Anal. Chem. 2001, 73, 4071-4078
Articles
Metathesis-Based Monoliths: Influence of Polymerization Conditions on the Separation of Biomolecules Betina Mayr,† Richard Tessadri,‡ Ekkehard Post,§ and Michael R. Buchmeiser*,†
Institute of Analytical Chemistry and Radiochemistry and Institute of Mineralogy and Petrography, University of Innsbruck, Innrain 52 a, A-6020 Innsbruck, Austria, and Netzsch-Gera¨tebau GmbH, Wittelsbacherstrasse 42, D-95100 Selb, Austria
Monolithic materials were prepared by transition metalcatalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8exo,endo-dimethanonaphthalene within the confines of surface-derivatized borosilicate columns in the presence of the porogenic solvents toluene and 2-propanol using Cl2(PCy3)2Ru(dCHPh) (1) as initiator. Relevant physicochemical data of the porous structure (specific surface area (σ), pore volume (Vp), volume fraction of pores (Ep), and intermicroglobule volume (Ez)) of the monolithic columns were determined by inverse size exclusion chromatography in tetrahydrofuran. Mean particle diameters were determined via electron microscopy. The influence of variations in polymerization conditions in terms of stoichiometry of the monomers and porogenic solvents on the chromatographic separation of the oligodeoxynucleotides dT12-dT18 and eight model proteins (ribonuclease A, insulin, cytochrome c, lysocyme, r-lactalbumin, r-chymotrypsinogen, β-lactoglobulin B, catalase) were studied. Also, the role of additional phosphine on the entire polymerization setup and the associated chromatographic separations was elucidated. Relevant chromatographic data as well as differences between the separation of oligodeoxynucleotides and proteins may directly be attributed to the above-mentioned physicochemical properties of the metathesis-based monoliths. Finally, DSCTGA-MS investigations on various monoliths of different composition and age were carried out in order to provide information on stability and oxidation behavior. For more than 30 years, columns packed with microparticulate sorbents have been successfully applied as separation media in HPLC. Despite many advantages, such microparticulate sorbents possess certain limitations such as comparably large void volumes between the packed particles and a slow diffusional mass transfer in particular of biomolecules from and to the stagnate mobile * Corresponding author. Phone: + 512-507-5184. Fax: + 512-507-2677. E-mail:
[email protected].. † Institute of Analytical Chemistry, University of Innsbruck. ‡ Institute of Mineralogy and Petrography, University of Innsbruck. § Netzsch-Gera ¨tebau GmbH. 10.1021/ac010452+ CCC: $20.00 Published on Web 08/07/2001
© 2001 American Chemical Society
phase present in the pores of the separation medium. Micropellicular supports that somehow overcome these problems show limitations in terms of column length due to the increasing back pressure proportional to the decreasing particle diameters; which additionally makes high demands on the packing procedure. One way to alleviate the problem of restricted mass transfer and intraparticle void volume is the concept of monolithic beds, where the separation medium consists of a continuous rod of a rigid, porous polymer.1-3 The first monolithic separation materials were already described in the late 1960s,4 but not before Hjerte´n et al. introduced the additionally compressed continuous supports, an intensive investigation on this area started.5-7 Since then, these materials have been further developed.8-11 So far, organic continuous beds are either based on methacrylates or poly(styrenedivinylbenzene)8,10-13 and are almost exclusively prepared by radical polymerization. An alternative approach for the preparation of functionalized polymer supports represents the use of ringopening metathesis polymerization (ROMP).14 We already have demonstrated the high versatility of ROMP for the preparation of sorbents suitable for separation sciences,15-25 including the synthesis of functionalized monolithic materials.26,27 In this con(1) Hansen, L. C.; Sievers, R. E. J. Chromatogr. 1974, 99, 123-133. (2) Rodrigues, A. E. J. Chromatogr., B 1997, 699, 47-61. (3) Xu, Y.; Liapis, A. I. J. Chromatogr., A 1996, 724, 13-25. (4) Kubin, M.; Spacek, P.; Chromecek, R. Collect. Czech. Chem. Commun. 1967, 32, 3881-3887. (5) Hjerte´n, S.; Li, Y.-M.; Liao, J.-L.; Mohammad, J.; Nakazato, K.; Pettersson, G. Nature 1992, 356, 810-811. (6) Hjerte´n, S.; Liao, J.-L.; Zhang, R. J. Chromatogr. 1989, 473, 273-275. (7) Maruska, A.; Ericson, C.; Ve´gva´ri, A.; Hjerte´n, S. J. Chromatogr., A 1999, 837, 25-33. (8) Peters, E. C.; Svec, F.; Fre´chet, J. M. J. Adv. Mater. 1999, 11, 1169-1181. (9) Svec, F.; Fre´chet, J. M. J. Science 1996, 273, 205-211. (10) Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Chem. Mater. 1996, 8, 744-750. (11) Viklund, C.; Ponte´n, E.; Glad, B.; Irgum, K.; Ho¨rstedt, P.; Svec, F. Chem. Mater. 1997, 9, 463-471. (12) Sykora, D.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1999, 852, 297304. (13) Wang, Q. C.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1993, 65, 2243-2248. (14) Buchmeiser, M. R. Chem. Rev. 2000, 100, 1565-1604. (15) Buchmeiser, M. R.; Atzl, N.; Bonn, G. K. J. Am. Chem. Soc. 1997, 119, 9166-9174. (16) Buchmeiser, M. R.; Tessadri, R.; Seeber, G.; Bonn, G. K. Anal. Chem. 1998, 70, 2130-2136. (17) Huber, C. G.; Buchmeiser, M. R. Anal. Chem. 1998, 70, 5288-5295.
Analytical Chemistry, Vol. 73, No. 17, September 1, 2001 4071
Figure 1. Pressure drop vs flow rate of different mobile phases measured on monolith 2 (100 × 3 mm i.d.) at room temperature. Mobile phase: (b) tetrahydrofuran, (9) water, (2) methanol, ([) acetonitrile.
tribution, we report in-depth investigations on the influence of polymerization parameters on the separation of oligodeoxynucleotides and proteins. RESULTS AND DISCUSSION Permeability.28 To allow the use of monolithic columns in a chromatographic system, their flow resistance must not exceed the upper back-pressure limits set for the equipment at the applied flow rates. Porous polymeric stationary phases in contact with organic solvents often lack sufficient mechanical strength, and the polymer may suffer deformation under the pressure gradient normally encountered in HPLC columns. To evaluate the mechanical stability of our column material, the pressure drop across the column was measured by perfusion with various solvents in a wide range of flow rates. Figure 1 summarizes the pressure drop at different flow rates for four different solvents. The excellent linear dependence obtained is indicated by a regression factor R2 better than 0.9999 for all graphs. This confirms that the rod is not compressed even at high flow rates. According to Darcy’s law (eq 1), for a given porous structure with a column permeability
u)
B0 ∆p η L
(1)
B0, the pressure drop through a column at a given flow rate is dependent only on the viscosity of the solvent. In eq 1, u represents the linear flow velocity, η is the viscosity of the solvent, L is the column length, and ∆p is the pressure drop across the column. (18) Sinner, F.; Buchmeiser, M. R.; Tessadri, R.; Mupa, M.; Wurst, K.; Bonn, G. K. J. Am. Chem. Soc. 1998, 120, 2790-2797. (19) Buchmeiser, M. R.; Mupa, M.; Seeber, G.; Bonn, G. K. Chem. Mater. 1999, 11, 1533-1540. (20) Seeber, G.; Brunner, P.; Buchmeiser, M. R.; Bonn, G. K. J. Chromatogr., A 1999, 848, 193-202. (21) Buchmeiser, M. R.; Sinner, F.; Mupa, M.; Wurst, K. Macromolecules 2000, 33, 32-39. (22) Buchmeiser, M. R.; Seeber, G.; Tessadri, R. Anal. Chem. 2000, 72, 25952602. (23) Mayr, B.; Sinner, F.; Buchmeiser, M. R. J. Chromatogr., A 2001, 907, 4756. (24) Eder, K.; Sinner, F.; Mupa, M.; Huber, C. G.; Buchmeiser, M. R. Electrophoresis 2001, 22, 109. (25) Mayr, B.; Buchmeiser, M. R. J. Chromatogr., A 2001, 907, 73-80. (26) Sinner, F.; Buchmeiser, M. R. Macromolecules 2000, 33, 5777-5786. (27) Sinner, F.; Buchmeiser, M. R. Angew. Chem. 2000, 112, 1491-1494. (28) Maa, Y.-F.; Horva´th, C. J. Chromatogr., A 1988, 445, 71-86.
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Thus, the back pressure at equal flow rate using water (η ) 1.00 × 10-3 kg/ms) as solvent is expected to be the highest, followed by methanol (η ) 0.60 × 10-3 kg/ms), tetrahydrofuran (THF) (η ) 0.49 × 10-3 kg/ms), and acetonitrile (η ) 0.36 × 10-3 kg/ms). This relationship is no longer valid if the pore structure and hence the permeability of the column changes depending on the used solvent, as appears to be the case for THF. As might be expected, THF, which represents an excellent polymer solvent, causes extensive swelling of the polymer rod; consequently, the permeability decreases and the back pressure is found to be significantly elevated. This is not really relevant, since THF is hardly used in HPLC separations. All other solvents investigated (water, methanol, acetonitrile) are common solvents for liquid chromatography and additionally represent poor polymer solvents. They do not cause any considerable swelling, perfectly follow Darcy’s law, and consequently give the expected order in back pressure. Porosity. Porosity and pore size distribution of each monolithic column was determined by inverse size exclusion chromatography (ISEC) in THF using polystyrene (PS) standards. We decided to use this method first reported by Hala´sz and Martin29 because it is a useful way to reveal differences in porosity between monoliths. Due to the fact that it is based on several assumptions, this method is not absolutely undisputed,30, 31 yet presents an appropriate way to perform such measurements as it works under conditions similar to those used in actual HPLC separations. Mercury intrusion (mercury porosimetry) and BET are competitive alternatives to analyze such rods. Particularly mercury intrusion is capable of providing data for the (here most relevant) macropores (>1000 Å). Nevertheless, these data are ambiguous again, since they are obtained in the dry state and may be hardly relevant for the rod under HPLC conditions. To obtain a representative set of monoliths that can be used to study structure-separation relationships, eight different monoliths with different values for t (34-73%), z (19-64%), and dp (1-4 µm) were synthesized. As already shown earlier,26,27 this variation in the volume fraction of the intermicroglobule void volume (z) and in due consequence in the volume fraction occupied by the mobile phase (t) may simply be achieved by the stoichiometry of the monomer (norbornene, NBE) and the cross-linker (1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene, DMN-H6). Representative illustrations of monoliths 1-8 are given in Figure 2. These eight different monoliths were characterized by ISEC. The (representative) ISEC calibration curve of monolith 2 (Figure 3) shows the retention volume for a set of narrow PS standards (PDI < 1.1 throughout) with defined molecular masses in the range of 92 (toluene)-1 250 000 Da (Table 1). Values for σ, σc, p, z, and t, were directly calculated from the 2610-1 250 000 molecular weight range. The principal shape of all curves was similar for all investigated monoliths yet quite different from calibration curves obtained on commercially available SEC columns. The fact, that there was nearly no separation in the range of molecular weights from 3000 to 90 000 clearly indicates that there are only few medium-sized pores. By way of contrast, there are many small-sized pores (1.5 nm), (29) Hala´sz, I.; Martin, K. Angew. Chem. 1978, 90, 954-961. (30) Knox, J. H.; Scott, H. P. J. Chromatogr. 1984, 316, 311-332. (31) Knox, J. H.; Ritchie, H. J. J. Chromatogr. 1987, 387, 65-84.
Figure 2. Electron microscope pictures of a monoliths 2-8. For their synthesis, refer to Table 1.
indicated by the high separation of toluene. This finding is in accordance with the extensive swelling of the polymer rod in THF. Influence of Polymerization Conditions on Chromatographic Behavior. Both standard oligodeoxynucleotides (dT)12-18 and proteins were used to elucidate the effects of variation in stoichiometry of the polymerization mixture on the chromatographic separation of the above-mentioned analytes. In a first experiment, a mixture of seven homologous oligothymidylic acids (dT12-dT18) ranging in mass from 3638 (dT12) to 5456 Da (dT18) was separated by ion-pair reversed-phase chromatography using a linear gradient of 11-16% acetonitrile in an eluent containing
100 mmol/L triethylammonium acetate. A representative separation is shown in Figure 4. As expected, elution order of oligodeoxynucleotides strongly correlates with their molecular mass since increasing molecular mass directly translates into an increase in hydrophobic interaction of the corresponding analyte with the rod. To check the overall reproducibility, three different monoliths with identical composition (2, 2b, 2c) were prepared in three independent experiments. Table 2 summarizes the results obtained and gives an overview over the values for tr, w0.5, R, and the standard deviations (σn-1) determined therefrom. As can be deduced from these data, values for the resolution (R) show an acceptable Analytical Chemistry, Vol. 73, No. 17, September 1, 2001
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Table 1. Physicochemical Data for Monoliths 1-5a no.
NBE (%)
DMN-H6 (%)
toluene (%)
2-PrOH (%)
1 2 3 4 5 6 7 8
15 20 25 10 30 25 25 25
15 20 25 30 10 25 25 25
10 10 10 10 10 10 10 10
60 50 40 50 50 40 40 40
PPh3 added (ppm)
σ (m2/g)
σc (m2)
p (%)
z (%)
t (%)
Fp (g/cm3)
Vp (mL)
dp (µm)
20 40 80
12 13 11 20 10 6 6 7
2 3 4 4 3 2 2 2
9 13 15 13 12 9 8 10
64 42 19 46 35 31 33 34
73 55 34 58 47 41 41 44
0.272 0.379 0.462 0.299 0.382 0.426 0.423 0.405
0.064 0.092 0.105 0.087 0.082 0.062 0.058 0.069
2 ( 0.5 2 ( 0.5 2 ( 0.5 2(1 nab 3(1 2.5 ( 1.5 4.5 ( 0.5
a Key: σ, specific surface; σ , surface area of monolith; , volume fraction of pores; , volume fraction of intermicroglobule void volume; , c p z t volume fraction occupied by mobile phase; Fp, apparent density; Vp, pore volume,V; dp, microglobule diameter. [Initiator (1)] ) 0.4 % throughout. b na, not available.
Table 2. Chromatographic Data Obtained from the Separation of Oligodeoxynucleotides (dT)12-18 on Three Identically Synthesized Monolithsa,b monolith 2b
(dT)12 (dT)13 (dT)14 (dT)15 (dT)16 (dT)17 (dT)18
monolith 2
monolith 2c
σn-1 (%)
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
1.10 1.36 1.65 1.94 2.21 2.48 2.72
0.08 0.09 0.10 0.10 0.11 0.11 0.11
1.77 1.71 1.65 1.47 1.46 1.34
0.99 1.25 1.54 1.84 2.15 2.44 2.70
0.08 0.09 0.11 0.11 0.12 0.13 0.15
1.73 1.69 1.60 1.59 1.37 1.10
1.48 1.82 2.17 2.48 2.78 3.06 3.32
0.11 0.12 0.13 0.13 0.12 0.12 0.12
1.74 1.63 1.39 1.37 1.33 1.22
22 20 19 17 15 13 12
15 15 14 13 6 8 16
1 3 9 8 5 10
aConditions: mobile phase, 100 mmol/L triethylammonium acetate at pH 7.0; linear gradient, 11-16 % acetonitrile in 10 min; flow rate, 2 mL/ min; temperature, 20 °C; detection, UV 264 nm; sample, (dT)12-18 , 0.1 µg total. b Values for tR and in w0.5 are given in minutes.
Figure 3. Inverse size exclusion chromatography calibration curve for monolith 2 (100 × 3 mm i.d.) obtained at room temperature: flow rate, 0.7 mL/min; mobile phase, THF; analytes, polystyrene standards and toluene; UV detection, 254 nm.
reproducibility (σn-1 ) 1-10%). In analogy, a mixture of eight proteins (Table 3) was injected and separated by reversed-phase chromatography using a linear gradient of 14.5-37% acetonitrile within 0.5 min and 37-46% acetonitrile within 1 min in an eluent containing 0.1% aqueous trifluoroacetic acid. A representative separation is shown in Figure 5. Again, to provide reproducibility data, separations were carried out on three different monoliths with identical composition (2, 2b, 2c) prepared in three independent experiments. The chromatographic data obtained therefrom are summarized in Table 4. As can be deduced, values for σn-1 are significantly elevated (2-20%) compared to those obtained with oligodeoxynucleotides, yet still good. In contrast to the separation of oligodeoxynucleotides, elution order of proteins is not solely governed by molecular mass. We tentatively ascribe this observation rather to differences in the set of amino acids 4074 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001
Figure 4. IP-RP-HPLC separation of a oligodeoxynucleotides (dT)12-18 on monolith 2: mobile phase, 100 mmol/L triethylammonium acetate at pH 7.0; linear gradient, 11-16% acetonitrile in 10 min; flow rate, 2 mL/min; T ) 20 °C; detection, UV 264 nm; sample, (dT)12-18 , 0.1 µg of each oligodeoxynucleotide.
than to differences in the secondary/tertiary structure of each protein (resulting from incomplete denaturation by trifluoroacetic acid). Variation in NBE + DMN-H6 to 2-Propanol Ratio. To investigate the influence of the relative ratios of NBE+DMN-H6 to 2-propanol on the polymerization system and consequently on the chromatographic behavior, three monolithic columns were prepared (monoliths 1-3). For these monoliths, a weight ratio of 1:1 for NBE to DMN-H6 (corresponding to a molar ratio of 6:4) was chosen. Table 5 summarizes the data obtained from the IP-
(3638-5456). Obviously, the hydrodynamic volume of dT12-dT18 is by far smaller than that of the proteins. In consequence, oligodeoxynucleotides are expected to be more affected by changes in the microporosity than the large proteins, which are too large to diffuse into these pores. Consequently, changes in the specific surface (which mainly stem from variations in microporosity) play a minor role in protein separation. The decrease in t, which is associated with the decreasing porogen content in monoliths 1-3, results in reduced retention times (tR). The optimum resolution was found on monolith 2. Variation in NBE to DMN-H6 Ratio. A comparison of the data obtained from the separation of oligodeoxynucleotides and proteins on monoliths 2, 4, and 5 allows the investigation of the influence of the NBE to DMN-H6 ratio thereon (Tables 7 and 8). As reported earlier,26 changes in the NBE to DMN-H6 ratio within a range of 0:50-35:15 do not result in any significant change in dp, z, or σ. Surprisingly, variations in the NBE to DMN-H6 ratio do lead to changes in the chromatographic properties of the resulting monoliths. Thus, the best oligodeoxynucleotide separation was obtained on monolith 2 based on a 1:1 ratio of NBE to DMN-H6. Higher (monolith 4) or lower (monolith 5) contents of DMN-H6 lead to a decrease in resolution. Thus, we were not able to achieve any separation of oligodeoxynucleotides on monolith 5. This result was confirmed by the separation of proteins (Table 8). Again, the best separation was achieved on monolith 2, while no separation was possible on monolith 5. Despite the similar physicochemical data, some significant differences among monoliths 1-5 may be deduced from the electron microscopy pictures (Figure 3). While monolith 2 possesses a well-structured microglobule network, in particular, monoliths 4 and 5 exhibit a more “molten” network which has an appearance similar to the one found in silica-based systems.32,33 In view of the general features of monolithic columns and of the fact the changes in the microporosity may not serve as a general explanation, this microglobule fine structure seems to represent (together with the parameters summarized in Table 1) another crucial parameter. Influence of Additional Phosphine. Grubbs’ catalystcatalyzed metathesis polymerizations proceed via a dissociative mechanism;34 i.e., dissociation of phosphine is required in order to resume polymerization (Figure 6). For the present catalytic system (Cl2Ru(PCy3)2(CHPh)), k1 ) 0.38 s-1, k-1 ) 0.62 s-1, and kB (rate constant for phosphine exchange) ) 0.16 s-1 (all at T ) 37 °C). This means that rebinding of phosphine is competitive with olefin coordination under the chosen reaction conditions. In
Table 3. Physical Properties and Source of Proteins 1-8 protein
Mw
source
isoelectric point
ribonuclease A insulin cytochrome c lysocyme R-lactalbumin R-chymotrypsinogen A β-lactoglobulin B catalase
13 682 5 734 12 360 14 305 14 180 25 656 18 277 57 586
bovine pancreas bovine pancreas horse heart chicken egg white bovine milk bovine pancreas bovine milk bovine liver
8.8 6.0 10.7 11.0 4.8 9.5 5.2 6
Figure 5. RP-HPLC separation of eight proteins on monolith 2: mobile phase, 0.1% aqueous trifluoroacetic acid; linear gradient, 14.5-37% acetonitrile in 0.5 min, 37-46% acetonitrile in 1 min; flow rate, 3 mL/min; T ) 20 °C; detection, UV (218 nm). samples, (1) ribonuclease A, (2) insulin, (3) cytochrome c, (4) lysozyme, (5) R-lactalbumin, (6) R-chymotrypsinogen A, (7) β-lactoglobulin B, and (8) catalase; inj.)ection volumes, 1-6, 22 µg; 7, 8, 44 µg.
RP separation of seven oligodeoxynucleotides on these three monolithic columns. As expected, increasing values for σc (Table 1) resulting from increasing NBE + DMN-H6 ratios lead to enhanced retention and resolution. In an analogous way, the separation of proteins was carried out on these columns. The corresponding data are summarized in Table 6. Not unexpected, the resulting chromatographic properties are hardly changed by the NBE + DMN-H6 to 2-propanol ratio. This is attributed to the different molecular weight range of the proteins (approximately 6000-58 000, Table 3) compared to that of oligodeoxynucleotides
Table 4. Chromatographic Data Obtained from the Separation of Proteins on Three Identical Synthesized Monolithsa monolith 2b
ribonuclease A insulin cytochrome c lysocyme R-lactalbumin R-chymotrypsinogen A β-lactoglobulin B catalase aValues
monolith 2
monolith 2c
σn-1
tRa
w0.5a
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
0.60 0.67 0.71 0.87 0.99 1.22 1.30 1.43
0.02 0.02 0.03 0.02 0.02 0.03 0.05 0.03
2.08 0.98 3.39 2.82 5.26 1.19 1.66
0.60 0.67 0.72 0.89 1.01 1.25 1.34 1.48
0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04
1.90 1.28 3.40 2.20 4.84 1.50 2.12
0.62 0.69 0.74 0.91 1.02 1.26 1.35 1.48
0.03 0.02 0.02 0.04 0.03 0.04 0.05 0.04
1.67 1.59 2.91 1.77 4.00 1.29 1.77
2 2 3 2 2 2 2 2
14 2 16 28 15 15 15 6
11 24 9 23 14 12 13
for tR and in w0.5 are given in minutes.
Analytical Chemistry, Vol. 73, No. 17, September 1, 2001
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Table 5. Chromatographic Data Obtained from the Separation of Oligodeoxynucleotides (dT)12-18 on Monoliths 1-3, 7, and 8a monolith 1 b
(dT)12 (dT)13 (dT)14 (dT)15 (dT)16 (dT)17 (dT)18
monolith 2
b
monolith 3
monolith 7
monolith 8
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
0.89 1.13 1.40 1.69 1.96 2.26 2.52
0.09 0.11 0.12 0.13 0.13 0.14 0.13
1.39 1.37 1.39 1.23 1.29 1.16
0.99 1.25 1.54 1.84 2.15 2.44 2.70
0.08 0.09 0.11 0.11 0.12 0.12 0.15
1.73 1.69 1.60 1.59 1.37 1.10
1.69 2.09 2.47 2.85 3.19 3.51 3.79
0.10 0.12 0.12 0.11 0.11 0.11 0.10
2.15 1.93 1.92 1.79 1.60 1.55
0.81 1.05 1.32 1.61 1.90 2.17 2.45
0.09 0.11 0.12 0.12 0.13 0.13 0.13
1.44 1.42 1.42 1.35 1.27 1.25
0.60 0.76 0.95 1.18 1.44 1.70 1.94
0.08 0.12 0.14 0.17 0.19 0.20 0.18
0.96 0.87 0.89 0.84 0.79 0.74
a Conditions: mobile phase, 100 mmol/L triethylammonium acetate at pH 7.0; linear gradient, 11-16 % acetonitrile in 10 min; flow rate, 2 mL/min; temperature, 20 °C; detection, UV 264 nm; sample, (dT)12-18 , 0.1 µg total. b Values for tR and in w0.5 are given in minutes.
Table 6. Chromatographic Data Obtained from the Separation of Proteins on Monoliths 1-3a monolith 1
ribonuclease A insulin cytochrome C lysocyme R-lactalbumin R-chymotrypsinogen A β-lactoglobulin B catalase
monolith 2
monolith 3
monolith 7
monolith 8
tRb
w0.5b
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
tR
w0.5
R
0.64 0.70 0.75 0.92 1.04 1.27 1.37 1.49
0.02 0.02 0.04 0.04 0.04 0.04 0.05 0.04
1.65 0.99 2.38 1.77 3.77 1.27 1.57
0.60 0.67 0.72 0.89 1.01 1.25 1.34 1.48
0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04
1.90 1.28 3.39 2.19 4.84 1.50 2.12
0.55 0.62 0.66 0.84 0.96 1.19 1.28 1.40
0.03 0.03 0.03 0.04 0.03 0.03 0.04 0.04
1.68 0.84 3.06 2.11 3.95 1.42 1.77
0.74 0.80 0.85 1.02 1.12 1.36 1.49 1.55
0.02 0.02 0.03 0.03 0.03 0.04 0.05 0.06
1.52 1.10 2.94 1.80 3.90 1.68 0.66
0.74 0.80 0.86 1.00 1.11 1.33 1.47 1.52
0.02 0.02 0.03 0.03 0.03 0.04 0.05 0.06
2.79 1.91 3.09 1.91 3.65 2.16 0.58
a Conditions: mobile phase, 0.1% aqueous trifluoroacetic acid; linear gradient, 14.5-37% acetonitrile in 0.5 min, 37-46% acetonitrile in 1 min; flow rate, 1.7 mL/min; temperature 20 °C; detection, UV 218 nm. b Values for tR and in w0.5 are given in minutes.
Table 7. Chromatographic Data Obtained from the Separation of Oligodeoxynucleotides on Monoliths 2 and 4a monolith 4
(dT)12 (dT)13 (dT)14 (dT)15 (dT)16 (dT)17 (dT)18
monolith 2
tRb
w0.5b
R
tR
w0.5
R
1.08 1.35 1.64 1.92 2.21 2.48 2.72
0.12 0.14 0.16 0.16 0.16 0.16 0.19
1.24 1.13 1.03 1.04 0.96 0.79
0.99 1.25 1.54 1.84 2.15 2.44 2.70
0.08 0.09 0.11 0.11 0.12 0.13 0.15
1.73 1.69 1.60 1.59 1.37 1.10
a Conditions: mobile phase, 100 mmol/L triethylammonium acetate at pH 7.0; linear gradient, 11-16% acetonitrile in 10 min; flow rate, 2 mL/min; temperature, 20 °C; detection, UV 264 nm; sample, (dT)12-18 , 0.1 µg total. b Values for tR and in w0.5 are given in minutes.
consequence, the presence of additional phosphine must be expected to have a drastic effect on the microstructure and hence on any subsequent separation. Since the presence of even small amounts (
