Unit 10-Size Exclusion Chromatography After Correction

Chromatographic Methods-III

UNIT 10

SIZE EXCLUSION CHROMATOGRAPHY

Structure 10.1

Introduction Objectives

10.2 10.3

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

10.4 10.5 10.6 10.7

Classification, Synthesis and Properties Variables Defining the Utility of Gel Unique Features of the Technique Some Applications Analytical Applications Preparative Applications Miscellaneous Applications

10.8 10.9 10.10

Summary Terminal Questions Answers

10.1 INTRODUCTION Before we discuss this chromatographic technique, technique, it may be d esirable 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 e xclusion chromatographic chromatographic technique appears in chromatographic chromatographic processes where liquid and solid are in equilibrium. Thus, very rightly the size exclusion chromatography chromatography is one of the important forms of liquid chromatographic 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 carbohydrates and acrylamide that have an open network formed by the cross linking of polymeric chains. Incidentally, this branch of chromatographic chromatographic science was also discovered in a B ioscience 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 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 p ermeation chromatography 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 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 chromatography. Reference to gels will also be made in Unit 12 on electrophoresis.

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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. chromatography. After explaining about the characteristics of gels needed for chromatographic purposes, a classification of important types of chromatographic 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 chromatography are explained. Finally, some of the important applications of the technique are discussed.

Size Exclusion Chromatography

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

discuss the principle responsible for separation of solutes,

•

describe gels and the characteristics required for their use for chromatographic 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 p acking 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.

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Chromatographic Methods-III

Fig. 10.1: Schematic of a chromatographic separation on a size size exclusion column. Large open crossed circles represent the stationary phase; large black circles represent molecules which do not penetrate K  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 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 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 V R be the retention volume for a solute with a chromatographic chromatographic column. Let V 0 be the interstitial volume (void volume), that is, the volume within the column which is available to the mobile phase. V L is the volume of water within the gel particles available for accepting solutes. On the lines of GLC, we can write the following equation: V R = V 0 + KV L

where, K  is  is some form of distribution coefficient. If the solute is completely excluded from the interior of gel then K  =  = 0 and V R = V 0. 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 V R = V 0 + V L. Taking the case of molecules which can penetrate the gel to some extent but not freely, K  values  values fall between 0 and 1. In cases where sieving is the only phenomenon responsible for fractionation, K  values  values greater than 1 would never be encountered. However 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 shows the typical behaviour of variation in the retention volume with the molecular weight of the solute.

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Size Exclusion Chromatography

Fig. 10.2: Relation between retention volume 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 chromatographically in the sloping region of the curve. By varying the degree of cross linking of the polymer, the curve shifts horizontally. There are materials available which fractionate molecules in various molecular weight ranges. For Sephadex G. 50, the fractionation range for peptides and globular protein is molecular weight 1,500 to 30,000 while the range for G. 150 is 5,000 to 400,000.

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

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

solute molecules do not enter the gel matrix

ii)

solute molecules enter the gel matrix

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

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

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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 macroreticular gels, the microstructure is strongly heterogenous 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 throughout the gel. They fractionate relatively lower molecular 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 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. 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 choice of gels

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

 Availability of gels with different particle size

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

vi)

 Mechanical rigidity 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 s ubstance; 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 a ggregates ggregates of macroreticular gel take care of the mechanical strength. These gels are called macro-microreticular macro-microreticular gels.

Size Exclusion Chromatography

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

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

10.4 CLASSIFICATION, 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 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 commonly by the trade names. A classification of different types of gels is given below. i)

Dextran gels (Sephadex)

ii)

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

iii)

Polyacrylamide gels (Bio-Gel)

iv)

Agar and Agrose gels

v)

Styrene-divinylbenzene Styrene-divinylbenzene gels (Styragel)

Besides the above classes inorganic materials like silica gel and porous glass are also used. Let us study the different different types of gels 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 d istribution (10 – 300) × 106. The raw material is purified, 45

Chromatographic Methods-III

partly hydrolysed hydrolysed and finally fractionated fractionated by ethanol precipitation to give a product 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 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 glyceryl links between b etween 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 representation of partial structure of Sephadex 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 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.

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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 exposure to oxidizing agents may cause an increase in the carboxyl group content. The increase of carboxyl group in the structure impairs the chromatographic chromatographic behaviour of the gel. It may be noted that initially Sephadex contains a very small amount of carboxyl groups. This amount is so low that most of the charge effects observed are attributed to Donnan effects caused by solutes.

Size Exclusion Chromatography

Sephadex gels were the first for which a close relationship between molecular size and elution behaviour was observed. In Sephadex 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. methylene-bisacrylamide. There are cross links not only in dextran but methylenebisacrylamide molecules also bind to each other. A h ypothetical structure of Sephacryl gel is shown Fig. 10.4.

Fig. 10.4: A schematic representation representation of partial structure of Sephacryl 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. a vailable. 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 s tructure is macroreticular, the slope of the selectivity curve is less than the microreticular Sephadex Sephadex gel. Due to la rge amount of methylene t he adsorption effects are more pronounced than with bis-acrylamide in the gel, the Sephadex. 47

Chromatographic Methods-III

3.

Polyacrylamide gels (Bio-Gel)

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

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

Bio-Gels are available from Bio-Rad B io-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 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.

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4.

Agar and agrose gels

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

Size Exclusion Chromatography

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

These gels are unique combining fractionation ranges at very high molecular masses with good mechanical stability. They are a good supplement supplement to the dextran gels and polyacrylamide gels. The agar gels with the lowest fractionation ranges correspond approximately in their fractionation properties to the dextran and polyacrylamide gels with the highest fractionation ranges. These gels can fractionate in the range intermediate between molecules and particles. Agrose gel for chromatographic work are available from Pharmacia Fine Chemicals (trade name Sepharose) and Bio-Rad (trade name Bio-Gel A). Sepharose is available in three normal types and three corresponding types crosslinked with 2, 3dibromopropanol. dibromopropanol. The normal types are Sepharose 2B, 4B and 6B and crosslinked types being Sepharose CL2B, CL2B, CL4B and CL6B. The numbers indicate the percentage 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 cur ve 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 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. 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.

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Chromatographic Methods-III

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

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

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

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

10.5 VARIABLES DEFINING THE UTILITY OF GEL After having learnt about the different categories of gels used for chromatographic 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 range. Sometimes, it is most important of the different properties is the fractionation range 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.

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

Size Exclusion Chromatography

For xerogels, the water regain is the common measure of the swelling capacity of the gel. Here, it may be important to tell more about xero gels and aerogels. Different gels react differently to the removal of liquid in them. The group of xerogels shrink on drying to a compact material containing only the gel matrix. The aerogels, on the other hand do not shrink; instead the surrounding air penetrates into the gel. Xerogels when brought in contact with the liquid, take up liquid and return to the gel state. In ae rogels 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, i t is percentage of gel matrix in the gel grain is taken instead of the water regain. While working with nonaqueous solvents, 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, 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 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 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 chromatographic properties provided the microbial growth is a voided.

SAQ 9 Mention three unique advantages of size e xclusion chromatography. chromatography. …………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

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Chromatographic Methods-III

10.7 SOME APPLICATIONS APPLICATIONS From the discussion in the preceding section, it is clear that the technique of size exclusion chromatography 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, chromatography, zone electrophoresis and HPLC. No doubt, these are not the direct applications of size exclusion chromatography 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 group separations One of the very common uses of gel filtration chromatography 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 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 spectrophotometric determination of protein. On a similar line, it is desirable to determine polysaccharides polysaccharides like insulin, amylose or dextran from low molecular mass sugar. Gel filtration chromatography 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 molecular weight interferences but this has not been used much.

ii)

 Analytical fractionation 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 d iffering number of sugar residues from each other. Maltooligosaccharides containing up to 15 glucose units and polymaltoses with chain length up to 21 glucose units can be separated from each other on Bio-Gel P2. Sephadex LH-20 provides a very good resolution of lipids and steroids. It has been possible to fractionate a very wide range of nonpolar lipids on LH-20 in chloroform and they were found to separate primarily according to their molecular size. The conventional liquid chromatography chromatography on

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

Size Exclusion Chromatography

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

 Measurement of protein binding and complex formation 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 bound to each other. The reaction mixture is fed to the column and the complex is separated chromatographically chromatographically from the reactant present p resent 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 d iabetics treated with insulin.

iv)

 Determination of molecular molecular masses It is rated as one of the most important applications of size exclusion chromatography. 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 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 recommended to make calibration curve with each bed. Adsorption phenomena phenomena limit the use of gel filtration chromatography for for large molecules and particles. This technique has lower accuracy for molecular mass measurement than some other available methods. On the other hand, this method of determination has the advantage of rapid measurement, is easy to perform and requires fairly inexpensive equipment.

10.7.2

Preparative Applications

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

Preparative group separation

ii)

Preparative fractionation.

Let us study about them briefly. i)

Preparative group separation

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

53

Chromatographic Methods-III

encountered. If distilled water is being used as an eluant, tailing may be more pronounced than at higher ionic strength. Moreover, dilution is another problem. It is proposed to use volatile electrolytes like pyridinium acetate and ethylenediamine acetate. The technique is not only confined to desalting of macromolecules but it has also been extended to desalting of virus. Gel filtration chromatography is frequently used for separation of macromolecules that have been modified chemically from the free reactant. The technique on a large scale has b een 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 removal of allergens from pharmaceutical preparations. Allergens are often compounds 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 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 in terest. The separation of an antibody from the antigens can be performed by gel filtration chromatography. chromatography. Fractionation of nucleic acids a cids and animal viruses has also been attained on agar gels. An important industrial example based on fractionation by gel filtration chromatography 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 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 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. chromatogram. This is because the gels usually crack on drying. This type of chromatography 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.

54

The most important application is the rapid determination of molecular masses of proteins and peptides. The technique is specially useful for this purpose as the sample can be chromatographed on the same plate as the reference substances.

Size Exclusion Chromatography

Another important application of gels is in capillary gel electrophoresis which is generally performed in a porous gel polymer matrix, the pores of which contain a buffer mixture in which the separation is carried out. This type of medium could provide a molecular sieving action which retards the migration of analyte species to various degrees depending upon 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 µ m diameter are used. They have a pore size of a few nm to several hundred nm. The controlled pore silica particles are coated with a hydrophilic phase to reduce adsorption of the solute.

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

Where the interference of a low molecular weight is removed before the determination of a high molecular weight compound. compound. ………………………………………………… ………………………… …………………………………………… ………………………................. …................. ……………………………………………………………………………………. ……………………………………………………………………………………. …………………………………………………………………………………….

ii)

Where the technique is used u sed 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 p principle rinciple 55

Chromatographic Methods-III

involved in separation is discussed. A general discussion on gels is presented which is followed by enumeration of properties which are required to make them suitable for chromatographic 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, chromatography, capillary gel electrophoresis and h igh performance liquid chromatography. chromatography.

10.9 TERMINAL QUESTIONS 1.

What is the basic equation for size exclusion chromatography? 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 chromatographic work.

4.

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

5.

What are the main disadvantages with Agrose gels?

6.

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

7.

How is gel filtration chromatography used for molecular weight determination of proteins? Comment on the utility of the method.

10.10

ANSWERS

Self Assessment Questions

56

1.

It is the pore size of the swollen gel which is responsible for the fractionation of molecules of different sizes by size exclusion chromatography. 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 f ractionation ranges are controlled by the microreticular structure.

5.

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

in the chromatographic behaviour of the gel primarily because of prominence of ion exchange mechanism. 6.

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

The basic structural unit is different. The Sephacryl is partly macroreticular in character.

ii)

Due to higher crosslinking in Sephacryl, the fractionation ranges extend to high molecular weight as compared to Sephadex.

iii)

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

iv)

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

7.

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

8.

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

9.

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

10.

Size Exclusion Chromatography

i)

The material to be separated does not suffer any destruction or denaturation.

ii)

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

iii)

The chromatographic columns usually need no regeneration. They can be used for a long duration without change in their chromatographic behavior.

i)

In the determination of protein in spinal fluid low molecular weight interferences are removed by gel filtration chromatography. 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 chromatography is V R = V 0 + KV L

where, V  R is retention volume, V 0 is the interstitial volume, K  is  is some form of distribution coefficient and V L is the volume of water within the gel particles. Normally, the K  value  value greater than 1 is not encountered. If it is encountered, it implies the occurrence of phenomena like adsorption, hydrogen hydrogen bonding and ion exchange between the gel matrix and the solute. 2.

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

Chromatographic Methods-III

macroreticular gels. The microreticular gels are supposed to be mechanically weaker than macroreticular gels. 3.

4.

5.

58

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

Matrix of gel should be inert.

ii)

Gel must be chemically stable.

iii)

It should have low content of ionic groups.

iv)

There should be a wide choice of availablility of gels with same general composition but different fractionational ranges.

v)

The gels should be available with different particle size.

vi)

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

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

Both gel types are macroreticular.

ii)

The selectivity curve of both types of gels are similar.

iii)

The mechanical properties of both types of gels are similar.

iv)

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

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

They contain a considerable amount of charged groups which cause problems due to ion exchange effects. To mitigate this problem, it is recommended 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 bound to each other, the resulting mixture is subjected to chromatographic 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 recommended to make calibration curve with each bed. This method is not as accurate as many other methods of mass measurement. But this method, based on size exclusion chromatography, has the advantages of rapid measurement, is easy to perform and requires fairly inexpensive experimental set up.

Further Reading 1.

North-Holland Gel Filtration Chromatography, Chromatography, By L. Fischer, Elsevier North-Holland Biomedical Press, Amsterdam, New York, Oxford.

2.

Quantitative Analysis, By R. A. Day Jr. and A. L. Underwood, Prentice and Hall of India Private Limited, New Delhi.

3.

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

Size Exclusion Chromatography

59

Chromatographic Methods-III

INDEX Agar and agrose gels 45, 49 Aluminosilicates 8 Amphoteric exchangers 7, 13 Applications of ion e xchange xchange 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 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 chromatography 40 Gels and their i mportant 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) Bio-Gel) 45, 48 Sephadex 46 Styrene-divinylbenzene gel (Styralgel) 49

Important properties of gels for chromatography 44 Utility of gel 50

Gel permeation chromatography 40 Ion exchange 5 Basic features of mechanism of 7 in mixed aqueous- organic media 24 Combined ion exchange solvent extraction (CIESE) 24

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

60

Separation of Actinide Actinide Elements 31, 33

Ion exchange mechanism 7

Size Exclusion Chromatography

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

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

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

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

Polyacrylamide gels (Bio-Gel) 45, 48 Resin properties 15 Capacity 17 Breakthrough (dynamic) capacity 17 Operating capacity 17 Sorption capacity 17 Total capacity of an ion exchange resin 17 Useful capacity 17

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

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

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

Chromatographic Methods-III

Sephadex G-200 46 Size exclusion chromatography 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 chromatography 40 Unique features of size exclusion chromatography 51 Utility of gel 50 Fractionation range 50

Zeolites 8

62