UNIT 5 LIQUID COLUMN

CHROMATOGRAPHY

Liquid Column
Chromatography

Structure
5.1

Introduction
Objectives

5.2

Recapitulation of Basics
Liquid-Solid Chromatography
Liquid-Liquid Chromatography

5.3

Experimental Set up
Equipment

5.4

Choice of Stationary and Mobile Phases

Stationary Phases Used in Liquid-Solid Column Chromatography
Stationary Phases Used in Liquid-Liquid Column Chromatography
Mobile Phases in Liquid Column Chromatography

5.5

Development Techniques
Frontal Analysis
Displacement Development
Elution Analysis

5.6
5.7
5.8
5.9
5.10

5.1

Basic Aspects of HPLC

Applications
Summary
Terminal Questions
Answers

INTRODUCTION

In the previous unit (Unit 4), a thorough discussion on the classification and general
principles of chromatography has been presented. While classifying the different
chromatographic techniques, the main criteria used was the nature of the mobile
phase. It was pointed out that a large number of diversifications are available in the
case of liquid chromatography. These are mainly due to shape of the support (column
and two dimensional), nature of support (simple and bonded) and the mechanism
(adsorption, partition, ion exchange and sieving) responsible for separations. In this
unit, it is proposed to discuss liquid column chromatography. Normally, in the
liquid column chromatography, the different mechanisms cited above should be
included but in the general parlance of chromatography, the technique includes only
two mechanisms, adsorption and partition. Thus, the discussion in this unit will
confine to liquid- solid adsorption and liquid- liquid partition chromatography. In this
course, separate units have been assigned for ion exchange and gel sieving
chromatography. Thus, we will be discussing only liquid-solid adsorption
chromatography (LSC) and liquid- liquid partition chromatography (LLC). The
situation is more or less similar to gas chromatography where we have GSC and GLC.
If we compare the two important types of chromatography, viz gas and liquid
chromatography, some of the advantages of liquid chromatography become very
apparent. The tremendous ability of gas chromatography to separate and analyze
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
chromatography. Liquid chromatography, on the other hand, is not limited by sample
volatility or thermal stability. Thus, liquid chromatography is ideally suited for the
separation of macromolecules, ionic species, labile material products and a wide
variety of other high molecular weight compounds. Liquid chromatography also
enjoys certain other advantages over gas chromatography in view of the fact that very
difficult separations are often more readily achieved by liquid chromatography than by

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Chromatographic
Methods-I

gas chromatography. The other advantage is about the sample recovery. Separated
fractions are easily collected and recovery is quantitative. The recovery of separated
components in gas chromatography is also possible but is generally less convenient
and less quantitative. It may be important here to point out that gas chromatography,
in general, is faster than liquid chromatography.
In this unit, we will first recapitulate some of the basics of liquid chromatography.
Some special features of liquid-solid adsorption and liquid-liquid partition column
chromatography will be discussed. This will be followed by a discussion on the
components of a liquid chromatography set up. The choice of stationary and mobile
phases is very important and the considerations involved will be discussed. This will
be followed by a discussion on the basic methods used for chromatographic column
development. In order to highlight the importance of the technique, some of the
applications to separate complex mixtures will be presented. Since the conventional
liquid chromatography has undergone a major development in the form of high
performance liquid chromatography, a brief idea about this will also be given at the
end.

Objectives
After studying this Unit, you should be able to

recapitulate some of the basic concepts of liquid-solid and liquid-liquid column

chromatography,

understand the functioning of the components of an improved version of a liquid

chromatographic set up,

appreciate the criteria used for the choice of stationary and mobile phase,

describe development techniques used,

get an idea about HPLC, and

enumerate some of the important applications of liquid column chromatography.

5.2

RECAPITULATION OF BASICS

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

chromatography (LSC) and liquid-liquid partition chromatography (LLC); both of
them being operated on a column. When a sample mixture is injected into a liquid
chromatographic column, it begins to migrate down the column under the influence of
the mobile phase. During this process, various components of the mixture will begin to
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
pass through the column and be eluted earlier. Thus, there will be peaks in the order in
the resulting chromatogram. The strongly retained components will elute later, the
relative separation being dependent upon the degree of retention by the stationary
phase for each sample component. Thus, the components pass down the column at
different speeds which can be related to the distribution of each component in the
stationary and mobile phases. The two forms of chromatography, (LSC) and (LLC),
basically differ in the mechanism responsible for separation. In one case adsorption is
responsible while partition is operative in the other. An idea about this is being given
below.

5.2.1 Liquid - Solid Chromatography

The liquid - solid chromatographic technique is based on adsorption phenomenon.
Consider a liquid solution of two compounds which has been brought into contact with
a porous adsorbent. The molecules from the solution enter the pores of adsorbent and

36

become attached to its surface. These molecules are held rather loosely by van der
Waals forces. Some pass back into the body of the solution, and their places in the
pores of the adsorbent are taken by other molecules. Thus, there is a continual
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
than the other, so that, when equilibrium is established, the concentration of this
component in the pores will be higher than its concentration in the surrounding liquid.
Assuming that the phases can be perfectly separated, the separation factor is defined
by the equation

Liquid Column
Chromatography

= ( X / Y )a / ( X/ Y)l
i.e., , the separation factor, is equal to the ratio of the mole fractions of the two
components, X over Y, in the adsorbed phase, a, divided by their ratio in the liquid
phase, l.

5.2.2 Liquid-liquid Chromatography

Liquid-liquid chromatography is sometimes called liquid partition chromatography.
Liquid-liquid chromatography is based on the separation of the solutes by their
differential partitioning between two immiscible phases. This usually involves a
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
mobile phase. In some circumstances, however, it is advantageous to reverse the roles
so that the stationary phase is less polar. This variation is known as reversed phase
partition chromatography. The process of liquid-liquid chromatography is similar to
simple batch extraction between two immiscible liquids in a separatory funnel. A
successive series of such extractions forms the basis of countercurrent distribution,
which is more efficient than simple one stage extraction. However, liquid-liquid
chromatography is many times faster and more efficient than countercurrent
extraction. This is the result of the large interface between moving and stationary
phases.
In liquid-liquid chromatography, equilibrium distribution of the solutes between the
mobile phase and the stationary phase takes place rapidly, and the separation of the
components of a mixture results from the resulting distributions of the various solute
molecules in the two immiscible phases. The distribution equilibria are described by
the distribution coefficient, often called partition coefficient K. For practical
chromatography, it is necessary to be able to predict a particular solvent-solute
relationship in order to obtain the required separation of a mixture. The distribution of
a solute between two phases is also defined in terms of capacity or retention factor, k.
The different terms and concept of theoretical plates and the rate theory have already
been explained in Unit 4. All these are applicable for both of these forms of column
chromatography.

SAQ 1
What is the basic difference in LSC and LLC? Which one will be generally faster?

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Chromatographic
Methods-I

5.3

EXPERIMENTAL SET UP

At this point, with the background that we already have, it may be important to discuss
the experimental set up of liquid column chromatography. Conventionally, in the
classical set up there is a simple column, packed with an adsorbent or a support
material coated with a stationary phase. The mixture is fed to the column which is then
irrigated with the mobile phase. The eluant is collected in small increments and put to
analysis. However, with time, several improvements have taken place.
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
and this amounts to a significant expense of both manpower and material. In classical
liquid chromatography, the sample application requires some skill and time on the part
of the operator. Solvent flow is achieved by gravity feeding of the column. Separations
require several hours. The detection and quantitation are done by the manual analysis
of individual fractions. Many fractions are collected normally and their processing
requires much time and effort. On the other hand, in modern liquid chromatography,
reusable columns are used so that a number of individual separations can be carried
out on a given column. Since the cost of an individual column can now be prorated
over a large number of samples, it is possible to use more expensive column packing.
Precise sample injection is achieved easily and rapidly in modern liquid
chromatography. Solvent flow is achieved by means of high pressure pumps with
controlled flow rate which results in more reproducible operations and better and
faster separations. The detection and quantitation are done with continuous detectors
of various types which yield a continuous chromatogram without intervention by the
operator.
Fig. 5.1(a) shows an improved version of a liquid chromatographic set up while a
more sophisticated set up is shown in Fig. 5.1 (b).

Fig. 5.1(a): An improved version of liquid chromatographic set up

38

Liquid Column
Chromatography

Fig. 5.1(b): A more sophisticated liquid chromatographic set up

1. Mobile phase reservoir 2. Slurring device 3. Lamp (Heating device) 4. Pump
5. Pressure monitoring device 6. Pump 7. Filter 8. Precolumn 9. Column 10. Pump
11. Injection port 12. Column thermostat 13. Detector 14. Recorder

5.3.1 Equipment
The equipment needed to carry out modern liquid chromatography is very different
from the relatively simple and unsophisticated equipment used for classical liquid
chromatography separations. The schematic of equipment used for modern liquid
chromatography is shown in Fig. 5.1 ( b). The components of this sophisticated set up
are discussed below.

i)

Mobile phase reservoir

It holds one litre of the mobile phase. This reservoir is made up of stainless steel
which is inert to most mobile phases and is not subject to breakage. Many
different forms of reservoirs have been used, and simple units may be
constructed from glass flasks or bottles of an appropriate size. Some reservoirs
are designed so that the mobile phase may be degassed in situ. Degassing is
required to eliminate dissolved gases, particularly oxygen. To permit in situ
degassing, reservoirs are sometimes equipped with a heater, a stirring
mechanism and inlets for applying vacuum and a nitrogen purge.

ii)

Pumps
One of the most important parts of modern liquid chromatography instrument is
the pumping system. In modern liquid chromatography, the resistance to flow of
the long, narrow columns packed with small particles is relatively high, and high
pressures are required. Pumps are grouped into two major categories:
mechanical pumps which deliver the mobile phase at a constant flow rate, and
pneumatic pumps which deliver the mobile phase with a constant pressure.

iii)

Filter
A filter is normally placed in the line following the pump to remove fine
particles of particulate material which can clog the inlet of the column.
Generally a 2 sintered stainless steel filter is adequate for this purpose.

iv)

Pressure monitoring device

A device for monitoring the column input pressure should be inserted in the line
between the pump and the chromatographic column. This pressure monitoring
device indicates if there has been a plugging of the column or a failure of the
pumping system.

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Chromatographic
Methods-I

v)

Sample introduction device

Sample introduction into a liquid chromatography column is a very important
factor in obtaining high column performance. The sample should be introduced
as an infinitely narrow band on to the chromatographic bed. The more defused is
the plug of sample in the mobile phase introduced into the column, the wider is
the separated component bands at the end of the column. The sample is injected
with a micro-syringe through a septum contained in a low volume inlet system.
Septum materials are generally made from silicone or Neoprene. It is generally
not feasible to make syringe injections above about 1500 psi through sampling
ports. Therefore, at high pressure, a stop flow injection technique is normally
used with syringe.

vi)

Column
The unpacked column must be constructed of materials that will withstand both
the pressures to be used and chemical action of the mobile phase. Most columns
are made up of stainless steel tubing. However heavy wall glass columns are
sometimes used. Columns that will withstand pressures up to 600 psi are
commercially available. For operation at high pressures, glass lined metal
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
the packing. Straight sections of liquid chromatography columns in lengths of
25-150 cm are normally preferred. Some columns may also be bent into a U
shape. Coiled columns are some times used, but are often less efficient than
columns prepared in straight sections. Precolumns generally are desirable. The
precolumn ensures that the mobile phase is completely saturated with the
stationary phase before it passes in to the carefully prepared analytical column.
The internal diameter of the column has a significant effect on the efficiency of
liquid chromatography columns. For analytical studies, columns 1-4 mm
internal diameter (i.d.) are normally used. Columns of larger internal diameter
are used for preparative work.

vii) Column thermostat

It is important to control the column temperature in liquid chromatography. The
temperature variations within the column should be maintained within 0.2 0C.
The larger changes in column temperature can result in significant variations in
retention time.

viii) Detectors
In liquid chromatography, the ideal detector should have high sensitivity, good
precision and predictable response to all solutes. It should be unaffected by
changes in temperature and carrier flow. It should not contribute to extra column
band broadening. It must be nondestructive of the solute. Two types of detectors
are in use in liquid chromatography, the bulk property or general detectors and
solute property or selective detectors. Bulk property detectors measure a change
in some overall physical property of the mobile phase plus that of the solute.
The solute property detectors are sensitive only to the solute.

ix)

Column packings
The packing of columns in liquid chromatography are described in terms of
adsorbent or stationary phase, the type of particle and particle size. Each of these
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 :

40

Dry packing of rigid solid material using tap-fill procedure.

Slurry packing technique for columns of hard gel.

Slurry sedimentation method for soft gels.

Liquid Column
Chromatography

SAQ 2
Why should the column temperature be maintained in a chromatographic set up?

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SAQ 3
What are the various techniques for column packing?

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5.4

CHOICE OF STATIONARY AND MOBILE PHASES

The success of a separation by liquid column chromatography depends upon a proper

choice of stationary and mobile phases. In LSC, the stationary phase is an adsorbent.
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,
the stationary phase is a liquid immobilized on an inert support. While considering the
liquid, its polarity and tendency to leach out become important. To counter act the
leaching, bonded phases have been developed. Moreover, the requirements of inert
supports on which the liquid is immobilized are equally important to know. Finally,
the requirements of mobile phase are to be properly understood. This section deals
with of the above mentioned different aspects.

5.4.1 Stationary Phases Used in Liquid-Solid Column Chromatography

Stationary phases of a great variety have been used in liquid chromatography.
Presumably any finely divided or porous solid which has the adsorption capacity and
which is not too soluble in the mobile phase may be used. Many different substances
have been used as adsorbents including activated alumina, silica gel, carbon,
magnesium oxide, magnesium carbonate, hydrated calcium silicate, talc, silver
sulphide, bauxite, activated clay from bentonite, fullers earth, sucrose, and powdered
cellulose.
i)

Adsorbent type
Various adsorbent types exhibit different selectivities towards different types of
compound. Polar adsorbents such as metal oxides, magnesium silicate etc.
selectively adsorb unsaturated, aromatics and polar molecules such as alcohols,
amines and acids. Polar adsorbents may be further sub-divided as acidic, basic
or neutral, according to the pH of the surface. Silica, magnesium silicate are
acidic and thus, they chemisorb bases. The alumina surface contains both acidic
and basic sites. Non-polar adsorbents such as graphitized carbon which is a
strong adsorbent and kieselguhr which is a weak adsorbent show no selectivity
for the adsorption of polar molecules.

ii)

Surface area
The surface area and pore diameter of a given adsorbent vary widely with the
method of manufacture. In adsorption chromatography, the separation depends
on the transport of the molecules through the system and on the interchange of
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Chromatographic
Methods-I

the 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
interchange between the phases and the amount of separation will be small. For
this reason, when dealing with large samples, it is important to select an
adsorbent with a large surface area.
iii)

Particle size
The effect of particle size of the adsorbent on the sharpness of chromatographic
separations has been noted by many investigators. For sharp separations, it is
recognized that a finely divided material is necessary. An adsorbent in the range
from 100 to 200 mesh (149 to 74 ) in particle size is specified. Some authors
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.,
below 50 in particle size, and columns that are poorly packed give rise to
zones that are irregular in shape. Some adsorbents are available only as finely
divided powders with particles below 10 in diameter, e.g. magnesium oxide. It
is necessary to mix these adsorbents with filter aids such as Celite or Hyflo
Super-Gel to obtain a practical rate of flow.

iv)

Activation and regeneration of adsorbent

The term activation refers to those processes which are used to enhance the
effectiveness of an adsorbent by improving the pore structure and increasing the
surface area. In general, with carbon, high temperature activation produces an
organophilic adsorbent, and low temperature activation, a hydrophilic adsorbent.
During adsorption, the pores of the adsorbent become filled with adsorptive
molecules. The term regeneration refers to the removal of the adsorbed
molecules and the return of the adsorbent to its original state. The regeneration
of the adsorbent can be carried out by gentle heating if the adsorbed molecules
are volatile. If they are non-volatile, then they may sometimes be removed by
elution or desorption with volatile solvent, which in turn, may be removed by
gentle heating. With some adsorbents overheating will destroy the pore structure
e.g. silica gel, should not be heated above 200oC. On the other hand, the rugged
adsorbents, fullers earth and bauxite, may be heated in an oxidizing atmosphere
to temperatures sufficient to burn off the adsorbed material near 540oC.

5.4.2 Stationary Phases Used in Liquid-Liquid Column

Chromatography
In liquid-liquid column chromatography, the stationary phase is a liquid that is
immobilized on a inert support. The stationary phases fall into two classes: the more
usual hydrophilic ones and the reversed phase. The stationary or supported liquid
phases have been of many kinds varying in polarity from water to paraffin
hydrocarbons. As a large number of liquid stationary phases can be held mechanically
on an inert support, such stationary phases have some disadvantages like leaching out
of the liquid stationary phase from the inert support. In order to eliminate such
disadvantages, surface-reacted or bonded stationary phases have been developed. The
advantages of these materials is that pre-columns and or presaturation of the two
phases is not required. In addition, packing with bonded stationary phases are quite
stable because there is no opportunity for the chemically bound stationary phase to
be eluted during use. A disadvantage of bonded-phase packing is a lack of systemic
information regarding the mode of retention for solutes. There are two types of
surface-reacted or bonded stationary phases that are now commercially available. The
first one is an esterified siliceous material e.g. Durapak and the second type is surface
reacted packing e.g. Bondpack, Vydac (organic coating is a monomolecular),
Permaphase ( organic coating is of many layers). Columns of bonded phase packings
have been used continuously for many months without changes in chromatographic
42

characteristics. These bonded phase materials are available with several types of
functional groups and used for separations of many types of solutes.
There are four different techniques now in use for preparing liquid coated packing for
liquid-liquid column chromatography:
1.

Solvent evaporation technique.

2.

In situ coating procedure.

3.

The solvent filtration technique.

4.

The equilibration technique.

Liquid Column
Chromatography

Supports for liquid-liquid partition chromatography

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

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

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

It should consist of particles as identical as possible which allow the most

uniform and reproducible packing.

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

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

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

When applied for routine analysis or for preparative purposes, it must be

relatively cheap, easily available and permit regeneration.

5.4.3 Mobile Phases in Liquid Column Chromatography

Various physical and chemical properties govern the choice of mobile phases. The
most important factor is the influence of the mobile phase on the selectivity of the
system. The solubility of the samples and influence of such properties as surface
tension and viscosity are also important. Solubility of some samples, especially
polymers, limits the choice of the mobile phase and the use of certain detectors
imposes constrains.
The choice of mobile phase in liquid column chromatography is all important. If
water-deactivated silica is used as an adsorbent, the solvent is then varied to give k
values in the optimum range (1< k <10). Solvent strength, which controls the k
values of all sample bands, is easily predicted in liquid-solid column chromatography.
It can be defined quantitatively by the solvent strength parameter o which are listed
for several pure solvents in Table 5.1. These values are for alumina as an adsorbent.
The solvents listed in Table 5.1 are arranged in order of increasing strengths which is
referred to as an elutropic series. If an initial solvent is too strong then a weaker
solvent is substituted. Similarly, if the initial solvent is too weak then a stronger
solvent is substituted. Thus, an elutropic series can be used to find out right solvent
strength by a rapid trial-and-error approach.

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Chromatographic
Methods-I

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
changes continuously with composition. Another advantage of binary solvents is that
solvent viscosity can be kept low.
While considering the choice of mobile phases in liquid-liquid column
chromatography, some basic characteristics of mobile phases must be considered. The
mobile phase must be immiscible with the stationary phase. It should have viscosity as
low as possible for higher column permeability and or efficiency. The detector can
also limit the phases which can be used. e.g. strongly UV-absorbing solvents should
be avoided with an ultraviolet photometric detector. The cost, toxicity, purity and
stability of a solvent also should be taken into account. Most important is the
selectivity of a liquid-liquid system for a given sample. In liquid-liquid column
chromatography, the k values of solutes are generally controlled by changing the
mobile phase. The scale of solvent polarity is defined by the Hildebrand solubility
parameter, . The parameter, , is a good measure of what is commonly called
polarity. Non-polar solvents have low values of , while polar solvents have large
values. The values of for different solvents are shown in Table 5.1.
Table 5.1: Solvent Strength and Polarity Data
Solvent

Hildebrand
solubility
parameter

Solvent
strength

Solvent

Hildebrand
solubility
parameter

Solvent
strength

n-Pentane

7.1

0.00

Ethylene
dichloride

9.7

0.44

Isooctane

7.0

0.01

Triethyl amine

7.5

0.54

0.01

Acetone

9.4

0.56

Cyclohexane

8.2

0.04

Dioxane

9.8

0.56

Cyclopentane

8.1

0.05

Tetrahydrofuran

9.1

0.57

Carbon
tetrachloride

8.6

0.18

Ethyl acetate

8.6

0.58

Xylene

8.8

0.26

Methyl acetate

9.2

0.60

i-Propyl ether

7.0

0.28

Nitromethane

11.0

0.64

Toluene

8.9

0.29

Acetonitrile

11.8

0.65

Benzene

9.2

0.32

Dimethyl
sulfoxide

11.5

0.75

Ethyl bromide

8.8

0.35

n-Propanol

10.2

0.82

Ethyl sulfide

8.6

0.38

Ethanol

11.2

0.88

Chloroform

9.1

0.40

Methanol

12.9

0.95

Methylene
chloride

11.9

0.42

Ethylene glycol

14.7

1.1

Petroleum ether

SAQ 4
What are the different techniques to prepare liquid coated support?

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44

SAQ 5
What is meant by solvent strength of a mobile phase?

Liquid Column
Chromatography

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SAQ 6
What is the advantage of using a binary solvent mixture as a mobile phase in LSC?

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SAQ 7
What does Hildebrand solubility parameter signify?

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5.5

DEVELOPMENT TECHNIQUES

After having learnt about the stationary and mobile phase, it is important to know as to
how the columns are developed. There are three basic methods of chromatographic
developments.
1.

Frontal analysis.

2.

Displacement development.

3.

Elution analysis.

Let us know them in a little more detail.

5.5.1 Frontal Analysis
In frontal analysis, a large sample in a suitable solvent is passed through a short
adsorption column previously saturated with solvent and the effluent is analyzed
continuously until its composition is identical with that of the original sample.
Consider a three component mixture containing equal quantities of each component
fed continuously onto a column. Because of the forces between solute and stationary
phase, each solute will be retained to a different extent as it comes into equilibrium
with the stationary phase while passing through the column. The first component to
elute will be that which is held least strongly in the stationary phase, then the second

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Chromatographic
Methods-I

component will elute but in conjunction with the first component, and finally, the most
strongly held of the three will elute in conjunction with the first and second
components. Subsequently, there will be no change in concentration of solute in the
mobile phase and the concentration of the respective solutes will be the same as the
feed mixture. The concentration profile resulting from frontal analysis is shown in
Fig. 5.2 (a). The continuous curve shows the total concentration of solutes in the
eluent, plotted against volume of mobile phase passed through the column, and the
dotted curves represent a similar concentration profile but for each individual
component. Frontal analysis was employed as a development procedure in the early
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
that no individual component is completely separated from the others in the mixture.

5.5.2 Displacement Development

Displacement development has the advantages of being able to accommodate large
samples and giving sharp separations. It depends on the competition between solutes
for the active sites of the adsorbent and is only really effective in separating very
strongly adsorbed materials. In displacement development, all the substances in the
sample will be held on the stationary phase so strongly that they cannot be eluted by
the mobile phase; they can, nevertheless, be displaced by substances that are held on
the surface by stronger forces. However, there will be competition between individual
solutes and, when the sample is placed on the column, all the immediately available
active sites of the adsorbent will be occupied by the most strongly held component.
As the band of the sample moves down the column, the next available sites will be
occupied by the next strongly retained component. Thus, all the components array
themselves along the column in order of their adsorption strength. To develop the
chromatogram, another substance called the displacer is introduced into the mobile
phase stream, the displacer has an even higher affinity for the adsorbent than any of
the components to be separated.
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
the next group of sites occupied by the next component which will then itself be
displaced. Thus, the displacer drives the adsorbed components progressively along
the column, each component displacing the one in front, until they are eluted in
the same order in which they were adsorbed on the column. The least strongly held
being eluted first. The concentration profile of displacement development is shown in
Fig. 5.2 (b). Displacement development has very limited applications as a separation
technique and is only very rarely used in quantitative analysis.

5.5.3 Elution Analysis

The elution analysis is the most common technique in chromatography. Employing
elution analysis, complete separation can be achieved. This technique is being widely
used for quantitative analysis. A relatively inert solvent is used in large quantities to
transport the components down the column.
The nature of the solvent influences the equilibrium between stationary and mobile
phases. The nature of the solvent can also affect the shape of the distribution
isotherms. Bands that might travel down the column with a symmetrical concentration
profile (Fig. 5.2 (c)) with one solvent might be quite unsymmetrical with another.
When elution analysis is used to separate materials with widely differing distribution
coefficients, the use of single elution solvent is not practical. If the eluent chosen is
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
hand, if the eluent is weak, it will be difficult to elute more strongly held materials.
46

Therefore, when the sample components have a wide range of distribution

coefficients, graded eluents are used. When utilizing graded eluents, the usual
procedure is to begin with the weakest solvent capable of eluting one of the sample
components and to change increasingly stronger solvents until all of the sample has
been eluted. The change from one member of the series to the next is made by a
stepwise increase in concentration of the stronger solvent. This technique is called as
gradient elution analysis. The gradient elution technique can be used not only with
mixed solvents but also with any phenomena that will give a solvent gradient in the
column, e.g. pH or ionic strength.

Liquid Column
Chromatography

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

47

Chromatographic
Methods-I

SAQ 8
What is meant by development of column? What are the different ways of
achieving it?

...
...
...
...
...

5.6

BASIC ASPECTS OF HPLC

Possibly, the most significant advancement in chromatography since 1966 is the

development of High Performance Liquid Chromatography (HPLC). The current
practice in HPLC is based on the application of pressure to liquid systems.
The HPLC is characterized by a long, narrow column packed with very fine powder
with spherical particles, operated at high liquid pressures, and possessing a continuous
monitoring facility for the common effluent.
Such chromatographs may be quite expensive, but in turn, it is possible to achieve
efficiencies comparable with those obtainable in gas chromatography, with separating
times of the same order and the ability to work with non-volatile materials.
Technically, the development work in HPLC has concentrated on the production of
pulse-free pumps for generating the high pressures required and sensitive detectors for
monitoring the column effluent.
In HPLC, the quantitative measurements are readily carried out on the chromatogram
since the amount of substance present is proportional to its peak area. The analysis of
standard samples is often necessary for comparative purposes. The advantages of
HPLC technique are as follows:
a)

Non-volatile materials can be handled.

b)

Ability to operate long columns at near optimum solvent velocity throughout

their length.

c)

The nature of mobile phases can be varied to produce either stepwise or gradient
elution or both.

The technique is discussed in detail in Unit 8.

5.7

APPLICATIONS

The liquid column chromatography has been used to separate a wide range of sample
types. Several interesting separations of compounds containing metal ions, isomers of
cobalt complexes involved in the synthesis of vitamin B-12 and metal--diketones
have been achieved. Steroids and related synthetically prepared compounds have been
separated by liquid-liquid column chromatography.
Various pesticides have also been separated by liquid-liquid column chromatography.
The stationary and mobile phases used for the separation of various compounds have
been shown in Table 5.2. The separations listed only give a birds eye view of the
variety of difficult separations that can be achieved by liquid chromatography.

48

Table 5.2: Applications of Liquid Column Chromatography

Sample Separation

Stationary Phase

Mobile Phase

Column
Parameters

Steroids containing
progesteron, androsteron,
testosteron, 19-nortestosteron

1% -oxydipropionitrile on
Zipax

Heptane

Flow 1
mL/min

Pesticide residues containing

methylparathion, parathion,

Diatomaceous earth
coated 2,2,4trimethylpentane

60.1 % water, 38.8

% etnanol,0.8 %
acetic acid, 0.21 %
NaOH,0.09 % KCl

18 0.27 cm
glass column

Vitamins ( mixture )

Permphase ODS

H2O to CH3OH 5
%

2 mL/min

Pesticides containing aldrin,

DDT, DDD, lindane, endrin

Corasil--II

n-hexane

Column 20
2.3 cm

Liquid Column
Chromatography

Flow rate 3.4

mL/hr

Flow rate 3.0

mL/mm
Barbiturates containing
hexobarbital, phenobarbital,
amobarbital, barbital

Vydac

2 % methanol in
heptane

Catecholamines from tisue,

plasma, urine

Acidified alumina

1.0 M acetic acid,

0.2 M perchloric
acid

Formaldehyde in air

Silica coated with

DNHP

Acetonitrile

Polycyclic aromatic
hydrocarbons

Alumina

Cyclohexanebenzene mixture

Coccidiostat in poultry
feedstuffs

Silica gel

Acetonitrilechloroform (1:1)

5.8

1m 2mm
stainless steel
column

SUMMARY

This unit embodies discussion on liquid column chromatography emphasizing on the

separations by adsorption and partition mechanism. To start with some of the
advantages of liquid chromatography (LSC, LLC) over gas chromatography (GSC,
GLC) have been highlighted. Some of the basics aspects relevant to LSC and LLC
have been recapitulated. The different components of a modern liquid column
chromatographic set up have been discussed. Since the success of a chromatographic
separation depends on the choice of stationary (adsorbent/ liquid coated support) and
mobile phases, the criteria used for their selection have been elaborated. The methods
used for the development of a column with their special features have been discussed.
Based on the background developed in the unit, an idea about HPLC is given. Finally,
the unit ends with a few typical applications of liquid column chromatography.

5.9

TERMINAL QUESTIONS

1.

In what particular respect, the liquid chromatography scores over gas

chromatography?

2.

What are the requirements of a good detector for liquid chromatographic set up?

49

Chromatographic
Methods-I

3.

What are the essential characteristics of a support material for liquid-liquid

partition chromatography?

4.

What is meant by activation and regeneration of an adsorbent?

5.

What are important requirements of an appropriate mobile phase?

6.

What is the role of displacer in the development of column by displacement

technique?

7.

Comment on the applications of liquid column chromatography in environmental

analysis by citing two examples.

5.10 ANSWERS
Self Assessment Questions
1.

The basic difference between LSC and LLC is that in LSC the separation takes
place due to difference in adsorption while in LLC the difference in partition is
responsible for the separation. Generally LLC is faster.

2.

The column temperatures should be controlled because large variations in

temperature can result in to significant variations in retention time.

3.

The various techniques used for column packing are:

4.

i)

Dry packing of rigid material using tap fill procedure.

ii)

Slurry packing technique for columns of hard gel.

iii)

Slurry sedimentation method for soft gel.

The different techniques to prepare liquid coated support are

i)

Solvent evaporation technique.

ii)

In situ coating procedure.

iii)

Solvent filtration technique.

iv)

Equilibration technique.

5.

The solvent strength, 0, controls the k values. These values are given with
respect to an adsorbent. The values for solvents arranged in increasing strengths
is referred as elutropic series. An elutropic series is used to find the right solvent
strength by trial and error approach.

6.

The advantage of using a binary solvent mixture in LSC is that solvent strength
changes continuously with the composition and the appropriate composition can
be selected. Moreover, the solvent viscosity can be controlled and kept low.

7.

The Hildebrand solubility parameter is a measure of the polarity of the

solvent. Non polar solvents have low values of while polar solvents have
large values.

8.

The development of a column essentially means separating the different

components of mixture fed to the column by irrigating it with a suitable mobile
phase. There are three basic methods for chromatographic development which
are as follows:
i)

50

Frontal analysis

ii)

Displacement development

iii)

Elution analysis.

Liquid Column
Chromatography

Terminal Questions
1.

Liquid chromatography scores over gas chromatography because the later is not
applicable in cases of compounds which are thermally unstable or not easy to
volatilize. In the case of GC, the quantitative recovery of the components is not
easily achieved while in LC it is easy to recover the samples quantitatively.

2.

A good detector should have high sensitivity, good precision and predictable
responsible to all solutes. It should be unaffected by temperature changes and
solvent flow. It should not contribute to extra band broadening. It should be
non-destructive to the solute.

3.

The essential characteristics of a support material for liquid-liquid partition

chromatography are as given under.
i)

It should be chemically inert. It should neither dissolve nor swell in the

stationary phase.

ii)

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

iii)

It should allow the column to have an acceptable pressure drop as regards

the mobile phase.

iv)

It should have sufficient mechanical strength and should not grind during
column packing, impregnation or regeneration of support material.

v)

If being used for routine analysis and commercial purposes it should be

relatively cheap, easily available and permit regeneration.

4.

Activation refers to those processes which are used to enhance the effectiveness
of an adsorbent by improving the pore structure and increasing the surface area.
The term regeneration refers to the removal of the adsorbed molecules and
bring the adsorbent to its original state.

5.

The requirements of an appropriate mobile phase are given as under:

6.

i)

It should have viscosity as low as possible for higher column permeability

and or efficiency.

ii)

It should give a retention/ capacity factor for solutes preferably between 2

and10.

iii)

The cost, toxicity, purity and stability should be given due consideration.

iv)

The detector response to the solutes should not be interfered with.

To develop a chromatogram by displacement technique, a displacer is

introduced in to the mobile phase stream. The displacer has even higher affinity
for the adsorbent than any of the components to be separated. Thus, on coming
in to contact with the sites occupied by the most strongly adsorbed component,
it will displace this component in the mobile phase and thus move on to the next
group of the sites occupied by the next component which will then itself be
displaced. Thus, the displacer drives the adsorbed components progressively
along the column, each component displacing the one in front until they are
eluted in the same order in which they were adsorbed on the column. The least
strongly held is eluted first.

51

Chromatographic
Methods-I

7.

There is a great awareness about the pesticide pollution. Organophosphorus

pesticides like methyl parathion and parathion are considered a little safer than
organochlorine like DDT. Liquid chromatography has been successful in
separating mixtures of organophosphorus and also that of organochlorine
pesticides. They can be subsequently quantified individually. Another class of
pollutants is polycyclic aromatic hydrocarbons. The technique has also been
successful in separating them.

Further Reading

52

1.

Chromatographic Methods, By R. Stock and C. B. F. Rice. Champman and

Hall and Science Paperback.

2.

Fundamentals of Chromatography, By H. G. Cassidy, Interscience Publishers.

3.

Chromatography: A Review of Principles and Applications, By E. Lederer and

M. Lederer, Elsevier Publishing Company.

4.

Principles of Adsorption Chromatography, By L. R. Snyder, Marcel Dekker,

Inc.

5.

Quantitative Analysis Using Chromatographic Techniques, By Elena Katz

(Editor), John Wiley & Sons.

6.

Introduction to Modern Liquid Chromatography, By L. R. Snyder and J. J.

Kirkland, A Wiley-Interscience Publication.

7.

Organic Trace Analysis, By Using Liquid Chromatography, By J. F.

Lawrence, Academic Press.