UV lamp is used to visualize the plates..

Paper ChromatographyPaper Chromatography

A method of partition chromatography using filter A method of partition chromatography using filter paper strips as carrier or inert supportpaper strips as carrier or inert support..

The factor governing separation of mixtures of solutes The factor governing separation of mixtures of solutes on filter paper is the on filter paper is the partition between two partition between two immiscible phasesimmiscible phases..

One is usually water adsorbed on cellulose fibres in One is usually water adsorbed on cellulose fibres in the paper (stationary phase)the paper (stationary phase)..

The second is the organic solvent flows past the The second is the organic solvent flows past the sample on the paper (stationary phase)sample on the paper (stationary phase)..

Partition occurs between the mobile phase and the Partition occurs between the mobile phase and the stationary aqueous phase bound by the cellulosestationary aqueous phase bound by the cellulose..

The isolation depends on partition coefficient of the The isolation depends on partition coefficient of the solutesolute..

( )

( )

c stationaryK

c mobile

General ProcedureGeneral Procedure

11 - -Choice of paper and solvent to be usedChoice of paper and solvent to be used..

22 - -Desalting of sampleDesalting of sample..

33 - -Application of the sampleApplication of the sample..

44 - -Equilibration of paperEquilibration of paper..

55 - -DevelopmentDevelopment..

66 - -DetectionDetection..

77 - -Identification of substancesIdentification of substances..

Techniques of development with various flow Techniques of development with various flow directionsdirections

Ascending development

Descending development

Radial development

Multiple chromatographyMultiple chromatography

Multiple chromatography includes all Multiple chromatography includes all procedures in which the development is procedures in which the development is repeated after one development is completedrepeated after one development is completed..

A- multiple development:A- multiple development: the chromatogram is the chromatogram is repeatedly developed in the same direction repeatedly developed in the same direction and thus the complete resolution of two or and thus the complete resolution of two or more substances which have Rmore substances which have Rff values close values close together can be obtainedtogether can be obtained..

As the mobile phase one can use either the same As the mobile phase one can use either the same solvent system or different solvent systemssolvent system or different solvent systems..

B- two-dimensional chromatographyB- two-dimensional chromatography::When large numbers of substances are to be separated When large numbers of substances are to be separated on a single chromatogramon a single chromatogram..

Development in a direction perpendicular to the first, Development in a direction perpendicular to the first, and with a solvent system different from that used and with a solvent system different from that used

initially is often necessaryinitially is often necessary . .

The sample is applied on one corner of a square piece of The sample is applied on one corner of a square piece of paper and after development with the first solvent, paper and after development with the first solvent, the paper is dried , rotated 90the paper is dried , rotated 90oo and developed in the and developed in the second directionsecond direction..

Usually, different types of solvents systems are used in Usually, different types of solvents systems are used in each direction. It is essential that the first solvent be each direction. It is essential that the first solvent be completely volatilecompletely volatile..

Columnar Chromatography (CC)Columnar Chromatography (CC)

This includes chromatographic methods in This includes chromatographic methods in whichwhich::

The stationary phase is packed into a columnThe stationary phase is packed into a column . .

The mobile phase is a moving liquid or gasThe mobile phase is a moving liquid or gas..

According to the mechanism of separation of According to the mechanism of separation of solutes, five major types of CC are solutes, five major types of CC are ditinguished. Usually, one mechanism ditinguished. Usually, one mechanism predominates but does not exclude the otherspredominates but does not exclude the others

Different Types of chromatography

Mode or typeStationary phaseMobile phaseMechanism

Adsorption Chromatography

Solid that attracts the solutes

Liquid or gasSolutes move at different rates according to the forces of attraction to the stationary phase.

Partition Chromatography

Thin film of liquid formed on the surface of a solid inert support

Liquid or gasSolutes equilibrate between the 2 phases according to their partition coefficients

Ion Exchange Chromatography

Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds

Liquid containing electrolytes

Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions.

Molecular Exclusion Chromatography

Porous gel with no attractive action on solute molecules

Liquid Molecules separate according to their size:

1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate.

Affinity Chromatography

Solid on which specific molecules are immobilized

Liquid or gasSpecial kind of solute molecules interact with those immobilized on the stationary phase

Column ChromatographyColumn Chromatography

Column Column chromatographychromatographyStationary phase is Stationary phase is held in a narrow held in a narrow tube through tube through which the mobile which the mobile phase is forced phase is forced under pressure or under pressure or under the effect under the effect of gravityof gravity

TermDefinition

SolventMobile liquid phase with no affinity to the stationary phase

(i.e. inert towards it) & no effect on solutes.

DeveloperAny liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move

them through the column.

EffluentAny liquid that passes out of the column.

EluentAny liquid that has lesser affinity to the stationary phase

than solutes but is capable to move them out of the column.

EluateFraction of eluent containing a required specific substance.

Retention volume (VR)

)or retardation volume :(Volume of mobile phase that passes out of the column, before elution of a specific

substance.

Open Column ChromatographyOpen Column Chromatography(Traditional column chromatography)(Traditional column chromatography)

Traditional column chromatography is Traditional column chromatography is characterized by addition of characterized by addition of mobile phasemobile phase under under atmospheric pressureatmospheric pressure and the and the stationary phasestationary phase is is packed in a glass packed in a glass columncolumn..

Packing & operating the columnPacking & operating the column1- Packing1- Packing

The selection of the method of packing The selection of the method of packing depends depends mainly mainly on theon the density of the soliddensity of the solid.Techniques .Techniques

used are the used are the wetwet, , drydry & & slurryslurry methods methods . .

In all cases In all cases avoid inclusion of air bubblesavoid inclusion of air bubbles

22 - -Sample ApplicationSample Application

Apply Apply evenly & in a concentrated solutionevenly & in a concentrated solution to to the top of the column which is protected the top of the column which is protected from disturbance (e.g. add glass wool or from disturbance (e.g. add glass wool or filter paper)filter paper)..

TechniqueProcedure

Isocratic elutionAddition of solvent mixture of fixed composition during the whole process.

Gradient elutionContinuous or linear elution: in which there is continuous change in the composition of the mobile phase over a period of time (e.g. polarity, pH or ionic strength).

Step wise or fractional elution: in which the change is not continuous i.e. a sudden change in the composition of the mobile phase is followed by a period where the mobile phase is held constant.

.1Elution techniques

44 - -DetectionDetection

On-column detectionOn-column detection for for coloredcolored or or fluorescent fluorescent compounds directly after compounds directly after developing the chromatogramdeveloping the chromatogram..

Monitoring of eluted fractions Monitoring of eluted fractions (PC or TLC)(PC or TLC)..

Using special detectorsUsing special detectors connected to the connected to the column such as refractive index, UV detectors, column such as refractive index, UV detectors,

etcetc……

Factors affecting solutes separation in CC) Factors affecting column efficiency(

FactorEffect

Particle size of solid stationary phase (or of support)

Decrease of size improves separation (but very small particles need high pressure).

Column dimensionsEfficiency increases as ratio length / width increases.

Uniformity of packingNon uniform packing results in irregular movement of solutes through column & less uniform zone formation, (i.e. band broadning or tailing).

Column temperatureIncrease in column temperature results in speed of elution but does not improve separation (tailing).

Eluting solventSolvents should be of low viscosity (to give efficient resolution) & h igh volatility (to get rapid recovery of the substances).

Solvent flow rateUniform & low flow rate gives better resolution.

Continuity of flowDiscontinuous flow disturbs resolution

Condition of adsorbentDeactivation of adsorbent decreases separation.

Concentration of solutesSubstances of high concentration move slowly.

Number of Theoretical PlatesNumber of Theoretical Plates(N)(N)

H = Theoretical Plate HeightL = Length of the Column.

N = L / H

As HETP decreases efficiency of the column increases.

Adsorption Column ChromatographyAdsorption Column Chromatography

AdsorbentsAdsorbents::The most common are The most common are Alumina Alumina & & Silica gelSilica gel in which the in which the interactionsinteractions with solute molecules is with solute molecules is due to OH groups due to OH groups presentpresent on their surfaceon their surface..

More polar moleculesMore polar molecules are are adsorbed moreadsorbed more strongly strongly & thus, will & thus, will elute more slowlyelute more slowly

Strength of adsorption of polar groups (solutes) on polar support Strength of adsorption of polar groups (solutes) on polar support is in the following orderis in the following order::

--C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -OH < -COOHOH < -COOHOlefins < Ethers < Esters < Lactones < Aldehydes < Amines < Olefins < Ethers < Esters < Lactones < Aldehydes < Amines < Phenols < AcidsPhenols < Acids..

Applications in separation of natural productsApplications in separation of natural products

AluminaAlumina: sterols, dyestuffs, vitamins, esters, : sterols, dyestuffs, vitamins, esters, alkaloids & inorganic compoundsalkaloids & inorganic compounds . .

Not used for compounds containing phenolic or Not used for compounds containing phenolic or carboxylic groupscarboxylic groups

Silica gel: Silica gel: sterols & amino acidssterols & amino acids..

Carbon: Carbon: peptides, carbohydrates & amino acidspeptides, carbohydrates & amino acids..

Calcium carbonate: Calcium carbonate: carotenoids & xanthophyllscarotenoids & xanthophylls..

Partition Column ChromatographyPartition Column Chromatography

In this type, the In this type, the packing consists of a packing consists of a theoretically inert support materialtheoretically inert support material coated coated

with a film of the liquid stationary phasewith a film of the liquid stationary phase . .

The The division into adsorption & partitiondivision into adsorption & partition is is only of only of theoreticaltheoretical significance as significance as in partition in partition chromatography the adsorption effects of chromatography the adsorption effects of the support can be feltthe support can be felt..

Selection of the solid supportSelection of the solid support

The support material shouldThe support material should : :adsorb & retain the mobile stationary phaseadsorb & retain the mobile stationary phase..

expose as large surface as possible to the mobile phaseexpose as large surface as possible to the mobile phasebe mechanically stablebe mechanically stable..be easy to packbe easy to pack..

not retard the solvent flownot retard the solvent flow

ExamplesExamples of solid supportsof solid supports::Silica gel, diatomaceous earth (as kieselguhr, celite etc.) Silica gel, diatomaceous earth (as kieselguhr, celite etc.) & cellulose& cellulose..

Selection of the mobile phaseSelection of the mobile phase

The The liquid stationary & mobile phasesliquid stationary & mobile phases should have a should have a considerable difference between their solvent strength considerable difference between their solvent strength parametersparameters..

Pure water > Methanol > Ethanol > Propanol > Pure water > Methanol > Ethanol > Propanol > Acetone > Ethyl acetate> Ether > Chloroform > Acetone > Ethyl acetate> Ether > Chloroform > Dichloromethane >Benzene > Toluene > Carbon Dichloromethane >Benzene > Toluene > Carbon tetrachloride > Cyclohexane > Hexane > Pentanetetrachloride > Cyclohexane > Hexane > Pentane..

e.g. if the e.g. if the stationary phasestationary phase is is waterwater, , pentanepentane would be would be the the eluent eluent of choiceof choice..

The The mobile phasemobile phase is usually is usually saturated saturated with the with the stationary phasestationary phase to to overcomeovercome " "strippingstripping" " (washing of the stationary phase from the (washing of the stationary phase from the column by the mobile phase)column by the mobile phase)..

Gel Permeation Chromatography (GPC)Gel Permeation Chromatography (GPC)

This type is also known asThis type is also known as::

Size Exclusion Chromatography (SEC)Size Exclusion Chromatography (SEC)

Molecular Exclusion Chromatography (MEC)Molecular Exclusion Chromatography (MEC)

Molecular Sieve Chromatography (MSC)Molecular Sieve Chromatography (MSC)

Gel Filtration Chromatography (GFC)Gel Filtration Chromatography (GFC)

Gel ChromatographyGel Chromatography..

Stationary phaseStationary phasePorous polymeric matrix:Porous polymeric matrix: formed of spongy particles, formed of spongy particles, with pores completely filled with the liquid mobile with pores completely filled with the liquid mobile phase phase (gel)(gel)..

The gels (polymers) consist of The gels (polymers) consist of openopen, , three-dimensional three-dimensional networksnetworks formed by cross-linking of long polymeric formed by cross-linking of long polymeric

chainschains . .

The The pore sizepore size varies with the degree of cross-linking varies with the degree of cross-linking.. The The diameter of the poresdiameter of the pores is critical asis critical as separation is separation is based on that based on that molecules above certain size are totally molecules above certain size are totally excluded from the poresexcluded from the pores because they can not enter because they can not enter the gelthe gel..

The The interior of the poresinterior of the pores is reachedis reached, partially or wholly, , partially or wholly, by smaller moleculesby smaller molecules . .

Affinity ChromatographyAffinity Chromatography

Mobile phaseMobile phase Mobile phase is a liquid as Mobile phase is a liquid as waterwater or or dilute acoholdilute acohol

Separation mechanismSeparation mechanismBased on difference between the solutes Based on difference between the solutes molecular weightsmolecular weights..

Molecules will distribute themselves outside & Molecules will distribute themselves outside & inside the pores according to their sizeinside the pores according to their size..

LargerLarger are excluded, are excluded, medium sizedmedium sized enter half- enter half-way & way & smallest smallest permeate all the waypermeate all the way . .

The The retention volume Voretention volume Vo of a substance is of a substance is inversely proportional to the molecular weight inversely proportional to the molecular weight

(M. Wt) of the solute(M. Wt) of the solute..

VoVo ~~ 1 / M.wt 1 / M.wt

Vo Vo = retention volume = retention volume

M.wt = Molecular WeightM.wt = Molecular Weight

Applications of GPC to natural productsApplications of GPC to natural products

Determination of M. wt. of peptides, proteins & polysaccharidesDetermination of M. wt. of peptides, proteins & polysaccharides.. Desalting of colloids e.g. desalting of albumin prepared with 2% (NHDesalting of colloids e.g. desalting of albumin prepared with 2% (NH44))22SOSO44..

Separation of mixture of mono- & polysaccharidesSeparation of mixture of mono- & polysaccharides..

Separation of amino acids from peptides & proteinsSeparation of amino acids from peptides & proteins..

Separation of proteins of different molecular weightsSeparation of proteins of different molecular weights..

Separation of mucopolysaccharides & soluble RNASeparation of mucopolysaccharides & soluble RNA..

Separation of myoglobin & haemoglobinSeparation of myoglobin & haemoglobin..

Separation of alkaloids & purification of enzymesSeparation of alkaloids & purification of enzymes..

Ion Exchange ChromatographyIon Exchange Chromatography

PrinciplePrincipleProcess by whichProcess by which ions ions of an of an electrolyte solutionelectrolyte solution are brought into are brought into contact with an contact with an ion exchange resinion exchange resin..

TheThe ion exchange resin ion exchange resin is an insoluble polymer consisting of a is an insoluble polymer consisting of a "matrix" "matrix" (Lattice or framework) that (Lattice or framework) that carries fixed charges carries fixed charges (not (not exchangeable) and mobile exchangeable) and mobile active ions "counter ions" active ions "counter ions" which are which are loosely attached to the matrixloosely attached to the matrix..

In In water, water, the the counter-ionscounter-ions move more or less freely in the move more or less freely in the frameworkframework && can be can be replaced by ions of the same sign present in replaced by ions of the same sign present in the surrounding solutionthe surrounding solution..

The The "matrix""matrix" (framework) (framework) of a "cation exchanger"of a "cation exchanger" is considered as is considered as a crystalline non-ionized "a crystalline non-ionized "polyanionpolyanion" & the " & the matrix of an "anion matrix of an "anion

exchanger"exchanger" as a non-ionized " as a non-ionized "polycationpolycation."."

Cation ExchangersCation Exchangers

Active ions (counter ions) are cationsActive ions (counter ions) are cations . .

The The polar groupspolar groups attached to theattached to the matrix are acidicmatrix are acidic (sulphonic acids, carboxylic acids, phenols, (sulphonic acids, carboxylic acids, phenols, phosphoric acids) e.g. a cation exchanger in the free phosphoric acids) e.g. a cation exchanger in the free carboxylic acid formcarboxylic acid form::

X-COOX-COO-- H H++

X = Frame work (matrix)X = Frame work (matrix)

--COOCOO-- = Fixed charge (anionic) = Fixed charge (anionic) , ,

Non-exchangeableNon-exchangeable

HH++ = Counter ion (cation), Exchangeable = Counter ion (cation), Exchangeable

They are usually (but not always) supplied in the NaThey are usually (but not always) supplied in the Na++ form: form: X-COOX-COO--NaNa++

or Na + , Where = Matrix

2 Na+ + Ca++ Cl2 Ca++ +2 Na+ Cl-

e.g. exchange with CaCl2 aqueous solution

Anion ExchangersAnion Exchangers

Active ions (counter ions) are anionsActive ions (counter ions) are anions..The polar groups attached to the matrix are The polar groups attached to the matrix are tertiary or quaternary ammonium groups (basic)tertiary or quaternary ammonium groups (basic)..

e.g. Anion exchanger in the quaternary e.g. Anion exchanger in the quaternary ammonium formammonium form::

X. NR3X. NR3++OHOH ––

X = Framework (matrix)X = Framework (matrix) --NR3 NR3 ++ = Fixed charge (cationic) = Fixed charge (cationic)

Non exchangeableNon exchangeable--OHOH–– = counter ion (anion), Exchangeable = counter ion (anion), Exchangeable

They are supplied as the chloride rather than the They are supplied as the chloride rather than the hydroxide as the chloride form is a more hydroxide as the chloride form is a more stable. Represented asstable. Represented as: X. NR3: X. NR3++ClCl --

or Cl - )where, is the matrix(

e.g. exchange with Na2SO4 solution

2 Cl - + Na2

+2 SO4-2 SO4

-2 + 2 Na+Cl -

Regeneration of the resinRegeneration of the resin

Ion exchange process is generally reversible e.g Ion exchange process is generally reversible e.g in the followingin the following::

22 NaNa++ + Ca + Ca++++ 2Cl 2Cl -- → Ca → Ca++++ + 2 Na + 2 Na++ Cl Cl ––

The cation exchanger could be exhausted after The cation exchanger could be exhausted after exchanging all Naexchanging all Na++ for Ca for Ca++++, the exchanger , the exchanger could be regenerated (loaded again with Nacould be regenerated (loaded again with Na++) ) by contacting it with excess Naby contacting it with excess Na++ ions e.g. a ions e.g. a solution of NaClsolution of NaCl..

Types of ExchangersTypes of Exchangers

Ion ExchangersIon ExchangersThese are eitherThese are either cation cation or or anionanion exchangersexchangers of either of either organicorganic or or inorganicinorganic nature nature..

A- Inorganic ion exchangersA- Inorganic ion exchangersCommon clays, soils, minerals e.g. zeolites used for Common clays, soils, minerals e.g. zeolites used for

"softening water"softening water."."Disadvantage:Disadvantage: low ion-exchange capacity low ion-exchange capacity..AdvantagesAdvantages::Great resistance to high temperaturesGreat resistance to high temperatures..High volume capacityHigh volume capacity..Great selectivity towards simple inorganic ionsGreat selectivity towards simple inorganic ions..

B- Organic exchangersB- Organic exchangers

They may be natural or syntheticThey may be natural or synthetic..

Preparation of organic synthetic ion exchangersPreparation of organic synthetic ion exchangers ::

Polycondensation of Polycondensation of phenolsphenols, , sulpho- sulpho- & & carboxy- carboxy- derivatives with formaldehyde derivatives with formaldehyde → → cationiccationic exchangersexchangers..

Polycondensation of Polycondensation of aromatic aminesaromatic amines with with formaldehydeformaldehyde → → anionic anionic exchangers exchangers..

These techniques yield products These techniques yield products linearlinear in structure & in structure & relatively relatively soluble in watersoluble in water which are now replaced which are now replaced by resin materials based on by resin materials based on styrene divinyl benzene styrene divinyl benzene copolymerscopolymers and and polyacrylate polyacrylate..

Applications of Ion Exchange Applications of Ion Exchange ChromatographyChromatography

11 - -Water softeningWater softening:: Removal of Ca2+, Mg2+ & other multivalent ions causing Removal of Ca2+, Mg2+ & other multivalent ions causing hardness of water by filtration through a layer of strong hardness of water by filtration through a layer of strong cation resincation resin..

22--Water demineralizationWater demineralization : :Removal of cations & anions dissolved in water. Usually Removal of cations & anions dissolved in water. Usually carried by the two step technique in which two columns carried by the two step technique in which two columns of strongly acid cation exchanger in [H+] form & of strongly acid cation exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in strongly basic anion exchanger in [OH-] form are used in sequencesequence..

33 - -NeutralizationNeutralization:: Cationic exchanger in [H+] can be used to neutralize alkali Cationic exchanger in [H+] can be used to neutralize alkali hydroxide & anionic exchanger in [OH+] form to hydroxide & anionic exchanger in [OH+] form to neutralize the acidityneutralize the acidity..

44 - -Separation of electrolytes from non-Separation of electrolytes from non-electrolyteselectrolytes..

55 - -Separation of carbohydrates & their Separation of carbohydrates & their derivativesderivatives::

Uronic acidsUronic acids separated on anion exchanger separated on anion exchanger..

SugarsSugars converted into ionized form by using converted into ionized form by using borate& separated on strong anion exchangerborate& separated on strong anion exchanger..

Hexosamines Hexosamines separated on strong cation separated on strong cation exchangerexchanger..

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