Journal of Chromatography, 544 (1991) 201-217
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`Elsevier Science Publishers B.V., Amsterdam CHROM. 22 946 Review Rigid macroporous copolymers as stationary phases in high- performance liquid chromatography LINDA L. LLOYD Po+ner
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`Laboratories Ltd.. Essex Road, Church Strerton. Shropshire SY6 6AX (U.K.)
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`ABSTRACT Rigid macroporous copolymers of styrene and divinylbenzene have been developed which have the high physical/mechanical stability necessary to operate under high-performance liquid chromatographic conditions of pressure and flow-rate. The matrices can be produced in a range of porosities, 100,300, 1000 and 4000 8, each with the controlled uniform pore geometry and pore size distribution essential for high efficiency separations. The small pore-high surface area macroporous poly(styreneedivinylbenzene) co- polymer, PLRP-S 100 A, can be used in the unmodified form for reversed-phase separations of small molecules or by increasing the pore size, PLRP-S 300 A or greater, for the analysis of biological macromol- ecules. The surface characteristics of the hydrophobic poly(styreneedivinylbenzene) matrix can be altered by derivatization or coating to produce ion exchange functionalities or a hydrophilic surface for gel filtration chromatography. The hydrophilic surface can then be further derivatized to be utilised as a base matrix for affinity chromatography. CONTENTS Introduction 202 Preparation and characterisation 202 2.1. Column efficiency . 206 2.2. Eluent compatability solvent strength 207 2.3. Macromolecule reversed-phase 208 Surface coatings/modifications 210 3.1. Ion-exchange 210 3.2. Gel filtration 212 3.3. Affinity chromatography 214 3.4. High speed separations 214 Conclusions 216 References 217 0021-9673/91/$03.50 0 1991 Elsevier Science Publishers B.V.
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`1. INTRODUCTION
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`L. L. LLOYD
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`The use of rigid macroporous copolymers for high-performance liquid chroma- tography (HPLC) has its origins in the work carried out by Peterson [l]. In 1954 he made the observation that proteins could under certain conditions be adsorbed by diethylaminoethyl (DEAE) derivatized cellulose and then subsequently eluted by increasing the ionic strength of the eluent. This example of the use of a polymeric anion exchanger for liquid chromatography was followed by the use of a carboxymethyl (CM) cellulose derivative for cation exchange chromatography [2] and a cross-linked polydextran gel which when swollen by aqueous eluents could be used to separate by size water-soluble biological macromolecules [3]. These materials are still commer- cially available for low/medium pressure liquid chromatography and are used extensively for preparative fractionations of biological macromolecules. However, these microporous polymer gels are lightly cross-linked copolymers in which the porosity of the particle is determined by the percentage of the cross-linking monomer used. The lower the percentage of cross-linker the bigger the porosity in the presence of a good solvent but the softer is the resultant polymer matrix. Therefore these lightly cross-linked semi-rigid polymer networks are easily compressed and can only be operated under low/medium pressure. With increasing pressure the polymer network collapses restricting the flow and reducing the column permeability. Polymers with a degree of cross-linking of 8% such as the sulphonated poly(styrenedivinylbenzene) matrices used for free amino acid [4] or oligosaccharide analysis [5] are used with HPLC equipment but only at elevated temperature and when packed in wide bore columns. Below this degree of cross-linking they are generally unsuitable for HPLC separations. In the 1970s silica became the matrix of choice for HPLC being a rigid macroporous support able to operate under high pressure/flow and being commer- cially available in a range of pore and particle sizes. The high flow-rates and favourable mass transfer characteristics enabled high efficiency separations to be achieved in a fraction of the time required when using microporous gels. The rigid macroporous polymers, copolymers with a high degree of cross-linking, were slower to develop than the inorganic matrices. Moore in 1964 [6] synthesised macroporous poly(styrene- divinylbenzene) materials with a high percentage of cross-linker, divinylbenzene. He carried out the polymerization in the presence of a porogen, a compound which is soluble in the monomers but insoluble in the polymer and produced rigid spherical particles containing large voids/pores. Porous materials similar to these have been used for organic phase gel permeation chromatography [7] and subsequently in microparticulate form for reversed-phase chromatography [8]. The range of rigid macroporous HPLC adsorbents produced by Polymer Labs. and discussed in this article are based on the technology originally developed by Moore [6]. The poly(styrene-divinylbenzene) matrices being used in an underivatized form for reversed-phase chromatography and after derivatization for ion-exchange, gel filtra- tion and affinity chromatography.
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`2. PREPARATION AND CHARACTERISATION
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`Macroporous polymer gels are usually prepared by suspension polymerization of the monomer or monomers and cross-linking monomer in a water-organic
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 203 two-phase reaction system [9]. The reaction mixture consisting of monomers, cross-linking monomer and polymerization initiator in the presence of a suitable porogen are stirred rapidly with water to produce organic phase droplets equivalent to the particle size of the polymer bead required. A suspension stabilizer may be required to prevent the droplets coalescing during polymerization. The polymerization proceeds in the droplets of the organic phase with the growing polymer chains precipitating in the droplet as they reach a critical size. The presence of the cross-linking monomer results in the formation of the rigid three dimensional structure. The porogen voids in the polymer network are the macropores. By suitable choice of porogen the pore size, pore size distribution and pore geometry can be optimized for HPLC separations. In size-exclusion chromatography (SEC), the separation mechanism is depen- dent upon the molecular size, hydrodynamic volume, of a solute in solution. Therefore an SEC calibration curve, plot of elution volume ver.suS molecular size may be related to the pore size distribution and pore volume of an HPLC adsorbent [lo]. Fig. 1 shows the SEC calibration curves for the range of poly(styrene-divinylbenzene) reversed- phase HPLC adsorbents, PLRP-S 100,300, 1000 and 4000 A. It can be seen that with the larger pore materials there is a sharp drop off in the calibration curve due to the absence of small pores. This reduces band broadening due to restricted diffusion for larger molecules which can occur when they are chromatographed using small pore materials. For macroporous packings the surface area, total pore volume and mean 10M 1M E .$ loon 3 a’ 3 8 g 10K 1K lOO* r- 4 5 8 8 Elution &ume lmll 9 $0 Fig. I. Size-exclusion chromatography calibration curves for polystyrene standards in tetrahydrofuran at ambient temperature. Column, 300 x 7.5 mm I.D.; flow-rate, 1 .O ml/min; detector, UV operated at 254 nm. (A) 100 A, 5 pm; (B) 300 A, 8 pm; (C) 1000 A, 8 pm; (D) 4000 A, 10 pm (K = kDa; M = 1000 kDa).
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`204 L. L. LLOYD TABLE I THE RELATIONSHIP BETWEEN PORE SIZE AND SURFACE AREA, AS DETERMINED BY BET NITROGEN ADSORPTION ISOTHERMS, FOR THE PLRP-S RANGE OF POLY(STYRENE- DIVINYLBENZENE) REVERSED-PHASE MATERIALS Adsorbent Pore size (A) Surface area (m*/g) PLRP-S 100 414 PLRP-S 300 384 PLRP-S 1000 261 PLRP-S 4000 139 pore diameter can be related. Typical HPLC silica based packing materials with a mean pore diameter of 300 8, and 100 A will have surface areas calculated from gas adsorption isotherms of approximately 200 and 300 m2/g, respectively [ 111. However, from the PLRP-S nitrogen adsorption isotherms, much higher surface areas were obtained, see Table 1. These high values are due to the biporous structure of the polymer matrix [ 121. The presence of pores of 20 A in radius or smaller as shown by the bimodel pore distribution obtained by nitrogen adsorption measurements suggests that micropores may be present in the network of cross-linked polymer chains which surround the macropores. These micropores will only be accessible to very small solutes but would be expected to influence selective binding/adsorption of small rigid molecules of a certain size or shape. Although it is not possible due to the nature of formation of the micropores to eliminate them entirely from the macroporous polvmer Fig. 250 2 4 6 8 Flow-Rate ImVmin] 2. Pressure versus flow velocity in acetonitrile-water (40:60, w/w) at ambient temperature. Column, x 4.6 mm I.D.; (A) 100 A, 5 pm; (B) 300 A, 8 pm; (C) 1000 A, 8 pm; (D) 4000 A, 10 pm.
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 205 the size and number can be minimized. From the SEC calibration curves for the PLRP-S range of materials there is no plateau evident at low molecular weight so suggesting that the micropores are only accessible to solutes with a hydrodynamic volume less than that of phenylhexane (molecular weight 162) which is the bottom point on the calibration curve. For HPLC separations the macroporous polymer must be able to operate at high flow-rates when packed in conventional HPLC column hardware of 4.6 mm I.D. This requires both mechanical rigidity and permeability. The stability of the column packing material may be monitored by examining the dependence of column pressure on eluent flow-rate. Fig. 2 shows the plots obtained for the range of PLRP-S adsorbents. A reasonable linear dependency is observed for the four adsorbents up to 4500 p.s.i. suggesting that little particle distortion/compression occurs below this pressure. The difference in the slope of plots is due to the particle size. Above 4500 p.s.i. the two larger pore materials start to compress and the permeability is reduced. Therefore the PLRP-S range of macroporous poly(styrene-divinylbenzene) matrices are all capable of operating under HPLC conditions of pressure and flow-rate. The influence of pore size and hence surface area on small molecule retention was determined for phenol, aniline, benzene and toluene as a function of eluent composition for the range of PLRP-S pore sizes. Fig. 3 shows the plots of log solute retentivity/capacity factor (k’) against mobile phase composition for the four solutes chromatographed using the four different pore sizes. In the case of both phenol and aniline the form of the plots indicates that the available surface area for solute interaction decreased with increasing pore size, i.e., at any given mobile phase composition the retentivity decreased as the pore size of the adsorbent increased. In the case of the benzene and toluene it was observed that the PLRP-S 1000 A adsorbent appeared to have less available surface area for solute interaction than the PLRP-S 4000 8, material as indicated by the lower solute retentivity. This may be due to differences in the microporous structure causing increased retention of the hydro- lu 3 ACN Fig. 3. Dependence of solute capacity factor (k’) on eluent composition at ambient temperature. Column, 150 x 4.6 mm I.D.; flow-rate, 1.0 ml/min; detector, UV operated at 254 nm. (A) 100 A, 5 pm; (B) 300 A, 8 pm; (C) 1000 A, 8 (D) 4000 A, 10 1 = Benzene; 2 = pm; pm. toluene; 3 = phenol; 4 = aniline.
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`Column efficiency
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`L. L. LLOYD phobic aromatics. Therefore it would appear that available surface area and hence pore size is an important variable in the control of retentivity with small molecules. Comparing the retentivity of the PLRP-S 100 8, material with small pore octadecyl (ODS) bonded silica with small molecules the hydrophobic poly(styrene-divinyl- benzene) matrix is found to be considerably more retentive [13]. Due to the higher available surface area and “apparent” increased hydrophobicity of the polymer compared to even high carbon load ODS silica materials it is exceptionally useful for pre-concentration work as for example in the determination of phenols [ 141 and for the reversed-phase analysis of water-soluble hydrophilic species for example vitamin C [ 151. The use of exceptionally hydrophobic solutes (where in order to obtain reasonable retention volumes, higher percentages of organic modifier or a stronger organic modifier are used in the eluent, for example in the determination of p-aminobenzoic acid in urine to evaluate exocrine pancreatic function [16]) enables eluents more compatible with sample solubility to be oxidized. 2.1.
`linear flow velocity produced for the high efficiency, small pore PLRP-S 100 A 5 pm material in acetonitrile-water (9: 1, w/w), Fig. 4, clearly shows that the plate height achieved with the polymer is comparable to that which would be expected for bonded silica packings but that the optimum linear velocity of the mobile phase is smaller. It has been shown that an increase in efficiency/decrease in the plate height value can be achieved by increasing the temperature [8]. However, it should be noted that increasing the temperature also reduces the retentivity. The improved efficiency has been tentatively attributed to an improvement in the restricted diffusion of a solute in the stationary phase and not to the earlier elution of the solute. Experiments have been performed where the temperature is increased but the retention held constant by increasing the aqueous content of the mobile phase (the decrease in liquid viscosity on increasing the temperature will be balanced by an increase in the aqueous content of the eluent, water being more viscous than the organic) therefore it is proposed that increased efficiency with increasing temperature is due to an improvement in the intraparticle mass transfer. 0.04 -z 3-
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`0.02- 0.01 ! 0:1 0:2 0:3 014 0% U kmk) Fig. 4. Dependence of HETP on flow velocity for PLRP-S 100 8, 5 pm at ambient temperature. Column, 200 x 4.6 mm I.D.; detector, UV operated at 254 nm; eluent acetonitrile-water (90:10, w/w); solute, nitrobenzene.
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 201 2.2. Eluent compatibility solvent strength There have been a number of solvent strength studies carried out using macroporous poly(styrene-divinylbenzene) adsorbents [17-211. Robinson et al. [ 181 working with XAD materials and benzo[a]pyrene and benz[a]anthracene as non-polar probes reported on eluotropic series with dimethyl sulphoxide (DMSO) being a strong solvent. However, in a subsequent study by Bowers and Pedigo [21] DMSO was shown to be a relatively poor solvent for poly(styrene-divinylbenzene) but a good solvent for benzo[a]pyrenes which influenced the results. In all studies there is agreement that the alcohols are weak solvents with acetonitrile and tetrahydrofuran increasing in strength. The solvent strength would appear to have a major influence on peak symmetry for non-polar small molecules. Bowers and Pedigo [21] correlated the peak symmetry with the column void volume for a series of eluents of different strength for acetonitrile and tetrahydrofuran, good solvents, the void volume was significantly smaller than for the poor solvents, alcohols and DMSO and the peak shape was significantly improved. This is interpreted as a swelling of the microporous structure of the particle polymer chain network by the good solvent. The asymmetrical peak shapes with poor solvents are more exaggerated as the aromatic nature of the solute increases, increased n--7~ interactions, however, a significant improvement in peak symmetry can be obtained by the use of a ternary eluent, water, good solvent and poor solvent. If approximately 10% of tetrahydrofuran is added to a poor solvent system then the peak shapes obtained are consistent with those of the good solvent. Fig. 5 shows the separation of a series of alkylphenones with a methanol-water (80:20, v/v) and acetonitrile-water (80:20, v/v) eluent. Increasing the strength of the mobile phase, replacing methanol, IA1 IBl 3 4 h- 5 2 3 k 4 5 I I 0 8 lmid 0 1lOlminl Fig. 5. Separation of alkylphenones using PLRP-S 100 A 5 pm material at ambient temperature. Column, 150 x 4.6 mm I.D.; flow-rate, 1.0 ml/min; detector, UV operated at 254 nm; eluent (A) acetonitrile-water (80:20, w/w); (B) methanol-water (80:20, w/w). Solutes, 1 = methylphenone; 2 = ethylphenone; 3 = propylphenone; 4 = butylphenone; 5 = pentylphenone.
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`a poor solvent, with acetonitrile, a good solvent, improves peak symmetry and reduces the analysis time. In the area of small molecule reversed-phase HPLC the efficiency of separation achieved with the poly(styrene-divinylbenzene) packings does not match that of silica based materials. However, the macroporous polymers do have significant advantages in the area of chemical stability where the silica base matrix is not stable in aqueous eluents above pH 8.0 [ 111 or where interactions with residual silanols can lead to peak tailing. At present polymeric materials tend to be used for reversed-phase separations when the separation is associated with difficulty on a silica-based packing, where chemical stability is required, such as basic compounds at high pH, or where the apparent greater hydrophobicity of the polymers can be utilized for sample pre-concentration/treatment and analysis of hydrophobic or hydrophilic species. 2.3.
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`Macromolecule reversed-phase
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`Reversed-phase chromatography of the biological macromolecules, polypeptides and proteins has gained in importance in recent years. However, the analysis system involves operating at extremes of pH under gradient elution conditions and therefore requires an HPLC stationary phase which is both chemically and physically stable. Also the interaction of charged peptides and proteins with accessible residual surface silanols on a silica-based matrix can result in a mixed-mode separation and peak tailing and broadening [22]. Therefore the mechanical stability, chemical stability and lack of surface silanols makes the PLRP-S, poly(styrene-divinylbenzene) matrix a suitable alternative for reversed-phase separations of biological macromolecules. These larger molecules will be excluded from the micropores so that the reduced efficiency compared with silica-based packings experienced with small non-polar molecules as previously discussed will not be evident. The suitability of the small pore high surface area, PLRP-S 100 A material in the high efficiency 5 pm particle size has been demonstrated for fingerprint peptide mapping [23]. The material exhibited predictable separations with equivalent selec- tivity and peak capacity to that of alkyl bonded phases. Synthetic peptide purifications have also been carried out utilizing the high surface area material for maximum sample load [24]. The high selectivity of the phase was utilized for the analysis of the glycopeptide antibiotic, vancomycin, and also to monitor changes which occur during its degradation [25]. With large molecule separations where diffusion coefliciencies have been measured at lo-” and lo-” m’/s [26] the pore size of the HPLC adsorbent is critical for high efficiency separations. The pore diffusion (solute transfer into and out of the porous structure of the packing material) further reduces the already low diffusion coefficients. It has been calculated that when the ratio of molecular diameter to pore diameter exceeds 0.2 the pore diffusion becomes restricted [27]. This results in a significant increase in band-spreading and hence a reduction in separation efficiency/resolution. Therefore the choice of the correct pore size PLRP-S material is critical in minimizing restricted diffusion of macromolecules. The PLRP-S 100 A material is suitable for peptides up to approximately 15-20 amino acids in length, the 300 A for medium-molecular-weight globular proteins, the 1000 A for larger globular proteins or those having fibrous type structures and the 4000 8, for very large or very fast separations. Fig. 6 compares the reversed-phase separations of collagen and
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 209 1 il:: 0 12lminl
`Fig. 6. Separation of fibrinogen and collagen at ambient temperature. Eluent (A) 0.2S*/0 trifluoroacetic acid in water;(B) 0.25% trifluoroacetic acid in 95% acetonitrile, 5% water; gradient, linear 20-60% B in 15 min; flow-rate, 1.0 ml/min; detector, UV operated at 220 nm. (A) Column, PLRP-S 300 A 8 pm 150 x 4.6 mm I.D.; (B) column, PLRP-S 1000 8. 8 pm 150 x 4.6 mm I.D. 1 = Collagen; 2 = fibrinogen. Fig. 7. Plot of retention versus acetonitrile concentration for the isocratic separation of myoglobin (M), neurotensin (A), neurotensin fragment 1-8 (0) and neurotensin fragment 8-13 (O), run on a PLRP-S 300 8,8 pm IS0 x 4.6 mm I.D. column. Eluent A, 0.1% trifluoroacetic acid in 1% acetonitrile, 99% water; eluent 8, 0.1% trifluoroacetic acid in 99% acetonitrile, I% water; flow-rate, 1.0 ml/min; detector, UV operated at 220 nm.
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`210 L. L. LLOYD fibrinogen, proteins which have rod-like conformations, using the PLRP-S 300 A and 1000 8, pore size materials. The increased band width due to restricted diffusion is clearly evident with the 300 8, material. Proteins are known to display steep elution isotherms in reversed-phase chromatography when using silica-based packings [28]. Fig. 7 shows the plot of retention
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`acetonitrile concentration generated using the PLRP-S 300 A for three peptides, neurotensin, neurotensin fragment 1-8, neurotensin fragment 8-13 and the protein myoglobin. As with the silica-based packings the elution isotherm for myoglobin is steep with only a 4% solvent strength window for interaction but with the three peptides the window is considerably larger, approximately 20%. Therefore for reversed-phase separations of proteins using the macroporous PLRP-S material the retention mechanism approaches that of an “on-off” retention model similar to the silica-based packings and therefore gradient separations are normally used. However, the smaller peptides which have adsorption isotherms similar to small molecules can be chromatographed in either an isocratic or gradient mode. When analysing the proteins it is essential for maximum efficiency/resolution that a sufficiently large pore sized HPLC adsorbent is used to minimize band broadening due to restricted diffusion within the porous matrix. 3. SURFACE COATINGS/MODIFICATIONS 3.1.
`The hydrophobic character of the poly(styrene-divinylbenzene) matrix is a serious limitation to its suitability for macromolecule separations other than is a reversed-phase mode. Even after derivatization the hydrophobic interactions between polymers and proteins can be sufficiently strong that the proteins may be denatured either when adsorbed onto the surface of the stationary phase or when subsequently eluted. Therefore it is essential that the hydrophobic character of the poly(styrene-divinylbenzene) matrix is masked for the chromatographic separation of biological macromolecules in all retention modes except reversed-phase. In the same way as a silica matrix can be derivatized and/or coated to alter surface characteristics so can a polymeric support. However, when a chemically stable polymer is coated it is possible to use chemistries which are themselves exceptionally stable and so an HPLC adsorbent is produced which is capable of withstanding aggressive eluents and extremes of pH. The polyethyleneimine coating technology, where a thin layer of the organic polyamine is adsorbed onto the surface of the silica through ion-pair formation between the silanols and amine groups and subsequent cross-linking, has been used to produce silica-based anion-exchangers for protein chromatography [29]. Subsequent studies have shown that the same coating process can be applied to negatively functionalized macroporous polymers [30,31]. The PL-SAX strong anion-exchange material has been produced by coating the PLRP-S poly(styrene-divinylbenzene) matrices which have been shown to have the required pore geometry for high efficiency separations of biological macromolecules with polyethyleneimine. This coating is then cross-linked in position to give chemical stability and quaternized to provide the strong anion-exchange functionality. The chemical stability of the matrix coupled with the quaternary amine functionality which is ionized over a broad pH range enables
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 211 TABLE 2 THE RELATIONSHIP BETWEEN PORE SIZE AND SPECIFIC IONIC CAPACITY, AS DETER- MINED BY FRONTAL ANALYSIS, FOR THE PL-SAX RANGE OF STRONG ANION- EXCHANGERS Adsorbent pore size Specific ionic capacity (A) (mmoles + /mlCV) 100 0.51 300 0.26 1000 0.23 4000 0.21 _ anion-exchange separations to be performed over a wider pH range than previously possible with weak ion-exchangers based on a silica matrix. With the polymer it is possible to strip denatured protein from the column or suppress the ionization of the quaternary amine group by flushing the column with strong alkali, sodium hydroxide or acid, hydrochloric acid. As the coating is not only adsorbed onto the surface of the matrix but covalently cross-linked in position, organic modifiers can also be used in the eluent or for column clean-up. The full range of PLRP-S adsorbents have been used as base matrices to produce polymeric strong anion-exchangers. Decreasing the adsorbent pore size increased the specific ionic capacity of the anion-exchanger as determined by frontal loading with sodium nitrate due to an increase in the total surface area, see Table 2. However, when evaluated for resolution and loading with a range of proteins of different molecular weight it was found that the small pore 100 A had much lower loadings compared with the wider pore materials. The surface area within the pore was not accessible. The maximum loading for the smallest protein used, P-lactoglobulin, was achieved with the 300-A pore material but unlike the lOOO- and 4000-A materials the 300-A was unable to resolve the A and B chains. The intraparticle diffusion even with this small globular protein is very hindered resulting in excessive band broadening. Table 3 summarizes the loadings achieved with the range of proteins evaluated on each pore size. It would TABLE 3 THE RELATIONSHIP BETWEEN PORE SIZE AND DYNAMIC PROTEIN LOAD, AS DETER- MINED BY FRONTAL ANALYSIS, FOR THE PL-SAX RANGE OF STRONG ANION- EXCHANGERS Protein Molecular Dynamic protein load (mg/mlCV) weight 100 A 300 A 1000 A 4000 A Thyroglobulin 669 000 6 IO 17 23 Catalase 232 000 14 35 20 IO y-Globulin 90 000 14 31 38 34 BSA 66 000 9 2 36 16 p-Lactoglobulin 35 000 22 108 46 20
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`appear that loading is related to available surface area with the PL-SAX 1000 A material being optimum for the medium-molecular-weight globular proteins and the PL-SAX 4000 8, material for the larger solutes such as thyroglobulin. The anomaly with catalase is due to it denaturing to sub-units of 57 500 molecular weight. Therefore for the analysis or purification of small/medium molecular weight proteins maximum loading coupled with good resolution would be achieved with the PL-SAX 1000 A material but for the analysis of larger molecules such as thyroglobulin or polynucleotides then the wider pore PL-SAX 4000 8, would be optimum. In addition to the PL-SAX strong anion-exchanger a strong cation-exchange material has been produced, PL-SCX, with sulphonic acid functionalities and a masked hydrophobic surface. With ion-exchange materials which are often used for preparative fractionations on a mg to kg scale in addition to analytical se two wide pore sized materials, PL-SCX 1000 8, and PL-SCX 4000 8: arations the , have been produced. As approximately 95% of the surface area of a porous adsorbent is located within the pore structure of the matrix it is essential that for high capacity the pores are sufficiently large to give free access to the internal surface area. As with the strong anion-exchanger the derivatization chemistries are covalent and therefore the PL-SCX matrix is able to withstand operation at extremes of pH, with high salt eluents and organic modifiers. The modifications carried out to the base poly(styrene-divinyl- benzene) matrix to produce the ion-exchangers do not weaken the polymer or reduce mechanical stability so enabling high pressures and flow-rates to be used as with the reversed-phase materials. 3.2. Gel
`For gel filtration chromatography (GFC) of biological molecules which has traditionally been carried out on polyacrylamide and polysaccharide soft gels or more recently diol coated silica matrices, surface hydrophilicity, homogeneity and reproduc- ibility are critical. Although the low cross-linked polyacrylamides and polysaccharides exhibit minimal non-specific interactions with a range of sample types the inherent softness of the polymers limits their suitability for high speed separations. The silica-based GFC packings have shallow calibration curves over a specific molecular size range for high resolution separations [32] but are limited due to lack of chemical stability, residual silanols that can function as cation-exchange groups, resulting in non-specific interactions, and increasing fragility as the pore size increases [33]. The chemistries used to produce the PL-GFC range of gel filtration columns produces an exceptionally hydrophilic surface with minimum ion-exchange functionalities. In order to probe the surface homogeneity of the PL-GFC matrix four proteins of different hydrophobicity and charge were used as chromatographic test probes myoglobin with an isoelectric point of 6.76-7.16, ovalbumin with an isoelectric point of 4.6, cc-chymotrypsinogen A at 9.1 and lysozyme at 11 .O. When chromatographed at pH 7 ovalbumin would have a nett negative charge, lysozyme and a-chymotryp- sinogen A a nett positive charge and myoglobin would be neutral. By measuring the retention volume of these four proteins in a low salt buffer with increasing concentrations of salt, ammonium sulphate, it is possible to probe the surface for both electrostatic functionalities and hydrophobicity. Fig. 8 shows the plots of retention
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`MACROPOROUS COPOLYMERS AS HPLC STATIONARY PHASES 213 I ‘6 64 0: 2 Concen~atio~(W-&$ (M) *IO I 16 a.4 0% 112 2:o Concentration (NH$~4 (M) I I& a’4 0:8 1.‘2 2:o Concentration (NH$Ed (M) Fig. 8. Plot of elution volume versus concentration of (NH&SO4 in 0.02 A4 KH2P04 at pH 7.0 for the proteins cc-chymotrypsinogen A (0). lysozyme (A), ovalbumin (W) and myoglobin (+), run on PL-GFC 300 A (A), PL-GFC 1000 8, (B) and PL-GFC 4000 8, (C). Column, 50 x 4.6 mm I.D. 8 pm; flow-rate,
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`ml/min; detector, UV operated at 220 nm. salt plots it is evident that there is no increase in retention at low salt concentrations indicating minimal non-specific interactions due to either cation- or anion-exchange functionalities and that hydrophobic interactions are not promoted even with a-chymotrypsinogen A until the ammonium sulphate concentration reaches 0.5 M. There is therefore a wide operating window of ionic strength where size separations could be expected to dominate making the material suitable for gel filtration chromatography of a wide range of water soluble polymers, neutral, cationic, anionic and hydrophobic. Three individual pore sizes have been produced, PL-GFC 300 8, with a pullulan polysaccharide resolving range of 100 to 200 kDa, PL-GFC 1000 A which resolves from 20 kDa to 2000 kDa and the PL-GFC 4000 8, resolving from 100 kDa to greater than 20 000 kDa. The materials have optimized pore size distributions to be used
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`individually or in the case of the 300 and 4000 A where there is a small overlap in the resolving ranges, in series for the analysis of broad distribution polymers. As with the previous polymeric materials discussed, the PL-GFC polymers are chemically stable enabling gel filtration chromatography to be carried out over a wide pH range with acid, alkali and methanol used as eluents, additives or in clean-up cycles without altering the surface characteristics. 3.3.
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`Affinity chromatography
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`Staphylococcus aureus
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`has been covalently attached to the surface of the PL-AFC matrix to produce an affinity material with a high specificity f