Sie sind auf Seite 1von 23

Chromatographic

Methods-III

UNIT 10

SIZE EXCLUSION
CHROMATOGRAPHY

Structure
10.1

Introduction
Objectives

10.2
10.3

Basic Principle
Gels and Their Important Properties
Important Properties of Gels for Chromatography

10.4
10.5
10.6
10.7

Classification, Synthesis and Properties


Variables Defining the Utility of Gel
Unique Features of the Technique
Some Applications
Analytical Applications
Preparative Applications
Miscellaneous Applications

10.8
10.9
10.10

Summary
Terminal Questions
Answers

10.1 INTRODUCTION
Before we discuss this chromatographic technique, it may be desirable to refer to
Unit 1 wherein classifications of separation methods have been dealt. In the scheme of
classification based on property resulting into separation, there is a distinct class in
which the separations are achieved on the basis of molecular geometry or molecular
dimensions. This size exclusion or gel filtration chromatography figures right in this
category. However, if we probe into another criteria of classification based on
equilibrium and rate processes, the exclusion chromatographic technique appears in
chromatographic processes where liquid and solid are in equilibrium. Thus, very
rightly the size exclusion chromatography is one of the important forms of liquid
chromatographic technique (Unit 4, sub-sec. 4.2.3).
The mobile phase is aqueous or organic and the stationary phase is a molecular sieve.
These sieves are generally polymeric carbohydrates and acrylamide that have an open
network formed by the cross linking of polymeric chains. Incidentally, this branch of
chromatographic science was also discovered in a Bioscience oriented laboratory.
This separation method originated in 1959 at the Biochemical Institute in Uppsala,
Sweden. Initially it was applied for the separation of water-soluble macromolecules of
biological importance. The technique was named as gel-filtration chromatography
(GFC). A few years later the technique was developed for synthetic polymers soluble
in organic solvents and it is was called as gel permeation chromatography (GPC).
This amounts to the fact that initially the names such as gel filtration chromatography
(mobile phase is water) used by biochemist and gel permeation chromatography
(mobile phase in organic solvent) used by polymer chemists described the technique.
Now the recommended or the most accepted name of the technique is size exclusion
chromatography (SEC). It is used in open column gravity fed for both analytical and
preparative separations and in high performance separations. The gel filtration also
finds use in thin layer chromatography and the technique is known as thin layer gel
filtration chromatography. Reference to gels will also be made in Unit 12 on
electrophoresis.

40

This unit on size exclusion chromatography first discusses the principle involved in
separations using gels. This is followed by a general discussion on gels and
characteristics required for the gels to be useful for chromatography. After explaining
about the characteristics of gels needed for chromatographic purposes, a classification
of important types of chromatographic gels is given. Along with this the methods of
preparation of the important categories of these gels on a broad basis are discussed and
the properties shown by them are highlighted. A brief note on the characteristics
which define the utility of a gel is also included. The unique features of this form of
chromatography are explained. Finally, some of the important applications of the
technique are discussed.

Size Exclusion
Chromatography

Objectives
After studying this Unit, you should be able to

discuss the principle responsible for separation of solutes,

describe gels and the characteristics required for their use for chromatographic
separations,

explain about the classification of gels, their method of preparation and


important properties of the different classes,

enumerate the variables which define the utility of a gel,

give the unique features of this chromatographic technique, and

cite some of the important applications of this method of separation.

10.2 BASIC PRINCIPLE


The packagings for size exclusion chromatography generally consist of cross linked
polymers of dextrans, polyacrylamides, styrene or silica. They have an open network.
On absorbing the solvent, swelling causes an opening of the structure. The degree of
cross linking will determine the size of the holes.
In this network of uniform pores, the solute and solvent molecules can diffuse. While
in the pores, molecules are effectively trapped and removed from the flow of the
mobile phase. The average residence time of the solute molecules depends upon their
effective size. The molecules which are significantly bigger than the average pore size
are excluded.
As a result of this, they suffer no retention and travel through the column at the rate of
mobile phase. The molecules that are appreciably smaller than the pore size can
penetrate throughout pore network and, thus, remain entrapped for the longest time.
As a result of this, the molecules which are able to penetrate the gel will spend part of
their time sheltered from the moving phase.
Between these two extremes are intermediate size molecules whose average
penetration into the pores of packing depends upon their diameters. Fractionation
within this group is directly related to the molecular size and, to some extent,
molecular shape.
Thus, there is a basis of separating molecules of different sizes. Fig. 10.1 shows
schematically three stages in the chromatographic separation of two extreme sizes of
molecules.

41

Chromatographic
Methods-III

Fig. 10.1: Schematic of a chromatographic separation on a size exclusion column. Large


open crossed circles represent the stationary phase; large black circles
represent molecules which do not penetrate K = 0; smaller black circles
represent molecules which penetrate the gel and are retarded in their
movement down the column.

It is important to note that size exlusions are different from other chromatographic
procedures. Here, there are no physical or chemical interactions between the analyte
and the stationary phase. As a matter of fact, efforts are made to avoid such
interactions because they may cause impaired column efficiencies. At this point, it
may be important to introduce the term exclusion limit. The exclusion limit is the
molecular weight of that molecule that will just permeate the gel and be retarded. This
can range from 1000 to several millions depending upon the gel. It should be kept in
mind that separation are based on molecular size and configuration rather than simply
its molecular weight but, generally, there is a correlation with molecular weight. Also,
generally the molecules smaller than the exclusion limit can be fractionated down to a
limiting size.
The entire picture of fractionation by size exclusion chromatography can be visualized
in some semiquantitative terms. Let VR be the retention volume for a solute with a
chromatographic column. Let V0 be the interstitial volume (void volume), that is, the
volume within the column which is available to the mobile phase. VL is the volume of
water within the gel particles available for accepting solutes. On the lines of GLC, we
can write the following equation:
VR = V0 + KVL
where, K is some form of distribution coefficient. If the solute is completely excluded
from the interior of gel then K = 0 and VR = V0. Such marker substances are available.
Now, if the solute can freely enter the gel, there should be no preference for water
inside or outside the gel and thus, K = 1, and VR = V0 + VL. Taking the case of
molecules which can penetrate the gel to some extent but not freely, K values fall
between 0 and 1.
In cases where sieving is the only phenomenon responsible for fractionation, K values
greater than 1 would never be encountered. However, sometimes these values are
obtained suggesting the occurrence of phenomena like adsorption, hydrogen bonding
and ion exchange between the gel and the solute.
Fig. 10.2 shows the typical
behaviour of variation in the retention volume with the molecular weight of the solute.

42

Size Exclusion
Chromatography

Fig. 10.2: Relation between retention volume and molecular weight of the solute. The
steep region between the arrows is the fractionation range.

If we refer to Fig. 10.2, it is clear that the molecules of differing in size can be
separated chromatographically in the sloping region of the curve. By varying the
degree of cross linking of the polymer, the curve shifts horizontally. There are
materials available which fractionate molecules in various molecular weight ranges.
For Sephadex G. 50, the fractionation range for peptides and globular protein is
molecular weight 1,500 to 30,000 while the range for G. 150 is 5,000 to 400,000.

SAQ 1
What particular property of gel is responsible for fractionation of solutes of different
molecular weights by size exclusion chromatography?
...
...
...

SAQ 2
What are the values of distribution coefficient if
i)

solute molecules do not enter the gel matrix

ii)

solute molecules enter the gel matrix

...
...
...

10.3 GELS AND THEIR IMPORTANT PROPERTIES


In everyday language, gel is a familiar name. It refers to a fairly soft, elastic material
containing water. In the scientific context, the term acquires a wider meaning. A gel
consists of a three dimensional network. The structural material, often consisting of
cross linked polymers, gives some mechanical stability. The space within the gel not
occupied by structural material is filled with liquid. Liquid occupies the main part of
gel. Some gels are soft and deform easily; this being the common picture. Others are
rigid or even brittle.

43

Chromatographic
Methods-III

There are many natural substances capable of forming gels and these include
polysaccharides from fruits and roots, proteins from animal tissues, inorganic silicates
and phosphates. If we look at gels from the chromatographic point of view and also
from general properties, two different types of gels can be distinguished. In the
macroreticular gels, the microstructure is strongly heterogenous with regions where
matrix material is aggregated and regions where there is very little gel matrix present.
The gel structure virtually free from gel matrix allows large molecules to enter. The
microreticular gels, on the other hand, show properties that the gel matrix is relatively
distributed throughout the gel. They fractionate relatively lower molecular mass
ranges than the macroreticular gels.

10.3.1

Important Properties of Gels for Chromatography

Out of different gels known only relatively few are suitable for chromatographic work.
Some essential requirements should be met by these gels to be useful for the said
purpose. These are as follows.
i)

Matrix of gel should be inert

Any interaction between the gel material and the solute may lead to irreversible
binding of the solute. The interaction may lead to chemical alteration of labile
substances. In biochemistry, there is a risk of denaturation of proteins and nucleic
acids. If the right material is there, gel filtration chromatography is one of the few
separation methods which is capable of giving quantitative yields.
ii)

Gel must be chemically stable

The gel should be stable over a wide range of pH and temperature. The gels that are
used in practice are stable over years and months. The leaching of material from bed
should be very low.
iii)

Low content of ionic groups

A low content of ionic groups is required to avoid ion exchange effects. Charged
groups will give bad yields of charged solute and asymmetric elution curve. It is
impossible to avoid the charged groups but the commercially available gels have very
low ionic groups.
iv)

Availability of wide choice of gels

For the adaption of a method of different problems, a wide choice of gels with same
general composition but different fractionation ranges should be available. For
microreticular gels, the fractionation range is mainly determined by the swelling
properties. Gels with low content of dry substance in the gel give access to larger
molecular weight molecules than those with high contents of dry substance.
With macroreticular gels, the content of the dry substance is no longer the only
variable which determines the fractionation range of the gel. The structure of the gel is
also important.
v)

Availability of gels with different particle size

The particle size distribution should be very carefully controlled. A column of small
particle size will generally give good resolution. If the particle size is increased, the
reasons for zone broadening are amplified. With large particles the diffusion in and out
of the particle takes longer. Flow pattern in large particles is inferior. On the other
hand, the resistance to flow in a bed of large particle is lower. Thus, a compromise
with regard to particle size should be reached giving maximum zone resolution under
the desired flow conditions. Generally, the commercial available gels are in bead
forms.
44

vi)

Mechanical rigidity of the gel particles

The mechanical rigidity of the gel grains should be as high as possible, otherwise they
tend to be deformed by the forces caused by the flow of liquid. The force may cause
the bead to compact reversibly or irreversibly, thus, increasing the flow through the
bed. The microreticular gels with small content of dry substance; a correspondingly
high exclusion limit, tend to be mechanically weak. One approach to solve the
problem is to synthesize gels with a macroreticular structure and with a microreticular
gel in the pores of macroreticular gel. The fractionation ranges are controlled by the
microreticular gel in the pores while the aggregates of macroreticular gel take care of
the mechanical strength. These gels are called macro-microreticular gels.

Size Exclusion
Chromatography

SAQ 3
What is a typical gel structure?
...
...
...
...

SAQ 4
What is a macro-microreticular gel? What is its special advantage for chromatographic
work?
...
...
...
...

10.4 CLASSIFICATION, SYNTHESIS AND PROPERTIES


In the previous section, we have discussed the characteristics which are needed for a
gel to be useful for chromatographic work. As such there are different types of gels
available but for general chromatographic separations, gels of some distinct categories
are used. These are commercially available in different sizes under different trade
names. They are known either by the basic unit of the polymer or more commonly by
the trade names. A classification of different types of gels is given below.
i)

Dextran gels (Sephadex)

ii)

Dextran-N, N -methylene- bisacrylamide gels (Sephacryl)

iii)

Polyacrylamide gels (Bio-Gel)

iv)

Agar and Agrose gels

v)

Styrene-divinylbenzene gels (Styragel)

Besides the above classes inorganic materials like silica gel and porous glass are also
used. Let us study the different types of gels in more detail.
1.

Dextran gels (Sephadex)

Dextran is a polysaccharide, built up from glucose residues. It is produced from the


fermentation of sucrose. The micro-organism that is used for fermentation is
Leuconostoc mesenteroides strain NRRLB512. The native dextran has high molecular
mass and a wide mass distribution (10 300) 106. The raw material is purified,
45

Chromatographic
Methods-III

partly hydrolysed and finally fractionated by ethanol precipitation to give a product of


suitable average mass and a narrow mass distribution. The glucose residues are joined
by -1, 6-glucosidic linkages. The chains are to a certain extent branched. The
branches are joined to the main chain by 1-2, 1-3 or 1-4 glucosidic linkages. If we
prepare dextran by Leuconostoc mesenteriodes strain B512, the branches are joined by
1-3-glucosidic linkages. Dextran dissolves in water but when it is cross linked to form
the gel, the polysaccharide chains of the gel form a three dimensional network. The
material, thus, becomes insoluble in water.
Sephadex is manufactured by a bead polymerization process. In this process, an
alkaline solution of dextran of suitable molecular mass distribution is suspended in an
organic solvent which is immiscible with water. Stabilizers are added to stabilize the
suspension. When the suspension is stirred to form an emulsion epichlorohydrin,
reacts with dextran matrix forming glyceryl links between the chains. After the
completion of the reaction, the product is washed thoroughly and allowed to shrink in
water-alcohol mixture and then dried. A partial structure of Sephadex is shown
in Fig. 10.3.

Fig. 10.3: A schematic representation of partial structure of Sephadex

Sephadex is available as a free-flowing powder consisting of regular bead from


Pharmacia Fine Chemicals. When this is suspended in water, the beads swell. Drying
and swelling of Sephadex is a reversible process. The material is capable of retaining
its chromatographic behaviour after repeated drying and swelling. Sephadex is
available in different types which differ in their degree of cross-linkage and, thus, the
swelling properties. The various types are characterized by the letter G followed by a
number. The range of the material available is Sephadex G 10 to Sephadex G 200.
The numbers correspond approximately to the water regain value of the gel multiplied
by a factor 10. Also, there are special kinds of Sephadex known as Sephadex LH-20
and LH-60 which are intended for use with polar organic solvents.

46

Sephadex is very chemically stable. The weakest points of attack in the structure are
the glucosides linkages which are hydrolysed at low pH. It is stable in alkaline
solutions. Its prolonged exposure to oxidizing agents may cause an increase in the
carboxyl group content. The increase of carboxyl group in the structure impairs the
chromatographic behaviour of the gel. It may be noted that initially Sephadex contains
a very small amount of carboxyl groups. This amount is so low that most of the charge
effects observed are attributed to Donnan effects caused by solutes.

Size Exclusion
Chromatography

Sephadex gels were the first for which a close relationship between molecular size and
elution behaviour was observed. In Sephadex series, there are gels available with
fractionation ranges distributed over a very wide interval of molecular masses. Some
differences are evident in the fractionation properties of different group of substances.
In most of the cases, these differences are relatively small and the general shape of the
fractionation curves is similar.
2.

Dextran N, N -methylene-bisacrylamide gels (Sephacryl)

Sephacryl is a dextran gel manufactured by cross linking allyl dextran with N, N methylene-bisacrylamide. There are cross links not only in dextran but methylenebisacrylamide molecules also bind to each other. A hypothetical structure of Sephacryl
gel is shown Fig. 10.4.

Fig. 10.4: A schematic representation of partial structure of Sephacryl

The cross linking reactions make the gel partly macroreticular and the gels can be
produced with fractionation ranges extending up to high molecular masses. They are
mechanically rigid and bear high pressures (upto 1M Pa) without compressing the bed.
These gels are intended mainly for use with aqueous eluants. Two types of Sephacryl
gels are available. They are Sephacryl-200 Superfine and Sephacryl S-300 Superfine.
The fractionation ranges cover the most common molecular masses of water soluble
proteins. Since in these gels, the structure is macroreticular, the slope of the selectivity
curve is less than the microreticular Sephadex gel. Due to large amount of methylene
bis-acrylamide in the gel, the adsorption effects are more pronounced than with
Sephadex.
47

Chromatographic
Methods-III

3.

Polyacrylamide gels (Bio-Gel)

Cross linked polyacrylamides form gels with water and are known as Bio-gels. They
are mainly used for biochemical work. Unlike the Sephadex gels, these are entirely
synthetic and made by acrylamide, H2C = CH CO NH2. It is synthesized by
copolymerizing acrylamide with the crosslinking agent N, N methylene-bisacrylamide (H2C = CH CO NH CH2 NH CO CH = CH2). The concentration
of the monomer can be varied to give different swelling characteristics and
chromatographic properties. A partial structure of Bio-Gel is shown in Fig. 10.5.

Fig. 10.5: A schematic representation of partial structure of Bio-Gel

Bio-Gels are available from Bio-Rad Laboratories. They are produced by bead
polymerization and are available as powder. They have a marked tendency to stick
together and form lumps. Like Sephadex, Bio Gel is xerogel.
When the dry powder is immersed in water, it swells to form the gel. Bio-Gel is quite
inert and the weakest point for chemical reaction are the amide groups which are
hydrolyzed at extremes of pH. On hydrolysis, the carboxyl groups formed impart the
ion exchange character.
Bio-Gels are available in eleven types with different swelling characteristics and
different fractionation ranges. The different types are characterized by the letter P.
There are gels from Bio-Gel P-2 to Bio Gel P-300. The number is intended to indicate
the exclusion limit.
There are striking similarities in the chromatographic behaviour of Bio-Gel and
Sephadex gel. Both the gel types are microreticular. Their selectivity curves are also
quite similar. The similarity is also reflected in their mechanical properties. Bio-Gel
beads like Sephadex beads with low water regain are brittle while the ones with high
water regain are very soft.

48

4.

Agar and agrose gels

Agar or agar-agar, as it was called earlier, is obtained from various species of seaweeds. It is a mixture of linear polysaccharides comprising mainly of D-galactose and
3,6-anhydro-L-galactose residues. A partial structure of agrose is shown in Fig.10.6.

Size Exclusion
Chromatography

Fig. 10.6: A schematic representation of partial structure of agrose

These gels are unique combining fractionation ranges at very high molecular masses
with good mechanical stability. They are a good supplement to the dextran gels and
polyacrylamide gels. The agar gels with the lowest fractionation ranges correspond
approximately in their fractionation properties to the dextran and polyacrylamide gels
with the highest fractionation ranges. These gels can fractionate in the range
intermediate between molecules and particles.
Agrose gel for chromatographic work are available from Pharmacia Fine Chemicals
(trade name Sepharose) and Bio-Rad (trade name Bio-Gel A). Sepharose is available
in three normal types and three corresponding types crosslinked with 2, 3dibromopropanol. The normal types are Sepharose 2B, 4B and 6B and crosslinked
types being Sepharose CL2B, CL4B and CL6B. The numbers indicate the percentage
of dry gel in the particles. Bio-Gel A is available in six types.
Unlike the gels discussed earlier, the macromolecules of gel matrix are not bound by
covalent bonds. They are supposed to be held together by hydrogen bonds. The
polysaccharide chains seem to aggregate in bundles. Between the bundles, there are
very large openings in the gel matrix. The structure is very open and at the same time
mechanically stable.
Agrose gel has some disadvantages as a chromatographic material. There is a
considerable amount of charged groups in the material. Because of the presence of
charged groups, it is recommended to work at high ionic strength to mitigate the
problem due to ion exchange effects. Another problem arises from the fact that agar
chains are not linked by covalent bonds. This makes the agar gels chemically unstable.
This amounts to the fact that they are less stable to pH extremes than the gels
described earlier. These gels maintain their structure if the water is substituted by
many organic solvents such as acetone or ethanol. The structure of agrose gels makes
it impractical to dry and reswell them. Therefore, once the gel has been prepared, it
should be stored in wet state. It may be important to emphasize again that agrose gel
have fractionation ranges at considerably higher molecular masses than expected by
comparison with dextran and polyacrylamide gels. The selectivity curve of these gels
is less steep than it is for the gels from dextran and polyacrylamide.
5.

Styrene-divinylbenzene gel (Styragel)

It is a class of polystyrene gels useful for purely non-aqueous separations in methylene


chloride, toluene, trichlorobenzene, tetrahydrofuran and so on. It cannot be used with
water, alcohol and acetone. These macroreticular gels have fractionation range from
1600 to 40 million. Styragel is available in 11 different types. These are manufactured
by Dowex Chemical Co. and sold by Waters Associate. Styragel is available
suspended in diethylbenzene. The beads are rigid and the solvent can be easily
exchanged even after the bed has been packed. The beads are supposed not to change
their volume with change of solvent.

49

Chromatographic
Methods-III

SAQ 5
What happens if the Sephadex gels are subjected to prolonged exposure to oxidizing
agents?
...
...
...
...
...

SAQ 6
In what important properties, the Sephadex and Sephacryl gels differ from each other?
...
...
...
...
...

SAQ 7
What are the weak points of chemical attack in Sephadex and Bio-Gels?
...
...
...
...
...

SAQ 8
What different classes of gels are not prepared from natural materials?
...
...
...
...

10.5 VARIABLES DEFINING THE UTILITY OF GEL


After having learnt about the different categories of gels used for chromatographic
work, the methods of their preparation and some of the important characteristics, it is
necessary to name a few variables which really characterize the utility of gel. The
most important of the different properties is the fractionation range. Sometimes, it is
given as the upper limit of fractionation or the exclusion limit. When the exclusion
limits or the fractionation ranges are given, it should be stated that for what type of
substances these data are obtained. These numbers vary with one type of substances to
another. For a given group of compounds, say globular proteins, polysaccharides, etc.

50

they are fairly constant. Another important variable of significance is the steepness of
the selectivity curve in the linear portion of the fractionation range.

Size Exclusion
Chromatography

For xerogels, the water regain is the common measure of the swelling capacity of the
gel. Here, it may be important to tell more about xero gels and aerogels. Different gels
react differently to the removal of liquid in them. The group of xerogels shrink on
drying to a compact material containing only the gel matrix. The aerogels, on the other
hand do not shrink; instead the surrounding air penetrates into the gel. Xerogels when
brought in contact with the liquid, take up liquid and return to the gel state. In aerogels
also, the air can be substituted by the liquid.
Getting back to water regain capacity, it is usually expressed as the amount of water
(in mL) imbibed by one gram of dry xerogel on swelling. It does not include the
interstitial liquid between the grains. In the case of dextran and polyacrylamide gels, it
has been observed that there is a close relationship between the water regain and the
fractionation properties. If the water regain is low, it is expected that the fractionation
range is at a low molecular mass. In the case of agrose gel, it is percentage of gel
matrix in the gel grain is taken instead of the water regain. While working with
nonaqueous solvents, the analogy is taken with water regain. Ultimately, the particle
size of the gel grain is an important variable. It affects the degree of zone broadening,
the resolution, the dilution and the flow rate. As a matter of fact, the above mentioned
information should be available to the user before a particular gel is put to use for
chromatographic work.

10.6 UNIQUE FEATURES OF THE TECHNIQUE


This simple technique of size exclusion chromatography rapidly became very popular
and almost became indispensable for biochemistry. Before we discuss some of its
important applications, it may be desirable to look into the reasons for its fast adoption
as a frontline chromatographic technique. The technique is simple to perform. It is
remarkably insensitive to the composition of the eluant and temperature. The added
advantage is that very liable compounds can be fractionated without the fear of their
destruction. The gel matrix generally does not cause denaturation and the experiment
can be performed in very mild conditions.
The size exclusion chromatography can fractionate substances of very high molecular
masses. By varying the contents of gel in gel matrix, the fractionation ranges can be
varied within wide limits. The most dense gels fractionate substances below molecular
mass 1000 while there are gels whose fractionation range extends to several millions.
Using this technique, certain problems in biochemistry can be resolved in a simple
way. Typical problems of this type are desalting of solutions of proteins and other high
molecular mass substances and determination of molecular weights of
macromolecules. The chromatographic columns usually need no regeneration. They
can be used over and again for a long duration of time without alteration in their
chromatographic properties provided the microbial growth is avoided.

SAQ 9
Mention three unique advantages of size exclusion chromatography.
...
...
...
...

51

Chromatographic
Methods-III

10.7 SOME APPLICATIONS


From the discussion in the preceding section, it is clear that the technique of size
exclusion chromatography has some unique advantages. Some of these features
strongly favour its use for biochemical work. Therefore, it finds extensive use for
separations in the said area. The applications are numerous and all of them cannot be
cited here. Typical examples from this very large field are enzyme purification,
purification and characterization of antibodies and separation and purification of
peptide and protein hormones.
The present section will only highlight some representative applications. It may not be
possible to give details of the procedures because of the constraints of space needed
for it. For the purposes of clarity and conciseness in the presentation, the applications
are subdivided in the following three heads.
1.

Analytical applications

2.

Preparative applications

3.

Miscellaneous applications

The miscellaneous applications include important applications which are not covered
in the first two heads particularly the use of gels in thin layer chromatography, zone
electrophoresis and HPLC. No doubt, these are not the direct applications of size
exclusion chromatography but they definitely reflect on the use of gels for separations.

10.7.1

Analytical Applications

It may be important to point out that within analytical applications, there are further
sub-divisions as follows:
i)

Analytical group separations


One of the very common uses of gel filtration chromatography is the removal of
interferences before the final determination. It is particularly important to
remove low molecular mass interferences before the determination of
macromolecular component in the mixture. The determination of protein in
spinal fluid is a typical example. The low molecular weight interferences are
removed for the spectrophotometric determination of protein.
On a similar line, it is desirable to determine polysaccharides like insulin,
amylose or dextran from low molecular mass sugar. Gel filtration
chromatography has been used for the purpose, say, the removal of glucose from
the mixture. The technique can also be used for the removal of high molecular
weight interferences but this has not been used much.

ii)

Analytical fractionation
This covers a wide range. It could be the separation of substances which can be
determined separately. The other end can be where elution curve profile can be
used to characterize the sample without the determination of concentration of
the different ingredients. A typical example of the first type is the separation of
sugars from cellulose hydrolysate. The technique is an extremely powerful tool
for the separation of oligosaccharides with differing number of sugar residues
from each other. Maltooligosaccharides containing up to 15 glucose units and
polymaltoses with chain length up to 21 glucose units can be separated from
each other on Bio-Gel P2. Sephadex LH-20 provides a very good resolution of
lipids and steroids. It has been possible to fractionate a very wide range of nonpolar lipids on LH-20 in chloroform and they were found to separate primarily
according to their molecular size. The conventional liquid chromatography on

52

silica and other materials is unsuccessful for this type of separation. Amino
acids can be separated on the tightly cross-linked types of gels.

Size Exclusion
Chromatography

The most important applications of analytical fractionation come closer to the


fingerprint end of the scale with substances overlapping to a large extent. Many
biological fluids have been characterized. The most important among these is
plasma. The elution behaviour of plasma proteins has been investigated. The
normal elution pattern changes strongly under the influence of certain diseases.
Thus, this form of chromatography acts as a diagnostic tool.
iii)

Measurement of protein binding and complex formation


Gel filtration chromatography has been very useful in the study of complexes
between proteins and low molecular mass solutes. The principle for the
measurement is simple if the reactants are irreversibly bound to each other. The
reaction mixture is fed to the column and the complex is separated
chromatographically from the reactant present in excess. One typical example of
complex formation between the proteins is the determination of heptoglobin
content of plasma. This exercise is carried out in clinical laboratories. Another
important example from clinical lab is the protein binding of insulin. It gives
information about the formation of insulin antibodies in diabetics treated with
insulin.

iv)

Determination of molecular masses


It is rated as one of the most important applications of size exclusion
chromatography. The determination of molecular masses of proteins is
particularly cited as an important example. The underlined principle is based on
the basic mechanism of the technique. It has been observed that for most
proteins a good close correlation between molecular mass and elution behaviour
is observed; very few proteins show anomalous behaviour. Relationship
between elution behaviour and molecular masses has been obtained for
carbohydrates and peptides. Sephadex G-100 and Sephadex G-200 have been
used most frequently. Agar and agrose gels have also been used and so is
Bio-Gel P. The eluant in use with proteins is usually buffer or saline. Long beds
should be used for better accuracy and precision of the measurement.
Although with some gels, calibration measurements are available but it is
recommended to make calibration curve with each bed. Adsorption phenomena
limit the use of gel filtration chromatography for large molecules and particles.
This technique has lower accuracy for molecular mass measurement than some
other available methods. On the other hand, this method of determination has
the advantage of rapid measurement, is easy to perform and requires fairly
inexpensive equipment.

10.7.2

Preparative Applications

In preparative applications, there are two distinct categories of uses.


i)

Preparative group separation

ii)

Preparative fractionation.

Let us study about them briefly.


i)

Preparative group separation


In a large number of biochemical preparations, desalting and exchange of
buffers is an important step. Theoretically, there is no problem in
achieving this separation but in actual practice, some problems may be
53

Chromatographic
Methods-III

encountered. If distilled water is being used as an eluant, tailing may be


more pronounced than at higher ionic strength. Moreover, dilution is
another problem. It is proposed to use volatile electrolytes like pyridinium
acetate and ethylenediamine acetate. The technique is not only confined to
desalting of macromolecules but it has also been extended to desalting of
virus.
Gel filtration chromatography is frequently used for separation of
macromolecules that have been modified chemically from the free
reactant. The technique on a large scale has been used in food industry for
the removal of sugars and low molecular weight contaminants from whey
proteins.
Another interesting example is the removal of undesirable molecular mass
contaminants. A typical example is the removal of allergens from
pharmaceutical preparations. Allergens are often compounds of high
molecular mass that is completely excluded on the gel types that are
interesting in industrial context. One example from this area is the
removal of penicillinoyated proteins from penicillin. In the method of
manufacture of penicillin, penicillinated proteins are formed. These are
highly allergenic and have to be removed.
ii)

Preparative fractionation
To a large extent, this technique has been used as a tool for separating the
proteins, their degradation products and their complexes.
Isolation of antibodies to specific antigens is a subject of great interest.
The separation of an antibody from the antigens can be performed by gel
filtration chromatography. Fractionation of nucleic acids and animal
viruses has also been attained on agar gels.
An important industrial example based on fractionation by gel filtration
chromatography is the preparation of tetanus toxoid for immunization
purposes. In order to make the extremely toxic tetanospasmin harmless so
that it can be injected, it is polymerized by treatment with formalin. The
polymer (toxoid) is separated from the monomer by gel filtration
chromatography on column packed with Sephadex G-100.
The examples of applications cited in the foregoing discussion pertain
exclusively to size exclusion chromatography. But there are many areas
where the gels used for size exclusion chromatography have applications
in other areas of separations.
The details of their applications are given in the respective Units.
However, a brief account of these is being given below under
miscellaneous applications.

10.7.3

Miscellaneous Applications

The gels find use in thin layer chromatography using the gravity flow. As the plates in
thin layer gel filtration chromatography may be difficult to dry, the chromatogram are
often developed, where the liquid in the layer is transferred to paper and developed on
the paper the same way as paper chromatogram. This is because the gels usually crack
on drying. This type of chromatography has been used mainly for the study of proteins
and peptides. The other types of fractionations which have been performed on these
plates are that of -amino acids, nuclopolysaccharides and antibodies.

54

The most important application is the rapid determination of molecular masses of


proteins and peptides. The technique is specially useful for this purpose as the sample
can be chromatographed on the same plate as the reference substances.

Size Exclusion
Chromatography

Another important application of gels is in capillary gel electrophoresis which is


generally performed in a porous gel polymer matrix, the pores of which contain a
buffer mixture in which the separation is carried out. This type of medium could
provide a molecular sieving action which retards the migration of analyte species to
various degrees depending upon the pore size of the polymer and the size of the
analyte ions. This is particularly helpful in separating macromolecules like proteins,
DNA fragments and oligonucleotides that have substantially same charge but differ in
size. These days most of the macroscale electrophoresis separations are carried on a
gel slab. Some capillary electrophoretic separations are carried in gels contained in
capillary tubes. The most common type of gel used for electrophoresis is based on
polyacrylamide polymer (Bio-Gel). The other gel like agrose, methyl cellulose and
polyethylene glycol have also been used for capillary gel electrophoresis.
For high performance analytical applications, small polystyrene or microporous silica
particles of 5-10 m diameter are used. They have a pore size of a few nm to several
hundred nm. The controlled pore silica particles are coated with a hydrophilic phase to
reduce adsorption of the solute.

SAQ 10
Give one example of each from the applications of size exclusion chromatography for
the following:
i)

Where the interference of a low molecular weight is removed before the


determination of a high molecular weight compound.
.................
.
.
.

ii)

Where the technique is used for diagnostic purposes.


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

iii)

Where this separation technique is employed for the purification of (a) medicine
(b) vaccine.
.................
.
.

10.8 SUMMARY
This unit on size exclusion chromatography begins with a brief introduction to the
technique highlighting the classification to which it belongs. The basic principle
55

Chromatographic
Methods-III

involved in separation is discussed. A general discussion on gels is presented which is


followed by enumeration of properties which are required to make them suitable for
chromatographic work. A section is devoted to the classification, synthesis and
properties of the gels used for separation purposes and are commercially available.
The different variables which help in defining the utility of a gel are cited. It is
followed by a discussion on some features of the technique which make it so
important for separations particularly of biochemical interest. At the end, some
representative applications of different types are enumerated. The applications of these
gels in areas of separations other than gel filtration chromatography are discussed.
These areas include thin layer chromatography, capillary gel electrophoresis and high
performance liquid chromatography.

10.9 TERMINAL QUESTIONS


1.

What is the basic equation for size exclusion chromatography? Under what
conditions the value of K, used in the equation, exceeds unity?

2.

What are two broad categories of gels based on their microstructure? Highlight
the differences between the two.

3.

Mention the characteristics which are important for a gel to be useful for
chromatographic work.

4.

What are the similarities in properties of Sephadex gels and Bio-Gels?

5.

What are the main disadvantages with Agrose gels?

6.

How is the information about protein binding and complex formation obtained
by size exclusion chromatography? How is this information useful? Cite an
example.

7.

How is gel filtration chromatography used for molecular weight determination


of proteins? Comment on the utility of the method.

10.10 ANSWERS
Self Assessment Questions

56

1.

It is the pore size of the swollen gel which is responsible for the fractionation of
molecules of different sizes by size exclusion chromatography.

2.

The value of distribution coefficient (K)


i)

for solute molecules which do not enter the gel matrix is zero

ii)

for solute molecules which enter the gel matrix is between 0 and 1.

3.

A gel consists of a three dimensional network often consisting of a crosslinked


polymer. The space within the gel not occupied by structural material is filled
with a liquid. The liquid occupies the main part of gel.

4.

A macro-microreticular gel consists of a macroreticular structure with


microreticular gel in the pores. The macroreticular structure takes care of
mechanical strength while the fractionation ranges are controlled by the
microreticular structure.

5.

If the Sephadex gels are subjected to oxidizing agents for longer duration, there
is an increase in the carboxyl group content of the gel. It leads to an impairment

in the chromatographic behaviour of the gel primarily because of prominence of


ion exchange mechanism.
6.

The Sephadex gels differ from the Sephacryl gels in the following respects
i)

The basic structural unit is different. The Sephacryl is partly


macroreticular in character.

ii)

Due to higher crosslinking in Sephacryl, the fractionation ranges extend to


high molecular weight as compared to Sephadex.

iii)

In Sephacryl, the slope of selectivity curve is less than Sephadex.

iv)

The adsorption effect is more prominent in Sephacryl than in Sephadex.

7.

In Sephadex, the weak point of chemical attack in the structure is glucoside


linkages which are hydrolysed at low pH. In Bio-Gel, the weakest point of
chemical attack is amide groups which are hydrolysed at extremes of pH.

8.

The Bio-Gels and styrene-divinylbenzene (Styragel) are not made from natural
materials. They are entirely synthetic.

9.

The three unique advantages of size exclusion chromatography are given below:

10.

Size Exclusion
Chromatography

i)

The material to be separated does not suffer any destruction or


denaturation.

ii)

By varying the contents of gel in gel matrix, the fractionation ranges can
be varied within wide limits.

iii)

The chromatographic columns usually need no regeneration. They can be


used for a long duration without change in their chromatographic
behavior.

i)

In the determination of protein in spinal fluid low molecular weight


interferences are removed by gel filtration chromatography.

ii)

The elution behaviour of plasma proteins changes strongly under the


influence of certain diseases. Thus, the technique acts as a diagnostic tool.

iii)

The separation technique is employed for the purification of


a)

penicillin from penicillinoyated proteins.

b)

Tetanus toxoid( polymer) from monomer.

Terminal Questions
1.

The basic equation for size exclusion chromatography is


VR = V0 + KVL

where, VR is retention volume, V0 is the interstitial volume, K is some form of


distribution coefficient and VL is the volume of water within the gel particles.
Normally, the K value greater than 1 is not encountered. If it is encountered, it
implies the occurrence of phenomena like adsorption, hydrogen bonding and ion
exchange between the gel matrix and the solute.
2.

The two classes of gels are macroreticular and microreticular. In macroreticular


gels, the microstructure is extremely heterogenous with regions where gel
material is aggregated and regions where there is very little gel matrix. The
regions free from gel matrix allows large molecules to enter. In the
microreticular structure, the gel matrix is relatively distributed throughout the
gel. They fractionate relatively lower molecular mass ranges than the
57

Chromatographic
Methods-III

macroreticular gels. The microreticular gels are supposed to be mechanically


weaker than macroreticular gels.
3.

4.

5.

58

The following are the important characteristics for a gel to be useful for
chromatographic work:
i)

Matrix of gel should be inert.

ii)

Gel must be chemically stable.

iii)

It should have low content of ionic groups.

iv)

There should be a wide choice of availablility of gels with same general


composition but different fractionational ranges.

v)

The gels should be available with different particle size.

vi)

The mechanical rigidity of the gel should be as high as possible.

The Sephadex gels and Bio-Gels show some striking similarities as given below:
i)

Both gel types are macroreticular.

ii)

The selectivity curve of both types of gels are similar.

iii)

The mechanical properties of both types of gels are similar.

iv)

The bead also shows similar behaviour. The bead with low water content
are brittle and the one with high content of water are soft.

There are some disadvantages with Agrose gels which are as follows:
i)

They contain a considerable amount of charged groups which cause


problems due to ion exchange effects. To mitigate this problem, it is
recommended to work at high ionic strength.

ii)

Agar chains are not linked by covalent bonds. This makes the gels
chemically unstable. These gels are less stable to pH extremes.

iii)

The structure of agrose gels makes it impractical to dry and reswell them.
Therefore, once the gel is prepared it has to be stored in a wet state.

iv)

The selectivity curve is less steep than it is for gels from dextran and
polyacrylamide.

6.

The technique has been very useful in the study of complexes between proteins
and between proteins and low molecular mass solutes. If the reactants are
irreversibly bound to each other, the resulting mixture is subjected to
chromatographic separation. The complex is separated from the reactants
present in excess. A typical example in this area is from clinical laboratory
where protein binding of insulin is investigated. The information is obtained
about the formation of insulin antibodies in diabetes treated with insulin.

7.

The determination of molecular masses of proteins is one of the most oft-cited


examples of size exclusion chromatography. The underlined principle is based
on the basic mechanism of the technique. For most of the proteins, a good close
relationship is observed between molecular mass and elution behaviour. A
variety of commercially available gels have been used for the said purpose. The
eluant generally used is buffer or saline. Long beds should be used for better
accuracy and precision of measurement. With some gels calibration
measurement data are available but it is recommended to make calibration curve
with each bed. This method is not as accurate as many other methods of mass
measurement. But this method, based on size exclusion chromatography, has the
advantages of rapid measurement, is easy to perform and requires fairly
inexpensive experimental set up.

Further Reading
1.

Gel Filtration Chromatography, By L. Fischer, Elsevier North-Holland


Biomedical Press, Amsterdam, New York, Oxford.

2.

Quantitative Analysis, By R. A. Day Jr. and A. L. Underwood, Prentice and Hall


of India Private Limited, New Delhi.

3.

Principles of Instrumental Analysis, By D. A. Skoog, F. J. Hoeller and T. A.


Nieman, Thomson.

Size Exclusion
Chromatography

59

Chromatographic
Methods-III

INDEX
Agar and agrose gels 45, 49
Aluminosilicates 8
Amphoteric exchangers 7, 13
Applications of ion exchange chromatography 31
Miscellaneous application 31, 33
Separation of ionized from nonionized 31, 32
Separation of metal ions and anions 31
Separation of organics 31,32
Separation of Actinide Elements 31, 33

Applications of Size exculpation of chromatography 52


Analytical 52
Preparative 52, 53
Miscellaneous 52, 54

Analcite 8
Anion exchangers 7, 8, 12
Basic principle of size exclusion chromatography 41
Exclusion limit 42

Batch method 18
Batch operation 23
Cation exchangers 7, 10
Chabazite 8
Chelates 26
Classification of ion exchangers 8
Inorganic 8
Natural 8
Organic 8
Synthetic 8

Classification, synthesis and properties 45


Column operation 23
Combined ion exchange solvent extraction (CIESE) 24
Dextran gels (sephadex) 45
Dextran-N, N methylene-bisacrylamide gels (Sephacryl) 45, 47
Dipicrylamine 26
Dowex A- 1 26
Exclusion limit 42
Feldspar 8
Gel filtration chromatography 40
Gels and their important properties 43
Classification, synthesis and properties 45
Agar and agrose gels 45, 49
Dextran gels (sephadex) 45
Dextran-N, N methylene-bisacrylamide gels (Sephacryl) 45
Polyacrylamide gels (Bio-Gel) 45, 48
Sephadex 46
Styrene-divinylbenzene gel (Styralgel) 49

Important properties of gels for chromatography 44


Utility of gel 50

Gel permeation chromatography 40


Ion exchange 5
Basic features of mechanism of 7
in mixed aqueous- organic media 24
Combined ion exchange solvent extraction (CIESE) 24

Ion exchange chromatography


Applications of 31
Miscellaneous application 31, 33
Separation of ionized from nonionized 31, 32
Separation of metal ions and anions 31
Separation of organics 31, 32

60

Separation of Actinide Elements 31, 33

Ion exchange mechanism 7

Size Exclusion
Chromatography

Amphoteric exchangers 7
Anion exchangers 7
Cation exchangers 7
Counter ions 7

Ion exchangers
Classification of 8
Inorganic 8
Natural 8
Organic 8
Synthetic 8

Kaolinite 8
Liquid ion exchangers 10
Miscellaneous applications 31, 33
Montmorillonite 8
Moving bed operations 24
Naturalite 8
Natural ion exchangers 8
Analcite 8
Aluminosilicates 8
Anion exchangers 8
Chabazite 8
Feldspar 8
Kaolinite 8
Montmorillonite 8
Naturalite 8
Zeolites 8

Nomenclature 15
Operating methods 23
Batch operation 23
Column operation 23
Moving bed operations 24

Polyacrylamide gels (Bio-Gel) 45, 48


Resin properties 15
Capacity 17
Breakthrough (dynamic) capacity 17
Operating capacity 17
Sorption capacity 17
Total capacity of an ion exchange resin 17
Useful capacity 17

Cross linkages 16
Distribution ratio 17
Batch method 18
Distribution coefficient 19

Equivalency of exchange 19
Moisture content 15
Particle Size 16
Resin selectivity 19
Apparent selectivity coefficient 22
Dowex 1 19
Dowex 50 19
Ionic forms of resin 21
Selectivity 19
Selectivity coefficient 19
Total solution ionic strength 21

Separation of organics 32
Separation of Actinide Elements 33
Separation of ionized from nonionzed 31, 32
Separation of metal ions and anions 31
Sephacryl 45
Sephadex G-10 46
61

Chromatographic
Methods-III

Sephadex G-200 46
Size exclusion chromatography 40
Applications 52
Analytical 52
Preparative 52, 53
Miscellaneous 52, 54

Basic principle 41
Unique features 51

Snake-cage polyelectroytes 14
Some applications 52
Analytical 52
Anaytical fractionation 52
Anaytical group separations 52
Determiniation of molecular masses 53
Measurement of protein binding and complex formation 53

Miscellaneous applications 54
Preparative applications 53
Preparative fractionation 54
Preparative group separation 53

Specific cation exchangers 25


Chelates 26
Dipicrylamine 26
Dowex A- 1 26

Styragel 45, 49
Synthesis of ion exchange resins 10
Amphoteric exchangers 13
Snake-cage polyelectroytes 14

Anion exchangers 12
Addition polymers 16
Condensation polymers 13

Cation exchangers 10
Addition polymers 11
Condensation polymers 11

Synthetic inorganic ion exchangers 27


Characteristics 27
Different types 27
Hydrous exides of polyvalent metals 27
Insoluble ferrocyanides 28
Insoluble salts of polyvalent metals 28
Miscellaneous inorganic ion exchangers 29
Salts of heteropoly acids 28
Synthetic aluminosilicates 29

Special properties and applications 29


Isotope generator 29
Radiation and thermal stability 29
Radionuclide generator 29
Unusual selectivity 30

Characteristics 27

Synthetic ion exchangers 9


Trade names and nomenclature 14
Nomenclature 15

Thin layer gel filtration chromatography 40


Unique features of size exclusion chromatography 51
Utility of gel 50
Fractionation range 50

Zeolites 8

62