Chromatographic Chromatographic Methods-III
Methods-III
UNIT 10
UNIT
10
SIZE
SIZE EXCLUSION
EXCLUSION
CHROMATOGRAPHY
CHROMATOGRAPHY
Structure Structure 10.1 10.1 IntroductionIntroduction Objectives Objectives 10.210.2 Basic PrincipleBasic Principle 10.3
10.3 Gels and Their Important PropertiesGels and Their Important Properties Important Properties of Gels for
Important Properties of Gels for ChromatographyChromatography 10.4
10.4 Classification, Synthesis and PropertiesClassification, Synthesis and Properties 10.5
10.5 Variables Defining the Utility of GelVariables Defining the Utility of Gel 10.6
10.6 Unique Features of the Unique Features of the TechniqueTechnique 10.7
10.7 Some ApplicationsSome Applications Analytical Applications Analytical Applications Preparative Applications Preparative Applications Miscellaneous Applications Miscellaneous Applications 10.8 10.8 SummarySummary 10.9
10.9 Terminal QuestionsTerminal Questions 10.10
10.10 AnswersAnswers
10.1
10.1 INTRODUCTION
INTRODUCTION
Before we discuss this chromatographic techniq
Before we discuss this chromatographic technique, it may be due, it may be desirable to refer toesirable to refer to Unit 1 wherein classifications of separation methods have been dealt. In the scheme of 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 classification based on property resulting into separation, there is a distinct class in which the separations are achieved on the basis of
which the separations are achieved on the basis of molecular geometrymolecular geometry or or molecularmolecular dimensions
dimensions. This. This size exclusionsize exclusion or or gel filtrationgel filtration chromatographychromatographyfigures right in thisfigures right in this category. However, if we probe into
category. However, if we probe into another criteria of classification based onanother criteria of classification based on equilibrium and rate processes, the e
equilibrium and rate processes, the e xclusion chromatogxclusion chromatographic technique appears inraphic technique appears in chromatograp
chromatographic processes where liquid and solid hic processes where liquid and solid are in equilibrium. Thus, veryare in equilibrium. Thus, very rightly the size
rightly the size exclusion chromatograpexclusion chromatography is one of hy is one of the important forms of liquidthe important forms of liquid chromatograp
chromatographic technique (Unit hic technique (Unit 4, sub-sec. 4.2.3).4, sub-sec. 4.2.3).
The mobile phase is aqueous or organic and the stationary phase is a molecular sieve. The mobile phase is aqueous or organic and the stationary phase is a molecular sieve. These sieves are
These sieves are generally polymeric carbohydgenerally polymeric carbohydrates and acrylamide that rates and acrylamide that have an openhave an open network formed by the cross linking of
network formed by the cross linking of polymeric chains. Incidentally, this branch ofpolymeric chains. Incidentally, this branch of chromatograp
chromatographic science was hic science was also discovered in a Balso discovered in a B ioscience oriented laboratory.ioscience oriented laboratory. This separation method originated in 1959 at
This separation method originated in 1959 at the Biochemical Institute in the Biochemical Institute in Uppsala,Uppsala, Sweden. Initially it was
Sweden. Initially it was applied for the separation of water-soluble macromolecules ofapplied for the separation of water-soluble macromolecules of biological importance. The technique was named as
biological importance. The technique was named as gel-filtration gel-filtration chromatogrchromatographyaphy (GFC). A few
(GFC). A few years later the technique was years later the technique was developed for synthetic polymers solubledeveloped for synthetic polymers soluble in organic solvents and it is was
in organic solvents and it is was called ascalled as gel pgel permeation chromatograpermeation chromatographyhy (GPC). (GPC). This amounts to the fact that initially
This amounts to the fact that initially the names such as gel filtration chromatographythe names such as gel filtration chromatography (mobile phase is water) used
(mobile phase is water) used by biochemist and gel by biochemist and gel permeation chromatogrpermeation chromatographyaphy (mobile phase in organic solvent) used by
(mobile phase in organic solvent) used by polymer chemists described the technique.polymer chemists described the technique. Now the recommended or the most accepted name of the technique is
Now the recommended or the most accepted name of the technique is size exclusionsize exclusion chromatography
chromatography (SEC). It is used in open column gravity fed for both analytical and(SEC). It is used in open column gravity fed for both analytical and preparative separations and in high performance separations. The gel filtration preparative separations and in high performance separations. The gel filtration alsoalso finds use in
finds use in thin layer chromatography and the technique is known asthin layer chromatography and the technique is known as thin layer gelthin layer gel filtration chromato
filtration chromatographygraphy. Reference to gels will also . Reference to gels will also be made in Unit 12 onbe made in Unit 12 on electrophoresis.
Size Exclusion Size Exclusion Chromatography Chromatography This unit on
This unit on size exclusion chromatography first discusses the principle involved insize exclusion chromatography first discusses the principle involved in separations using gels. This is followed by a general discussion on gels and
separations using gels. This is followed by a general discussion on gels and characteristics required for the gels
characteristics required for the gels to be useful to be useful for chromatograpfor chromatography. After explaininghy. After explaining about the characteristics of
about the characteristics of gels needed for chromatographic purposes, a classificationgels needed for chromatographic purposes, a classification of important types of
of important types of chromatogchromatographic gels is raphic gels is given. Along with this the given. Along with this the methods ofmethods of preparation of the important categories of these gels on a broad basis are discussed and 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 the properties shown by them are highlighted. A brief note on the characteristics which define the utility of a gel
which define the utility of a gel is also included. The unique features of this form ofis also included. The unique features of this form of chromatograp
chromatography are explained. Finally, some hy are explained. Finally, some of the important applications of of the important applications of thethe technique are discussed.
technique are discussed. Objectives
Objectives
After studying this Unit, you should be able to After studying this Unit, you should be able to
•
• discuss the principle responsible for separation of solutes,discuss the principle responsible for separation of solutes, •
• describe gels and the characteristics required for their use for describe gels and the characteristics required for their use for chromatogchromatographicraphic
separations, separations,
•
• explain about the classification of gels, their method of preparation andexplain about the classification of gels, their method of preparation and
important properties of the different classes, important properties of the different classes,
•
• enumerate the variables which define the utility of a gel,enumerate the variables which define the utility of a gel, •
• give the unique features of give the unique features of this chromatographic technique, andthis chromatographic technique, and •
• cite some of the important applications of this method of separation.cite some of the important applications of this method of separation.
10.2
10.2 BASIC PRINCIPLE
BASIC PRINCIPLE
The packagings for size exclusion chromatography generally consist of cross linked The packagings for size exclusion chromatography generally consist of cross linked polymers of dextrans, polyacrylamides, styrene or silica. They have
polymers of dextrans, polyacrylamides, styrene or silica. They have an open network.an open network. On absorbing the solvent, swelling causes
On absorbing the solvent, swelling causes an opening of the structure. an opening of the structure. The degree ofThe degree of cross linking will determine the size of the
cross linking will determine the size of the holes.holes.
In this network of uniform pores, the solute and solvent molecules can diffuse. While 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 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 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 effective size. The molecules which are significantly bigger than the average pore size are excluded.
are excluded. As a result of this,
As a result of this, they suffer no retention and travel through the column at the rate ofthey suffer no retention and travel through the column at the rate of mobile phase. The molecules that are appreciably smaller than the pore size
mobile phase. The molecules that are appreciably smaller than the pore size cancan penetrate throughout pore network and, thus, remain entrapped for the longest penetrate throughout pore network and, thus, remain entrapped for the longest time.time. As a result of this,
As a result of this, the molecules which are able to penetrate the gel the molecules which are able to penetrate the gel will spend part ofwill spend part of their time sheltered from the moving phase.
their time sheltered from the moving phase.
Between these two extremes are intermediate size molecules whose average Between these two extremes are intermediate size molecules whose average penetration into the pores of p
penetration into the pores of packing depends upon their diameters. Fractionationacking depends upon their diameters. Fractionation within this group is directly related to the
within this group is directly related to the molecular size and, to some extent,molecular size and, to some extent, molecular shape.
molecular shape.
Thus, there is a basis of
Thus, there is a basis of separating molecules of different sizes. Fig. 10.1 showsseparating molecules of different sizes. Fig. 10.1 shows schematically three stages in the chromatographic separation of two extreme sizes of schematically three stages in the chromatographic separation of two extreme sizes of molecules.
Chromatographic Chromatographic Methods-III Methods-III
Fig. 10.1:
Fig. 10.1: Schematic of a chromatographic separation on a sizSchematic of a chromatographic separation on a size exclusion column. Largee exclusion column. Large open crossed circles represent the stationary phase; large black circles
open crossed circles represent the stationary phase; large black circles represent molecules which do not
represent molecules which do not penetratepenetrate K K = 0; smaller black circles = 0; smaller black circles represent molecules which penetrate the gel and are retarded in their represent molecules which penetrate the gel and are retarded in their movement down the column.
movement down the column. It is important
It is important to note that to note that size exlusions are different from other size exlusions are different from other chromatogchromatographicraphic procedures. Here, there are no physical or chemical interactions between the analyte procedures. Here, there are no physical or chemical interactions between the analyte and the stationary phase. As a matter of fact,
and the stationary phase. As a matter of fact, efforts are made to avoid suchefforts are made to avoid such
interactions because they may cause impaired column efficiencies. At this point, it interactions because they may cause impaired column efficiencies. At this point, it may be important to introduce the term
may be important to introduce the term exclusion limit exclusion limit . The. The exclusion limitexclusion limit is the is the molecular weight of that molecule that will just
molecular weight of that molecule that will just permeate the gel and be retarded. Thispermeate the gel and be retarded. This can range from 1000 to several millions depending upon the gel. It should be kept in can range from 1000 to several millions depending upon the gel. It should be kept in mind that separation are based
mind that separation are based on molecular size and on molecular size and configuratioconfiguration rather than n rather than simplysimply its molecular weight but, generally, there is a
its molecular weight but, generally, there is a correlation with molecular weight. Also,correlation with molecular weight. Also, generally the molecules smaller than the exclusion limit can be fractionated down to a generally the molecules smaller than the exclusion limit can be fractionated down to a limiting size.
limiting size.
The entire picture of
The entire picture of fractionation by size exclusion chromatography can be visualizedfractionation by size exclusion chromatography can be visualized in some semiquantitative terms. Let
in some semiquantitative terms. Let V V RR be be the retention volume for a solute with athe retention volume for a solute with a
chromatograp
chromatographic column. hic column. LetLet V V 00 be the interstitial volume (void volume), that is, be the interstitial volume (void volume), that is, thethe
volume within the column which is available to the
volume within the column which is available to the mobile phase.mobile phase. V V LL is the volume of is the volume of
water within the gel particles available
water within the gel particles available for accepting solutes. On the lines of GLC, wefor accepting solutes. On the lines of GLC, we can write the
can write the following equation:following equation: V
V RR = = V V 00 + + KV KV LL
where,
where, K K is some form of distribution coefficient. If the solute is is some form of distribution coefficient. If the solute is completely excludedcompletely excluded from the interior of gel then
from the interior of gel then K K = 0 and = 0 and V V RR = = V V 00. Such marker substances are available.. Such marker substances are available.
Now, if the solute can freely enter
Now, if the solute can freely enter the gel, there should be no preference for waterthe gel, there should be no preference for water inside or outside the gel and thus,
inside or outside the gel and thus, K K = 1, and = 1, and V V RR = = V V 00 + + V V LL. Taking the case of. Taking the case of
molecules which can penetrate the gel to some extent but not
molecules which can penetrate the gel to some extent but not freely,freely, K K values fall values fall between 0 and 1.
between 0 and 1.
In cases where sieving is
In cases where sieving is the only phenomenon responsible for fractionation,the only phenomenon responsible for fractionation, K K values values greater than 1 would never be
greater than 1 would never be encountered. Howeverencountered. However, sometimes these , sometimes these values arevalues are obtained suggesting the occurrence of
obtained suggesting the occurrence of phenomena like adsorption, hydrogen bondingphenomena like adsorption, hydrogen bonding and
and ion ion exchange exchange between between the the gel gel and and the the solute. solute. Fig. 10.2 sFig. 10.2 shows hows the the typicaltypical
behaviour of variation in the retention volume with the molecular weight of the solute. behaviour of variation in the retention volume with the molecular weight of the solute.
Size Exclusion Size Exclusion Chromatography Chromatography
Fig. 10.2:
Fig. 10.2: Relation between retention voRelation between retention volume and molecular weight of the solute. Thelume and molecular weight of the solute. The steep region between the arrows is the fractionation range.
steep region between the arrows is the fractionation range. If we refer to Fig. 10.2,
If we refer to Fig. 10.2, it is clear it is clear that the molecules of differing in size can bethat the molecules of differing in size can be separated chromatographic
separated chromatographically in the ally in the sloping region of the sloping region of the curve. By varying thecurve. By varying the degree of cross linking of the polymer, the curve shifts horizontally. There are degree of cross linking of the polymer, the curve shifts horizontally. There are materials available which fractionate
materials available which fractionate molecules in various molecular weight ranges.molecules in various molecular weight ranges. For Sephadex G. 50, the
For Sephadex G. 50, the fractionation range for peptides and globular protein isfractionation range for peptides and globular protein is molecular weight 1,500 to 30,000 while the
molecular weight 1,500 to 30,000 while the range for G. 150 is range for G. 150 is 5,000 to 400,000.5,000 to 400,000. SAQ 1
SAQ 1
What particular property of gel is
What particular property of gel is responsible for fractionation of solutes of differentresponsible for fractionation of solutes of different molecular weights by
molecular weights by size exclusion chromatography?size exclusion chromatography?
………... ………... ………... ………... ………... ………... SAQ 2 SAQ 2
What are the values of distribution coefficient if What are the values of distribution coefficient if i)
i) solute molecules do not enter the gel matrixsolute molecules do not enter the gel matrix ii)
ii) solute molecules enter the gel matrixsolute molecules enter the gel matrix
………... ………... ………... ………... ………... ………...
10.3
10.3 GELS AND THEIR IMPORTANT PROPERTIES
GELS AND THEIR IMPORTANT PROPERTIES
In everyday language, gel is a familiar name. It
In everyday language, gel is a familiar name. It refers to a fairly soft, elastic refers to a fairly soft, elastic materialmaterial containing water. In the scientific context, the term acquires a
containing water. In the scientific context, the term acquires a wider meaning. A gelwider meaning. A gel consists of a
consists of a three dimensional network. The structural material, often three dimensional network. The structural material, often consisting ofconsisting of cross linked polymers, gives some mechanical stability. The space within the gel not cross linked polymers, gives some mechanical stability. The space within the gel not occupied by structural material is filled with liquid.
occupied by structural material is filled with liquid. Liquid occupies the main part ofLiquid occupies the main part of gel. Some gels are soft and deform easily; this
gel. Some gels are soft and deform easily; this being the common picture. Others arebeing the common picture. Others are rigid or even brittle.
Chromatographic Chromatographic Methods-III
Methods-III There are many natural There are many natural substances capable of forming gels and these polysaccharides from fruits and roots, proteins from animal tissues, inorganic silicatespolysaccharides from fruits and roots, proteins from animal tissues, inorganic silicatessubstances capable of forming gels and these includeinclude and phosphates. If we look at
and phosphates. If we look at gels from the chromatographic point of view gels from the chromatographic point of view and alsoand also from general properties, two different types of gels
from general properties, two different types of gels can be distinguished. In thecan be distinguished. In the macroreticular
macroreticular gelsgels, the microstructure is , the microstructure is strongly heterogenoustrongly heterogenous with regions wheres with regions where matrix material is aggregated and regions where there is very little
matrix material is aggregated and regions where there is very little gel matrix present.gel matrix present. The gel structure virtually free from gel matrix allows large molecules to enter. The gel structure virtually free from gel matrix allows large molecules to enter. TheThe microreticular gels
microreticular gels, on the other hand, show properties that the gel matrix is , on the other hand, show properties that the gel matrix is relativelyrelatively distributed throug
distributed throughout hout the gel. They fractionate relatively lower moleculathe gel. They fractionate relatively lower molecular massr mass ranges than the macroreticular gels.
ranges than the macroreticular gels. 10.3.1
10.3.1 Important Properties of Gels for ChromatographyImportant Properties of Gels for Chromatography Out of different gels known only relatively few
Out of different gels known only relatively few are suitable for chromatographic work.are suitable for chromatographic work. Some essential requirements should be met by these gels to be useful for the
Some essential requirements should be met by these gels to be useful for the saidsaid purpose. These are as follows.
purpose. These are as follows. i)
i) Matrix of gel shou Matrix of gel should be inertld be inert
Any interaction between the gel material and the solute may lead
Any interaction between the gel material and the solute may lead to irreversibleto irreversible binding of the solute. The interaction may lead to chemical alteration
binding of the solute. The interaction may lead to chemical alteration of labileof labile substances. In biochemistry, there is
substances. In biochemistry, there is a risk of denaturation of proteins a risk of denaturation of proteins and nucleicand nucleic acids. If the right material is
acids. If the right material is there, gel filtration chromatography is one of the fewthere, gel filtration chromatography is one of the few separation methods which is capable of
separation methods which is capable of giving quantitative yields.giving quantitative yields. ii)
ii) Gel must be chemically stableGel must be chemically stable
The gel should be stable over a wide range of
The gel should be stable over a wide range of p pH and temperature. The gels that areH and temperature. The gels that are used in practice are stable
used in practice are stable over years and monthover years and months. The leaching of material from beds. The leaching of material from bed should be very low.
should be very low. iii)
iii) Low content of ionic Low content of ionic groupsgroups A low content of
A low content of ionic groups is required to ionic groups is required to avoid ion exchange effects. Chargedavoid ion exchange effects. Charged groups will give bad yields of charged solute and asymmetric elution curve. It is groups will give bad yields of charged solute and asymmetric elution curve. It is impossible to avoid the
impossible to avoid the charged groups but the commercially available gels have charged groups but the commercially available gels have veryvery low ionic
low ionic groups.groups. iv)
iv) Availability of wide cho Availability of wide choice of gelsice of gels
For the adaption of a method of different problems, a wide choice of gels with sa For the adaption of a method of different problems, a wide choice of gels with sa meme general composition but different fractionation ranges should be available. For general composition but different fractionation ranges should be available. For microreticular gels, the fractionation range is
microreticular gels, the fractionation range is mainly determined by the swellingmainly determined by the swelling properties. Gels with low content of dry substance in the gel give access to properties. Gels with low content of dry substance in the gel give access to largerlarger molecular weight molecules than those with
molecular weight molecules than those with high contents of dry substance.high contents of dry substance. With macroreticular gels, the content of the dry substance is no
With macroreticular gels, the content of the dry substance is no longer the onlylonger the only
variable which determines the fractionation range of the gel. The structure of the gel is variable which determines the fractionation range of the gel. The structure of the gel is also important.
also important. v)
v) Availability of gels with Availability of gels with different particle sizedifferent particle size
The particle size distribution should be very carefully controlled. A column of small The particle size distribution should be very carefully controlled. A column of small particle size will generally give good resolution. If the particle size
particle size will generally give good resolution. If the particle size is increased, theis increased, the reasons for zone broadening are amplified. With large
reasons for zone broadening are amplified. With large particles the diffusion in particles the diffusion in and outand out of the particle takes longer. Flow pattern in large particles
of the particle takes longer. Flow pattern in large particles is inferior. On the otheris inferior. On the other hand, the resistance to flow in a
hand, the resistance to flow in a bed of large particle is lower. Thus, a compromisebed of large particle is lower. Thus, a compromise with regard to particle
with regard to particle size should be reached giving maximum zone size should be reached giving maximum zone resolution underresolution under the desired flow conditions. Generally, the commercial available gels are in bead the desired flow conditions. Generally, the commercial available gels are in bead forms.
Size Exclusion Size Exclusion Chromatography Chromatography vi)
vi) Mechanical rigidi Mechanical rigidity of the gel particlesty of the gel particles
The mechanical rigidity of the gel grains should be as high as
The mechanical rigidity of the gel grains should be as high as possible, otherwise theypossible, otherwise they tend to be deformed by the forces caused by the flow of liquid.
tend to be deformed by the forces caused by the flow of liquid. The force may causeThe force may cause the bead to
the bead to compact reversibly or irreversibly, thus, increasing the flow compact reversibly or irreversibly, thus, increasing the flow through thethrough the bed. The microreticular gels with
bed. The microreticular gels with small content of dry ssmall content of dry s ubstance; a correspondinglyubstance; a correspondingly high exclusion limit, tend to be mechanically weak. One approach to solve the 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
problem is to synthesize gels with a macroreticular structure and with a microreticularmicroreticular gel in the pores of
gel in the pores of macroreticular gel. The fractionation ranges are controlled by themacroreticular gel. The fractionation ranges are controlled by the microreticular gel in the pores while the a
microreticular gel in the pores while the a ggregateggregates of macroreticular gel take care ofs of macroreticular gel take care of the mechanical strength. These gels are called
the mechanical strength. These gels are called macro-micromacro-microreticular reticular gelsgels.. SAQ 3
SAQ 3
What is a typical gel structure? What is a typical gel structure?
………... ………... ………... ………... ………... ………... ………... ………... SAQ 4 SAQ 4 What is a
What is a macro-microreticmacro-microreticular gel? What ular gel? What is its is its special advantage for chromatographicspecial advantage for chromatographic work? work? ………... ………... ………... ………... ………... ………... ………... ………...
10.4
10.4 CLASSIFICATION
CLASSIFICATION,
, SYNTHESIS AND
SYNTHESIS AND PROPERTIES
PROPERTIES
In the previous section, we have discussed the characteristics which are needed for a 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 gel to be useful for chromatographic work. As such there are different types of gels available but for
available but for general chromatogrageneral chromatographic separations, gels of phic separations, gels of some distinct categoriessome distinct categories are used. These are commercially available in different sizes under different trade are used. These are commercially available in different sizes under different trade names. They are known either by the basic unit of
names. They are known either by the basic unit of the polymer or more commonlthe polymer or more commonly byy by the trade names. A classification of different types of
the trade names. A classification of different types of gels is given below.gels is given below. i)
i) Dextran gels (Sephadex)Dextran gels (Sephadex) ii)
ii) Dextran-Dextran- N N ,, N N ′′-methylene- bisacrylamide gels (Sephacryl)-methylene- bisacrylamide gels (Sephacryl)
iii)
iii) Polyacrylamide gels (Bio-Gel)Polyacrylamide gels (Bio-Gel) iv)
iv) Agar and Agrose gelsAgar and Agrose gels v)
v) Styrene-divinylbenzene gels Styrene-divinylbenzene gels (Styragel)(Styragel)
Besides the above classes inorganic materials like silica
Besides the above classes inorganic materials like silica gel and porous glass are alsogel and porous glass are also used.
used. Let us study the difLet us study the different types of gferent types of gels in more detail.els in more detail. 1.
1. Dextran gels (Sephadex)Dextran gels (Sephadex)
Dextran is a polysaccharide, built up from glucose residues. It is produced from the Dextran is a polysaccharide, built up from glucose residues. It is produced from the fermentation of sucrose. The micro-organism that is used
fermentation of sucrose. The micro-organism that is used for fermentation isfor fermentation is Leuconostoc mesenteroides strain NRRLB512. The native dextran has high
Leuconostoc mesenteroides strain NRRLB512. The native dextran has high molecularmolecular mass and a wide mass d
Chromatographic Chromatographic Methods-III
Methods-III partly hydrolysed partly hydrolysuitable average mass and a suitable average mass and a narrow mass distribution. The glucose residues are joinedsed and finally fractioand finally fractionated by ethanol precipitation to give a produnarrow mass distribution. The glucose residues are joinednated by ethanol precipitation to give a product ofct of by
byαα-1, 6-glucosidic linkages. The chains are to a certain extent branched. The-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 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 prepare dextran by Leuconostoc mesenteriodes strain B512, the branches are joined by 1-3-glucosi
1-3-glucosidic linkages. Dextran dissolves in dic linkages. Dextran dissolves in water but when it water but when it is cross linked is cross linked to formto form the gel, the polysaccharide chains of the gel form a
the gel, the polysaccharide chains of the gel form a three dimensional network. Thethree dimensional network. The material, thus, becomes insoluble in water.
material, thus, becomes insoluble in water.
Sephadex is manufactured by a bead polymerization process. In
Sephadex is manufactured by a bead polymerization process. In this process, anthis process, an alkaline solution of dextran of
alkaline solution of dextran of suitable molecular mass distribution is suspended in suitable molecular mass distribution is suspended in anan organic solvent which is immiscible with water. Stabilizers are added to stabilize the organic solvent which is immiscible with water. Stabilizers are added to stabilize the suspension. When the suspension is stirred to form
suspension. When the suspension is stirred to form an emulsion epichlorohydrin,an emulsion epichlorohydrin, reacts with dextran matrix
reacts with dextran matrix forming glyceryforming glyceryl links bl links between the chains. After theetween the chains. After the completion of the reaction, the
completion of the reaction, the product is washed thoroughly and allowed to shrink inproduct is washed thoroughly and allowed to shrink in water-alcohol mixture and then dried. A
water-alcohol mixture and then dried. A partial structure of Sephadex is partial structure of Sephadex is shownshown in Fig. 10.3.
in Fig. 10.3.
Fig. 10.3:
Fig. 10.3: A schematic rA schematic representation of partial structure of Sepresentation of partial structure of Sephadexephadex Sephadex is available as
Sephadex is available as a free-flowing powder consisting of regular bead froma free-flowing powder consisting of regular bead from Pharmacia Fine Chemicals. When this is suspended in water, the beads swell.
Pharmacia Fine Chemicals. When this is suspended in water, the beads swell. DryingDrying and swelling of Sephadex is a reversible process. The material is capable of
and swelling of Sephadex is a reversible process. The material is capable of retainingretaining its chromatographic behaviour after repeated drying and swelling. Sephadex is
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 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 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
number. The range of the material available is Sephadex G – 10 to Sephadex G – 200.Sephadex G – 200. The numbers correspond approx
The numbers correspond approximately to the imately to the water regain value of water regain value of the gel multipliedthe gel multiplied by a factor
by a factor 10. Also, there are 10. Also, there are special kinds of Sephadex known as Sephadex LH-20special kinds of Sephadex known as Sephadex LH-20 and LH-60 which are intended for use
Size Exclusion Size Exclusion Chromatography Chromatography Sephadex is very chemically stable. The weakest points of attack in
Sephadex is very chemically stable. The weakest points of attack in the structure arethe structure are the glucosides linkages which are
the glucosides linkages which are hydrolysed at lowhydrolysed at low p pH. It is stable in alkalineH. It is stable in alkaline solutions. Its prolonged exposur
solutions. Its prolonged exposure to oxidizing agents may cause an increase in e to oxidizing agents may cause an increase in thethe carboxyl group content. The increase of carboxyl group in
carboxyl group content. The increase of carboxyl group in the structure impairs thethe structure impairs the chromatograp
chromatographic behaviour of the gel. It may be hic behaviour of the gel. It may be noted that initially Sephadex containsnoted that initially Sephadex contains a very small amount of carboxyl groups. This amount is so low that most of
a very small amount of carboxyl groups. This amount is so low that most of the chargethe charge effects observed are attributed to
effects observed are attributed to Donnan effects caused by solutes.Donnan effects caused by solutes.
Sephadex gels were the first for which a close relationship between molecular size and Sephadex gels were the first for which a close relationship between molecular size and elution behaviour was observed. In Sephade
elution behaviour was observed. In Sephadex series, there are gels x series, there are gels available withavailable with fractionation ranges distributed over a very wide
fractionation ranges distributed over a very wide interval of molecular masses. Someinterval of molecular masses. Some differences are evident in
differences are evident in the fractionation properties of different group of substances.the fractionation properties of different group of substances. In most of the cases,
In most of the cases, these differences are relatively small and the general shape of thethese differences are relatively small and the general shape of the fractionation curves is similar.
fractionation curves is similar. 2.
2. Dextran –Dextran – N N ,, N N ′′-methylene-bisacrylamide gels (Sephacryl)-methylene-bisacrylamide gels (Sephacryl)
Sephacryl is a dextran gel manufactured by cross linking allyl dextran with
Sephacryl is a dextran gel manufactured by cross linking allyl dextran with N N ,, N N ′′
--methylene-bisacryl
methylene-bisacrylamide. There are amide. There are cross links not only in cross links not only in dextran but methylene-dextran but methylene-bisacrylamide molecules also bind to
bisacrylamide molecules also bind to each other. A heach other. A hypothetical structure of Sephacrylypothetical structure of Sephacryl gel is shown Fig. 10.4.
gel is shown Fig. 10.4.
Fig. 10.4:
Fig. 10.4: A schematic representaA schematic representation of partial structure of Setion of partial structure of Sephacrylphacryl The cross linking reactions make the gel partly macroreticular and the gels can be The cross linking reactions make the gel partly macroreticular and the gels can be produced with fractionation ranges extending up to high molecular masses.
produced with fractionation ranges extending up to high molecular masses. They areThey are mechanically rigid and bear high pressures (upto 1M
mechanically rigid and bear high pressures (upto 1M Pa) without compressing the bed.Pa) without compressing the bed. These gels are
These gels are intended mainly for use with aqueous eluants. intended mainly for use with aqueous eluants. Two types of SephacrylTwo types of Sephacryl gels are a
gels are available. They are Sephacryl-200 Superfine and Sephacryl S-300 Superfine.vailable. They are Sephacryl-200 Superfine and Sephacryl S-300 Superfine. The fractionation ranges cover the most
The fractionation ranges cover the most common molecular masses of water solublecommon molecular masses of water soluble proteins. Since in these gels, the s
proteins. Since in these gels, the s tructure is macroreticular, the slope of the selectivitytructure is macroreticular, the slope of the selectivity curve is less than the
curve is less than the microreticular Sephamicroreticular Sephadex gel. Due to ladex gel. Due to la rge amount of methylenerge amount of methylene bis
bis-acrylamide in the gel, t-acrylamide in the gel, the adsorption effects are more pronounced than withhe adsorption effects are more pronounced than with Sephadex.
Chromatographic Chromatographic Methods-III
Methods-III 3.3. Polyacrylamide gels (Bio-Gel)Polyacrylamide gels (Bio-Gel)
Cross linked polyacrylamides form gels with water and
Cross linked polyacrylamides form gels with water and are known as Bio-gels. Theyare known as Bio-gels. They are mainly used for biochemical work. Unlike
are mainly used for biochemical work. Unlike the Sephadex gels, these are the Sephadex gels, these are entirelyentirely synthetic and made by acrylamide, H
synthetic and made by acrylamide, H22C = CH – CO – NHC = CH – CO – NH22. It is synthesized by. It is synthesized by
copolymerizing acrylamide with the c
copolymerizing acrylamide with the c rosslinking agentrosslinking agent N N ,, N N ′′–methylene-–methylene-bis
bis--acrylamide (H
acrylamide (H22C = CH – CO – NH – CHC = CH – CO – NH – CH22 – NH – CO – CH = CH – NH – CO – CH = CH22). The concentration). The concentration
of the monomer can be varied to give different swelling characteristics and of the monomer can be varied to give different swelling characteristics and chromatograp
chromatographic properties. A partial hic properties. A partial structure of Bio-Gel is structure of Bio-Gel is shown in Fig. 10.5.shown in Fig. 10.5.
Fig. 10.5:
Fig. 10.5: A schematic repA schematic representation of partial structure of Bresentation of partial structure of Bio-Gelio-Gel Bio-Gels are available from B
Bio-Gels are available from Bio-Rad Laboratories. They are produced by beadio-Rad Laboratories. They are produced by bead polymerization and are available as powder. They have a marked tendency to stick polymerization and are available as powder. They have a marked tendency to stick together and form lumps. Like Sephadex, Bio
together and form lumps. Like Sephadex, Bio Gel is xerogel.Gel is xerogel. When the dry powder is immersed in water, it swells
When the dry powder is immersed in water, it swells to form the gel. Bio-Gel is quiteto form the gel. Bio-Gel is quite inert and the weakest point for chemical reaction are
inert and the weakest point for chemical reaction are the amide groups which arethe amide groups which are hydrolyze
hydrolyzed at d at extremes ofextremes of p pH. On hydrolysis, the carboxyl groups formed impart theH. On hydrolysis, the carboxyl groups formed impart the ion exchange character.
ion exchange character.
Bio-Gels are available in eleven types with different swelling characteristics and Bio-Gels are available in eleven types with different swelling characteristics and different fractionation ranges. The different types are characterized by
different fractionation ranges. The different types are characterized by the letter P.the letter P. There are gels from Bio-Gel P-2 to Bio Gel
There are gels from Bio-Gel P-2 to Bio Gel P-300. The number is intended to indicateP-300. The number is intended to indicate the exclusion limit.
the exclusion limit.
There are striking similarities
There are striking similarities in the chromatographic behaviour of Bio-Gel andin the chromatographic behaviour of Bio-Gel and Sephadex gel. Both the gel types are microreticular. Their selectivity curves are also Sephadex gel. Both the gel types are microreticular. Their selectivity curves are also quite similar. The similarity is also
quite similar. The similarity is also reflected in their mechanical properties. Bio-Gelreflected in their mechanical properties. Bio-Gel beads like Sephadex beads with low water regain are brittle while the ones
beads like Sephadex beads with low water regain are brittle while the ones with highwith high water regain are very soft.
Size Exclusion Size Exclusion Chromatography Chromatography 4.
4. Agar and agrose gelsAgar and agrose gels
Agar or agar-agar, as it was called e
Agar or agar-agar, as it was called e arlier, is obtained from various species of sea-arlier, is obtained from various species of sea-weeds. It is a
weeds. It is a mixture of linear polysaccharides comprmixture of linear polysaccharides comprising mainly of D-galactose andising mainly of D-galactose and
3,6-anhydro-3,6-anhydro-L-galactose residues. A partial structure of L-galactose residues. A partial structure of agrose is shown in Fig.10.6.agrose is shown in Fig.10.6.
Fig. 10.6:
Fig. 10.6: A schematic represenA schematic representation of partial structure of atation of partial structure of agrosegrose These gels are
These gels are unique combining fractionation ranges at very high molecular massesunique combining fractionation ranges at very high molecular masses with good mechanical stability. They are a
with good mechanical stability. They are a good supplegood supplement to the dextran gels andment to the dextran gels and polyacrylamide gels. The agar gels with
polyacrylamide gels. The agar gels with the lowest fractionation ranges correspondthe lowest fractionation ranges correspond approximately in their fractionation properties to the dextran
approximately in their fractionation properties to the dextran and polyacrylamide gelsand polyacrylamide gels with the highest fractionation ranges. These gels can fractionate in the range
with the highest fractionation ranges. These gels can fractionate in the range intermediate between molecules and particles.
intermediate between molecules and particles.
Agrose gel for chromatographic work are available from Pharmacia
Agrose gel for chromatographic work are available from Pharmacia Fine ChemicalsFine Chemicals (trade name Sepharose) and Bio-Rad (trade name
(trade name Sepharose) and Bio-Rad (trade name Bio-Gel A). Sepharose is availableBio-Gel A). Sepharose is available in three normal
in three normal types and three corresponding types crosslinked with 2, 3-types and three corresponding types crosslinked with 2, 3-dibromopr
dibromopropanol. The normal types are opanol. The normal types are Sepharose 2B, 4B and 6B Sepharose 2B, 4B and 6B and crosslinkedand crosslinked types being Sepharose CL2
types being Sepharose CL2B, CL4B and CL6B. B, CL4B and CL6B. The numbers indicate the perceThe numbers indicate the percentagentage of dry gel in the particles. Bio-Gel A is available in six types.
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
Unlike the gels discussed earlier, the macromolecules of gel matrix are not bound bybound by covalent bonds. They are supposed to be
covalent bonds. They are supposed to be held together by hydrogen bonds. Theheld together by hydrogen bonds. The polysaccharide chains seem to aggregate in bundles.
polysaccharide chains seem to aggregate in bundles. Between the bundles, there areBetween the bundles, there are very large openings in the gel matrix. The structure is
very large openings in the gel matrix. The structure is very open and at the same timevery open and at the same time mechanically stable.
mechanically stable.
Agrose gel has some disadvantages as a chromatographic material. There is a Agrose gel has some disadvantages as a chromatographic material. There is a considerable amount of charged groups in the material.
considerable amount of charged groups in the material. Because of the presence ofBecause of the presence of charged groups, it is recommended to work at high ionic strength to mitigate the 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 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. 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
This amounts to the fact that they are less stable to p pH extremes than the gelsH extremes than the gels described earlier. These gels maintain their structure if the water
described earlier. These gels maintain their structure if the water is substituted byis substituted by many organic solvents such as acetone or ethanol. The structure of agrose gels makes many organic solvents such as acetone or ethanol. The structure of agrose gels makes it impractical to dry and reswell
it impractical to dry and reswell them. Therefore, once the gel has been prepared, itthem. Therefore, once the gel has been prepared, it should be stored in wet state. It
should be stored in wet state. It may be important to emphasize again that agrose gelmay be important to emphasize again that agrose gel have fractionation ranges at considerably higher molecular masses than
have fractionation ranges at considerably higher molecular masses than expected byexpected by comparison with dextran and polyacrylamide gels. The selectivity cur
comparison with dextran and polyacrylamide gels. The selectivity cur ve of these gelsve of these gels is less steep
is less steep than it is for the than it is for the gels from dextran and polyacrylamide.gels from dextran and polyacrylamide. 5.
5. Styrene-divinylbenzene gel (Styragel)Styrene-divinylbenzene gel (Styragel) It is
It is a class a class of polystyrene gels useful for purely non-aqueous separations in methyleneof polystyrene gels useful for purely non-aqueous separations in methylene chloride, toluene, trichlorobenzene, tetrahydr
chloride, toluene, trichlorobenzene, tetrahydrofuran and so on. ofuran and so on. It cannot be used withIt cannot be used with water, alcohol and acetone. These
water, alcohol and acetone. These macroreticular gels have fractionation range frommacroreticular gels have fractionation range from 1600 to 40 million. Styragel is available in 11
1600 to 40 million. Styragel is available in 11 different types. These are manufactureddifferent types. These are manufactured by Dowex Chemical Co. and sold by Waters Associate. Styragel is available
by Dowex Chemical Co. and sold by Waters Associate. Styragel is available suspended in diethylbenzen
suspended in diethylbenzene. The beads are rigid and the e. The beads are rigid and the solvent can be easilysolvent can be easily
exchanged even after the bed has been packed. The beads are supposed not to change exchanged even after the bed has been packed. The beads are supposed not to change their volume with change of solvent.
Chromatographic Chromatographic Methods-III
Methods-III SAQ 5SAQ 5
What happens if the
What happens if the Sephadex gels are subjected to prolonged exposure to oxidizingSephadex gels are subjected to prolonged exposure to oxidizing agents? agents? ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 6 SAQ 6
In what important properties, the Sephadex and
In what important properties, the Sephadex and Sephacryl gels differ from each other?Sephacryl gels differ from each other? ………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 7 SAQ 7
What are the weak points of chemical attack
What are the weak points of chemical attack in Sephadex and Bio-Gels?in Sephadex and Bio-Gels?
………... ………... ………... ………... ………... ………... ………... ………... ………... ………... SAQ 8 SAQ 8
What different classes of gels are not prepared from natural materials? What different classes of gels are not prepared from natural materials?
………... ………... ………... ………... ………... ………... ………... ………...
10.5
10.5 VARIABLES DEFINING THE UTILITY OF GEL
VARIABLES DEFINING THE UTILITY OF GEL
After having learnt about the different
After having learnt about the different categories of gels used for categories of gels used for chromatograchromatographicphic work, the methods of their preparation and some of the important characteristics, it is 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
necessary to name a few variables which really characterize the utility of gel. Thegel. The most important of the different properties is the
most important of the different properties is the fractionation ran fractionation rangege. Sometimes, it is. Sometimes, it is given as the upper limit of fractionation or the exclusion limit. When the
given as the upper limit of fractionation or the exclusion limit. When the exclusionexclusion limits or the fractionation ranges are given, it should be stated
limits or the fractionation ranges are given, it should be stated that for what type ofthat for what type of substances these data are obtained. These numbers vary with one type of substances to substances these data are obtained. These numbers vary with one type of substances to another. For a
Size Exclusion Size Exclusion Chromatography Chromatography they are fairly constant. Another important variable of significance is the
they are fairly constant. Another important variable of significance is the steepness ofsteepness of the selectivity curve
the selectivity curve in the in the linear portion of the fractionation range.linear portion of the fractionation range.
For xerogels, the water regain is the common measure of the swelling capacity of the For xerogels, the water regain is the common measure of the swelling capacity of the gel. Here, it may be
gel. Here, it may be important to tell more about xero gels and aerogels. Different gelsimportant to tell more about xero gels and aerogels. Different gels react differently to the removal of liquid in them.
react differently to the removal of liquid in them. The group of xerogels shrink onThe group of xerogels shrink on drying to a compact material containing only the gel matrix. The
drying to a compact material containing only the gel matrix. The aerogels, on the otheraerogels, on the other hand do not shrink; instead
hand do not shrink; instead the surrounding air penetrates into the the surrounding air penetrates into the gel. Xerogels whengel. Xerogels when brought in contact with the liquid, take up liquid and
brought in contact with the liquid, take up liquid and return to the gel state. In aereturn to the gel state. In aerogelsrogels also, the air
also, the air can be substituted by the liquid.can be substituted by the liquid. Getting back to water regain capacity, it is
Getting back to water regain capacity, it is usually expressed as the amount of waterusually expressed as the amount of water (in mL) imbibed by one gram of dry xerogel on swelling. It does not include the (in mL) imbibed by one gram of dry xerogel on swelling. It does not include the interstitial liquid between the grains. In the case
interstitial liquid between the grains. In the case of dextran and polyacrylamide gels, itof dextran and polyacrylamide gels, it has been observed that there is a
has been observed that there is a close relationship between the water regain and theclose relationship between the water regain and the fractionation properties. If the water regain is low, it is
fractionation properties. If the water regain is low, it is expected that the fractionationexpected that the fractionation range is at a
range is at a low molecular mass. In the case of agrose gel, ilow molecular mass. In the case of agrose gel, i t is percentage of gelt is percentage of gel matrix in the gel grain is
matrix in the gel grain is taken instead of the water regain. While working withtaken instead of the water regain. While working with nonaqueous solve
nonaqueous solvents, the analogy is taken with water nts, the analogy is taken with water regain. Ultimately, the particleregain. Ultimately, the particle size of the
size of the gel grain is an important variable. It affects the degree of gel grain is an important variable. It affects the degree of zone broadeningzone broadening,, the resolution, the dilution and the flow rate. As
the resolution, the dilution and the flow rate. As a matter of fact, the a matter of fact, the above mentionedabove mentioned information should be available to the user before a particular gel is
information should be available to the user before a particular gel is put to use forput to use for chromatograp
chromatographic hic work.work.
10.6
10.6 UNIQUE FEATURES OF THE TECHNIQUE
UNIQUE FEATURES OF THE TECHNIQUE
This simple technique of
This simple technique of size exclusion chromatography rapidly became very popularsize exclusion chromatography rapidly became very popular and almost became indispensable for biochemistry. Before we
and almost became indispensable for biochemistry. Before we discuss some of itsdiscuss some of its important applications, it may be desirable to look into the reasons for
important applications, it may be desirable to look into the reasons for its fast adoptionits fast adoption as a frontline chromatographic technique. The technique is simple to perform. It is as a frontline chromatographic technique. The technique is simple to perform. It is remarkably insensitive to the composition of
remarkably insensitive to the composition of the eluant and temperature. The the eluant and temperature. The addedadded advantage is that very liable compounds can be fractionated without the fear of their advantage is that very liable compounds can be fractionated without the fear of their destruction. The gel matrix
destruction. The gel matrix generally does not cause denaturation and the generally does not cause denaturation and the experimentexperiment can be performed in
can be performed in very mild conditions.very mild conditions. The size exclusion
The size exclusion chromatogrchromatography can fractionate substances of very aphy can fractionate substances of very high molecularhigh molecular masses. By varying the contents of gel in
masses. By varying the contents of gel in gel matrix, the fractionation ranges can begel matrix, the fractionation ranges can be varied within wide limits. The most dense gels
varied within wide limits. The most dense gels fractionate substances below molecularfractionate substances below molecular mass 1000 while there are gels whose fractionation range extends to several millions. 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 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 way. Typical problems of this type are desalting of solutions of proteins and other high molecular mass substances and determination of molecular weights of
molecular mass substances and determination of molecular weights of macromolecules. The chromatographic columns usually need no
macromolecules. The chromatographic columns usually need no regeneration. Theyregeneration. They can be used over and again for a
can be used over and again for a long duration of time without alteration in theirlong duration of time without alteration in their chromatograp
chromatographic properties provided the hic properties provided the microbial growth is amicrobial growth is a voided.voided. SAQ 9
SAQ 9
Mention three unique
Mention three unique advantages of size eadvantages of size e xclusion chromatograxclusion chromatography.phy.
………... ………... ………... ………... ………... ………... ………... ………...
Chromatographic Chromatographic Methods-III
Methods-III
10.7 SOME
10.7
SOME APPLICA
APPLICATIONS
TIONS
From the discussion in the preceding section, it is
From the discussion in the preceding section, it is clear that the technique of sizeclear that the technique of size exclusion chromatogr
exclusion chromatography has some unique advantages. Some of aphy has some unique advantages. Some of these featuresthese features strongly favour its use for biochemical work. Therefore, it
strongly favour its use for biochemical work. Therefore, it finds extensive use forfinds extensive use for separations in the said area. The applications are numerous and all of
separations in the said area. The applications are numerous and all of them cannot bethem cannot be cited here. Typical examples from this very large field are
cited here. Typical examples from this very large field are enzyme purification,enzyme purification, purification and characterization of antibodies and separation and purification of purification and characterization of antibodies and separation and purification of peptide and protein hormones.
peptide and protein hormones.
The present section will only highlight some representative applications. It may not be 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 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 for it. For the purposes of clarity and conciseness in the presentation, the applications are subdivided in the
are subdivided in the following three heads.following three heads. 1.
1. Analytical applicationsAnalytical applications 2.
2. Preparative applicationsPreparative applications 3.
3. Miscellaneous applicationsMiscellaneous applications
The miscellaneous applications include important applications which are not
The miscellaneous applications include important applications which are not coveredcovered in the first two heads particularly the use of
in the first two heads particularly the use of gels in thin layer chromatographygels in thin layer chromatography, zone, zone electrophoresis and HPLC. No doubt, these are not the direct applications of size electrophoresis and HPLC. No doubt, these are not the direct applications of size exclusion chromatogr
exclusion chromatography but they definitely reflect aphy but they definitely reflect on the use on the use of gels for separations.of gels for separations. 10.7.1
10.7.1 Analytical ApplicationsAnalytical Applications It may be important to point out that
It may be important to point out that within analytical applications, there are furtherwithin analytical applications, there are further sub-divisions as follows:
sub-divisions as follows: i)
i) Analytical grou Analytical group separationsp separations One of the
One of the very common uses of gel filtration very common uses of gel filtration chromatogchromatography is the raphy is the removal ofremoval of interferences before the final determination. It is particularly important to interferences before the final determination. It is particularly important to remove low molecular mass interferences before the determination of remove low molecular mass interferences before the determination of macromolecular compone
macromolecular component in the nt in the mixture. The determination of protein inmixture. The determination of protein in spinal fluid is a
spinal fluid is a typical example. The low molecular weight interferences aretypical example. The low molecular weight interferences are removed for the
removed for the spectrophotomspectrophotometric determination of etric determination of protein.protein. On a similar line,
On a similar line, it is desirable to it is desirable to determine polysaccharidetermine polysaccharides like insulin,des like insulin, amylose or dextran from low molecular
amylose or dextran from low molecular mass sugar. Gel filtrationmass sugar. Gel filtration chromatograp
chromatography has been used hy has been used for the purpose, say, the for the purpose, say, the removal of glucose fromremoval of glucose from the mixture. The technique can also be
the mixture. The technique can also be used for the removal of high mused for the removal of high molecularolecular weight interferences but this has not been used much.
weight interferences but this has not been used much. ii)
ii) Analytical fraction Analytical fractionationation
This covers a wide range. It could be the
This covers a wide range. It could be the separation of substances which can beseparation of substances which can be determined separately. The other end can be where elution curve profile can be determined separately. The other end can be where elution curve profile can be used to characterize the
used to characterize the sample without the determination of sample without the determination of concentration ofconcentration of the different ingredients. A typical example of the first type is
the different ingredients. A typical example of the first type is the separation ofthe separation of sugars from cellulose hydrolysate. The technique is an
sugars from cellulose hydrolysate. The technique is an extremely powerful toolextremely powerful tool for the separation of oligosaccharides with d
for the separation of oligosaccharides with d iffering number of sugar residuesiffering number of sugar residues from each other. Maltooligosaccharides containing up to 15
from each other. Maltooligosaccharides containing up to 15 glucose units andglucose units and polymaltoses with chain length up to 21 glucose units can be separated from polymaltoses with chain length up to 21 glucose units can be separated from each other on Bio-Gel
each other on Bio-Gel P2. Sephadex LH-20 provides a very good resolution ofP2. Sephadex LH-20 provides a very good resolution of lipids and steroids. It has been possible to fractionate a very
lipids and steroids. It has been possible to fractionate a very wide range of non-wide range of non-polar lipids on LH-20 in
polar lipids on LH-20 in chloroform and they were found to separate primarilychloroform and they were found to separate primarily according to their molecular size.
Size Exclusion Size Exclusion Chromatography Chromatography silica and other materials is unsuccessful for this type of separation. Amino
silica and other materials is unsuccessful for this type of separation. Amino acids can be separated on the tightly cross-linked types of gels.
acids can be separated on the tightly cross-linked types of gels.
The most important applications of analytical fractionation come closer to the The most important applications of analytical fractionation come closer to the fingerprint end of the scale with substances overlapping to a large extent. Many fingerprint end of the scale with substances overlapping to a large extent. Many biological fluids have been characterized. The
biological fluids have been characterized. The most important among these ismost important among these is plasma. The elution behaviour of plasma proteins
plasma. The elution behaviour of plasma proteins has been investigated. Thehas been investigated. The normal elution pattern changes strongly under the influence of
normal elution pattern changes strongly under the influence of certain diseases.certain diseases. Thus, this form of
Thus, this form of chromatogrchromatography acts as aphy acts as a diagnostic tool.a diagnostic tool. iii)
iii) Measurement Measurement of protein binding of protein binding and complex fand complex formationormation Gel filtration chromatography has been very useful in
Gel filtration chromatography has been very useful in the study of complexesthe study of complexes between proteins and low molecular mass solutes.
between proteins and low molecular mass solutes. The principle for theThe principle for the measurement is simple if the reactants are
measurement is simple if the reactants are irreversibly bounirreversibly bound to each other. Thed to each other. The reaction mixture is fed to the column and the
reaction mixture is fed to the column and the complex is separatedcomplex is separated chromatogra
chromatographically from the reactant pphically from the reactant present in excess. One resent in excess. One typical example oftypical example of complex formation between the proteins is
complex formation between the proteins is the determination of heptoglobinthe determination of heptoglobin content of plasma. This exercise is carried out in
content of plasma. This exercise is carried out in clinical laboratories. Anotherclinical laboratories. Another important example from clinical lab is the protein binding of insulin. It gives important example from clinical lab is the protein binding of insulin. It gives information about the formation of insulin antibodies in d
information about the formation of insulin antibodies in d iabetics treated withiabetics treated with insulin.
insulin. iv)
iv) Determination of m Determination of molecular massesolecular masses It is rated as
It is rated as one of the most important applications of size exclusionone of the most important applications of size exclusion chromatogra
chromatography. The determination of molecular masses of phy. The determination of molecular masses of proteins isproteins is
particularly cited as an important example. The underlined principle is based on 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
the basic mechanism of the technique. It has been observed that for most proteins a good close correlation between molecular
proteins a good close correlation between molecular mass and elution behaviourmass and elution behaviour is observed; very few
is observed; very few proteins show anomalous behaviour. Relationshipproteins show anomalous behaviour. Relationship between elution behaviour and molecular masses has been obtained for between elution behaviour and molecular masses has been obtained for carbohydr
carbohydrates and ates and peptides. Sephadex G-100 and peptides. Sephadex G-100 and Sephadex G-200 have beenSephadex G-200 have been used most frequently. Agar and agrose gels have also been used and so is used most frequently. Agar and agrose gels have also been used and so is Bio-Gel P. The eluant in use
Bio-Gel P. The eluant in use with proteins is usually buffer or saline. Long bedswith proteins is usually buffer or saline. Long beds should be used for better
should be used for better accuracy and precision of the measurement.accuracy and precision of the measurement. Although with some gels, calibration measurements are available but it is Although with some gels, calibration measurements are available but it is recommende
recommended to d to make calibration curve with each bed. make calibration curve with each bed. Adsorption phenomAdsorption phenomenaena limit the use of gel
limit the use of gel filtration chromatography ffiltration chromatography for large molecules and particles.or large molecules and particles. This technique has lower accuracy for
This technique has lower accuracy for molecular mass measurement than somemolecular mass measurement than some other available methods. On the
other available methods. On the other hand, this method of other hand, this method of determination hasdetermination has the advantage of rapid measurement, is
the advantage of rapid measurement, is easy to perform and requires fairlyeasy to perform and requires fairly inexpensive equipment.
inexpensive equipment. 10.7.2
10.7.2 Preparative ApplicationsPreparative Applications
In preparative applications, there are two distinct categories of uses. In preparative applications, there are two distinct categories of uses. i)
i) Preparative group separationPreparative group separation ii)
ii) Preparative fractionation.Preparative fractionation. Let us study about them briefly. Let us study about them briefly.
i)
i) Preparative group separationPreparative group separation In a
In a large number of biochemical preparations, desalting and exchange oflarge number of biochemical preparations, desalting and exchange of buffers is an important step. Theoretically, there is no problem in
buffers is an important step. Theoretically, there is no problem in achieving this separation but in actual practice, some problems may be achieving this separation but in actual practice, some problems may be