Unit 4 General Principles of Chromatography

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General Principles of General Principles of Chromatography Chromatography

UNIT

UNIT 4

4 GENERAL

GENERAL PRINCIPLES

PRINCIPLES OF

OF

CHROMATOGRAPHY

CHROMATOGRAPHY

Structure Structure 4.1 4.1 IntroductionIntroduction Objectives Objectives 4.2

4.2 Definition and ClassificationDefinition and Classification Definition Definition Classification Classification Liquid Chromatography Liquid Chromatography Gas Chromatography Gas Chromatography

Supercritical Fluid Chromatography Supercritical Fluid Chromatography Conclusions

Conclusions 4.3

4.3 Elution on ColumnsElution on Columns 4.4

4.4 Migration RatesMigration Rates Distribution Constant Distribution Constant Retention Time Retention Time Retention Factor Retention Factor

Selectivity Factor (Separation Factor) Selectivity Factor (Separation Factor) Shapes of Peaks

Shapes of Peaks 4.5

4.5 Concept of Theoretical PlatesConcept of Theoretical Plates 4.6

4.6 Rate TheoryRate Theory

Eddy Diffusion Term Eddy Diffusion Term

Longitudinal Diffusion Term Longitudinal Diffusion Term

Non-equilibrium in Mass Transfer Term Non-equilibrium in Mass Transfer Term van Deemter Plot and Zone Broadening van Deemter Plot and Zone Broadening 4.7

4.7 ResolutionResolution 4.8

4.8 SummarySummary 4.9

4.9 Terminal QuestionsTerminal Questions 4.10

4.10 AnswersAnswers

4.1

INTRODUCTION

4.1

INTRODUCTION

In the beginning of this course in Unit 1 of B

In the beginning of this course in Unit 1 of B lock 1, we have realized thatlock 1, we have realized that chromatograp

chromatography as hy as a collective science which a collective science which encompasses a number of importantencompasses a number of important separation techniques. The subject was introduced to the scientific world in a very separation techniques. The subject was introduced to the scientific world in a very modest way by M. Tswett in 1906. He employed the technique to separate various modest way by M. Tswett in 1906. He employed the technique to separate various pigments such as chlorophylls and xanthophylls by passing a solution

pigments such as chlorophylls and xanthophylls by passing a solution of theseof these compounds throug

compounds through a glass h a glass column packed with finely divided calcium column packed with finely divided calcium carbonate.carbonate. Later on this technique was named as

Later on this technique was named as liquid-solid adsorption liquid-solid adsorption chromatogrchromatographyaphy. About. About the same time, Thompson and Way had realized the

the same time, Thompson and Way had realized the ion exchange propeion exchange properties of soils.rties of soils. Almost after three decades,

Almost after three decades, in 1935, Adams and Holmes observed ion ein 1935, Adams and Holmes observed ion e xchangexchange character in crushed phonograph. This particular observation opened up the field for character in crushed phonograph. This particular observation opened up the field for synthetic organic resin exchangers. Thereafter, the subject of

synthetic organic resin exchangers. Thereafter, the subject of ion exchangeion exchange chromatography

chromatography  started expanding in its utility. The  started expanding in its utility. The concept ofconcept of gas-liquidgas-liquid chromatography

chromatography was first introduced by Martin and Synge in 1941. They were also was first introduced by Martin and Synge in 1941. They were also responsible for the

responsible for the developments in liquid-liquid partition chromatography. Moreover,developments in liquid-liquid partition chromatography. Moreover, in 1944, from Martin’s laboratory, the separation of

in 1944, from Martin’s laboratory, the separation of amino acids byamino acids by paper paper chromatogra

chromatography was phy was reported reported . This made people to realize the . This made people to realize the importance of such aimportance of such a simple technique for tedious separations. Simultaneously the efforts were

simple technique for tedious separations. Simultaneously the efforts were being madebeing made to understand the theoretical aspects

to understand the theoretical aspects of chromatographyof chromatography. The . The high point of all high point of all thesethese activities reached in

activities reached in 1952 when the contributions of Martin and Synge were1952 when the contributions of Martin and Synge were recognized and they were awarded the

recognized and they were awarded the Nobel Prize for their Nobel Prize for their work in chromatographicwork in chromatographic science.

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Chromatographic Chromatographic Methods-I

Methods-I The analytical scientists all around the world got actively engaged in improving theThe analytical scientists all around the world got actively engaged in improving theparticipating phases, detectors and the instrumentation in participating phases, detectors and the instrumentation in general. In 1959, a techniquegeneral. In 1959, a technique known as

known as gel filtragel filtration chromatographytion chromatography to separate high and low molecular weight to separate high and low molecular weight substances, originated at Biochemical Institute in

substances, originated at Biochemical Institute in Uppsala, Sweden. In the 1960s,Uppsala, Sweden. In the 1960s, further improvements in the conventional liquid chromatography led to the

further improvements in the conventional liquid chromatography led to the development of

development of high performance high performance liquid chromatographyliquid chromatography. The following decade of. The following decade of the seventies saw an

the seventies saw an important improvemeimportant improvement in nt in adsorption chromatoadsorption chromatography in thegraphy in the form of

form of affinity affinity chromatograchromatographyphy which was  which was mainly based on biological interactions.mainly based on biological interactions. The researches for developments in chromatography were still being carried out The researches for developments in chromatography were still being carried out and aand a new type of chromatography using superficial fluids appeared on the scene.

new type of chromatography using superficial fluids appeared on the scene. Supercritical fluid

Supercritical fluid chromatogchromatographyraphy is  is a hybrid of gas and a hybrid of gas and liquid chromatography andliquid chromatography and combines the advantageous features of the two.

combines the advantageous features of the two. The above description on the growth of

The above description on the growth of chromatograpchromatography clearly points out that hy clearly points out that onceonce the potential of

the potential of some simpler versions of chromatography for separations was realized,some simpler versions of chromatography for separations was realized, the researches took place on a phenomenal pace. It will not

the researches took place on a phenomenal pace. It will not be wrong to say that thebe wrong to say that the entire twentieth century can be

entire twentieth century can be named as the named as the century of chromatograpcentury of chromatography. Even today,hy. Even today, the efforts are being made to bring different types of improvements. At this point, one the efforts are being made to bring different types of improvements. At this point, one should not forget that the

should not forget that the developmedevelopments are not nts are not only because of variety ofonly because of variety of physicochemical principles that can be

physicochemical principles that can be incorporaincorporated in chromatographic techniques toted in chromatographic techniques to bring out separations, but are partly due to the compelling need of the scientific bring out separations, but are partly due to the compelling need of the scientific community to achieve a variety of tedious separations particularly related with life community to achieve a variety of tedious separations particularly related with life sciences.

sciences.

The present unit aims

The present unit aims at proposing a classification of at proposing a classification of chromatographichromatographic techniques. Anc techniques. An effort will also be made to e

effort will also be made to explain some of the general principles which lead toxplain some of the general principles which lead to separation of compounds present in a mixture. B

separation of compounds present in a mixture. B efore discussing the theory or theefore discussing the theory or the concepts, it may be necessary to familiarize you with

concepts, it may be necessary to familiarize you with the terms which are used tothe terms which are used to assess the separation efficiency of a process.

assess the separation efficiency of a process.

Objectives Objectives

After studying this Unit, you should be able to After studying this Unit, you should be able to •

discribe the different ways chromatographic techniques can be classified,discribe the different ways chromatographic techniques can be classified,

explain processes taking place during elution on explain processes taking place during elution on columns,columns,

• realize the importance of terms like migration rate, distribution constant,realize the importance of terms like migration rate, distribution constant, retention time, retention factor, selectivity factor

retention time, retention factor, selectivity factor and resolution,and resolution, •

explain the concept of theoretical plates and its importance, andexplain the concept of theoretical plates and its importance, and

• discuss the rate theorydiscuss the rate theory..

4.2

4.2

DEFINITION

DEFINITION AND

AND CLASSIFICATION

CLASSIFICATION

In order to classify the different forms of chromatography, we will have to once again In order to classify the different forms of chromatography, we will have to once again look into the definition of

look into the definition of chromatograchromatography. An analysis of phy. An analysis of the definition will help uthe definition will help uss to visualize the different ways the techniques can be classified and finally evolve a to visualize the different ways the techniques can be classified and finally evolve a system of classification so as to be

system of classification so as to be able to include most of the forms ofable to include most of the forms of chromatography.

chromatography.

4.2.1

4.2.1 DefinitionDefinition

Chromatography

Chromatography  is a  is a method of separation in which the componemethod of separation in which the components to be nts to be separatedseparated are distributed between two phases, one of these is called a

are distributed between two phases, one of these is called a stationary phasestationary phase and the and the other a

other a mobile phasemobile phase which moves on the stationary phase in a  which moves on the stationary phase in a definite direction. Thedefinite direction. The components of a mixture redistribute themselves between two phases b

components of a mixture redistribute themselves between two phases b y a processy a process which may be

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General Principles of General Principles of Chromatography Chromatography phase can be a

phase can be a solid or a liquid and the mobile phase a solid or a liquid and the mobile phase a liquid, gas or a supercriticalliquid, gas or a supercritical fluid. The stationary liquid is held on an

fluid. The stationary liquid is held on an inert solid support or coated on the walls of ainert solid support or coated on the walls of a capillary. The solid whether it is support or an

capillary. The solid whether it is support or an active material is held in active material is held in the form of athe form of a column, coated over a plate or presented as a

column, coated over a plate or presented as a paper. The latter two types come underpaper. The latter two types come under the head of

the head of two dimensional chromatographytwo dimensional chromatography..

4.2.2

4.2.2 ClassificationClassification

If we look into the definition of chromatography and the situation spelt out above, If we look into the definition of chromatography and the situation spelt out above, chromatograp

chromatography can be hy can be classified on the basis classified on the basis of the of the following:following: •

• The shape of the solid support.The shape of the solid support. •

• The nature of the mobile phase.The nature of the mobile phase. •

• The mechanism responsible for separation.The mechanism responsible for separation. If we go by the

If we go by the shape of support shape of support  in chromatography, there will be  in chromatography, there will be three dimensionalthree dimensional (column) and two dimensional (thin layer

(column) and two dimensional (thin layer and paper) chromatograpand paper) chromatography. In twohy. In two dimensional chromatogra

dimensional chromatography, there will only phy, there will only bebe twotwotypes of types of chromatograchromatography, thinphy, thin layer and paper and all the other

layer and paper and all the other forms of chromatograforms of chromatography will fall under the head ofphy will fall under the head of column chromatogra

column chromatography. This is too simple a phy. This is too simple a classification and does not take intoclassification and does not take into consideration the nature of the

consideration the nature of the mobile phase and the process mobile phase and the process responsible forresponsible for separation. Moreover,

separation. Moreover, two dimensional chromatographytwo dimensional chromatography is operated using the liquid is operated using the liquid only as a mobile phase.

only as a mobile phase.

If the classification is done purely on the basis

If the classification is done purely on the basis of the nature of mobile phase, there willof the nature of mobile phase, there will be three types of

be three types of chromatogchromatographic techniques –raphic techniques – liquid liquid ,, gasgas and and supercriticalsupercritical fluid fluid chromatogra

chromatography. Here, the first two are well phy. Here, the first two are well known and several advancemeknown and several advancements havents have already been made in

already been made in these two forms of these two forms of chromatograchromatography.phy. Supercritical fluidSupercritical fluid chromatography

chromatography  is relatively new and does not figure so frequently. Inspite of some is relatively new and does not figure so frequently. Inspite of some advantages it has still to

advantages it has still to become popular. Again, in this type of classification, a largebecome popular. Again, in this type of classification, a large number of chromatographic techniques with varied modes of separations fall under the number of chromatographic techniques with varied modes of separations fall under the head of

head of liquid chromatographyliquid chromatography.. In case the

In case the mechanism responsible for mechanism responsible for separationseparation is taken as the criteria for is taken as the criteria for classification, there will b

classification, there will be four distinct types e four distinct types of chromatographyof chromatography vizviz,, adsorptionadsorption,,  partition

 partition,, ionion--exchangeexchange and and sizesize exclusionexclusion. But we will . But we will have to separately mention thehave to separately mention the competing phases.

competing phases.

The above discussion clearly points out that no single criteria by itself

The above discussion clearly points out that no single criteria by itself will be able towill be able to effectively classify the various chromatographic techniques. We will have

effectively classify the various chromatographic techniques. We will have to take oneto take one as the main criteria

as the main criteria and make a sub-classification based on the other yardstick. Aand make a sub-classification based on the other yardstick. A simple approach seems to be to take the

simple approach seems to be to take the nature of the mobile phase as the nature of the mobile phase as the main criteriamain criteria and propose a sub-classification based on the

and propose a sub-classification based on the various mechanisms responsivarious mechanisms responsible forble for separation. If any special feature exists within a sub-classification it may be

separation. If any special feature exists within a sub-classification it may be pointedpointed out right there.

out right there.

For summing up the c

For summing up the c lassification of chromatographic techniques, it emerges thatlassification of chromatographic techniques, it emerges that there will be

there will be three types of chromatographic techniques.three types of chromatographic techniques. i)

i) Liquid Liquid chromatograchromatography.phy. ii)

ii) Gas chromatography, andGas chromatography, and iii)

iii) Supercritical fluid chromatograpSupercritical fluid chromatography.hy. We will discuss each one in

We will discuss each one in brief and take into consideration the other competitingbrief and take into consideration the other competiting phase, the mechanism responsible for separation and the

phase, the mechanism responsible for separation and the nature and shape of surface.nature and shape of surface. The following sub-sections will be devoted to discussion on these lines. Finally, the The following sub-sections will be devoted to discussion on these lines. Finally, the conclusion will be presented in the form of a t

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Chromatographic Chromatographic Methods-I

Methods-I SAQ 1SAQ 1 Based on the

Based on the shape of support, what will bshape of support, what will be the main e the main categories of chromatographycategories of chromatography??

………... ………... ………... ………... ………... ………... ………... ………... 4.2.3

4.2.3 Liquid ChromatographyLiquid Chromatography

The simplest one with liquid as a

The simplest one with liquid as a mobile phase seems to be based on partition betweenmobile phase seems to be based on partition between the liquid supported on an inert support and the other liquid acting as a

the liquid supported on an inert support and the other liquid acting as a mobile phase.mobile phase. This is known as

This is known as liquid- liquid partition chroliquid- liquid partition chromatography. The support holding thematography. The support holding the liquid can be in the form of three

liquid can be in the form of three dimensions (dimensions (columncolumn) or two dimensions () or two dimensions (thinthin layer layer  and

and paper  paper ). In the two dimensional form, the mobile phase can move from top to). In the two dimensional form, the mobile phase can move from top to bottom or bottom to top and thus, known as

bottom or bottom to top and thus, known as descendingdescending or or ascendingascending chromatographychromatography .. The two types of

The two types of partition chromatography are also distinguishable on the basis ofpartition chromatography are also distinguishable on the basis of relative polarities of the mobile and stationary phases.

relative polarities of the mobile and stationary phases. ConventionallyConventionally, the highly polar, the highly polar solvent is held on the support and the mobile phase is

solvent is held on the support and the mobile phase is relatively non-polar. This isrelatively non-polar. This is known as

known as normal-phase chromatographynormal-phase chromatography. If the stationary phase is non-polar and the. If the stationary phase is non-polar and the mobile phase relatively polar, it is termed as

mobile phase relatively polar, it is termed as reversed-phase chromatographyreversed-phase chromatography. An. An important modification in terms of stationary phase

important modification in terms of stationary phase is introduced by loading theis introduced by loading the extractant used for solvent extraction on

extractant used for solvent extraction on a hydrophobizea hydrophobized inert d inert support and irrigatingsupport and irrigating the support with aqueous solvents. This

the support with aqueous solvents. This is known asis known as extraction extraction chromatochromatographygraphy. At. At this point, it

this point, it may be important to may be important to introduce liquid chromatograpintroduce liquid chromatography on bonded phases.hy on bonded phases. Initially, the partition chromatography was only liquid-liquid type but now

Initially, the partition chromatography was only liquid-liquid type but now chromatograp

chromatography with hy with bonded phase has gained prominence.bonded phase has gained prominence.

In liquid-liquid type, a liquid stationary phase is held on the support packing by In liquid-liquid type, a liquid stationary phase is held on the support packing by physical adsorption while in bonded phase the

physical adsorption while in bonded phase the stationary phase is bonded chemicallystationary phase is bonded chemically to the support. One of the main disadvantages of the liquid-liquid system is the loss of to the support. One of the main disadvantages of the liquid-liquid system is the loss of stationary phase by dissolution in the

stationary phase by dissolution in the mobile phase. This requires periodic recoating ofmobile phase. This requires periodic recoating of the support particles. Furthermore, the solubility of

the support particles. Furthermore, the solubility of the stationary phase prohibits thethe stationary phase prohibits the use of liquid-phase packing for gradient elution. However, with bonded-phase, these use of liquid-phase packing for gradient elution. However, with bonded-phase, these problems are mitigated to a large extent. In

problems are mitigated to a large extent. In a majority of cases, the bonded- phasea majority of cases, the bonded- phase packings for partition chromatography are prepared from rigid silica or silica packings for partition chromatography are prepared from rigid silica or silica basedbased compositions. Bonded phase packings are classified as reversed phase

compositions. Bonded phase packings are classified as reversed phase when thewhen the bonded coating is non polar and as the normal phase if the

bonded coating is non polar and as the normal phase if the coating contains polarcoating contains polar functional groups. Mostly in reverse

functional groups. Mostly in reversed phase the coating is a d phase the coating is a CC88chain (chain (nn-octyl) or a C-octyl) or a C1818 chain (

chain (nn-octyldecyl). For normal phase polar bonded phases that have -octyldecyl). For normal phase polar bonded phases that have a diola diol (-C

(-C33HH66OCHOCH22CHOHCHCHOHCH22OH), cyano (-COH), cyano (-C22HH44CN), amino (-CCN), amino (-C33HH66NHNH22) and) and dimethylamino (-C

dimethylamino (-C33HH66N(CHN(CH33))22) functional groups are commercially available. We) functional groups are commercially available. We have seen that the nature of the

have seen that the nature of the stationary phase and the way it is held on stationary phase and the way it is held on the supportthe support are responsible for quite a

are responsible for quite a few diversifications in liquid partition chromatography. Iffew diversifications in liquid partition chromatography. If the solid support instead of holding the liquid acts as an a

the solid support instead of holding the liquid acts as an a ctive adsorbent, the relatedctive adsorbent, the related chromatography is known as

chromatography is known as liquid-solid adsorption liquid-solid adsorption chromatogrchromatographyaphy. This statement. This statement is true for

is true for both column and two dimensional chromatography. Sometimeboth column and two dimensional chromatography. Sometimes, it s, it becomesbecomes difficult to distinguish whether it is the partition or

difficult to distinguish whether it is the partition or the adsorption which is dominatingthe adsorption which is dominating in the separation.

in the separation.

An advancement in liquid adsorption chromatography was reported in the form of An advancement in liquid adsorption chromatography was reported in the form of affinity chromatograp

affinity chromatography. In this the column material, a hy. In this the column material, a gel like material, is gel like material, is attachedattached with a biospecific material through a spacer arm. This biospecific material is

with a biospecific material through a spacer arm. This biospecific material is known asknown as a ligand. The ligand selectively picks up a biological molecule from the

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General Principles of General Principles of Chromatography Chromatography form a complex. The desired biological molecule can be eluted by modest changes

form a complex. The desired biological molecule can be eluted by modest changes such as pH or ionic strength in the

such as pH or ionic strength in the eluant.eluant.

An important branch of liquid chromatography is

An important branch of liquid chromatography is ion ion exchange chromatographyexchange chromatography. This. This involves exchange of ions, cations or

involves exchange of ions, cations or anions, between an insoluble matrix and theanions, between an insoluble matrix and the solution which comes in contact with it. The

solution which comes in contact with it. The insoluble material which carriesinsoluble material which carries exchangeable cations and anions is known as

exchangeable cations and anions is known as ion exchanger. Those carryingion exchanger. Those carrying exchangeable cations are known as

exchangeable cations are known as cation exchangerscation exchangers and those with  and those with exchangeableexchangeable anions are known as

anions are known as anion exchangersanion exchangers. There are. There are amphoteric exchangersamphoteric exchangers which are which are capable of both cation

capable of both cation and anion exchange. Ion exchange, with a and anion exchange. Ion exchange, with a very few exceptions,very few exceptions, is a reversible process. In contrast to adsorption, ion exchange is a

is a reversible process. In contrast to adsorption, ion exchange is a stoichiometricstoichiometric process. Every ion removed from the solution is

process. Every ion removed from the solution is replaced by an equivalent amount ofreplaced by an equivalent amount of another ionic species of

another ionic species of the same sign. the same sign. Ion exchangers are organic and inorganic,Ion exchangers are organic and inorganic, natural and synthetic. Most of the ion exchangers in daily use are o

natural and synthetic. Most of the ion exchangers in daily use are o rganic resins.rganic resins. There is another form of

There is another form of chromatograchromatography which is synonymous to ion- exchangephy which is synonymous to ion- exchange chromatography and is known as

chromatography and is known as ion-pair ion-pair  (or (or paired-ion paired-ion)) chromatographychromatography. It is a type. It is a type of reversed phase chromatography and used for the separation of

of reversed phase chromatography and used for the separation of ionic species. In this,ionic species. In this, the mobile phase consists of

the mobile phase consists of an aqueous buffer containing an organic solventan aqueous buffer containing an organic solvent (methanol/ acetonitrile) and an organic compound containing a counter ion

(methanol/ acetonitrile) and an organic compound containing a counter ion of oppositeof opposite in charge to the analyte. The

in charge to the analyte. The counter ion combines with the analyte to form a neutralcounter ion combines with the analyte to form a neutral ion pair. The neutral ion pair is

ion pair. The neutral ion pair is retained on the non- polar stationary phase. It isretained on the non- polar stationary phase. It is subsequently eluted with an aqueous solution of

subsequently eluted with an aqueous solution of methanol or other water solublemethanol or other water soluble organic solvents. Ionic or partial i

organic solvents. Ionic or partial ionic compounds can be chromatographed ononic compounds can be chromatographed on reversed phase columns by using ion pairing reagents. Typically these

reversed phase columns by using ion pairing reagents. Typically these reagents arereagents are long-chain alkyl anions or cations. When

long-chain alkyl anions or cations. When these reagents are used in these reagents are used in dilute solutions,dilute solutions, the retention of the analytical ion

the retention of the analytical ion can increase. Ccan increase. C55 to C to C1010 alkyl sulphonates are alkyl sulphonates are commonly used cationic compounds. Tetra alkyl ammonium salts

commonly used cationic compounds. Tetra alkyl ammonium salts (tetramethyl-,(tetramethyl-, tetrabutyl-, and ammonium salt or

tetrabutyl-, and ammonium salt or triethyl- (Ctriethyl- (C55- C- C88) alkyl ammonium salts are) alkyl ammonium salts are generally used in the case of anionic solutes.

generally used in the case of anionic solutes. The applications of ion

The applications of ion pair chromatography frequpair chromatography frequently overlap with those of ently overlap with those of ion- ion-exchange chromatography

exchange chromatography. Ion . Ion chromatogchromatography is raphy is generally used for the generally used for the separationsseparations which are difficult to attain with

which are difficult to attain with ion exchange chromation exchange chromatography. A typical example isography. A typical example is the separation of chlorate and nitrate which is difficult to

the separation of chlorate and nitrate which is difficult to achieve on an ion exchangeachieve on an ion exchange but easily attained

but easily attained by ion pair chromatography.by ion pair chromatography. Size

Size exclusion chromatographyexclusion chromatography is  is a very useful form of a very useful form of liquid chromatographyliquid chromatography employed for the separation of high molecular weight substances. This is also called employed for the separation of high molecular weight substances. This is also called gel permeation

gel permeation or or gel filtrationgel filtration chromatographychromatography . Two important types of column. Two important types of column packing material used for chromatography in aqueous phase are

packing material used for chromatography in aqueous phase are cross-linked dextranscross-linked dextrans and

and polyacrylamide polyacrylamide. The process responsible for the separation is due to the. The process responsible for the separation is due to the distribution of solute between the aqueous phase with in the gel particles and

distribution of solute between the aqueous phase with in the gel particles and externalexternal water. The selectivity is achieved on the

water. The selectivity is achieved on the basis of pore size of the basis of pore size of the gel. A solute which isgel. A solute which is small enough may enter the gel phase. In a

small enough may enter the gel phase. In a column, this will have retarding effect. Thecolumn, this will have retarding effect. The molecules which have penetrated the gel, will spend their time she

molecules which have penetrated the gel, will spend their time she ltered from theltered from the moving phase. At the other extreme if the molecule is

moving phase. At the other extreme if the molecule is large enough not to be able tolarge enough not to be able to enter the pores will spend all the

enter the pores will spend all the time in the time in the mobile phase and move rapidly througmobile phase and move rapidly throughh the column. In between there will be molecules of

the column. In between there will be molecules of intermediate size which canintermediate size which can penetrate in some degree and their movement down the column will be somewhat penetrate in some degree and their movement down the column will be somewhat retarded. This forms the basis for separating the molecules of different sizes. retarded. This forms the basis for separating the molecules of different sizes. While concluding the discussion on liquid chromatography, it will

While concluding the discussion on liquid chromatography, it will be important tobe important to point out that the most significant advancement in liquid chromatography came in the point out that the most significant advancement in liquid chromatography came in the form of

form of high performancehigh performance or or high pressure high pressure liquid chromatographliquid chromatographyy. This improved. This improved version is more or

version is more or less available for dless available for d ifferent forms of liquid chromatography. Theifferent forms of liquid chromatography. The separation is performed on a

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Chromatographic Chromatographic Methods-I

Methods-I particles of very small size, 3 particles of very small size, 3 -10 µm. that permit partition, ion that permit partition, ion exchange or molecular permeation. The pressure generallyexchange or molecular permeation. The pressure generally-10 µm. The material may be an adsorbent or materialsThe material may be an adsorbent or materials used is around 6000 psi. The mobile phase may be composed of one solvent or a used is around 6000 psi. The mobile phase may be composed of one solvent or a mixture of solvents with or

mixture of solvents with or without modifiers. Now high pressure thin layerwithout modifiers. Now high pressure thin layer chromatograp

chromatography (HPTLC) has hy (HPTLC) has also gained prominence.also gained prominence.

SAQ 2 SAQ 2

What is the

What is the main advantage of reversed phase liquid chromatography?main advantage of reversed phase liquid chromatography?

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 3 SAQ 3 What is

What is extraction chromatographyextraction chromatography??

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 4 SAQ 4

What particular property of the gel

What particular property of the gel is responsible for separations in gelis responsible for separations in gel chromatography? chromatography? ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... 4.2.4

4.2.4 Gas ChromatographyGas Chromatography

In gas chromatography

In gas chromatography, the mobile phase is , the mobile phase is a gas. The gas does not react a gas. The gas does not react with thewith the molecules of the analyte. It only carries the

molecules of the analyte. It only carries the sample through the column. Two types ofsample through the column. Two types of gas chromatography are

gas chromatography are encounteredencountered •

• Gas-solid chromatography (GSC).Gas-solid chromatography (GSC). •

Gas-liquid chromatography (GLC).Gas-liquid chromatography (GLC). In GSC, the retention of the analyte is due

In GSC, the retention of the analyte is due toto physical adsor physical adsorptionption on the column on the column material. In GLC, the separation takes place because of

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General Principles of General Principles of Chromatography Chromatography mobile phase and the liquid immobilized on the surface of an inert

mobile phase and the liquid immobilized on the surface of an inert solid. GSC is not assolid. GSC is not as popular as GLC because of some limitations of the former mode. In GSC,

popular as GLC because of some limitations of the former mode. In GSC, thethe retention time may be unusually large and high temperatures may be required to retention time may be unusually large and high temperatures may be required to desorb the species. There may be severe tailing due to

desorb the species. There may be severe tailing due to non-linear character ofnon-linear character of adsorption. The active adsorbent may catalyze the adsorbed species and change it to adsorption. The active adsorbent may catalyze the adsorbed species and change it to some other compound. As a result of all these GSC has

some other compound. As a result of all these GSC has a limited utility and gasa limited utility and gas chromatograp

chromatography more or hy more or less refers less refers to gas-liquid partition chromatography. Generally,to gas-liquid partition chromatography. Generally, two types of

two types of columns are used in gas columns are used in gas chromatograchromatography, packed and open tubular orphy, packed and open tubular or capillary columns. The chromatographic columns vary in length from less

capillary columns. The chromatographic columns vary in length from less than 2m tothan 2m to 50m or more. They are constructed of stainless steel, aluminium glass or fused glass or 50m or more. They are constructed of stainless steel, aluminium glass or fused glass or teflon. Open tubular columns are two

teflon. Open tubular columns are two basic types, wall-coated open tubular andbasic types, wall-coated open tubular and support-coated open tubular. Wall coated columns are capillary tubes

support-coated open tubular. Wall coated columns are capillary tubes coated with thincoated with thin layer of stationary phase. In

layer of stationary phase. In support coated open tubular columns the inner surface issupport coated open tubular columns the inner surface is lined with a thin

lined with a thin film of support material. This support material holds the stationaryfilm of support material. This support material holds the stationary phase. The most

phase. The most commonly used stationary phases are polydimethyl siloaxanes.commonly used stationary phases are polydimethyl siloaxanes. Organic species bonded to a solid surface are also used as

Organic species bonded to a solid surface are also used as stationary phases.stationary phases.

SAQ 5 SAQ 5

What are the

What are the two types of columns used two types of columns used in gas chromatography?in gas chromatography?

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... 4.2.5

4.2.5 Supercritical Fluid ChromatographySupercritical Fluid Chromatography

During the last

During the last quarter of the twentieth century, quarter of the twentieth century, another type of chromatography withanother type of chromatography with an entirely new type of mobile phase, supercritical fluid, has come up on t

an entirely new type of mobile phase, supercritical fluid, has come up on t he forefront.he forefront. It is basically partition chromatography. Here, instead of a liquid or a

It is basically partition chromatography. Here, instead of a liquid or a gas, we use agas, we use a supercritical fluid. In order to

supercritical fluid. In order to understand aunderstand a supercritical fluid supercritical fluid  we will have  we will have to knowto know critical temperature

critical temperature and and pressure pressure of a substance. The of a substance. The critical temperaturecritical temperature of a of a substance is the temperature above which a distinct liquid phase cannot exist substance is the temperature above which a distinct liquid phase cannot exist regardless of pressure. The vapour pressur

regardless of pressure. The vapour pressure of a substance at e of a substance at its critical temperature isits critical temperature is its

its critical pressurecritical pressure. At temperature and pressure above its critical temperature and. At temperature and pressure above its critical temperature and pressure (

pressure (critical pointcritical point), a substance is known as), a substance is known as supercritical fluidsupercritical fluid. The properties. The properties like density, viscosity and other

like density, viscosity and other characteristics of supercritical fluids are characteristics of supercritical fluids are intermediateintermediate between the gaseous and liquid state. The most well known compound used as a between the gaseous and liquid state. The most well known compound used as a supercritical fluid in chromatography is CO

supercritical fluid in chromatography is CO22. The other compounds in use . The other compounds in use are nitrousare nitrous oxide, ammonia and

oxide, ammonia and nn- butane. There are - butane. There are about two dozen compounds which haveabout two dozen compounds which have been used as

been used as mobile phases in supercritical chromatography. The critical temperaturemobile phases in supercritical chromatography. The critical temperature and pressure at these temperatures is well within the operating conditions used in and pressure at these temperatures is well within the operating conditions used in HPLC.

HPLC.

One remarkable property of supercritical fluids which is related to their high densities One remarkable property of supercritical fluids which is related to their high densities (0.2- 0.5 g/cm

(0.2- 0.5 g/cm33) is ) is their capability to dissolve ltheir capability to dissolve large non-volatile molecules. Anotherarge non-volatile molecules. Another property which is associated with many supercritical fluids is that the dissolved property which is associated with many supercritical fluids is that the dissolved analyte can be easily recovered by allowing the solutions to equilibrate with the analyte can be easily recovered by allowing the solutions to equilibrate with the atmosphere at a relatively low temperature. This is i

atmosphere at a relatively low temperature. This is i mportant if the analyte happens tomportant if the analyte happens to be thermally unstable. A significant point which goes in the favour of many

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Chromatographic Chromatographic Methods-I

Methods-I supercritical fluids is that they are inexpensive and non- toxic. They can be easilysupercritical fluids is that they are inexpensive and non- toxic. They can be easilyallowed to evaporate in aallowed to evaporate in atmosphere with no harmful effects.tmosphere with no harmful effects. As a matter of fact

As a matter of fact supercritical fluid chromatogsupercritical fluid chromatography is a hybrid of gas and liquidraphy is a hybrid of gas and liquid chromatograp

chromatography. It combines some of hy. It combines some of the advantages of both liquid and the advantages of both liquid and gasgas chromatograp

chromatography. In supercritical fluid chromatography, the mobile has hy. In supercritical fluid chromatography, the mobile has a low viscositya low viscosity and high self diffusion coefficient. The

and high self diffusion coefficient. The mobile phase can be further modified bymobile phase can be further modified by addition of polar modifier in

addition of polar modifier in small amounts. In this small amounts. In this type of chromatography bothtype of chromatography both packed and open tubular columns are used.

packed and open tubular columns are used. In open tubular columns, the internalIn open tubular columns, the internal coating is of

coating is of siloaxanes of various types and that siloaxanes of various types and that of bonded phases.of bonded phases.

Like gas chromatography, supercritical fluid chromatography is faster than liquid Like gas chromatography, supercritical fluid chromatography is faster than liquid chromatograp

chromatography because of lower viscosity. Diffusion rates hy because of lower viscosity. Diffusion rates in supercritical fluidin supercritical fluid chromatograp

chromatography are intermediate between those in gases and in hy are intermediate between those in gases and in liquids. As a result ofliquids. As a result of this, the band broadening is greater in supercritical fluids than in liquids but lesser this, the band broadening is greater in supercritical fluids than in liquids but lesser thanthan in gases. In nutshell, the intermediate diffusivities and viscosities of supercritical in gases. In nutshell, the intermediate diffusivities and viscosities of supercritical fluids may lead to

fluids may lead to faster separations than are obtained with faster separations than are obtained with liquid chromatographyliquid chromatography accompanied by lower zone

accompanied by lower zone broadening than observed in broadening than observed in gas chromatographygas chromatography.. This new type of

This new type of chromatographchromatography has started finding applications for y has started finding applications for a wide variety ofa wide variety of materials like natural products, foods, pesticides,

materials like natural products, foods, pesticides, polymers, fossil fuels and explosives.polymers, fossil fuels and explosives. In spite of

In spite of gaining prominencegaining prominence, this particular , this particular techniqutechnique has e has not been discussed innot been discussed in detail in this course. It is

detail in this course. It is still to match still to match in popularity with other separation techniquesin popularity with other separation techniques included in the course.

included in the course.

SAQ 6 SAQ 6

Mention two important advantages of

Mention two important advantages of supercritical fluid chromatography.supercritical fluid chromatography.

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... 4.2.6 4.2.6 ConclusionsConclusions

To sum up the discussion on classification of chromatographic techniques, it can be To sum up the discussion on classification of chromatographic techniques, it can be said that a

said that a large number of techniques fall under the large number of techniques fall under the head of liquid chromatography.head of liquid chromatography. This is mainly because of the fact

This is mainly because of the fact that a large number of mechanisms operate with athat a large number of mechanisms operate with a liquid as a mobile phase. Moreover, there is the

liquid as a mobile phase. Moreover, there is the availability of a variety of types ofavailability of a variety of types of surfaces as stationary phases. Also, there have been some improvements in the surfaces as stationary phases. Also, there have been some improvements in the methodolog

methodology. The most y. The most important in this regard is important in this regard is the introduction of highthe introduction of high performance liquid chromatography

performance liquid chromatography. Gas . Gas chromatograchromatography, inspite of phy, inspite of being a being a valuablevaluable tool for separations, remains mostly confined to

tool for separations, remains mostly confined to gas-liquid chromatoggas-liquid chromatography because ofraphy because of certain limitations of

certain limitations of its gas-solid version. Supercritical fluid chromatography has toits gas-solid version. Supercritical fluid chromatography has to still catch

still catch up liquid and gas up liquid and gas chromatograchromatography in their phy in their popularitypopularity. The . The entire discussionentire discussion on classification is being summed up in Table 4.1.

on classification is being summed up in Table 4.1. It is not possible to

It is not possible to include all the different features discussed in the text in include all the different features discussed in the text in the tablethe table because it may become too congested. However, the salient features of the

because it may become too congested. However, the salient features of the classifications are being presented in the table.

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General Principles of General Principles of Chromatography Chromatography Table 4.1:

Table 4.1: Classification of Classification of Chromatographic MethodsChromatographic Methods General

General Category Category StationaStationary ry Phase Phase Mechanism Mechanism ModesModes Liquid Liquid Chromatography Chromatography (LC)[Mobile (LC)[Mobile Phase: liquid] Phase: liquid] Liquid adsorbed on Liquid adsorbed on a solid or organic a solid or organic bonded to solid bonded to solid surface surface Partition

Partition Column, Column, two two dimensional;dimensional; normal, reversed phase; normal, reversed phase; loaded extractant; ion pair loaded extractant; ion pair formation

formation Solid

Solid adsorbent adsorbent Adsorption Adsorption Column, Column, two two dimensional,dimensional, adsorbent, biospecific adsorbent, biospecific molecule attached to molecule attached to surface surface Ion Exchanger Ion Exchanger Liquid in Liquid in interstices of a gel interstices of a gel Ion Exchange Ion Exchange Partition/ Sieving Partition/ Sieving

Column, two dimensional, Column, two dimensional, batch, column, thin layer batch, column, thin layer

Gas Gas Chromatography Chromatography (GC)[Mobile (GC)[Mobile phase: gas] phase: gas] Liquid adsorbed on Liquid adsorbed on a solid or organic a solid or organic bonded to solid bonded to solid surface surface Solid adsorbent Solid adsorbent Partition Partition Adsorption Adsorption Column Column Column Column Supercritical Supercritical Fluid Fluid Chromatography Chromatography (SFC)[Mobile (SFC)[Mobile phase: supercritical phase: supercritical fluid] fluid] Organic species Organic species bonded to solid bonded to solid surface surface Partition Column Partition Column Note

Note: The different forms of liquid chromatography can be carried in the high: The different forms of liquid chromatography can be carried in the high performance liquid chromatography mode; the exception being if the paper is used as performance liquid chromatography mode; the exception being if the paper is used as the support.

the support.

4.3

4.3

ELUTION

ELUTION OF

OF COLUMNS

COLUMNS

After having learnt the classification of chromatographic methods, it is important to After having learnt the classification of chromatographic methods, it is important to look into some concepts which

look into some concepts which may explain the separation process. From themay explain the separation process. From the discussion, it can be noted that the

discussion, it can be noted that the separations are conducted on columnseparations are conducted on columns or on planars or on planar surfaces. The equilibria upon which the

surfaces. The equilibria upon which the two types of chromatography are based aretwo types of chromatography are based are identical. The theory developed for column

identical. The theory developed for column chromatogchromatography is adapted to raphy is adapted to planarplanar chromatography.

chromatography.

To start with we look into a simple picture as to how the two substances X and Y are To start with we look into a simple picture as to how the two substances X and Y are separated on a column by elution with a liquid as a mobile phase. Actually, elution separated on a column by elution with a liquid as a mobile phase. Actually, elution involves washing a species through a column by continuous addition of fresh solvent. involves washing a species through a column by continuous addition of fresh solvent. The different stages of separation are schematically shown in Fig. 4.1. The sample is The different stages of separation are schematically shown in Fig. 4.1. The sample is introduced at the head of column (time

introduced at the head of column (time t t 00) where the components of the sample) where the components of the sample distribute themselves between the two phases. Further addition of mobile phase pushes distribute themselves between the two phases. Further addition of mobile phase pushes the solvent containing a part of the sample down the column. Here further partition the solvent containing a part of the sample down the column. Here further partition between the mobile phase and fresh portion of stationary phase occurs (time between the mobile phase and fresh portion of stationary phase occurs (time t t 11).). Simultaneously, partitioning between the fresh solvent and the stationary phase is Simultaneously, partitioning between the fresh solvent and the stationary phase is taking place at the site of the original sample.

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Chromatographic Chromatographic Methods-I Methods-I (a) (a) (b) (b) Fig. 4.1:

Fig. 4.1: (a) Column separation of a mixture of components X a(a) Column separation of a mixture of components X and Y; and (b) Output ofnd Y; and (b) Output of signal detector at various stages

signal detector at various stages

In this way, continued addition of solvent carries the solute molecules down the In this way, continued addition of solvent carries the solute molecules down the column in a continuous series of

column in a continuous series of transfers between the mobile and stationary phases.transfers between the mobile and stationary phases. The solute movement can only occur in the mobile phase, therefore, the average rate at The solute movement can only occur in the mobile phase, therefore, the average rate at which a solute zone migrates down the column depends upon fraction of time it

which a solute zone migrates down the column depends upon fraction of time it spends in that phase. This fraction of time spent i

spends in that phase. This fraction of time spent i s small for solutes which are stronglys small for solutes which are strongly retained by the stationary phase. If we refer to Fig. 4.1,

retained by the stationary phase. If we refer to Fig. 4.1, this is the situation for Y. this is the situation for Y. OnOn the other hand, the retention of X is more

the other hand, the retention of X is more in the mobile phase. As a in the mobile phase. As a result of thisresult of this difference in retention rates, the

difference in retention rates, the components of mixture separate into bands or zones,components of mixture separate into bands or zones, (time t

(time t22). If sufficient quantity of solvent is passed, the two ). If sufficient quantity of solvent is passed, the two species are isolated asspecies are isolated as individual zones and then pass out of the column where they can be detected or individual zones and then pass out of the column where they can be detected or collected. (time t

collected. (time t33 and time t and time t44).).

It should be noticed that during the separation procedure, the dilution of analyte takes It should be noticed that during the separation procedure, the dilution of analyte takes place. The analytes get more dilute

place. The analytes get more dilute than they were present in the original mixture.than they were present in the original mixture. Therefore, the detectors employed for the

Therefore, the detectors employed for the separated componentseparated components should be s should be invariablyinvariably more sensitive than would be required if the separation was not needed. The detector more sensitive than would be required if the separation was not needed. The detector responds to the concentration of the solute coming out of the column. When its signal responds to the concentration of the solute coming out of the column. When its signal is plotted against time or

is plotted against time or the volume of the mobile phase a series of the volume of the mobile phase a series of peaks are plotted.peaks are plotted. Such a plot is known as a

Such a plot is known as a chromatogramchromatogram. The chromatogram is useful for both. The chromatogram is useful for both qualitative and quantitative analysis. The position of the peak on time axis

qualitative and quantitative analysis. The position of the peak on time axis helps tohelps to identify the components and the areas

identify the components and the areas under the peak provide the aunder the peak provide the amount of eachmount of each component.

component.

Fig. 4.2:

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General Principles of General Principles of Chromatography Chromatography In the separation of X and Y,

In the separation of X and Y, the species Y is the species Y is more strongly held by the column andmore strongly held by the column and thus it lags. During the

thus it lags. During the movememovement in the nt in the column the distance between the two zonescolumn the distance between the two zones increases. At the same time,

increases. At the same time, broadening of two zones takes place. This lowers thebroadening of two zones takes place. This lowers the efficiency of the separation method. There can be two approaches to increase the efficiency of the separation method. There can be two approaches to increase the resolution of species.

resolution of species. 1.

1. One component moves at a faster pace and the other at One component moves at a faster pace and the other at a slower rate.a slower rate. 2.

2. The rate of zone broadening for the two species is decreased.The rate of zone broadening for the two species is decreased. The two situations are illustrated in Fig. 4.3.

The two situations are illustrated in Fig. 4.3.

Fig. 4.3: Two approaches to improve the binary separation; (i) initial c

Fig. 4.3: Two approaches to improve the binary separation; (i) initial c hromatogram withhromatogram with overlapping peaks improved by; (ii) increase in band separation; and (iii)

overlapping peaks improved by; (ii) increase in band separation; and (iii) decrease in band width

decrease in band width

4.4

4.4

MIGRATION

MIGRATION RATES

RATES

From the above example, it becomes clear that

From the above example, it becomes clear that the effectiveness of a column inthe effectiveness of a column in separating the two solutes depends in part on the relative rate

separating the two solutes depends in part on the relative rate at which the two areat which the two are eluted from the

eluted from the column. The rates depend upon the magnitude of column. The rates depend upon the magnitude of the equilibriumthe equilibrium constants of reactions by which the solutes distribute themselves between the mobile constants of reactions by which the solutes distribute themselves between the mobile and stationary phases.

and stationary phases.

4.4.1

4.4.1 Distribution ConstantDistribution Constant

In chromatography

In chromatography, the , the distribution equilibrium is often simple and is distribution equilibrium is often simple and is described by thedescribed by the equations that involve the transfer of an analyte between the mobile and stationary equations that involve the transfer of an analyte between the mobile and stationary phase. Thus, for the solute X, we may write

phase. Thus, for the solute X, we may write X

Xmobilemobile ⇔⇔ X Xstationarystationary The equilibrium constant,

The equilibrium constant, K K , for the reaction is , for the reaction is known asknown as distributiondistribution constant constant  and is and is given by the

given by the following expressiofollowing expressionn K  K == ..  M   M  S  S  C  C  C  C  … … (4.1)(4.1) Where,

Where, C C S S  is the  is the molar concentration of the solute in the stationary phase andmolar concentration of the solute in the stationary phase and C C  M  M  is its is its molar concentration in the mobile

molar concentration in the mobile phase. Ideally,phase. Ideally, C C S S  is directly proportional to is directly proportional to C C  M  M  over over a wide range of concentration. If such a situation exists, the

a wide range of concentration. If such a situation exists, the chromatograchromatography is knownphy is known as

as linear linear chromatogrchromatographyaphy and results in symmetric Gaussian and results in symmetric Gaussian type peaks. The retentiontype peaks. The retention times are

times are independent of the amount of the independent of the amount of the compound injected. Ideal behaviour, on thecompound injected. Ideal behaviour, on the other hand, is not attainable in

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Chromatographic Chromatographic Methods-I

Methods-I as non-linear chromatographynon-ideal behaviour which operates and non-ideal behaviour which operates and the corresponding chromatogasnon-linear chromatography.. the corresponding chromatography is knownraphy is known

SAQ 7 SAQ 7

What conditions will lead

What conditions will lead to linear ideal to linear ideal chromatograpchromatography?hy?

………... ………... ………... ………... ………... ………... ………... ………... 4.4.2

4.4.2 Retention TimeRetention Time

In the preceding sub-section, we have

In the preceding sub-section, we have introduced a term known asintroduced a term known as retentionretention timetime. It is. It is time taken after sample injection for the

time taken after sample injection for the analyte peak to reach the detector. It isanalyte peak to reach the detector. It is denoted by the symbol

denoted by the symbol t t  R Rand in Fig. 4.4 is represented.and in Fig. 4.4 is represented.

Fig. 4.4:

Fig. 4.4: Small peak at the left represents a species that is not retained and reachSmall peak at the left represents a species that is not retained and reaches thees the detector immediately.

detector immediately. t t M  M  is approximately equal to the ti is approximately equal to the ti me needed for theme needed for the molecules of the mobile phase to pass thr

molecules of the mobile phase to pass thr ough the columnough the column The small peak in the beginning on the left

The small peak in the beginning on the left is for the species that is is for the species that is not retained at thenot retained at the column. Sometimes, the sample may contain an unretained species. If it is the

column. Sometimes, the sample may contain an unretained species. If it is the re it mayre it may be added because it helps in peak identification. The time

be added because it helps in peak identification. The time ttMM for the unretained species for the unretained species is the same as

is the same as the average rate of migration of mobile phase molecules. If L is the average rate of migration of mobile phase molecules. If L is thethe length of the column, the average rate of migration of solute molecules is

length of the column, the average rate of migration of solute molecules is given by thegiven by the following expression: following expression: vv==  R  R t  t   L  L … … (4.2)(4.2) Similarly, the average linear rate of migration of mobile phase molecule

Similarly, the average linear rate of migration of mobile phase molecule u u==  M   M  t  t   L  L … … (4.3)(4.3) where,

where, t t  M  M  is called dead  is called dead time. It is the time. It is the time required for the average molecules of thetime required for the average molecules of the mobile phase to pass throug

mobile phase to pass through the column. h the column. After getting familiar with the termsAfter getting familiar with the terms distribution constant and retention time, it may be important to look into the distribution constant and retention time, it may be important to look into the relationship between the two terms. We express migration rate of a solute relationship between the two terms. We express migration rate of a solute vv = = uu × fraction of time solute  × fraction of time solute spends in the mobile phasespends in the mobile phase

… (4.4) … (4.4) This fraction is equal to average number of moles of a solute in the mobile phase at This fraction is equal to average number of moles of a solute in the mobile phase at any instant divided by the total number of moles of the solute

any instant divided by the total number of moles of the solute in the column.in the column.

vv== uu × ×  Moles of the solute i Moles of the solute in mobile phasen mobile phase … … (4.5)(4.5) Total moles in the column

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General Principles of General Principles of Chromatography Chromatography

The number of moles of the solute in the mobile and stationary phases are the The number of moles of the solute in the mobile and stationary phases are the product of their concentration and respective volumes

product of their concentration and respective volumes

vv== uu × × S  S  S  S   M   M   M   M   M   M   M   M  V  V  C  C  V  V  C  C  V  V  C  C  + + … … (4.6)(4.6) where,

where, C C  and and V V  denote the concentration and  denote the concentration and volume, respectivelyvolume, respectively

vv = = uu × ×  M   M   M   M  S  S  S  S V V  /C  /C  V V  C  C  + + 1 1 1 1 … … (4.7)(4.7) We know that We know that KK ==  M   M  S  S  C  C  C  C  Thus, we have a

Thus, we have a relation between solute migration and distribution constantrelation between solute migration and distribution constant

v = u × v = u ×  M   M  S  S  /V  /V  KV  KV  + + 1 1 1 1 … (4.8) … (4.8) 4.4.3

4.4.3 Retention FactorRetention Factor

There is another term known as retention factor or capacity factor which is commonly There is another term known as retention factor or capacity factor which is commonly used to describe the migration rates of the solutes.

used to describe the migration rates of the solutes. For a solute X, the retentionFor a solute X, the retention factor

factork k  X  X '' is expressed asis expressed as ''  X   X  k  k  ==  M   M  S  S   X   X  V  V  V  V  K  K  … (4.9) … (4.9) K 

K XX= distribution constant for X= distribution constant for X

Substituting Eq. 4.9 in Eq. 4.8, we get Substituting Eq. 4.9 in Eq. 4.8, we get

vv== uu × ×  X   X  k  k  + + 1 1 1 1 … …(4.10)(4.10)

Now, substituting Eqs. 4.3 and 4.2 into Eq. 4.10, we get Now, substituting Eqs. 4.3 and 4.2 into Eq. 4.10, we get

 R  R t  t   L  L = =  M   M  t  t   L  L  ×  ×  X   X  k  k  + + 1 1 1 1 … … (4.11)(4.11)

Rearranging the above equation, we get Rearranging the above equation, we get

k  k  X  X '' ==  M   M   M   M   R  R t  t  t  t  t  t  −− … … (4.12)(4.12) We can determine

We can determine t t  R R and and t t  M  M  from a chromatogram. If the retention factor is much less from a chromatogram. If the retention factor is much less than unity, elution occurs so rapidly that the accurate determination of retention factor than unity, elution occurs so rapidly that the accurate determination of retention factor is difficult. It the

is difficult. It the retention factor is larger, somewhere 20 or 30, the elution times areretention factor is larger, somewhere 20 or 30, the elution times are unusually long. Ideally separations of solutes are

unusually long. Ideally separations of solutes are carried out for which the carried out for which the retentionretention factors are between 2 and 10.

factors are between 2 and 10.

SAQ 8 SAQ 8

What are the problems if the

What are the problems if the retention factor is too small or too large?retention factor is too small or too large?

………... ………... ………... ………... ………... ………...

Figure

Updating...

References