Anal. Chem. 1002, 64, 1232-1238
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Polymeric Porogens Used in the Preparation of Novel Monodispersed Macroporous Polymeric Separation Media for High-Performance Liquid Chromatography Q. Ching Wang, Ken Hosoya, Frantisek Svec, and Jean M. J. Fr6chet’ Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301
A novel approach to monodzed macroporous polymerlc uparatlonmedla wlth vastly enhanced pore dze dktrlbutlons and chromatographlc propertieshas been developed. Key to thk approachIsthe comblned use of monodkpcMedpolymeric partlcles and wltable solvents as porogens In the copolymerlzatlon of styrene and dlvlnylbenzene. Followlng polymerlzatlon, the polymorlc porogen Is dkrolved, Ieavlng behlnd the monodzed b a d 8 wlth a controlled pore structure. The exact pore dze and pore dze dlstrlbutlon of the flnal beads are largely controlled by the amount of soluble polymer In the polymerlzlng mlxture: the larger the proportion of soluble polymer In the system, the larger the porw. The unlformly slzed macroporousbeads prepared wlth an opthnlzed ratlo of polymwlc and low molecular welght porogens proved to be very efflclent even In short columns for the separation of polystyrene standards In the SEC mode and the separation of protelns In the reversed-phase mode. The relatlonshlp between pore slze and spoclflc wrface area, on one hand, and chromatographlc properties of the statlonary phase, on the other, have been clearly documented.
INTRODUCTION Since their introduction 1954l polymeric separation media have attracted attention due to their chemical stability in the entire pH range. The rigid, highly cross-linked styrene copolymers were first used for SEC chromatography by Moore.2 The macroporous copolymers currently available are not only chemically stable but also more resistant to the mechanical forces that prevail in a column and therefore are a better match to the traditional packings based on silica gel. Most of the commercial polymeric packings are based on styrene-divinylbenzene copolymers and are available in a broad range of mean pore sizes, particle sizes, and surface chemistries.34 Most polymer separation media are still produced by a classicalsuspension polymerization.3~6This technique affords beads that have rather broad particle size distributions and, therefore, which cannot be used directly for chromatography. In the case of polydisperse column packings, the permeability of the column is determined by the smallest particles, while its efficiency is controlled by the largest onesa7 Dewaele and Verzele have documented experimentally8 the strong dependence of back-pressure on the particle size (1)Sober, H. A.; Peterson, E. A. J. Am. Chem. SOC. 1954, 76, 1711. (2)Moore, J. C. J. Poly?. Sci., Part A 1964, 2, 835. (3)Pietrzyk, D. J. In Hyh-Performonce Liquid Chromatography; Brown, P. R., Hartwick, R. A., Eds.;J. Wiley: New York, 1989;p 223. (4)Tanaka, N.; Araki, M. Adu. Chromatogr. 1989, 30, 81. (5)Lee, D.P. J. Chromatogr. 1988,443, 143. (6)Lloyd, L. L. J. Chromatogr. 1991,544, 201. (7)Collin, H.In High-Performance Liquid Chromatography; Brown, P. R., Hartwick, R. A,, Eds.; J. Wiley: New York, 1989 p 455. (8)Dewaele, C.; Verzele, M. J . Chromatogr. 1983, 260, 13.
distribution. To reduce the back-pressure and increase the efficiency of the packed column, tedious size fractionation of the beads obtained by suspension polymerization has to be done to afford a narrow fraction of useful particles, while the remainder is waste. The fraction thus obtained has areduced polydispersity; however, it is never strictly uniform. In the search for uniformly sized beads, Ugelstad developed a technique he termed the “activated multi-step swelling and polymerization”method.9 In fad, this procedure is a standard suspension polymerization in which the size of the starting droplets is not determined by the stirring conditions but by the use of an aqueous dispersion of preformed monosized seed particles in a swollen state. The swollen particles containing appropriate monomers are then subjected to suspension polymerization in a process that excludes coalescence of the droplets. The method was first used for the preparation of monodispersed porous styrene-divinylbenzene particles which were only recently employed in size exclusion (SEC) and ion-exchangechromatography.lOJ1 The SEC column exhibited a rather high efficiency,and the ratio between the inner pore volume Vi and the interstitial void volume V,-, which affects the resolution12 was about 1. Somewhat similar materials prepared by analogous means were also tested in normal-phase and reversed-phaseHPLC13.14 as well as in high-performance affinity chromatography.15 The pore size of the packing is very important for the highly efficient separation of macromolecules such as proteins or synthetic polymers. Unger16 has calculated that when the ratio of the molecular diameter of the eluted molecule to the pore diameter of the packing exceeds 0.2, the pore diffusion becomes restricted. This causes peak broadening and reduces resolution. Materials with a mean pore size up to 100nm are therefore recommended for the separation of large globular proteins.6 Macroporosity or the development of permanent pores in polymer beads occurs when the polymerization mixture contains inert porogens and cross-linkingmonomers. Macroporous polymers with very large pores (over 50 nm) have been prepared by classical suspension polymerization in the presence of solution of an inert polymer dissolved in monomers of monomel-solvent mixtures. Though this approach17J8does not produce monodispersed materials, it is interesting as the (9)Ugelstad, J.; Kaggerud, K. H.; Hansen, F. K.; Berger, A. Makromol. Chem. 1979, 180,737. (10)Kulin, L.I.; Flodin, P.; Ellingsen, T.; Ugelstad, J. J. Chromatogr. 1990,514, 1. (11)Ellingsen, T.; Aune, 0.; Ugelstad, J.; Hagen, S. J. Chromatogr. 1990,535, 147. (12)Chang, H.; Gooding, K. M.; Regnier, F. E. J.Chromatogr. 1976, 125, 103. (13)Rapp, W.;Bayer, E. 15th International Symposium on Column Liquid Chromatography, Basel, June 2-7, 1991. (14)Hosoya, K.; Frbchet, J. M. J. J. Liq. Chromatogr., submitted for publication. (15)Clonis, Y.D.;Lowe, C. R. J. Chromatogr. 1991,540, 103. (16)Unger, K. K.; Janzen, R.; Jilge, G. Chromatographia 1987, 24, 144. (17)Guyot, A.; Bartholin, M. Progr. Polym. Sci. 1982,8, 277.
0003-2700/92/0384-1232$03.00/0 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
Table I. Composition of Polymerization Mixture Used for the PreDaration of Polymeric Porogen Beads.
a
A
B
C
D
E
F
0.06 0.60 6.40 0.32 5.3
0.06 0.60 5.10 0.26 5.1
0.06 0.6 3.80
0.06 0.60 2.60 0.13 4.2
0.06 0.60 1.30 0.07 3.6
0.06 0.60 0 0 2.5
0.19 4.7
Table 11. Composition of Polymerization Mixture for Preparation of Monodispersed Polymeric Separation
Median
experiment polymer seeds, g 1-chlorodecane,mL styrene, mL benzoylperoxide, g porogenbead size, pm
1239
experiment ~
A styrene, mL divinylbenzene,mL dibutylphthalate, mL benzoylperoxide, g calcd bead size,pm
B
5.0 5.0 5.0 5.0 0 1.3 0.10 0.10 7.43 7.39
C
D
E
F
5.0 5.0 2.6
5.0 5.0 3.8 0.10 7.42
5.0 5.0 5.1 0.10 7.41
5.0 5.0 6.4
0.10
7.40
0.10
7.34
For other conditions see Experimental Section. For other conditions see Experimental Section.
pore size and pore size distribution are controlled by the molecular weight of the porogenic polymer and by its concentration in the organic phase. This report describes a novel approach to monodispersed polymeric separation media involving a modified two-step procedure with solvents and polymers as porogens. It demonstrates the influence of the composition of the porogen mixture on the chromatographicproperties of the uniform macroporous styrene-divinylbenzene beads.
EXPERIMENTAL SECTION Materials. The styrene (Aldrich) and divinylbenzene (80% divinyl monomer, Dow Chemicals)were extracted with 10% aqueous sodium hydroxide and water, dried over magnesium sulfate, and distilled under vacuum. All other materials were used as received. All solvents were of HPLC purity. Preparation of Uniform Porous Beads. Preparation of the Primary Seeds. The monodisperse polystyrene seeds (1.0-pm diameter, 12% solid in water) were prepared by emulsifier-free emulsion polymerization, as described elsewhere.19 Preparation of the Polymeric Porogen. The primary seed particles (0.50-mL dispersion) were swollen by absorption of 0.6 mL of 1-chlorodecanefor 30 h. The swelling was continued for another 5 h in a mixture containing varying amounts of styrene (Table I) and benzoyl peroxide (5% wlv of styrene) emulsified by sonication in 0.25 wt 5% aqueous sodium dodecy1 sulfate (SDS)solution. To the dispersion of swollen particles was added a sufficient amount of a 5% solution of poly(viny1 alcohol) (Aldrich, MW 85 000-146 000, 88% hydrolyzed) to adjust to 1wt % the total concentration of poly(vinylalcohol) in the mixture. The polymerization was carried out in a round-bottom sealed glass reactor (Buchi BEP 280). The oxygen was removed from the mixture by purging with nitrogen for 15 min, and the polymerization was initiated by heating to 70 "C. After 24 h of polymerization the contents of the reactor were cooled to ambient temperature. The enlarged seed particles were then used in the next step without purification. Preparation of the HPLC Separation Media. A 10-mL mixture of equal volumes of styrene and divinylbenzene,ben(18)Revillon,A.;Guyot, A.;Yuan, Q.; da Prato, P. React.Polym. 1989, 10, 11.
(19)Smigol, V.; Svec, F.; Hosoya, K.; Wang, Q. C.; FrBchet, J. M. J. Angew. Makromol. Chem. 1992,195,151. (20)Guyot, A. In Syntheses and Separations Using Functional Polymers; Sherrinaon, D. C., Hodge, P., Eds.;J. Wiley: New York, 1989; P 1. (21)Margel, S.;Nov, E.; Fisher, I. J. Polym. Sci., Polym. Chem. Ed. 1991,29,347. (22)Vanderhoff, J. W.;El-Aasser,M. S.; Micale, F. J.; Sudd, E. D.; Tseng,C. M.; Silwanowitz,A.; Kornfield,D.M.; Vincente,J. J.Dispersion Sci. Technol. 1984,5,231. (23)Sing, S.W. Pure Appl. Chem. 1979,51, 1. (24)Seidl,J.;Malinsky, J.; Dusek, K.;Heitz,W. Adu.Polym.Sci. 1968, 5, 113. (25)Tanaka, N.;Hashizume, K.; Araki, M.;Tsuchiya, H.; Okuno, A,; Iwaguchi, K.; Ohnishi, S.; Takai, N. J. Chromatogr. 1988,448,95.
zoyl peroxide (1% wlv of monomers), and varying amounts of dibutyl phthalate (Table 11)was emulsified in 0.25 wt % aqueous SDS solution and added to the aqueous dispersion of the polymeric porogen (Table I) in the reactor. The mixture was stirred for 5 hat room temperature. This stirring resulted in the transfer of all of the emulsifiedliquid into the polymer porogen particles. The dispersion of the organic phase in the water phase was deaerated with nitrogen and the reactor sealed. After the temperature was increased to 70 "C, the polymerizationwas allowed to proceed for 24 h. The contents of the reactor were transferred into a large beaker, and the beads were decanted repeatedly with methanol until the supernatant remained clear. The beads were then extracted with toluene for 48 h in a Soxhlet apparatus to remove the porogens and dried in air. The calculated bead size of all the polymers described in this report was about 7.4 pm; some size variations were observed as the percentage of polymeric porogen is increased. Characterization of the Properties. Surface morphology and particle size of the beads were investigated by scanning electron microscopy (JEOL JSM 35CF). The specific surface area was calculated from the BET isotherm of nitrogen; the pore size distribution in the dry state was determined by mercury porosimetryusing a custommade combined BET-sorptometer and mercury porosimeter from Porous Materials, Inc., Ithaca, NY. Chromatography. The macroporous beads were slurrypacked with 80% aqueous acetonitrile into a stainless steel column (150-mm X 4.6-mm i.d.) under a constant pressure of 10 MPa. Approximately 35 mL of solvent was pumped through the column. Chromatography was carried out using a Nicolet LC 9560 ternary gradient liquid chromatograph. The samples were injected through a Rheodyne C7125 valve loop injector (20 pL). The column was thermostated to 30 "C. The separation was monitored using a Hewlett-Packard 1050UV detector at 254 nm for polystyrene standards and at 218 nm for proteins. Size exclusion chromatographywas performed in THF with benzene and polystyrene standards with molecular weights ranging from 1250 to 2 950 000 (Polymer Laboratories). The number of theoretical plates, N, was calculated according to the equation N = 5.5(tr/tw1/~)2, where t, is the retention time and twl/zis the peak width at half-height. The resolution Rs of two peaks was obtained using the equation
R, = 2At,!(tw1 + tw,) (1) The specific resolution R,, in size exclusion chromatography (SEC) was calculated according to the equation26 R,, = 0.576/D2u (2) where 02 is the slope of the straight-line portion of the calibration curve in SEC and u is the peak standard deviation (26) Yau, W. W.; Kirkland, J. J.; Bly, D. D.; Stoklosa, H. J. J. Chromatogr. 1976, 125, 219.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
in milliliters. The value 0 2 was calculated from
whereM1and M2 are the molecular weights of the polystyrene standards and v R 1 and V R are ~ the retention volumes. The values of B were calculated according to the equation B = (A/hZ?r) (4) where A is peak area and h is peak height. The model protein mixture composed of cytochrome c (4 mg/mL), lysozyme (0.8 mg/mL), myoglobin (4 mg/mL), and chicken egg albumin (8 mg/mL) (Sigma, St. Louis) was separated by reversed-phase chromatography using a linear gradient from 20 to 60 vol % of acetonitrile in water containing 0.1 vol % trifluoroacetic acid in 20 min.
RESULTS AND DISCUSSION Preparation of the Monodispersed Beads. While the Ugelstad method of preparation+ll of uniformly sized porous polymer beads has been extremely successful in the preparation of monodispersed but conventional separation media, its use of low molecular weight porogens does not allow the versatility that is required for some separations based on size exclusion. In contrast, beads with pores larger than 50 nm can be obtained using polymeric porogens in ordinary suspension polymerizations. A combination of the Ugelstad and the polymeric porogen approaches would be desirable, as it would lead to uniform beads with large pores. Unfortunately, such a combination is not readily achieved. In the Ugelstad method, size uniformity results from the use of uniform seed particles, 1pm or less in size, which are first “activated” with a solvent and then swollen with monomers and low molecular weight porogens. The shape of the initial seed particle is maintained throughout the process as the porogens and monomers diffuse through the aqueous phase to the “activated” seeds. In the case of polymeric porogens, it is not possible to achieve the diffusion of the porogen through the aqueous phase; therefore traditional approaches17J8 have involved the formation of nonuniform droplets of varying sizes. The seed particles advocated by Ugelstad are too small to effectively function as porogens. For example the enlargement of a 1-pm bead to 7 pm represents a 343-fold increase in volume; therefore the concentration of the original seed in the final droplet is less than 0.3 % . Guyot has suggested that in order to function as a polymeric porogen, the concentration of soluble polymer in the droplet should exceed 10%.20 Obviously,even this amount is not sufficient,as porositiesof 40-60 % are usually preferable, this requires the use of approximately 40450% of porogens in the polymerization mixture. Dependingon the separation to be performed and to achieve the benefits of both types of porogens, we advocate the control of pore size distribution by balancing the porogenic mixture with low molecular weight solvents for small pore sizes and polymers for large pores. Our approach is therefore to prepare sufficientlylarge monodispersed polymeric porogen particles and enlarge them to the final sizewith a mixture of monomers and, if desired, low molecular weight porogens. For example, the use of polymer porogen beads 5.6 pm in size is required to obtain HPLC beads 7 pm in size with 50% porosity. As these figures do not take into account the shrinkage that occurs during polymerization,the actual size of the final beads may be less after polymerization than in the swollen droplets. Since emulsifier-free emulsion polymerization cannot be used to produce monodispersed polymeric porogen particles much larger than 1pm, other techniques such as dispersion
I _ _ -
Flgure 1. Scanning electron micrograph of 7.4-pm porous beads D.
polymerization21 or seeded polymerization9-l1p22 should be used. For example, Table I lists experimental conditions used in the preparation of polymeric porogen particles using Ugelstad’s “activated swelling and polymerization method” in the absence of a cross-linking monomer. The 1.0-pm monodispersed polystyrene seeds used for these preparations were obtained by emulsifier-free emulsion polymerization, as described previo~s1y.l~The data shown in Table I report bead sizes obtained for polymeric porogen particles that still contain any unreacted monomer or low molecular weight solvent used in the polymerization. For example, experiment A affords 5.3-pm beads largely composed of polystyrene but containing about 9 % 1-chlorodecane. In contrast, blank experiment F carried out without any monomer is a simple swelling of the seed beads with the solvent to afford 2.5-pm particles containing only 10% of the polymer in l-chlorodecane solution. The monodispersed polystyreneporogen particles described in Table I, or equivalent particles obtained by another technique such as dispersion polymerization, are the starting materials used to prepare the final cross-linked porous beads. They will transfer their size monodispersity to the final beads, while the soluble polymer they contain will be used in the control of pore formation. For example, the particles listed in Table I were swollenwith a mixture of emulsifiedmonomers and additional low molecularweight porogen in varying ratios, as shown in Table 11. Once the swelling was completed, freeradical polymerization was initiated by heating. After completing the polymerization and a standard workup procedure including the removal of the soluble polymer, porous polymer beads approximately 7.4 pm in size were obtained. The scanning electron micrograph of Figure 1 documents the uniformity of beads, confirmingthat the overall process has not degraded the size monodispersity of the starting polymeric porogen particles. Properties of the Pores. As may be expected, the pore structure characteristics of the HPLC separation media prepared above varies greatly with the nature of the porogen used in their preparation. This is confirmed by surface area and pore size measurements reported in Table 111. The table combines results of two independent methods, BET and mercury porometry. As the methods cover different range of pores, the data obtained for the same beads do not fully correspond. The BET method is the most accurate for measurements in the range of very small pores from about 2 to 20 nm, while the mercury porometry best describes larger mesopores with a diameter over 10 nm as well as macropores
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, l9Q2
Table 111.
ProDerties of the Pores of the Size-Monodispersed HPLC Beads ~
beads A
B C D E F a
% polymer porogena
100 80 60 40 20 0
50 nm
0.299 0.252 0.313 0.365 0.383 0.383
0.374 0.531 0.506 0.374 0.152 0.192
~~~
BET
mercury porometry median pore diameter, nm
37 66 53 43 21 23
pore
mL/g 10-50 nm 0.025 0.016 0.130 0.050 0.191 0.191
