Unit 5 Liquid Column Chromatography

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Liquid Column Liquid Column Chromatography Chromatography

UNIT

UNIT 5

5 LIQUID

LIQUID COLUMN

COLUMN

CHROMATOGRAPHY

Structure Structure 5.1 5.1 IntroductionIntroduction Objectives Objectives 5.2

5.2 Recapitulation of BasicsRecapitulation of Basics Liquid-Solid Chromatography Liquid-Solid Chromatography Liquid-Liquid Chromatography Liquid-Liquid Chromatography 5.3

5.3 Experimental Set upExperimental Set up Equipment

Equipment 5.4

5.4 Choice of Stationary and Mobile PhasesChoice of Stationary and Mobile Phases Stationary Phases Used in Liquid-Solid Column C

Stationary Phases Used in Liquid-Solid Column C hromatographyhromatography Stationary Phases Used in Liquid-Liquid Column C

Stationary Phases Used in Liquid-Liquid Column C hromatographyhromatography Mobile Phases in Liquid Column Chromatography

Mobile Phases in Liquid Column Chromatography 5.5

5.5 Development TechniquesDevelopment Techniques Frontal Analysis Frontal Analysis Displacement Development Displacement Development Elution Analysis Elution Analysis 5.6

5.6 Basic Aspects of HPLCBasic Aspects of HPLC 5.7

5.7 ApplicationsApplications 5.8

5.8 SummarySummary 5.9

5.9 Terminal QuestionsTerminal Questions 5.10

5.10 AnswersAnswers

5.1

5.1 INTRODUCTION

INTRODUCTION

In the previous unit

In the previous unit (Unit 4), a (Unit 4), a thorough discussiothorough discussion on the n on the classification and generalclassification and general principles of chromatography has been presented. While classifying the different principles of chromatography has been presented. While classifying the different chromatograp

chromatographic techniques, the main hic techniques, the main criteria used was criteria used was thethe nature of the mobilenature of the mobile phase

phase. It was pointed out that a . It was pointed out that a large number of diversifications are available in thelarge number of diversifications are available in the case of liquid chromatography. These are mainly due to

case of liquid chromatography. These are mainly due to shape of the shape of the support support (column (column and two

and two dimensional),dimensional), nature of support nature of support (simple and bonded) and the (simple and bonded) and the mechanismmechanism (adsorption, partition, ion exchange and sieving) responsible for separations. In this (adsorption, partition, ion exchange and sieving) responsible for separations. In this unit, it is

unit, it is proposed to discussproposed to discuss liquid column chromatographyliquid column chromatography. Normally, in the. Normally, in the liquid column chromatography, the different mechanisms cited above should be liquid column chromatography, the different mechanisms cited above should be included but in the

included but in the general parlance of chromatographygeneral parlance of chromatography, the , the technique includes onlytechnique includes only two mechanisms,

two mechanisms, adsorptionadsorption and and partition partition. Thus, the discussion in this unit will. Thus, the discussion in this unit will confine to

confine to liquid- solid liquid- solid adsorptioadsorptionn and and liquid- liquid liquid- liquid partition chromatographypartition chromatography. In this. In this course, separate units have been

course, separate units have been assigned forassigned for ion exchangeion exchange and and gel sievinggel sieving chromatography

chromatography . Thus, we . Thus, we will be discussing only liquid-solid adsorptionwill be discussing only liquid-solid adsorption chromatograp

chromatography (LSC) and hy (LSC) and liquid- liquid partition liquid- liquid partition chromatograchromatography (LLC). Thephy (LLC). The situation is more or less similar

situation is more or less similar to gas chromatography where we have GSC and GLC.to gas chromatography where we have GSC and GLC. If we compare the

If we compare the two important types of chromatography,two important types of chromatography, vizviz gas and liquid gas and liquid chromatograp

chromatography, some of hy, some of the advantages of the advantages of liquid chromatography become veryliquid chromatography become very apparent. The tremendous ability of gas

apparent. The tremendous ability of gas chromatogrchromatography to separate and aphy to separate and analyzeanalyze complex mixtures is widely appreciated but the drawback of this technique is that only complex mixtures is widely appreciated but the drawback of this technique is that only 20% of known organic compounds can be handled satisfactorily by gas

20% of known organic compounds can be handled satisfactorily by gas chromatograp

chromatography. Liquid chromatography, on the other hand, is hy. Liquid chromatography, on the other hand, is not limited by not limited by samplesample volatility or thermal stability. Thus, liquid chromatography is ideally suited for the volatility or thermal stability. Thus, liquid chromatography is ideally suited for the separation of macromolecules, ionic species, labile material products and a wide separation of macromolecules, ionic species, labile material products and a wide variety of other

variety of other high molecular weight compounds. Liquid high molecular weight compounds. Liquid chromatogchromatography alsoraphy also enjoys certain other advantages over gas chromatography in view of

enjoys certain other advantages over gas chromatography in view of the fact that the fact that veryvery difficult separations are often more readily a

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Chromatographic Chromatographic Methods-I

Methods-I gas chromatography. The other gas chromatography_{fractions are easily collected}_{fractions are easily collected and recovery is quantitative. The}. The other advantage is about the sample _{and recovery is quantitative. The recovery of separated}advantage is about the sample recovery. Separatedrecovery. Separated_{recovery of separated} components in gas chromatography is also possible but is

components in gas chromatography is also possible but is generally less convenientgenerally less convenient and less quantitative. It may be important here to point out

and less quantitative. It may be important here to point out that gas chromatographthat gas chromatography,y, in general, is

in general, is faster than liquid chromatography.faster than liquid chromatography. In this unit, we will

In this unit, we will first recapitulate some of the basics of liquid chromatography.first recapitulate some of the basics of liquid chromatography. Some special features of

Some special features of liquid-solid adsorptioliquid-solid adsorption and liquid-liquid partition columnn and liquid-liquid partition column chromatograp

chromatography will be hy will be discussed. This will be discussed. This will be followed by a discussion on thefollowed by a discussion on the components of a liquid chromatography set up. The

components of a liquid chromatography set up. The choice of stationary and mobilechoice of stationary and mobile phases is very important and

phases is very important and the considerations involved will be discussed. This willthe considerations involved will be discussed. This will be followed by a

be followed by a discussion on the basic methods used for discussion on the basic methods used for chromatogchromatographic columnraphic column development. In order to highlight the importance of

development. In order to highlight the importance of the technique, some of thethe technique, some of the applications to separate complex mixtures will

applications to separate complex mixtures will be presented. Since the be presented. Since the conventionconventionalal liquid chromatography has underg

liquid chromatography has undergone a major one a major development in the form of highdevelopment in the form of high performance liquid chromat

performance liquid chromatography, a brief idea about this will also be ography, a brief idea about this will also be given at thegiven at the end.

end.

Objectives Objectives

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

•• recapitulate some of the recapitulate some of the basic concepts of liquid-solid and liquid-liquid columnbasic concepts of liquid-solid and liquid-liquid column chromatography,

chromatography,

•• understand the functioning of the components of an understand the functioning of the components of an improved version of a liquidimproved version of a liquid chromatograp

chromatographic set hic set up,up,

•• appreciate the criteria used for the choice of stappreciate the criteria used for the choice of st ationary and mobile phase,ationary and mobile phase,

•• describe development techniques used,describe development techniques used,

•• get an idea about HPLC, andget an idea about HPLC, and

•• enumerate some of the enumerate some of the important applications of liquid column chromatography.important applications of liquid column chromatography.

5.2

5.2 RECAPITULATION OF

RECAPITULA

TION OF BASICS

BASICS

As stated earlier in this unit,

As stated earlier in this unit, we are going to confine ourselves to liquid-solidwe are going to confine ourselves to liquid-solid chromatograp

chromatography (LSC) and hy (LSC) and liquid-liquid partition chromatograpliquid-liquid partition chromatography (LLC); both hy (LLC); both ofof them being operated on a column. When a sample mixture is injected

them being operated on a column. When a sample mixture is injected into a liquidinto a liquid chromatograp

chromatographic column, it hic column, it begins to migrate down the begins to migrate down the column under the influence ofcolumn under the influence of the mobile phase. During this

the mobile phase. During this process, various components of the mixture will begin process, various components of the mixture will begin toto separate depending upon their affinity for the stationary phase in the presence of the separate depending upon their affinity for the stationary phase in the presence of the mobile phase. The components that are weakly retained by the stationary phase will mobile phase. The components that are weakly retained by the stationary phase will pass through the column and be eluted earlier. Thus, there will be peaks

pass through the column and be eluted earlier. Thus, there will be peaks in the order inin the order in the resulting chromatogram. The strongly retained components will elute la

the resulting chromatogram. The strongly retained components will elute la ter, theter, the relative

relative separation being depenseparation being dependent upon the degree of retentident upon the degree of retention by the stationaryon by the stationary phase for each sample

phase for each sample component. Thus, the components pass down the column atcomponent. Thus, the components pass down the column at different speeds which can be related to the distribution of each component in the different speeds which can be related to the distribution of each component in the stationary and mobile phases. The

stationary and mobile phases. The two forms of chromatography, (LSC) and (LLC),two forms of chromatography, (LSC) and (LLC), basically differ in the

basically differ in the mechanism responsible for separation. In one case mechanism responsible for separation. In one case adsorption isadsorption is responsible while partition is operative in the other. An idea about this is

responsible while partition is operative in the other. An idea about this is being givenbeing given below.

below.

5.2.1

5.2.1 Liquid - Solid ChromatographyLiquid - Solid Chromatography

The liquid - solid

The liquid - solid chromatogrchromatographic technique is based aphic technique is based onon adsorption phenomenonadsorption phenomenon.. Consider a liquid solution of

Consider a liquid solution of two compounds which has been brought into contact withtwo compounds which has been brought into contact with a porous adsorbent. The molecules from the

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Liquid Column Liquid Column Chromatography Chromatography become attached to its surface. These molecules are held rather

become attached to its surface. These molecules are held rather loosely by van derloosely by van der Waals’ forces. Some pass back into the body of the solution, and their places Waals’ forces. Some pass back into the body of the solution, and their places in thein the pores of the adsorbent are taken by other molecules. Thus, there is a

pores of the adsorbent are taken by other molecules. Thus, there is a continualcontinual interchange between the molecules in the body of the solution and those in the pores interchange between the molecules in the body of the solution and those in the pores of the adsorbent. Usually, one component tends to be more firmly held on the surface of the adsorbent. Usually, one component tends to be more firmly held on the surface than the other, so that, when equilibrium is established, the

than the other, so that, when equilibrium is established, the concentration of thisconcentration of this component in the pores will

component in the pores will be higher than its be higher than its concentration in the surrounding liquid.concentration in the surrounding liquid. Assuming that the phases can be perfectly separated, the separation factor is defined Assuming that the phases can be perfectly separated, the separation factor is defined by the equation

by the equation

α

α = ( = ( X / Y X / Y ))aa / ( X/ Y) / ( X/ Y)ll

i.e

i.e.,., α α _{, the}_{, the separation factor}_{separation factor , is equal to the ratio of the mole fractions of the two}_{, is equal to the ratio of the mole fractions of the two}

components, X over Y, in the adsorbed phase,

components, X over Y, in the adsorbed phase, aa, divided by their ratio in the liquid, divided by their ratio in the liquid phase,

phase, ll..

5.2.2

5.2.2 Liquid-liquid ChromatographyLiquid-liquid Chromatography

Liquid-liquid chromatogra

Liquid-liquid chromatography is phy is sometimes calledsometimes called liquid partition liquid partition chromatochromatographygraphy.. Liquid-liquid chromatog

Liquid-liquid chromatography is based on raphy is based on the separation of the the separation of the solutes by theirsolutes by their differential partitioning

differential partitioning between two immiscible phases. This usually involves a between two immiscible phases. This usually involves a stationary phase coated on an inert solid support, normally silica gel and an stationary phase coated on an inert solid support, normally silica gel and an

immiscible mobile phase. Most commonly the stationary phase is more polar than the immiscible mobile phase. Most commonly the stationary phase is more polar than the mobile phase. In some circumstances, however, it is advantageous to reverse the roles mobile phase. In some circumstances, however, it is advantageous to reverse the roles so that the stationary phase is

so that the stationary phase is less polar. This variation is known asless polar. This variation is known as reversed phasereversed phase partition chromat

partition chromatographyography. The process of . The process of liquid-liquid chromatliquid-liquid chromatography is similar toography is similar to simple batch extraction between two immiscible liquids in a separatory funnel. A simple batch extraction between two immiscible liquids in a separatory funnel. A successive series of such

successive series of such extractions forms the basis of countercurrent distribution,extractions forms the basis of countercurrent distribution, which is

which is more more efficient than efficient than simple osimple one stage ne stage extraction. extraction. However, However, liquid-liquidliquid-liquid chromatograp

chromatography is many hy is many times faster and more times faster and more efficient than countercurrentefficient than countercurrent extraction. This is the result of

extraction. This is the result of the large interface between moving and stationarythe large interface between moving and stationary phases.

phases.

In liquid-liquid chromatography

In liquid-liquid chromatography, equilibrium distribution of the solutes b, equilibrium distribution of the solutes b etween theetween the mobile phase and the stationary phase takes place rapidly, and the

mobile phase and the stationary phase takes place rapidly, and the separation of theseparation of the components of a mixture results

components of a mixture results from the resulting distributions of the from the resulting distributions of the various solutevarious solute molecules in the two immiscible phases. The distribution equilibria are described by molecules in the two immiscible phases. The distribution equilibria are described by the

the distribution coefficient distribution coefficient , often called, often called partition coefficient K partition coefficient K . For practical. For practical chromatograp

chromatography, it is necessary to be hy, it is necessary to be able to predict a particular solvent-soluteable to predict a particular solvent-solute

relationship in order to obtain the required separation of a mixture. The distribution of relationship in order to obtain the required separation of a mixture. The distribution of a solute between two phases is also defined in

a solute between two phases is also defined in terms ofterms of capacitycapacity or or retention factor retention factor ,, k k ´.´. The different terms and concept of theoretical plates and the rate

The different terms and concept of theoretical plates and the rate theory have alreadytheory have already been explained in Unit 4. All these

been explained in Unit 4. All these are applicable for both of these forms of columnare applicable for both of these forms of column chromatography.

chromatography.

SAQ 1 SAQ 1

What is the basic di

What is the basic di fference in LSC and LLC? Which one will be generally faster?fference in LSC and LLC? Which one will be generally faster?

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Methods-I

5.3

5.3 EXPERIMENTAL SET UP

EXPERIMENTAL SET UP

At this point, with the background that we already have, it may be

At this point, with the background that we already have, it may be important to discussimportant to discuss the experimental set up of

the experimental set up of liquid column chromatographyliquid column chromatography. Conventionally, in the. Conventionally, in the classical set up there is

classical set up there is a simple column, packed with an adsorbent or a supporta simple column, packed with an adsorbent or a support material coated with a stationary phase. The mixture is

material coated with a stationary phase. The mixture is fed to the column which is thenfed to the column which is then irrigated with the mobile phase. The eluant is

irrigated with the mobile phase. The eluant is collected in small increments and put tocollected in small increments and put to analysis. However, with time, several

analysis. However, with time, several improvements have taken place.improvements have taken place. In classical liquid chromatography, the column is used only once and is then In classical liquid chromatography, the column is used only once and is then discarded. Therefore, the packing in a column has to be refilled for each separation discarded. Therefore, the packing in a column has to be refilled for each separation and this amounts to a significant expense of both manpower and material. In classical and this amounts to a significant expense of both manpower and material. In classical liquid chromatography

liquid chromatography, the sample , the sample application requires some skill and application requires some skill and time on the time on the partpart of the operator. Solvent flow is

of the operator. Solvent flow is achieved by gravity feeding of the column. achieved by gravity feeding of the column. SeparationsSeparations require several hours. The detection and quantitation

require several hours. The detection and quantitation are done by the are done by the manual analysismanual analysis of individual fractions. Many fractions are collected

of individual fractions. Many fractions are collected normally and their processingnormally and their processing requires much time and effort.

requires much time and effort. On the other hand, in On the other hand, in modern liquid chromatogramodern liquid chromatography,phy, reusable columns are used so that a number of individual separations can be carried reusable columns are used so that a number of individual separations can be carried out on a given column. Since the cost of

out on a given column. Since the cost of an individual column can now be proratedan individual column can now be prorated over a large number of samples, it is possible

over a large number of samples, it is possible to use more expensive column packingto use more expensive column packing.. Precise sample injection is achieved easily and rapidly in modern liquid

Precise sample injection is achieved easily and rapidly in modern liquid chromatograp

chromatography. Solvent flow is hy. Solvent flow is achieved by means of high achieved by means of high pressure pumps withpressure pumps with controlled flow rate which results in

controlled flow rate which results in more reproducible operatiomore reproducible operations and better ns and better andand faster separations. The detection and quantitation a

faster separations. The detection and quantitation a re done with continuous detectorsre done with continuous detectors of various types which yield a

of various types which yield a continuous chromatogcontinuous chromatogram without intervention by theram without intervention by the operator.

operator.

Fig. 5.1(a) shows an improved version of a

Fig. 5.1(a) shows an improved version of a liquid chromatograpliquid chromatographic set up hic set up while awhile a more sophisticated set up is shown in Fig. 5.1 (b).

more sophisticated set up is shown in Fig. 5.1 (b).

Fig. 5.1(a):

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Liquid Column Liquid Column Chromatography Chromatography

Fig. 5.1(b):

Fig. 5.1(b): A more sophisticated A more sophisticated liquid chromatographic set uliquid chromatographic set upp 1.

1. Mobile pMobile phase resehase reservoir 2. rvoir 2. Slurring Slurring device 3. device 3. Lamp Lamp (Heating d(Heating device) 4. evice) 4. PumpPump 5.

5. Pressure monitoring device Pressure monitoring device 6. P6. Pump 7. ump 7. Filter 8. Filter 8. Precolumn 9. Precolumn 9. Column Column 10. Pump10. Pump 11.

11. Injection port 12. Injection port 12. Column thermostat Column thermostat 13. D13. Detectetector or 14. Recorder14. Recorder 5.3.1

5.3.1 EquipmentEquipment

The equipment needed to carry out

The equipment needed to carry out modern liquid chromatogramodern liquid chromatography is very differentphy is very different from the relatively simple

from the relatively simple and unsophisticated equipment used for classical liquidand unsophisticated equipment used for classical liquid chromatograp

chromatography separations. The hy separations. The schematic of equipment used schematic of equipment used for modern liquidfor modern liquid chromatograp

chromatography is shown in Fig. 5.1 ( hy is shown in Fig. 5.1 ( b). The components of this sophisticated set upb). The components of this sophisticated set up are discussed below.

are discussed below.

i)

i) Mobile phase reservoirMobile phase reservoir It holds one litre of the

It holds one litre of the mobile phase. This reservoir is made up of stainless steelmobile phase. This reservoir is made up of stainless steel which is inert to most mobile phases and i

which is inert to most mobile phases and i s not subject to breakage. Manys not subject to breakage. Many different forms of reservoirs have been used, and

different forms of reservoirs have been used, and simple units may besimple units may be constructed from glass flasks or bottles of

constructed from glass flasks or bottles of an appropriate size. Some reservoirsan appropriate size. Some reservoirs are designed so that the mobile phase may be degassed

are designed so that the mobile phase may be degassed in situin situ. Degassing is. Degassing is required to eliminate dissolved gases, particularly oxygen. To

required to eliminate dissolved gases, particularly oxygen. To permitpermit in situin situ degassing, reservoirs are sometimes equipped with a heater,

degassing, reservoirs are sometimes equipped with a heater, a stirringa stirring mechanism and inlets for

mechanism and inlets for applying vacuum and a nitrogen purge.applying vacuum and a nitrogen purge.

ii)

ii) PumpsPumps One of the

One of the most important parts of modern liquid chromatography instrument ismost important parts of modern liquid chromatography instrument is the pumping system. In modern liquid chromatography, the resistance to

the pumping system. In modern liquid chromatography, the resistance to flow offlow of the long, narrow columns packed with small particles is relatively high, and high the long, narrow columns packed with small particles is relatively high, and high pressures are required. Pumps are grouped into two

pressures are required. Pumps are grouped into two major categories:major categories: mechanical pumps

mechanical pumps which deliver the mobile phase at a which deliver the mobile phase at a constant flow rate, andconstant flow rate, and pneumatic pum

pneumatic pumpsps which deliver the mobile phase with a which deliver the mobile phase with a constant pressure.constant pressure.

iii)

iii) FilterFilter

A filter is normally placed in

A filter is normally placed in the line following the pump to remove finethe line following the pump to remove fine particles of particulate material which can clog the

particles of particulate material which can clog the inlet of the column.inlet of the column. Generally a 2

Generally a 2µµ sintered stainless steel sintered stainless steel filter is adequate for this purpose.filter is adequate for this purpose.

iv)

iv) Pressure monitoring devicePressure monitoring device

A device for monitoring the column input pressure shoul

A device for monitoring the column input pressure should be inserted in the d be inserted in the lineline between the pump

between the pump and the chromatographic column. This and the chromatographic column. This pressure monitoringpressure monitoring device indicates if there has been a plugging of the

device indicates if there has been a plugging of the column or a failure of thecolumn or a failure of the pumping system.

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Methods-I v)v) Sample introduction deviceSample introduction device Sample introduction into a liquid

Sample introduction into a liquid chromatogchromatography column is a raphy column is a very importantvery important factor in obtaining high column performance. The sample

factor in obtaining high column performance. The sample should be introducedshould be introduced as an infinitely narrow band on to the chromatographic bed. The more defused is as an infinitely narrow band on to the chromatographic bed. The more defused is the plug of sample in the

the plug of sample in the mobile phase introduced into the column, the wider ismobile phase introduced into the column, the wider is the separated component bands at the end of the column. The sample is

the separated component bands at the end of the column. The sample is injectedinjected with a micro-syringe through a septum contained in a low volume inlet system. with a micro-syringe through a septum contained in a low volume inlet system. Septum materials are

Septum materials are generally made from silicone or Neoprene. It is generally made from silicone or Neoprene. It is generallygenerally not feasible to

not feasible to make syringe injections above about 1500 psi tmake syringe injections above about 1500 psi t hrough samplinghrough sampling ports. Therefore, at high pressure, a

ports. Therefore, at high pressure, a stop flow injection technique is stop flow injection technique is normallynormally used with syringe.

used with syringe.

vi)

vi) ColumnColumn

The unpacked column must be constructed of

The unpacked column must be constructed of materials that will materials that will withstand bothwithstand both the pressures to be used and chemical action of

the pressures to be used and chemical action of the mobile phase. Most columnsthe mobile phase. Most columns are made up of stainless steel

are made up of stainless steel tubing. However heavy wall glass columns aretubing. However heavy wall glass columns are sometimes used. Columns that will withstand pressures up to 600 psi are sometimes used. Columns that will withstand pressures up to 600 psi are commercially available. For operation at high

commercially available. For operation at high pressures, glass lined metalpressures, glass lined metal columns also can be used. Column end fittings should be designed with columns also can be used. Column end fittings should be designed with

minimum dead volume. Porous plugs are used in the ends of columns to retain minimum dead volume. Porous plugs are used in the ends of columns to retain the packing. Straight sections of

the packing. Straight sections of liquid chromatograpliquid chromatography columns in lengths ofhy columns in lengths of 25-150 cm are normally preferred. Some colum

25-150 cm are normally preferred. Some columns may also be bent ns may also be bent into a “U”into a “U” shape.

shape. Coiled columns arCoiled columns are some times used, but ae some times used, but are often less efficient thanre often less efficient than columns prepared in straight sections. Precolumns generally are desirable. columns prepared in straight sections. Precolumns generally are desirable. TheThe precolumn ensures that the mobile phase is completely saturated with the precolumn ensures that the mobile phase is completely saturated with the stationary phase before it passes in to the

stationary phase before it passes in to the carefully prepared analytcarefully prepared analytical column.ical column. The internal diameter of the column has a si

The internal diameter of the column has a si gnificant effect on the efficiency ofgnificant effect on the efficiency of liquid chromatography colum

liquid chromatography columns. For analytical studies, ns. For analytical studies, columns 1-4 mmcolumns 1-4 mm internal diameter (i.d.) are

internal diameter (i.d.) are normally used. Columns of larger internal diameternormally used. Columns of larger internal diameter are used for preparative work.

are used for preparative work.

vii)

vii) Column thermostatColumn thermostat It is i

It is important to control the column mportant to control the column temperature in liquid chromatography. Thetemperature in liquid chromatography. The temperature variations within the column should be

temperature variations within the column should be maintained withinmaintained within±± 0.2 0.200C.C. The larger changes in column temperature can result in significant variations in The larger changes in column temperature can result in significant variations in retention time.

retention time.

viii)

viii) DetectorsDetectors

In liquid chromatography, the ideal detector should have

In liquid chromatography, the ideal detector should have high sensitivityhigh sensitivity,, goodgood precision

precision andand predictable respons predictable responsee to all solutes. It should be unaffected byto all solutes. It should be unaffected by changes in temperature and carrier flow.

changes in temperature and carrier flow. It should not contribute to extra It should not contribute to extra columncolumn band broadening. It must be nondestructive of the

band broadening. It must be nondestructive of the solute. Two types of detectorssolute. Two types of detectors are in use in l

are in use in liquid chromatograpiquid chromatography, thehy, the bulk propertybulk property or or general detectorsgeneral detectors and and solute property

solute property oror selective detectorsselective detectors. Bulk property detectors measure a . Bulk property detectors measure a changechange in some overall physical property of the mobile phase plus that of the solute. in some overall physical property of the mobile phase plus that of the solute. The solute property detectors are sensitive only to the solute.

The solute property detectors are sensitive only to the solute.

ix)

ix) Column packingsColumn packings

The packing of columns in

The packing of columns in liquid chromatography are described in terms ofliquid chromatography are described in terms of adsorbent or stationary phase, the type of particle and particle size. Each of adsorbent or stationary phase, the type of particle and particle size. Each of thesethese particle characteristics has an important effect on the performance and use of a particle characteristics has an important effect on the performance and use of a given packing material. The column are packed by various techniques such as : given packing material. The column are packed by various techniques such as :

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Liquid Column Liquid Column Chromatography Chromatography •• Slurry packing technique for columns of hard gel.Slurry packing technique for columns of hard gel.

•• Slurry sedimentation method for soft gels.Slurry sedimentation method for soft gels.

SAQ 2 SAQ 2

Why should the column temperature be maintained in

Why should the column temperature be maintained in a chromatographic set up?a chromatographic set up?

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What are the

What are the various techniques for column packing?various techniques for column packing?

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5.4

5.4 CHOICE OF STATIONARY AND MOBILE PHASES

CHOICE OF STATIONARY AND MOBILE PHASES

The success of a

The success of a separation by liquid column chromatography dependseparation by liquid column chromatography depends upon a propers upon a proper choice of stationary and mobile phases. In LSC, the stationary phase is an

choice of stationary and mobile phases. In LSC, the stationary phase is an adsorbent.adsorbent. And once we talk about the adsorbent, some of its properties, viz adsorbent type, And once we talk about the adsorbent, some of its properties, viz adsorbent type, surface area, particle size and activation and regeneration become relevant. In LLC, surface area, particle size and activation and regeneration become relevant. In LLC, the stationary phase is a l

the stationary phase is a liquid immobilized on an inert support. While considering theiquid immobilized on an inert support. While considering the liquid, its polarity and tendency to leach out become important. To counter act

liquid, its polarity and tendency to leach out become important. To counter act thethe leaching, bonded phases have been developed. Moreover, the requirements of inert leaching, bonded phases have been developed. Moreover, the requirements of inert supports on which the liquid

supports on which the liquid is immobilized are equally important to is immobilized are equally important to know. Finally,know. Finally, the requirements of mobile phase are

the requirements of mobile phase are to be properly understood. This section dealsto be properly understood. This section deals with of the

with of the above mentioned different aspects.above mentioned different aspects.

5.4.1

5.4.1 Stationary Phases Stationary Phases Used in Used in Liquid-Solid Liquid-Solid Column ChromatographyColumn Chromatography

Stationary phases of a great

Stationary phases of a great variety have been used variety have been used in liquid chromatography.in liquid chromatography. Presumably any finely divided or porous solid which has

Presumably any finely divided or porous solid which has the adsorption capacity andthe adsorption capacity and which is not too soluble in the

which is not too soluble in the mobile phase may be used. Many different substancesmobile phase may be used. Many different substances have been used as adsorbents including activated alumina, silica gel, carbon,

have been used as adsorbents including activated alumina, silica gel, carbon, magnesium oxide, magnesium carbonate, hydrated calcium silicate, talc, magnesium oxide, magnesium carbonate, hydrated calcium silicate, talc, silversilver sulphide, bauxite, activated clay from bentonite,

sulphide, bauxite, activated clay from bentonite, fuller’s earth, sucrose, and powderedfuller’s earth, sucrose, and powdered cellulose.

cellulose. i)

i) Adsorbent typeAdsorbent type

Various adsorbent types exhibit different selectivities towards different types of Various adsorbent types exhibit different selectivities towards different types of compound.

compound. Polar adsorbentsPolar adsorbents such as metal oxides, magnesium silicate etc. such as metal oxides, magnesium silicate etc. selectively adsorb unsaturated, aromatics and polar molecules such as

selectively adsorb unsaturated, aromatics and polar molecules such as alcohols,alcohols, amines and acids.

amines and acids. Polar adsorbents may be further sub-divided asPolar adsorbents may be further sub-divided as acidicacidic,, basicbasic or

or neutralneutral, according to the pH of the surface. Silica, , according to the pH of the surface. Silica, magnesium silicate aremagnesium silicate are acidic and thus, they chemisorb bases. The alumina surface contains both acidic acidic and thus, they chemisorb bases. The alumina surface contains both acidic and basic sites.

and basic sites. Non-polar adsorbeNon-polar adsorbents such as nts such as graphitized carbon which is agraphitized carbon which is a strong adsorbent and kieselguhr which is a

strong adsorbent and kieselguhr which is a weak adsorbent show no selectivityweak adsorbent show no selectivity for the adsorption of polar molecules.

for the adsorption of polar molecules. ii)

ii) Surface areaSurface area

The surface area and pore diameter of a given adsorbent vary widely with the The surface area and pore diameter of a given adsorbent vary widely with the method of manufacture. In

method of manufacture. In adsorption chromatogradsorption chromatography, the separation aphy, the separation dependsdepends on the transport of the molecules through the system and on the interchange of on the transport of the molecules through the system and on the interchange of

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Methods-I the molecules between an adsorbed phase and a liquid phase. If the volume ofthe molecules between an adsorbed phase and a liquid phase. If the volume of_{the adsorbed phase per unit quantity of the adsorbent is low, both the amount of}_{the adsorbed phase per unit quantity of the adsorbent is low, both the amount of} interchange between the phases and the amount of separation will be small. For interchange between the phases and the amount of separation will be small. For this reason, when dealing with large samples, it is

this reason, when dealing with large samples, it is important to select animportant to select an adsorbent with a large surface area.

adsorbent with a large surface area. iii)

iii) Particle sizeParticle size

The effect of particle

The effect of particle size of the size of the adsorbent on the sharpness of chromatographicadsorbent on the sharpness of chromatographic separations has been noted by

separations has been noted by many investigators. For sharp separations, it ismany investigators. For sharp separations, it is recognized that a finely divided material is necessary. An adsorbent in the range recognized that a finely divided material is necessary. An adsorbent in the range from 100 to 200 mesh (149 to 74

from 100 to 200 mesh (149 to 74 µµ) in particle size is ) in particle size is specified. Some authorsspecified. Some authors recommend the use of more finely divided materials. However, it is more recommend the use of more finely divided materials. However, it is more difficult to pack columns uniformly if the adsorbent is very finely divided i.e., difficult to pack columns uniformly if the adsorbent is very finely divided i.e., below 50

below 50 µµ in particle size, in particle size, and columns that are poorly packed give rise toand columns that are poorly packed give rise to zones that are irregular in shape. Some adsorbents are available only as finely zones that are irregular in shape. Some adsorbents are available only as finely divided powders with particles below 10

divided powders with particles below 10µµ in diameter, e.g. magnesium oxide. It in diameter, e.g. magnesium oxide. It is necessary to mix these adsorbents with filter aids

is necessary to mix these adsorbents with filter aids such as Celite or Hyflosuch as Celite or Hyflo Super-Gel to obtain a practical rate of flow.

Super-Gel to obtain a practical rate of flow. iv)

iv) Activation and regeneration of adsorbentActivation and regeneration of adsorbent The term

The term activationactivation refers to those processes which are used to enhance the refers to those processes which are used to enhance the effectiveness of an adsorbent by improving the pore st

effectiveness of an adsorbent by improving the pore st ructure and increasing theructure and increasing the surface area. In

surface area. In general, with carbon, high temperature activation produces angeneral, with carbon, high temperature activation produces an organophilic adsorbent, and low temperature

organophilic adsorbent, and low temperature activation, a hydrophilic adsorbent.activation, a hydrophilic adsorbent. During adsorption, the pores of the adsorbent become filled

During adsorption, the pores of the adsorbent become filled with adsorptivewith adsorptive molecules. The term

molecules. The term regenerationregeneration refers to the removal of the adsorbed refers to the removal of the adsorbed molecules and the return of the adsorbent to its original state.

molecules and the return of the adsorbent to its original state. The regenerationThe regeneration of the adsorbent can be carried out by gentle heating if

of the adsorbent can be carried out by gentle heating if the adsorbed moleculesthe adsorbed molecules are volatile. If they are non-volatile, then they may sometimes be

are volatile. If they are non-volatile, then they may sometimes be removed byremoved by elution or desorption with volatile solvent, which in turn, may be removed by elution or desorption with volatile solvent, which in turn, may be removed by gentle heating. With some a

gentle heating. With some adsorbents overheating will destroy the pore structuredsorbents overheating will destroy the pore structure e.g. silica gel, should not be heated

e.g. silica gel, should not be heated above 200above 200ooC. On the other hand, the ruggedC. On the other hand, the rugged adsorbents, fuller’s earth and bauxite, may be

adsorbents, fuller’s earth and bauxite, may be heated in an oxidizing atmosphereheated in an oxidizing atmosphere to temperatures sufficient to burn off

to temperatures sufficient to burn off the adsorbed material near 540the adsorbed material near 540ooC.C.

5.4.2

5.4.2 Stationary PhaStationary Phases Used in ses Used in Liquid-Liquid Liquid-Liquid ColumnColumn Chromatography

Chromatography

In liquid-liquid column chromatography

In liquid-liquid column chromatography, the stationary phase , the stationary phase is a is a liquid that isliquid that is immobilized on a inert support. The stationary phases fall into two classes: the immobilized on a inert support. The stationary phases fall into two classes: the moremore usual

usual hydrophilic oneshydrophilic ones and the and the reversed phasereversed phase. The stationary or . The stationary or supported liquidsupported liquid phases have been of

phases have been of many kinds varying in polarity from water to many kinds varying in polarity from water to paraffinparaffin hydrocarb

hydrocarbons. As a ons. As a large number of liquid stationary phases can large number of liquid stationary phases can be held mechanicallybe held mechanically on an inert

on an inert support, such stationary phases have some disadvantages like leaching outsupport, such stationary phases have some disadvantages like leaching out of the liquid stationary phase from the inert support. In order to eliminate such

of the liquid stationary phase from the inert support. In order to eliminate such disadvantages, surface-reacted or bonded stationary phases have

disadvantages, surface-reacted or bonded stationary phases have been developed. Thebeen developed. The advantages of these materials is that pre-colum

advantages of these materials is that pre-columns and ns and or presaturation of the twoor presaturation of the two phases is not required.

phases is not required. In addition, packing with bonded stationary phases are quiteIn addition, packing with bonded stationary phases are quite stable because

stable because there is there is no oppno opportunity ortunity for the for the chemically bochemically bound stationaund stationary phase ry phase toto be eluted during use. A

be eluted during use. A disadvantagdisadvantage of bonded-phase packing is a e of bonded-phase packing is a lack of systemiclack of systemic information regarding the mode of retention for solutes. There

information regarding the mode of retention for solutes. There are two types ofare two types of surface-reacted or bonded stationary phases that are now commercially available. surface-reacted or bonded stationary phases that are now commercially available. TheThe first one is an esterified siliceous material e

first one is an esterified siliceous material e .g. Durapak .g. Durapak �� and the second type is and the second type is surfacesurface reacted packing e.g. Bondpack

reacted packing e.g. Bondpack ��, Vydac, Vydac�� (organic coating is (organic coating is a monomolecular),a monomolecular), Permaphase

Permaphase�� ( organic coating is ( organic coating is of many layers). Columns of bonded phase packingsof many layers). Columns of bonded phase packings have been used

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Liquid Column Liquid Column Chromatography Chromatography characteristics. These bonded phase materials are available with several types of

characteristics. These bonded phase materials are available with several types of functional groups and used for separations of many types of

functional groups and used for separations of many types of solutes.solutes.

There are four different techniques now in use for preparing liquid coated packing for There are four different techniques now in use for preparing liquid coated packing for liquid-liquid column

liquid-liquid column chromatogrchromatography:aphy: 1.

1. Solvent evaporation technique.Solvent evaporation technique. 2.

2. In situ coating procedure.In situ coating procedure. 3.

3. The solvent filtration technique.The solvent filtration technique. 4.

4. The equilibration t echnique.The equilibration technique.

Supports for liquid-liquid partition chromatography Supports for liquid-liquid partition chromatography

In liquid-liquid partition chromatography, the stationary liquid phase is

In liquid-liquid partition chromatography, the stationary liquid phase is supported onsupported on an inert support. An ideal support material has to meet

an inert support. An ideal support material has to meet the following requirementsthe following requirements..

•• It should be chemically inert and it must not It should be chemically inert and it must not dissolve or swell in the stationarydissolve or swell in the stationary phase.

phase.

•• It should display good wetability by the stationary phase and it should neitherIt should display good wetability by the stationary phase and it should neither dissolve nor react with the mobile phase.

dissolve nor react with the mobile phase.

•• It should consist of particles as identical as It should consist of particles as identical as possible which allow the mostpossible which allow the most uniform and reproducible packing.

uniform and reproducible packing.

•• It should have large enough surface to retain the stationary phase as a It should have large enough surface to retain the stationary phase as a thinthin uniform film. Porous supports generally meet this requirement.

uniform film. Porous supports generally meet this requirement.

•• It should allow the columns to have an acceptable pressure drop as regards theIt should allow the columns to have an acceptable pressure drop as regards the mobile phase.

mobile phase.

•• It should have sufficient mechanical stability. It should have sufficient mechanical stability. It must not It must not grind during columngrind during column packing, impregnation of stationary phase or regeneration of support material. packing, impregnation of stationary phase or regeneration of support material.

•• When applied for routine analysis or for When applied for routine analysis or for preparative purposes, it must bepreparative purposes, it must be relatively cheap, easily available

relatively cheap, easily available and permit regeneration.and permit regeneration.

5.4.3

5.4.3 Mobile Phases in Liquid Column ChromatographyMobile Phases in Liquid Column Chromatography

Various physical and chemical properties govern the choice

Various physical and chemical properties govern the choice of mobile phases. Theof mobile phases. The most important factor is the influence of the mobile phase on the

most important factor is the influence of the mobile phase on the selectivity of theselectivity of the system. The solubility of the samples and influence of such properties as surface system. The solubility of the samples and influence of such properties as surface tension and viscosity are a

tension and viscosity are also important. Solubility of some lso important. Solubility of some samples, especiallysamples, especially polymers, limits the choice of the mobile phase and

polymers, limits the choice of the mobile phase and the use of certain detectorsthe use of certain detectors imposes constrains.

imposes constrains.

The choice of mobile phase in liquid column chromatography is all important. If The choice of mobile phase in liquid column chromatography is all important. If water-deactivated silica is used as an

water-deactivated silica is used as an adsorbent, the solvent is then varied to giveadsorbent, the solvent is then varied to give k k ´´ values in the optimum range (1<

values in the optimum range (1< k k ´ <10). Solvent strength, which controls the´ <10). Solvent strength, which controls the k k ´´ values of all

values of all sample bands, is easily sample bands, is easily predicted in liquid-solid column chromatography.predicted in liquid-solid column chromatography. It can be defined quantitatively by the solvent strength parameter

It can be defined quantitatively by the solvent strength parameterε ε oo_{which are listed}_{which are listed}

for several pure solvents in Table 5.1. These values are for alumina

for several pure solvents in Table 5.1. These values are for alumina as an adsorbent.as an adsorbent. The solvents listed in Table 5.1 are

The solvents listed in Table 5.1 are arranged in order of increasing strengarranged in order of increasing strengths which isths which is referred to as an elutropic series. If an

referred to as an elutropic series. If an initial solvent is too strong then a initial solvent is too strong then a weakerweaker solvent is substituted. Similarly, if the initial

solvent is substituted. Similarly, if the initial solvent is too weak then a solvent is too weak then a strongerstronger solvent is substituted. Thus, an elutropic series can be used to

solvent is substituted. Thus, an elutropic series can be used to find out right solventfind out right solvent strength by a

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Methods-I Binary solvent mixtures are often used Binary solvent mixtures are often used in liquid solid column chromatography. There_{are several advantages in the use of binary solvent mixtures e.g. solvent strength}_{are several advantages in the use of binary solvent mixtures e.g. solvent strength}in liquid solid column chromatography. There changes continuously with composition. Another advantag

changes continuously with composition. Another advantage of e of binary solvents is thatbinary solvents is that solvent viscosity can be kept low.

solvent viscosity can be kept low. While considering the choice of

While considering the choice of mobile phases in liquid-liquid columnmobile phases in liquid-liquid column chromatograp

chromatography, some basic characteristics of hy, some basic characteristics of mobile phases must be mobile phases must be considered. Theconsidered. The mobile phase must be immiscible with the stationary phase. It should have viscosity as mobile phase must be immiscible with the stationary phase. It should have viscosity as low as possible for h

low as possible for h igher column permeability and or efficiency. The detector igher column permeability and or efficiency. The detector cancan also limit the

also limit the phases which can be phases which can be used. e.g. strongly UV-absorbing solvents shouldused. e.g. strongly UV-absorbing solvents should be avoided with an ultraviolet photometric detector. The cost, toxicity, purity and be avoided with an ultraviolet photometric detector. The cost, toxicity, purity and stability of a solvent also should be taken into a

stability of a solvent also should be taken into a ccount. Most important is theccount. Most important is the selectivity of a

selectivity of a liquid-liquid system for a given sample. In liquid-liquid system for a given sample. In liquid-liquid columnliquid-liquid column chromatograp

chromatography, the k´ hy, the k´ values of solutes are values of solutes are generally controlled by changing thegenerally controlled by changing the mobile phase. The scale

mobile phase. The scale of solvent polarity is defined by of solvent polarity is defined by the Hildebrand solubilitythe Hildebrand solubility parameter,

parameter,δ δ _{. The parameter,}_{. The parameter,}δ δ _{, is a}_{, is a good measure of what is commonly called}_{good measure of what is commonly called}

polarity. Non-polar solvents have low values of

polarity. Non-polar solvents have low values ofδ δ _{, while polar solvents have large}_{, while polar solvents have large}

values. The values of

values. The values ofδ δ _{for different solvents are shown in Table 5.1.}_{for different solvents are shown in Table 5.1.}

Table 5.1: Solvent Strength and Polarity Data Table 5.1: Solvent Strength and Polarity Data Solvent Hildebrand Solvent Hildebrand solubility solubility parameter parameter δ δ δ δδ δ δ δ Solvent Solvent strength strength ε ε ε ε ε ε ε ε oo Solvent Hildebrand Solvent Hildebrand solubility solubility parameter parameter δ δ δ δ δ δ δ δ Solvent Solvent strength strength ε ε ε ε ε ε ε ε oo n

n-Pentane -Pentane 7.1 7.1 0.00 0.00 EthyleneEthylene dichloride dichloride

9.7 0.44

Isooctane

Isooctane 7.0 7.0 0.01 0.01 Triethyl Triethyl amine amine 7.5 7.5 0.540.54 Petroleum

Petroleum ether ether 0.01 0.01 Acetone Acetone 9.4 9.4 0.560.56 Cyclohexane

Cyclohexane 8.2 8.2 0.04 0.04 Dioxane Dioxane 9.8 9.8 0.560.56 Cyclopentane

Cyclopentane 8.1 8.1 0.05 0.05 Tetrahydrofuran Tetrahydrofuran 9.1 9.1 0.570.57 Carbon

Carbon tetrachloride tetrachloride

8.6

8.6 0.18 0.18 Ethyl Ethyl acetate acetate 8.6 8.6 0.580.58

Xylene

Xylene 8.8 8.8 0.26 0.26 Methyl Methyl acetate acetate 9.2 9.2 0.600.60 ii-Propyl -Propyl ether ether 7.0 7.0 0.28 0.28 Nitromethane Nitromethane 11.0 11.0 0.640.64 Toluene

Toluene 8.9 8.9 0.29 0.29 Acetonitrile Acetonitrile 11.8 11.8 0.650.65 Benzene

Benzene 9.2 9.2 0.32 0.32 DimethylDimethyl sulfoxide sulfoxide

11.5 0.75

Ethyl

Ethyl bromide bromide 8.8 8.8 0.35 0.35 n-Propanol n-Propanol 10.2 10.2 0.820.82 Ethyl

Ethyl sulfide sulfide 8.6 8.6 0.38 0.38 Ethanol Ethanol 11.2 11.2 0.880.88 Chloroform

Chloroform 9.1 9.1 0.40 0.40 Methanol Methanol 12.9 12.9 0.950.95 Methylene

Methylene chloride chloride

11.9

11.9 0.42 0.42 Ethylene Ethylene glycol glycol 14.7 14.7 1.11.1

SAQ 4 SAQ 4

What are the

What are the different techniques to prepare liquid coated support?different techniques to prepare liquid coated support?

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

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Liquid Column Liquid Column Chromatography Chromatography SAQ 5 SAQ 5

What is meant by solvent strength of a

What is meant by solvent strength of a mobile phase?mobile phase?

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 6 SAQ 6

What is the advantage of using a

What is the advantage of using a binary solvent mixture as a mobile phase in LSC?binary solvent mixture as a mobile phase in LSC?

………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 7 SAQ 7

What does Hildebrand solubility

What does Hildebrand solubility parameter signify?parameter signify?

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

5.5

5.5 DEVELOPMENT TECHNIQUES

DEVELOPMENT TECHNIQUES

After having learnt about the stationary and mobile phase, it is important to

After having learnt about the stationary and mobile phase, it is important to know as toknow as to how the columns are developed.

how the columns are developed. There are three basic There are three basic methods of chromatographimethods of chromatographicc developments.

developments. 1.

1. Frontal analysis.Frontal analysis. 2.

2. Displacement development.Displacement development. 3.

3. Elution analysis.Elution analysis.

Let us know them in a little more detail. Let us know them in a little more detail. 5.5.1

5.5.1 Frontal AnalysisFrontal Analysis

In frontal analysis, a large sample in

In frontal analysis, a large sample in a suitable solvent is passed through a shorta suitable solvent is passed through a short adsorption column previously saturated with solvent and the effluent is

adsorption column previously saturated with solvent and the effluent is analyzedanalyzed continuously until its composition is identical with that of the original sample. continuously until its composition is identical with that of the original sample. Consider a three component mixture containing equal quantities

Consider a three component mixture containing equal quantities of each componentof each component fed continuously onto a column. Because of

fed continuously onto a column. Because of the forces between solute and the forces between solute and stationarystationary phase, each solute will be

phase, each solute will be retained to a different extent as it retained to a different extent as it comes into equilibriumcomes into equilibrium with the stationary phase while

with the stationary phase while passing through the column. The first component topassing through the column. The first component to elute will be that

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Methods-I component will elute but in conjunction with the first component, and finally, the mostcomponent will elute but in conjunction with the first component, and finally, the most_{strongly held of the three will elute in}_{strongly held of the three will elute in conjunction with the first and second}_{conjunction with the first and second} components. Subsequ

components. Subsequently, there will be ently, there will be no change in concentration of solute no change in concentration of solute in thein the mobile phase and the concentration of the respective solutes will be the

mobile phase and the concentration of the respective solutes will be the same as thesame as the feed mixture. The concentration profile resulting from frontal analysis is

feed mixture. The concentration profile resulting from frontal analysis is shown inshown in Fig. 5.2 (a). The continuous curve shows the total concentration of solutes in the Fig. 5.2 (a). The continuous curve shows the total concentration of solutes in the eluent, plotted against volume of

eluent, plotted against volume of mobile phase passed through the column, and themobile phase passed through the column, and the dotted curves represent a similar

dotted curves represent a similar concentration profile but for each individualconcentration profile but for each individual component. Frontal analysis was employed as a

component. Frontal analysis was employed as a development procedure in the earlydevelopment procedure in the early stages of chromatography and before detection procedures were fully effective. It is stages of chromatography and before detection procedures were fully effective. It is not often used today, and certainly not for quantitative analysis. The reason for this is not often used today, and certainly not for quantitative analysis. The reason for this is that no individual component is completely separated from the others in the mixture. that no individual component is completely separated from the others in the mixture.

5.5.2

5.5.2 Displacement DevelopmentDisplacement Development

Displacement development has the advantages of being able

Displacement development has the advantages of being able to accommodate largeto accommodate large samples and giving sharp separations. It depends on

samples and giving sharp separations. It depends on the competition between solutesthe competition between solutes for the active sites of

for the active sites of the adsorbent and is only really effective in separating verythe adsorbent and is only really effective in separating very strongly adsorbed materials. In displacement development, all the

strongly adsorbed materials. In displacement development, all the substances in thesubstances in the sample will be held on the stationary phase so strongly that they cannot be

sample will be held on the stationary phase so strongly that they cannot be eluted byeluted by the mobile phase; they can, nevertheless, be displaced by substances that are held on the mobile phase; they can, nevertheless, be displaced by substances that are held on the surface by stronger forces. However, there will

the surface by stronger forces. However, there will be competition between individualbe competition between individual solutes and, when the sample is placed on the

solutes and, when the sample is placed on the column, all the immediately availablecolumn, all the immediately available active sites of the

active sites of the adsorbent will be occupied by the most strongly held componenadsorbent will be occupied by the most strongly held component.t. As the band of the sample moves down the column, the next a

As the band of the sample moves down the column, the next a vailable sites will bevailable sites will be occupied by the next s

occupied by the next strongly retained componetrongly retained component. Thus, all nt. Thus, all the components arraythe components array themselves along the column in

themselves along the column in order of their adsorption strength. To develop theorder of their adsorption strength. To develop the chromatogram, another substan

chromatogram, another substance called ce called thethe displacer displacer is introduced into the mobile is introduced into the mobile phase stream, the displacer has an even higher affinity for the adsorbent than any of phase stream, the displacer has an even higher affinity for the adsorbent than any of the components to be separated.

the components to be separated.

Thus, on coming into contact with the sites occupied by the most strongly adsorbed Thus, on coming into contact with the sites occupied by the most strongly adsorbed component, it will displace this component into the mobile phase and thus, move onto component, it will displace this component into the mobile phase and thus, move onto the next group of sites occupied by the next component which will then itself be the next group of sites occupied by the next component which will then itself be displaced. Thu

displaced. Thus, the displacer s, the displacer drives the adsdrives the adsorbed comporbed components onents progressively progressively alongalong the

the column, column, each each component displacing component displacing the the one one in in front, front, until until they they are are eluted eluted inin the same order in which they were adsorbed on the column. The least st

the same order in which they were adsorbed on the column. The least st rongly heldrongly held being eluted first. The

being eluted first. The concentration profile of displacement development is shown inconcentration profile of displacement development is shown in Fig. 5.2 (b). Displacement development has very limited applications as a separation Fig. 5.2 (b). Displacement development has very limited applications as a separation technique and is only very rarely used in quantitative analysis.

technique and is only very rarely used in quantitative analysis.

5.5.3

5.5.3 Elution AnalysisElution Analysis

The elution analysis is

The elution analysis is the most common technique in chromatography. Employingthe most common technique in chromatography. Employing elution analysis, complete separation can be

elution analysis, complete separation can be achieved. This technique is being achieved. This technique is being widelywidely used for quantitative analysis. A relatively inert solvent is used in

used for quantitative analysis. A relatively inert solvent is used in large quantities tolarge quantities to transport the components down the column.

transport the components down the column. The nature of

The nature of the solvent influences the equilibrium between stationary and the solvent influences the equilibrium between stationary and mobilemobile phases. The nature of the solvent can also affect the

phases. The nature of the solvent can also affect the shape of the distributionshape of the distribution

isotherms. Bands that might travel down the column with a symmetrical concentration isotherms. Bands that might travel down the column with a symmetrical concentration profile (Fig. 5.2 (c)) with

profile (Fig. 5.2 (c)) with one solvent might be quite one solvent might be quite unsymmetrical with another.unsymmetrical with another. When elution analysis is used to separate materials with widely differing distribution When elution analysis is used to separate materials with widely differing distribution coefficients, the use of single elution solvent is not practical.

coefficients, the use of single elution solvent is not practical. If the eluent chosen isIf the eluent chosen is strong enough to remove the most strongly held materials in a reasonable time, it will strong enough to remove the most strongly held materials in a reasonable time, it will carry the more weakly held materials through the column too rapidly. On the other carry the more weakly held materials through the column too rapidly. On the other hand, if the eluent is weak, it

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Liquid Column Liquid Column Chromatography Chromatography Therefore, when the sample components have a

Therefore, when the sample components have a wide range of distributionwide range of distribution coefficients, graded eluents are used.

coefficients, graded eluents are used. When utilizing graded eluents, the usualWhen utilizing graded eluents, the usual procedure is to begin with the weakest solvent capable of eluting one of the

procedure is to begin with the weakest solvent capable of eluting one of the samplesample components and to change increasingly stronger solvents until all

components and to change increasingly stronger solvents until all of the sample hasof the sample has been eluted. The change from one member of the series to

been eluted. The change from one member of the series to the next is made by athe next is made by a stepwise increase in concentration of the strong

stepwise increase in concentration of the stronger solvent. er solvent. This technique is called asThis technique is called as gradient elution analysis

gradient elution analysis. The . The gradient egradient elution technilution technique can que can be usebe used not d not only only withwith mixed solvents but also with any phenomena that will give a solvent gradient in mixed solvents but also with any phenomena that will give a solvent gradient in thethe column,

column, e.g.e.g. pH or ionic strength. pH or ionic strength.

Fig. 5.2: (a) Frontal analysis; (b) Displacement development; and (c) Elution analysis Fig. 5.2: (a) Frontal analysis; (b) Displacement development; and (c) Elution analysis