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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
10.8
10.9
10.10
Summary
Terminal Questions
Answers
10.1 INTRODUCTION
Before we discuss this chromatographic technique, it may be desirable to refer to
Unit 1 wherein classifications of separation methods have been dealt. In the scheme of
classification based on property resulting into separation, there is a distinct class in
which the separations are achieved on the basis of molecular geometry or molecular
dimensions. This size exclusion or gel filtration chromatography figures right in this
category. However, if we probe into another criteria of classification based on
equilibrium and rate processes, the exclusion chromatographic technique appears in
chromatographic processes where liquid and solid are in equilibrium. Thus, very
rightly the size exclusion chromatography is one of the important forms of liquid
chromatographic technique (Unit 4, sub-sec. 4.2.3).
The mobile phase is aqueous or organic and the stationary phase is a molecular sieve.
These sieves are generally polymeric carbohydrates and acrylamide that have an open
network formed by the cross linking of polymeric chains. Incidentally, this branch of
chromatographic science was also discovered in a Bioscience oriented laboratory.
This separation method originated in 1959 at the Biochemical Institute in Uppsala,
Sweden. Initially it was applied for the separation of water-soluble macromolecules of
biological importance. The technique was named as gel-filtration chromatography
(GFC). A few years later the technique was developed for synthetic polymers soluble
in organic solvents and it is was called as gel permeation chromatography (GPC).
This amounts to the fact that initially the names such as gel filtration chromatography
(mobile phase is water) used by biochemist and gel permeation chromatography
(mobile phase in organic solvent) used by polymer chemists described the technique.
Now the recommended or the most accepted name of the technique is size exclusion
chromatography (SEC). It is used in open column gravity fed for both analytical and
preparative separations and in high performance separations. The gel filtration also
finds use in thin layer chromatography and the technique is known as thin layer gel
filtration chromatography. Reference to gels will also be made in Unit 12 on
electrophoresis.
40
This unit on size exclusion chromatography first discusses the principle involved in
separations using gels. This is followed by a general discussion on gels and
characteristics required for the gels to be useful for chromatography. After explaining
about the characteristics of gels needed for chromatographic purposes, a classification
of important types of chromatographic gels is given. Along with this the methods of
preparation of the important categories of these gels on a broad basis are discussed and
the properties shown by them are highlighted. A brief note on the characteristics
which define the utility of a gel is also included. The unique features of this form of
chromatography are explained. Finally, some of the important applications of the
technique are discussed.
Size Exclusion
Chromatography
Objectives
After studying this Unit, you should be able to
describe gels and the characteristics required for their use for chromatographic
separations,
41
Chromatographic
Methods-III
It is important to note that size exlusions are different from other chromatographic
procedures. Here, there are no physical or chemical interactions between the analyte
and the stationary phase. As a matter of fact, efforts are made to avoid such
interactions because they may cause impaired column efficiencies. At this point, it
may be important to introduce the term exclusion limit. The exclusion limit is the
molecular weight of that molecule that will just permeate the gel and be retarded. This
can range from 1000 to several millions depending upon the gel. It should be kept in
mind that separation are based on molecular size and configuration rather than simply
its molecular weight but, generally, there is a correlation with molecular weight. Also,
generally the molecules smaller than the exclusion limit can be fractionated down to a
limiting size.
The entire picture of fractionation by size exclusion chromatography can be visualized
in some semiquantitative terms. Let VR be the retention volume for a solute with a
chromatographic column. Let V0 be the interstitial volume (void volume), that is, the
volume within the column which is available to the mobile phase. VL is the volume of
water within the gel particles available for accepting solutes. On the lines of GLC, we
can write the following equation:
VR = V0 + KVL
where, K is some form of distribution coefficient. If the solute is completely excluded
from the interior of gel then K = 0 and VR = V0. Such marker substances are available.
Now, if the solute can freely enter the gel, there should be no preference for water
inside or outside the gel and thus, K = 1, and VR = V0 + VL. Taking the case of
molecules which can penetrate the gel to some extent but not freely, K values fall
between 0 and 1.
In cases where sieving is the only phenomenon responsible for fractionation, K values
greater than 1 would never be encountered. However, sometimes these values are
obtained suggesting the occurrence of phenomena like adsorption, hydrogen bonding
and ion exchange between the gel and the solute.
Fig. 10.2 shows the typical
behaviour of variation in the retention volume with the molecular weight of the solute.
42
Size Exclusion
Chromatography
Fig. 10.2: Relation between retention volume and molecular weight of the solute. The
steep region between the arrows is the fractionation range.
If we refer to Fig. 10.2, it is clear that the molecules of differing in size can be
separated chromatographically in the sloping region of the curve. By varying the
degree of cross linking of the polymer, the curve shifts horizontally. There are
materials available which fractionate molecules in various molecular weight ranges.
For Sephadex G. 50, the fractionation range for peptides and globular protein is
molecular weight 1,500 to 30,000 while the range for G. 150 is 5,000 to 400,000.
SAQ 1
What particular property of gel is responsible for fractionation of solutes of different
molecular weights by size exclusion chromatography?
...
...
...
SAQ 2
What are the values of distribution coefficient if
i)
ii)
...
...
...
43
Chromatographic
Methods-III
There are many natural substances capable of forming gels and these include
polysaccharides from fruits and roots, proteins from animal tissues, inorganic silicates
and phosphates. If we look at gels from the chromatographic point of view and also
from general properties, two different types of gels can be distinguished. In the
macroreticular gels, the microstructure is strongly heterogenous with regions where
matrix material is aggregated and regions where there is very little gel matrix present.
The gel structure virtually free from gel matrix allows large molecules to enter. The
microreticular gels, on the other hand, show properties that the gel matrix is relatively
distributed throughout the gel. They fractionate relatively lower molecular mass
ranges than the macroreticular gels.
10.3.1
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)
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)
The gel should be stable over a wide range of pH and temperature. The gels that are
used in practice are stable over years and months. The leaching of material from bed
should be very low.
iii)
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)
For the adaption of a method of different problems, a wide choice of gels with same
general composition but different fractionation ranges should be available. For
microreticular gels, the fractionation range is mainly determined by the swelling
properties. Gels with low content of dry substance in the gel give access to larger
molecular weight molecules than those with high contents of dry substance.
With macroreticular gels, the content of the dry substance is no longer the only
variable which determines the fractionation range of the gel. The structure of the gel is
also important.
v)
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)
The mechanical rigidity of the gel grains should be as high as possible, otherwise they
tend to be deformed by the forces caused by the flow of liquid. The force may cause
the bead to compact reversibly or irreversibly, thus, increasing the flow through the
bed. The microreticular gels with small content of dry substance; a correspondingly
high exclusion limit, tend to be mechanically weak. One approach to solve the
problem is to synthesize gels with a macroreticular structure and with a microreticular
gel in the pores of macroreticular gel. The fractionation ranges are controlled by the
microreticular gel in the pores while the aggregates of macroreticular gel take care of
the mechanical strength. These gels are called macro-microreticular gels.
Size Exclusion
Chromatography
SAQ 3
What is a typical gel structure?
...
...
...
...
SAQ 4
What is a macro-microreticular gel? What is its special advantage for chromatographic
work?
...
...
...
...
ii)
iii)
iv)
v)
Besides the above classes inorganic materials like silica gel and porous glass are also
used. Let us study the different types of gels in more detail.
1.
Chromatographic
Methods-III
46
Sephadex is very chemically stable. The weakest points of attack in the structure are
the glucosides linkages which are hydrolysed at low pH. It is stable in alkaline
solutions. Its prolonged exposure to oxidizing agents may cause an increase in the
carboxyl group content. The increase of carboxyl group in the structure impairs the
chromatographic behaviour of the gel. It may be noted that initially Sephadex contains
a very small amount of carboxyl groups. This amount is so low that most of the charge
effects observed are attributed to Donnan effects caused by solutes.
Size Exclusion
Chromatography
Sephadex gels were the first for which a close relationship between molecular size and
elution behaviour was observed. In Sephadex series, there are gels available with
fractionation ranges distributed over a very wide interval of molecular masses. Some
differences are evident in the fractionation properties of different group of substances.
In most of the cases, these differences are relatively small and the general shape of the
fractionation curves is similar.
2.
Sephacryl is a dextran gel manufactured by cross linking allyl dextran with N, N methylene-bisacrylamide. There are cross links not only in dextran but methylenebisacrylamide molecules also bind to each other. A hypothetical structure of Sephacryl
gel is shown Fig. 10.4.
The cross linking reactions make the gel partly macroreticular and the gels can be
produced with fractionation ranges extending up to high molecular masses. They are
mechanically rigid and bear high pressures (upto 1M Pa) without compressing the bed.
These gels are intended mainly for use with aqueous eluants. Two types of Sephacryl
gels are available. They are Sephacryl-200 Superfine and Sephacryl S-300 Superfine.
The fractionation ranges cover the most common molecular masses of water soluble
proteins. Since in these gels, the structure is macroreticular, the slope of the selectivity
curve is less than the microreticular Sephadex gel. Due to large amount of methylene
bis-acrylamide in the gel, the adsorption effects are more pronounced than with
Sephadex.
47
Chromatographic
Methods-III
3.
Cross linked polyacrylamides form gels with water and are known as Bio-gels. They
are mainly used for biochemical work. Unlike the Sephadex gels, these are entirely
synthetic and made by acrylamide, H2C = CH CO NH2. It is synthesized by
copolymerizing acrylamide with the crosslinking agent N, N methylene-bisacrylamide (H2C = CH CO NH CH2 NH CO CH = CH2). The concentration
of the monomer can be varied to give different swelling characteristics and
chromatographic properties. A partial structure of Bio-Gel is shown in Fig. 10.5.
Bio-Gels are available from Bio-Rad Laboratories. They are produced by bead
polymerization and are available as powder. They have a marked tendency to stick
together and form lumps. Like Sephadex, Bio Gel is xerogel.
When the dry powder is immersed in water, it swells to form the gel. Bio-Gel is quite
inert and the weakest point for chemical reaction are the amide groups which are
hydrolyzed at extremes of pH. On hydrolysis, the carboxyl groups formed impart the
ion exchange character.
Bio-Gels are available in eleven types with different swelling characteristics and
different fractionation ranges. The different types are characterized by the letter P.
There are gels from Bio-Gel P-2 to Bio Gel P-300. The number is intended to indicate
the exclusion limit.
There are striking similarities in the chromatographic behaviour of Bio-Gel and
Sephadex gel. Both the gel types are microreticular. Their selectivity curves are also
quite similar. The similarity is also reflected in their mechanical properties. Bio-Gel
beads like Sephadex beads with low water regain are brittle while the ones with high
water regain are very soft.
48
4.
Agar or agar-agar, as it was called earlier, is obtained from various species of seaweeds. It is a mixture of linear polysaccharides comprising mainly of D-galactose and
3,6-anhydro-L-galactose residues. A partial structure of agrose is shown in Fig.10.6.
Size Exclusion
Chromatography
These gels are unique combining fractionation ranges at very high molecular masses
with good mechanical stability. They are a good supplement to the dextran gels and
polyacrylamide gels. The agar gels with the lowest fractionation ranges correspond
approximately in their fractionation properties to the dextran and polyacrylamide gels
with the highest fractionation ranges. These gels can fractionate in the range
intermediate between molecules and particles.
Agrose gel for chromatographic work are available from Pharmacia Fine Chemicals
(trade name Sepharose) and Bio-Rad (trade name Bio-Gel A). Sepharose is available
in three normal types and three corresponding types crosslinked with 2, 3dibromopropanol. The normal types are Sepharose 2B, 4B and 6B and crosslinked
types being Sepharose CL2B, CL4B and CL6B. The numbers indicate the percentage
of dry gel in the particles. Bio-Gel A is available in six types.
Unlike the gels discussed earlier, the macromolecules of gel matrix are not bound by
covalent bonds. They are supposed to be held together by hydrogen bonds. The
polysaccharide chains seem to aggregate in bundles. Between the bundles, there are
very large openings in the gel matrix. The structure is very open and at the same time
mechanically stable.
Agrose gel has some disadvantages as a chromatographic material. There is a
considerable amount of charged groups in the material. Because of the presence of
charged groups, it is recommended to work at high ionic strength to mitigate the
problem due to ion exchange effects. Another problem arises from the fact that agar
chains are not linked by covalent bonds. This makes the agar gels chemically unstable.
This amounts to the fact that they are less stable to pH extremes than the gels
described earlier. These gels maintain their structure if the water is substituted by
many organic solvents such as acetone or ethanol. The structure of agrose gels makes
it impractical to dry and reswell them. Therefore, once the gel has been prepared, it
should be stored in wet state. It may be important to emphasize again that agrose gel
have fractionation ranges at considerably higher molecular masses than expected by
comparison with dextran and polyacrylamide gels. The selectivity curve of these gels
is less steep than it is for the gels from dextran and polyacrylamide.
5.
49
Chromatographic
Methods-III
SAQ 5
What happens if the Sephadex gels are subjected to prolonged exposure to oxidizing
agents?
...
...
...
...
...
SAQ 6
In what important properties, the Sephadex and Sephacryl gels differ from each other?
...
...
...
...
...
SAQ 7
What are the weak points of chemical attack in Sephadex and Bio-Gels?
...
...
...
...
...
SAQ 8
What different classes of gels are not prepared from natural materials?
...
...
...
...
50
they are fairly constant. Another important variable of significance is the steepness of
the selectivity curve in the linear portion of the fractionation range.
Size Exclusion
Chromatography
For xerogels, the water regain is the common measure of the swelling capacity of the
gel. Here, it may be important to tell more about xero gels and aerogels. Different gels
react differently to the removal of liquid in them. The group of xerogels shrink on
drying to a compact material containing only the gel matrix. The aerogels, on the other
hand do not shrink; instead the surrounding air penetrates into the gel. Xerogels when
brought in contact with the liquid, take up liquid and return to the gel state. In aerogels
also, the air can be substituted by the liquid.
Getting back to water regain capacity, it is usually expressed as the amount of water
(in mL) imbibed by one gram of dry xerogel on swelling. It does not include the
interstitial liquid between the grains. In the case of dextran and polyacrylamide gels, it
has been observed that there is a close relationship between the water regain and the
fractionation properties. If the water regain is low, it is expected that the fractionation
range is at a low molecular mass. In the case of agrose gel, it is percentage of gel
matrix in the gel grain is taken instead of the water regain. While working with
nonaqueous solvents, the analogy is taken with water regain. Ultimately, the particle
size of the gel grain is an important variable. It affects the degree of zone broadening,
the resolution, the dilution and the flow rate. As a matter of fact, the above mentioned
information should be available to the user before a particular gel is put to use for
chromatographic work.
SAQ 9
Mention three unique advantages of size exclusion chromatography.
...
...
...
...
51
Chromatographic
Methods-III
Analytical applications
2.
Preparative applications
3.
Miscellaneous applications
The miscellaneous applications include important applications which are not covered
in the first two heads particularly the use of gels in thin layer chromatography, zone
electrophoresis and HPLC. No doubt, these are not the direct applications of size
exclusion chromatography but they definitely reflect on the use of gels for separations.
10.7.1
Analytical Applications
It may be important to point out that within analytical applications, there are further
sub-divisions as follows:
i)
ii)
Analytical fractionation
This covers a wide range. It could be the separation of substances which can be
determined separately. The other end can be where elution curve profile can be
used to characterize the sample without the determination of concentration of
the different ingredients. A typical example of the first type is the separation of
sugars from cellulose hydrolysate. The technique is an extremely powerful tool
for the separation of oligosaccharides with differing number of sugar residues
from each other. Maltooligosaccharides containing up to 15 glucose units and
polymaltoses with chain length up to 21 glucose units can be separated from
each other on Bio-Gel P2. Sephadex LH-20 provides a very good resolution of
lipids and steroids. It has been possible to fractionate a very wide range of nonpolar lipids on LH-20 in chloroform and they were found to separate primarily
according to their molecular size. The conventional liquid chromatography on
52
silica and other materials is unsuccessful for this type of separation. Amino
acids can be separated on the tightly cross-linked types of gels.
Size Exclusion
Chromatography
iv)
10.7.2
Preparative Applications
ii)
Preparative fractionation.
Chromatographic
Methods-III
Preparative fractionation
To a large extent, this technique has been used as a tool for separating the
proteins, their degradation products and their complexes.
Isolation of antibodies to specific antigens is a subject of great interest.
The separation of an antibody from the antigens can be performed by gel
filtration chromatography. Fractionation of nucleic acids and animal
viruses has also been attained on agar gels.
An important industrial example based on fractionation by gel filtration
chromatography is the preparation of tetanus toxoid for immunization
purposes. In order to make the extremely toxic tetanospasmin harmless so
that it can be injected, it is polymerized by treatment with formalin. The
polymer (toxoid) is separated from the monomer by gel filtration
chromatography on column packed with Sephadex G-100.
The examples of applications cited in the foregoing discussion pertain
exclusively to size exclusion chromatography. But there are many areas
where the gels used for size exclusion chromatography have applications
in other areas of separations.
The details of their applications are given in the respective Units.
However, a brief account of these is being given below under
miscellaneous applications.
10.7.3
Miscellaneous Applications
The gels find use in thin layer chromatography using the gravity flow. As the plates in
thin layer gel filtration chromatography may be difficult to dry, the chromatogram are
often developed, where the liquid in the layer is transferred to paper and developed on
the paper the same way as paper chromatogram. This is because the gels usually crack
on drying. This type of chromatography has been used mainly for the study of proteins
and peptides. The other types of fractionations which have been performed on these
plates are that of -amino acids, nuclopolysaccharides and antibodies.
54
Size Exclusion
Chromatography
SAQ 10
Give one example of each from the applications of size exclusion chromatography for
the following:
i)
ii)
iii)
Where this separation technique is employed for the purification of (a) medicine
(b) vaccine.
.................
.
.
10.8 SUMMARY
This unit on size exclusion chromatography begins with a brief introduction to the
technique highlighting the classification to which it belongs. The basic principle
55
Chromatographic
Methods-III
What is the basic equation for size exclusion chromatography? Under what
conditions the value of K, used in the equation, exceeds unity?
2.
What are two broad categories of gels based on their microstructure? Highlight
the differences between the two.
3.
Mention the characteristics which are important for a gel to be useful for
chromatographic work.
4.
5.
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.
10.10 ANSWERS
Self Assessment Questions
56
1.
It is the pore size of the swollen gel which is responsible for the fractionation of
molecules of different sizes by size exclusion chromatography.
2.
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.
4.
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
The Sephadex gels differ from the Sephacryl gels in the following respects
i)
ii)
iii)
iv)
7.
8.
The Bio-Gels and styrene-divinylbenzene (Styragel) are not made from natural
materials. They are entirely synthetic.
9.
The three unique advantages of size exclusion chromatography are given below:
10.
Size Exclusion
Chromatography
i)
ii)
By varying the contents of gel in gel matrix, the fractionation ranges can
be varied within wide limits.
iii)
i)
ii)
iii)
b)
Terminal Questions
1.
Chromatographic
Methods-III
4.
5.
58
The following are the important characteristics for a gel to be useful for
chromatographic work:
i)
ii)
iii)
iv)
v)
vi)
The Sephadex gels and Bio-Gels show some striking similarities as given below:
i)
ii)
iii)
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)
ii)
Agar chains are not linked by covalent bonds. This makes the gels
chemically unstable. These gels are less stable to pH extremes.
iii)
The structure of agrose gels makes it impractical to dry and reswell them.
Therefore, once the gel is prepared it has to be stored in a wet state.
iv)
The selectivity curve is less steep than it is for gels from dextran and
polyacrylamide.
6.
The technique has been very useful in the study of complexes between proteins
and between proteins and low molecular mass solutes. If the reactants are
irreversibly bound to each other, the resulting mixture is subjected to
chromatographic separation. The complex is separated from the reactants
present in excess. A typical example in this area is from clinical laboratory
where protein binding of insulin is investigated. The information is obtained
about the formation of insulin antibodies in diabetes treated with insulin.
7.
Further Reading
1.
2.
3.
Size Exclusion
Chromatography
59
Chromatographic
Methods-III
INDEX
Agar and agrose gels 45, 49
Aluminosilicates 8
Amphoteric exchangers 7, 13
Applications of ion exchange chromatography 31
Miscellaneous application 31, 33
Separation of ionized from nonionized 31, 32
Separation of metal ions and anions 31
Separation of organics 31,32
Separation of Actinide Elements 31, 33
Analcite 8
Anion exchangers 7, 8, 12
Basic principle of size exclusion chromatography 41
Exclusion limit 42
Batch method 18
Batch operation 23
Cation exchangers 7, 10
Chabazite 8
Chelates 26
Classification of ion exchangers 8
Inorganic 8
Natural 8
Organic 8
Synthetic 8
60
Size Exclusion
Chromatography
Amphoteric exchangers 7
Anion exchangers 7
Cation exchangers 7
Counter ions 7
Ion exchangers
Classification of 8
Inorganic 8
Natural 8
Organic 8
Synthetic 8
Kaolinite 8
Liquid ion exchangers 10
Miscellaneous applications 31, 33
Montmorillonite 8
Moving bed operations 24
Naturalite 8
Natural ion exchangers 8
Analcite 8
Aluminosilicates 8
Anion exchangers 8
Chabazite 8
Feldspar 8
Kaolinite 8
Montmorillonite 8
Naturalite 8
Zeolites 8
Nomenclature 15
Operating methods 23
Batch operation 23
Column operation 23
Moving bed operations 24
Cross linkages 16
Distribution ratio 17
Batch method 18
Distribution coefficient 19
Equivalency of exchange 19
Moisture content 15
Particle Size 16
Resin selectivity 19
Apparent selectivity coefficient 22
Dowex 1 19
Dowex 50 19
Ionic forms of resin 21
Selectivity 19
Selectivity coefficient 19
Total solution ionic strength 21
Separation of organics 32
Separation of Actinide Elements 33
Separation of ionized from nonionzed 31, 32
Separation of metal ions and anions 31
Sephacryl 45
Sephadex G-10 46
61
Chromatographic
Methods-III
Sephadex G-200 46
Size exclusion chromatography 40
Applications 52
Analytical 52
Preparative 52, 53
Miscellaneous 52, 54
Basic principle 41
Unique features 51
Snake-cage polyelectroytes 14
Some applications 52
Analytical 52
Anaytical fractionation 52
Anaytical group separations 52
Determiniation of molecular masses 53
Measurement of protein binding and complex formation 53
Miscellaneous applications 54
Preparative applications 53
Preparative fractionation 54
Preparative group separation 53
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
Characteristics 27
Zeolites 8
62