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Chapter 5 High performance size exclusion chromatography
CHAPTER 5
High performance size exclusion chromatography
5.1. Introduction One of the earliest concepts to be realised by researchers was the possibility of separating molecules from complex mixtures on the basis of their molecular size. Thus the techniques of ultrafiltration and dialysis have been developed to achieve such separations. However, higher resolution and selectivity can be obtained using size exclusion chromatography (SEC). The development of suitable chromatographic supports was addressed by biochemists and organic chemists independently, who developed two separate types of stationary phase and also two sets of nomenclature. Gel filtration, as defined by the biochemist, refers to macroporous cross-linked dextrans and acrylamides compatible with aqueous buffers (Porath and Flodin, 1959). Gel permeation, as defined by the organic chemist, refers to the use of rigid, polymerbased resins such as polymethacrylate, polydivinyl benzene and microparticulate silica resins which are stable in both organic and aqueous buffers at pH values less than 8.0. The term size exclusion is used in this text to encompass the forms of chromatography otherwise known as gel filtration and gel permeation. The development of high performance (implying faster analysis, better resolution) size exclusion supports, particularly for proteins, has been relatively slow, mainly because of the difficulties inherent in the principle upon which the separation is based.
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
57
5.2. Principles If a mixture of molecules with different sizes is applied to a column which has been packed with porous material of a specific pore size, only a certain percentage of the molecules will be small enough to enter the pores, the rest being excluded. The larger molecules will therefore spend less time in the pores and will pass through the column more rapidly. The separation is based on the physical parameters of the molecules themselves and not on their chemical properties and it is this principle which differentiates SEC from the other forms of chromatography. Separation of two compounds occurs because of the differences in the distribution of the molecules between the mobile phase and the stationary phase. Mathematical treatments of the theory of size exclusion chromatography are usually based upon models which consider the solute molecules as spherical and the stationary phase as a rigid gel (Ambler and MacIntyre, 1975), although in reality not all the molecules are the same shape in solution (Table 5.1). It has been demonstrated that a polymer of a given molecular weight can have a radius equivalent to that of a globular protein of much higher molecular weight (Ui, 1979). A theoretical analysis of SEC can be derived from the chromatographic elution profile. The retention of a given molecule is expressed as the distribution coefficient Kd (Fig. 5.1), where V, is the volume of the solvent outside the particles of the stationary phase and V, is the solvent inside the particles which is available for chromatography. TABLE 5.1 Illustration of the mass (kDa) and Stokes radii (A) of femtin, catalase and bovine albumin, and the mass of a polymer of an equivalent radius
Mass
Femtin Catalase Bovine albumin
Radius
(kW
(4
480 240 61
156 104 70
Equivalent polymer mass (kW 57.3 28.7 14.3
APPLICATIONS OF HPLC IN BIOCHEMISTRY
58
. II
V.A
,
I
-
J
vi
_
~
Elution volume
_
_
Fig. 5.1. Diagrammatic illustration of the parameters involved in size exclusion chromatography. V,, volume of solvent outside the particles; y , volume of solvent inside the particles available for chromatography, V, (A), elution volume of a sample A; V , (B), elution volume of a sample B. From this, the retention coefficient Kd can be derived for each sample:
Reproduced from LKB technical brochure, with permission.
allows the determination of the elution volume ( V , ) of a given molecule of interest. If the solute is large and excluded from the gel then V, = V, and K , = 0. If the solute is small enough to penetrate and completely into all the accessible pore volume, then V, = V, Kd = 1. In practice the value of Kd varies between 0 and 1 since not all the pore volume is ever completely accessible. For any given column there will be a fixed relationship between these parameters and the elution volume; however, the value of Kd is independent of the column parameters (i.e. height, diameter).
+
~
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
59
5.3.Factors affecting resolution 5.3.1. Selectivity Since the principle of SEC is different from the other forms of chromatography the parameters of the resolution equation are not normally applicable. However the control of selectivity in SEC is simple and predictable once the parameters of the stationary phase are optimised. The critical features of the stationary phase are pore volume, pore size distribution and particle shape. It is also important that the support is inert to both ionic and hydrophobic interactions with the sample molecules and mobile phase. Developments to optimise particle shape and size distribution have led to the increasingly popular use of small particles (5-100 pm), spherical in shape and with a minimum size distribution of plus or minus 10%(Chapter 10). Strict control of these criteria is essential for the manipulation of the physical aspects of column efficiency. An excess in pore size distribution results in increased peak broadening (Werner and Halasz, 1980). Soft dextrans and agaroses which lack mechanical stability are not suitable for use in HPLC because of the pressures applied. A list of commercially available supports currently being used can be found in the literature (Lesec, 1985; Wehr, 1984). The two most common stationary phases for aqueous SEC are cross-linked polymer-based polyether or polyester and silica-based phases. Each of these stationary phases have hydroxyl groups covalently linked to the surface (Regnier and Noel, 1976). For non-aqueous SEC the silica-based phases are popular, e.g. ZORBAX SEC, a ‘silanised’ porous silica microsphere (Yau et al., 1978). Alternatively, polystyrene divinyl benzene stationary phases have been used and are now available in a wide range of efficiencies and pore sizes, e.g. TSK H (Cooper et al., 1975). At present the two most popular stationary phases are silica based to whch an organic phase has been attached, i.e. glycerol-propyl type, Waters 1 series, Synchropak GPC, TSK SW types (Kato et al., 1980). These stationary phases possess properties suitable for use with aqueous buffers. The most popular stationary phases for use with non-aqueous buffers are of the type PSDV (Spheron, Shodex) and
APPLICATIONS OF HPLC IN BIOCHEMISTRY
60
methacrylate (Spheron P series), which may also be used with aqueous solvents. TSK PW polyether supports are very popular f9r separation of proteins and polynucleotides. Pore sizes of 25-4000 A are available. Pore sizes larger than this tend to show reduced resolution due to decreased plate number. 5.3.2. Column capacity
The disadvantage of SEC compared to the other forms of chromatography is the relatively low sample capacity. In analytical separations where optimum resolution is required, the total load volume should not exceed 1-248, of the total column volume (Roumeliotis and Unger, 1979). This need not apply in all circumstances since when using SEC for the purpose of changing buffers or desalting a sample up to 10% of the column volume is easily accommodated. 5.3.3. Pore size distribution A critical feature affecting selectivity in SEC is the minimisation of pore size distribution. Control of this parameter facilitates separation of molecules with a particular size distribution. Stationary phases are available over a wide range of pore sizes (Table 5.2) and often these columns can be used in series (Mori, 1979). Thus, effective selection within a broad range is accomplished by the first column and fractionation within a more defined range is achieved on the second column. TABLE5.2 The molecular weight range (in Daltons) for the separation of proteins and 'random coil' molecules using currently available supports Support TSK 2000 TSK 3000 TSK 4000
Globular proteins 1,000- 30,000 2,000- 80,000 20,000-1,000,000
Random coil 500- 8,000 1,000- 30,000 20,000-150,000
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
61
5.3.4. Pore volume
Pore volume ( V , ) is an important parameter affecting resolution in SEC. This parameter presents difficulties in the design of stationary phases since a large pore volume is usually achieved using an open lattice bead structure which will be less mechanically stable to increased pressures at high flow rates. The compromise between pore volume and mechanical stability has been the major difficulty in the development of supports suitable for macromolecules. Thus, analytical size exclusion columns generally are longer than those used in the other chromatographic modes, so that the amount of stationary phase and thus the effective pore volume available for chromatography is increased. The migration of molecules between the stationary phase and the mobile phase is driven by random movement or diffusion, a factor which is deleterious to high resolution in all forms of chromatography. Since resolution in SEC is solely dependent on diffusion, unlike the other forms of chromatography, optimisation of stationary phase particles is important to improve mass transfer. Factors deleterious to mass transfer can be divided into three separate types: those attributable to stagnant mobile phase in the pores of the particles, those caused by differential penetration of the solute molecules into the stationary phase and, finally, longitudinal diffusion between the particles (Snyder and Kirkland, 1979). Even w i t h the best supports available there will be some irregularities in the particle shapes which will lead to non-uniform channels through which the molecules will permeate. Optimisation of the physical parameters of the column, the type of stationary phase and the method of packing are normally directed at overcoming these effects.
5.4. Mobile phase effects 5.4.1. p H
The mobile phase can affect resolution through direct interaction with both the stationary phase and the solute. Silica columns are best used
62
APPLICATIONS OF HPLC IN BIOCHEMISTRY
at pH values less than 7.0 to minimise the formation of silanolate anions which could then participate as weak cation exchangers. Silanisation of silica-based stationary phases is essential to prevent any interactions of either aqueous mobile phases or sample molecules with free silanolate anions. 5.4.2. Ionic strength
SEC of macromolecules is generally carried out using buffers which contain counter-ions to stabilise charged residues and thus maintain the structural integrity of the solute. This is particularly important for proteins, which contain a wide variety of interacting groups. However, an excessively high ionic strength will tend to promote hydrophobic interactions and therefore a low ionic strength buffer (0.1 M) is normally recommended. The inclusion of suitable counter-ions (Na+, K ' , NH:), will also offset some of the problems associated with high pH values by masking reactive silanolate anions. By altering the pH in conditions of low ionic strength, the polarity of the stationary phase can be altered to suit the particular requirements of the solute. This has been called 'non-ideal' SEC and is useful for certain proteins (Kopaciewicz and Regnier, 1982). 5.4.3. Flow rate
Mobile phase flow rates of around 0.5-1.0 ml/min are recommended for the resolution of a variety of macromolecules on a number of stationary phases. However, flow rates of 0.1-0.5 ml/min are recommended for use with TSK SW and H types. In general, for larger molecules (polynucleotides, proteins), the mass transfer term is much larger and the flow rate has to be correspondingly reduced to maintain resolution.
5.5. Molecular weight determination The size of the solute molecules is characterised by their hydrodynamic radius (Stokes radius) in a particular solvent. Using SEC it is
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
63
t
I
I
I
I
I
20
25
30
35
40
45
Elution v o l u m e ,
ml
Fig. 5.2. A plot of the retention volume versus the logarithm of the molecular weight of a series of standards (proteins) of known molecular weight. Chromatographic conditions: columns, TSK-Gel G2000 SW, G3000 SW and G4000 SW ( 6 0 0 x 7 3 mm I.D.); mobile phase, 1/15 phosphate buffer, 1/10 M KCI; flow rate, 1 ml/min; temperature, ambient. Reproduced from TSK technical bulletin, with permission.
possible to estimate the molecular weight of an unknown protein or polymer by comparing its retention volume with a plot of retention volume versus the logarithm of molecular weight for a series of standards of known molecular weight (Fig. 5.2). These plots can provide accurate determinations of molecular weights for polymers and proteins provided the unknowns adopt a similar conformation in solution to that of the standard. T h s implies an inherent error and calculations based solely on this type of analysis require further confirmation. For a more accurate assessment, strong denaturants such as 0.1% sodium dodecylsulphate (SDS) or 6 M guanidine hydrochloride can be included in the mobile phase in aqueous SEC. This tends to promote uniform conformation of the components of the solute and will be closer to the ideal situation. The inclusion of denaturants has the added benefit of reducing any chemical interactions of solutes
64
APPLICATIONS OF HPLC IN BIOCHEMISTRY
the stationary phase. The addition of detergent to the mobile phase promotes random coil conformation and therefore a corresponding increase in the Stokes radius occurs. This has been useful for the estimation of molecular weights of both fibrous and globular proteins (Barden, 1983), although some subunits of multimeric enzymes will dissociate under these conditions. A comparison of SEC in SDS and SDS-polyacrylamide gels for the determination of molecular weight concluded that the former is more useful when a detailed knowledge of the solute is available (Josic et al., 1984). Alternatively, charge interactions can be neutralised completely by chemical modification, and by also disrupting hydrophobic interactions, the solute should behave ideally (Meredith, 1984). Early supports for hgh performance SEC proved disappointing in terms of stability, resolution and recovery. Recent advances have offset many of these problems and offer researchers another powerful fractionation tool. There are distinct advantages over the other forms of chromatography since recovery is generally in excess of 90%, the mobile phase is simple (requiring no complex buffer change or gradient), the profile is highly reproducible and the elution order is predictable.
High performance size exclusion chromatography
5.1. Introduction One of the earliest concepts to be realised by researchers was the possibility of separating molecules from complex mixtures on the basis of their molecular size. Thus the techniques of ultrafiltration and dialysis have been developed to achieve such separations. However, higher resolution and selectivity can be obtained using size exclusion chromatography (SEC). The development of suitable chromatographic supports was addressed by biochemists and organic chemists independently, who developed two separate types of stationary phase and also two sets of nomenclature. Gel filtration, as defined by the biochemist, refers to macroporous cross-linked dextrans and acrylamides compatible with aqueous buffers (Porath and Flodin, 1959). Gel permeation, as defined by the organic chemist, refers to the use of rigid, polymerbased resins such as polymethacrylate, polydivinyl benzene and microparticulate silica resins which are stable in both organic and aqueous buffers at pH values less than 8.0. The term size exclusion is used in this text to encompass the forms of chromatography otherwise known as gel filtration and gel permeation. The development of high performance (implying faster analysis, better resolution) size exclusion supports, particularly for proteins, has been relatively slow, mainly because of the difficulties inherent in the principle upon which the separation is based.
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
57
5.2. Principles If a mixture of molecules with different sizes is applied to a column which has been packed with porous material of a specific pore size, only a certain percentage of the molecules will be small enough to enter the pores, the rest being excluded. The larger molecules will therefore spend less time in the pores and will pass through the column more rapidly. The separation is based on the physical parameters of the molecules themselves and not on their chemical properties and it is this principle which differentiates SEC from the other forms of chromatography. Separation of two compounds occurs because of the differences in the distribution of the molecules between the mobile phase and the stationary phase. Mathematical treatments of the theory of size exclusion chromatography are usually based upon models which consider the solute molecules as spherical and the stationary phase as a rigid gel (Ambler and MacIntyre, 1975), although in reality not all the molecules are the same shape in solution (Table 5.1). It has been demonstrated that a polymer of a given molecular weight can have a radius equivalent to that of a globular protein of much higher molecular weight (Ui, 1979). A theoretical analysis of SEC can be derived from the chromatographic elution profile. The retention of a given molecule is expressed as the distribution coefficient Kd (Fig. 5.1), where V, is the volume of the solvent outside the particles of the stationary phase and V, is the solvent inside the particles which is available for chromatography. TABLE 5.1 Illustration of the mass (kDa) and Stokes radii (A) of femtin, catalase and bovine albumin, and the mass of a polymer of an equivalent radius
Mass
Femtin Catalase Bovine albumin
Radius
(kW
(4
480 240 61
156 104 70
Equivalent polymer mass (kW 57.3 28.7 14.3
APPLICATIONS OF HPLC IN BIOCHEMISTRY
58
. II
V.A
,
I
-
J
vi
_
~
Elution volume
_
_
Fig. 5.1. Diagrammatic illustration of the parameters involved in size exclusion chromatography. V,, volume of solvent outside the particles; y , volume of solvent inside the particles available for chromatography, V, (A), elution volume of a sample A; V , (B), elution volume of a sample B. From this, the retention coefficient Kd can be derived for each sample:
Reproduced from LKB technical brochure, with permission.
allows the determination of the elution volume ( V , ) of a given molecule of interest. If the solute is large and excluded from the gel then V, = V, and K , = 0. If the solute is small enough to penetrate and completely into all the accessible pore volume, then V, = V, Kd = 1. In practice the value of Kd varies between 0 and 1 since not all the pore volume is ever completely accessible. For any given column there will be a fixed relationship between these parameters and the elution volume; however, the value of Kd is independent of the column parameters (i.e. height, diameter).
+
~
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
59
5.3.Factors affecting resolution 5.3.1. Selectivity Since the principle of SEC is different from the other forms of chromatography the parameters of the resolution equation are not normally applicable. However the control of selectivity in SEC is simple and predictable once the parameters of the stationary phase are optimised. The critical features of the stationary phase are pore volume, pore size distribution and particle shape. It is also important that the support is inert to both ionic and hydrophobic interactions with the sample molecules and mobile phase. Developments to optimise particle shape and size distribution have led to the increasingly popular use of small particles (5-100 pm), spherical in shape and with a minimum size distribution of plus or minus 10%(Chapter 10). Strict control of these criteria is essential for the manipulation of the physical aspects of column efficiency. An excess in pore size distribution results in increased peak broadening (Werner and Halasz, 1980). Soft dextrans and agaroses which lack mechanical stability are not suitable for use in HPLC because of the pressures applied. A list of commercially available supports currently being used can be found in the literature (Lesec, 1985; Wehr, 1984). The two most common stationary phases for aqueous SEC are cross-linked polymer-based polyether or polyester and silica-based phases. Each of these stationary phases have hydroxyl groups covalently linked to the surface (Regnier and Noel, 1976). For non-aqueous SEC the silica-based phases are popular, e.g. ZORBAX SEC, a ‘silanised’ porous silica microsphere (Yau et al., 1978). Alternatively, polystyrene divinyl benzene stationary phases have been used and are now available in a wide range of efficiencies and pore sizes, e.g. TSK H (Cooper et al., 1975). At present the two most popular stationary phases are silica based to whch an organic phase has been attached, i.e. glycerol-propyl type, Waters 1 series, Synchropak GPC, TSK SW types (Kato et al., 1980). These stationary phases possess properties suitable for use with aqueous buffers. The most popular stationary phases for use with non-aqueous buffers are of the type PSDV (Spheron, Shodex) and
APPLICATIONS OF HPLC IN BIOCHEMISTRY
60
methacrylate (Spheron P series), which may also be used with aqueous solvents. TSK PW polyether supports are very popular f9r separation of proteins and polynucleotides. Pore sizes of 25-4000 A are available. Pore sizes larger than this tend to show reduced resolution due to decreased plate number. 5.3.2. Column capacity
The disadvantage of SEC compared to the other forms of chromatography is the relatively low sample capacity. In analytical separations where optimum resolution is required, the total load volume should not exceed 1-248, of the total column volume (Roumeliotis and Unger, 1979). This need not apply in all circumstances since when using SEC for the purpose of changing buffers or desalting a sample up to 10% of the column volume is easily accommodated. 5.3.3. Pore size distribution A critical feature affecting selectivity in SEC is the minimisation of pore size distribution. Control of this parameter facilitates separation of molecules with a particular size distribution. Stationary phases are available over a wide range of pore sizes (Table 5.2) and often these columns can be used in series (Mori, 1979). Thus, effective selection within a broad range is accomplished by the first column and fractionation within a more defined range is achieved on the second column. TABLE5.2 The molecular weight range (in Daltons) for the separation of proteins and 'random coil' molecules using currently available supports Support TSK 2000 TSK 3000 TSK 4000
Globular proteins 1,000- 30,000 2,000- 80,000 20,000-1,000,000
Random coil 500- 8,000 1,000- 30,000 20,000-150,000
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
61
5.3.4. Pore volume
Pore volume ( V , ) is an important parameter affecting resolution in SEC. This parameter presents difficulties in the design of stationary phases since a large pore volume is usually achieved using an open lattice bead structure which will be less mechanically stable to increased pressures at high flow rates. The compromise between pore volume and mechanical stability has been the major difficulty in the development of supports suitable for macromolecules. Thus, analytical size exclusion columns generally are longer than those used in the other chromatographic modes, so that the amount of stationary phase and thus the effective pore volume available for chromatography is increased. The migration of molecules between the stationary phase and the mobile phase is driven by random movement or diffusion, a factor which is deleterious to high resolution in all forms of chromatography. Since resolution in SEC is solely dependent on diffusion, unlike the other forms of chromatography, optimisation of stationary phase particles is important to improve mass transfer. Factors deleterious to mass transfer can be divided into three separate types: those attributable to stagnant mobile phase in the pores of the particles, those caused by differential penetration of the solute molecules into the stationary phase and, finally, longitudinal diffusion between the particles (Snyder and Kirkland, 1979). Even w i t h the best supports available there will be some irregularities in the particle shapes which will lead to non-uniform channels through which the molecules will permeate. Optimisation of the physical parameters of the column, the type of stationary phase and the method of packing are normally directed at overcoming these effects.
5.4. Mobile phase effects 5.4.1. p H
The mobile phase can affect resolution through direct interaction with both the stationary phase and the solute. Silica columns are best used
62
APPLICATIONS OF HPLC IN BIOCHEMISTRY
at pH values less than 7.0 to minimise the formation of silanolate anions which could then participate as weak cation exchangers. Silanisation of silica-based stationary phases is essential to prevent any interactions of either aqueous mobile phases or sample molecules with free silanolate anions. 5.4.2. Ionic strength
SEC of macromolecules is generally carried out using buffers which contain counter-ions to stabilise charged residues and thus maintain the structural integrity of the solute. This is particularly important for proteins, which contain a wide variety of interacting groups. However, an excessively high ionic strength will tend to promote hydrophobic interactions and therefore a low ionic strength buffer (0.1 M) is normally recommended. The inclusion of suitable counter-ions (Na+, K ' , NH:), will also offset some of the problems associated with high pH values by masking reactive silanolate anions. By altering the pH in conditions of low ionic strength, the polarity of the stationary phase can be altered to suit the particular requirements of the solute. This has been called 'non-ideal' SEC and is useful for certain proteins (Kopaciewicz and Regnier, 1982). 5.4.3. Flow rate
Mobile phase flow rates of around 0.5-1.0 ml/min are recommended for the resolution of a variety of macromolecules on a number of stationary phases. However, flow rates of 0.1-0.5 ml/min are recommended for use with TSK SW and H types. In general, for larger molecules (polynucleotides, proteins), the mass transfer term is much larger and the flow rate has to be correspondingly reduced to maintain resolution.
5.5. Molecular weight determination The size of the solute molecules is characterised by their hydrodynamic radius (Stokes radius) in a particular solvent. Using SEC it is
Ch. 5
SIZE EXCLUSION CHROMATOGRAPHY
63
t
I
I
I
I
I
20
25
30
35
40
45
Elution v o l u m e ,
ml
Fig. 5.2. A plot of the retention volume versus the logarithm of the molecular weight of a series of standards (proteins) of known molecular weight. Chromatographic conditions: columns, TSK-Gel G2000 SW, G3000 SW and G4000 SW ( 6 0 0 x 7 3 mm I.D.); mobile phase, 1/15 phosphate buffer, 1/10 M KCI; flow rate, 1 ml/min; temperature, ambient. Reproduced from TSK technical bulletin, with permission.
possible to estimate the molecular weight of an unknown protein or polymer by comparing its retention volume with a plot of retention volume versus the logarithm of molecular weight for a series of standards of known molecular weight (Fig. 5.2). These plots can provide accurate determinations of molecular weights for polymers and proteins provided the unknowns adopt a similar conformation in solution to that of the standard. T h s implies an inherent error and calculations based solely on this type of analysis require further confirmation. For a more accurate assessment, strong denaturants such as 0.1% sodium dodecylsulphate (SDS) or 6 M guanidine hydrochloride can be included in the mobile phase in aqueous SEC. This tends to promote uniform conformation of the components of the solute and will be closer to the ideal situation. The inclusion of denaturants has the added benefit of reducing any chemical interactions of solutes
64
APPLICATIONS OF HPLC IN BIOCHEMISTRY
the stationary phase. The addition of detergent to the mobile phase promotes random coil conformation and therefore a corresponding increase in the Stokes radius occurs. This has been useful for the estimation of molecular weights of both fibrous and globular proteins (Barden, 1983), although some subunits of multimeric enzymes will dissociate under these conditions. A comparison of SEC in SDS and SDS-polyacrylamide gels for the determination of molecular weight concluded that the former is more useful when a detailed knowledge of the solute is available (Josic et al., 1984). Alternatively, charge interactions can be neutralised completely by chemical modification, and by also disrupting hydrophobic interactions, the solute should behave ideally (Meredith, 1984). Early supports for hgh performance SEC proved disappointing in terms of stability, resolution and recovery. Recent advances have offset many of these problems and offer researchers another powerful fractionation tool. There are distinct advantages over the other forms of chromatography since recovery is generally in excess of 90%, the mobile phase is simple (requiring no complex buffer change or gradient), the profile is highly reproducible and the elution order is predictable.