40

Chromatographic

Methods-III UNIT 10 SIZE EXCLUSION

CHROMATOGRAPHY

Structure

10.1 Introduction Objectives

10.2 Basic Principle

10.3 Gels and Their Important Properties Important Properties of Gels for Chromatography

10.4 Classification, Synthesis and Properties

10.5 Variables Defining the Utility of Gel

10.6 Unique Features of the Technique

10.7 Some Applications Analytical Applications

Preparative Applications

Miscellaneous Applications

10.8 Summary

10.9 Terminal Questions

10.10 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.

41

Size Exclusion

Chromatography 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.

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.

42

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.

43

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?

…………………………………………………………………………………………...

…………………………………………………………………………………………...

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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.

44

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.

45

Size Exclusion

Chromatography 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.

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,

46

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 α-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.

47

Size Exclusion

Chromatography 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.

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 methylene-

bisacrylamide 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.

48

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-bis-

acrylamide (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.

49

Size Exclusion

Chromatography 4. Agar and agrose gels

Agar or agar-agar, as it was called earlier, is obtained from various species of sea-

weeds. 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.

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, 3-

dibromopropanol. 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.

50

Chromatographic

Methods-III SAQ 5

What happens if the Sephadex gels are subjected to prolonged exposure to oxidizing

agents?

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

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SAQ 6

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

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

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SAQ 7

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

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

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SAQ 8

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

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

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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.

51

Size Exclusion

Chromatography they are fairly constant. Another important variable of significance is the steepness of

the selectivity curve in the linear portion of the fractionation range.

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.

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

…………………………………………………………………………………………...

52

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 non-

polar lipids on LH-20 in chloroform and they were found to separate primarily

according to their molecular size. The conventional liquid chromatography on

53

Size Exclusion

Chromatography silica and other materials is unsuccessful for this type of separation. Amino

acids can be separated on the tightly cross-linked types of gels.

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

54

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 α -amino acids, nuclopolysaccharides and antibodies.

55

Size Exclusion

Chromatography 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.

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 µm 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

56

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

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

57

Size Exclusion

Chromatography 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:

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.

10. 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

58

Chromatographic

Methods-III macroreticular gels. The microreticular gels are supposed to be mechanically

weaker than macroreticular gels.

3. 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.

4. 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.

5. 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.

59

Size Exclusion

Chromatography 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.

60

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

61

Size Exclusion

Chromatography

Separation of Actinide Elements 31, 33

Ion exchange mechanism 7 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

62

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