Monodispersemacroporous Particles

As mentioned before, the monodisperse-macroporous particles are suitable as chromatographic packing materials, particularly in the HPLC applications. Ugel-

FACING PAGE

FIG. 12 Binding of several PCMS particles onto one DNA molecule via the interaction between primary amine groups of PEI and phosphate groups of DNA. (Reprinted with permission from Ref. 24, Copyright © 2001, VSP International Science Publishers.)

stad et al. were the first to propose the use of monodisperse-macroporous styrene-divinylbenzene-based particles as a chromatographic support in the separation of proteins by reversed-phase liquid chromatography (RPLC) [26]. Styrene-divi-nylbenzene-based particles produced by a shape-template polymerization were also successfully tried as chromatographic packing material in size exclusion chromatography (SEC) [27,28]. The SEC calibration curves were almost identical for all of the beads produced with different concentrations of the cross-linking agent [27]. However, the more cross-linked particles exhibited better mechanical and hydrodynamic properties in chromatographic studies [27]. The same particles were also used as a stationary phase in protein separation RPLC [28]. These macroporous particles were obtained by using a porogen mixture composed of a low molecular weight organic agent and the linear polystyrene derived from the seed latex [28]. Frechet's group developed a shape-template polymerization method for the production of monodisperse-macroporous glyci-dyl methacrylate beads [29-32]. The effects of production conditions (i.e., the type and concentration of the swelling agent, inert porogenic solvent, and the cross-linking agent) on both the size and porosity properties and the chromato-graphic behavior of the columns including these particles were extensively investigated [29-32]. The pore size distribution of these particles could be controlled by using a chain transfer agent in the repolymerization stage [29]. Poly(dihydroxypropyl methacrylate-co-ethylene glycol dimethacrylate) particles obtained by the acidic hydrolysis of poly(glycidyl methacrylate-co-ethylene gly-col dimethacrylate)-poly(GMA-co-EGDMA) beads were tried as packing material in normal-phase HPLC [32]. The columns obtained with the hydrolyzed particles exhibited an excellent chromatographic performance in the separation of positional isomers of benzene derivatives and hydrophilic poly(ethylene ox-ide)s differing only in their chain length [32]. The monodisperse-macroporous particles with reasonably polar character were obtained in the form of poly-(methacrylic acid-co-ethylene glycol dimethacrylate) and poly(2-hydroxyethyl methacrylate-co-ethylene glycol dimethacrylate) [33]. The chiral supports obtained from these beads were successfully used as stationary phase for the enan-tiomeric separation of amino acids by normal-phase HPLC [33]. Recently, thermoresponsive chromatographic supports in form of poly(N-isopropylacry-lamide)-grafted macroporous poly(ethylene glycol dimethacrylate) particles were obtained by Frechet et al. [34]. In their study, temperature-dependent SEC calibration curves were obtained with the produced supports by using dextran standards [34]. A pore size-specific functionalization process was applied to the monodisperse and macroporous poly(GMA-co-EGDMA) particles for the synthesis of a separation media for the complete separation of complex samples that require a combination of size exclusion or ion exchange with reversed-phase chromatographic modes [35]. In the synthesis of the separation medium, the large pores of the poly(GMA-co-EGDMA) particles were selectively hydro-

lyzed to diol groups whereas the small pores were modified by highly hydropho-bic octadecylamine [35]. The pore size-specific functionalized particles were successfully used in the complete separation of samples including both hydro-philic proteins and hydrophobic drugs [35]. A similar study on the pore size-specific functionalization of poly(GMA-co-EGDMA) particles by a different protocol was also performed by the same group [36].

In our studies on the production of monodisperse-macroporous particles suitable for HPLC applications, poly(styrene-co-divinylbenzene)-poly(S-co-DVB)-based materials were predominantly investigated [37-40]. Following the extraction with tetrahydrofuran (THF), the poly(styrene-co-divinylbenzene) particles with different size and porosity properties were slurry charged into the stainless steel HPLC columns (30 x 7.8 mm) under a pressure of 150 atm. The chromato-graphic performance of these columns was tested both in SEC and RPLC. In the chromatographic studies performed with the SEC mode, the polystyrene standards in the MW range of 2100-1,460,000 were injected into the columns containing monodispersed polystyrene-divinylbenzene beads. The liquid chro-matograms obtained with the poly(S-co-DVB) beads 7 pm in size with an average pore size of approximately 200 nm were exemplified in Fig. 14. Here THF

A

4

A !

^ , 1 1 \

Time (minutes)

FIG. 14 Liquid chromatograms of polystyrene standards in the average MW range of 2100-1,460,000. Order of elution: 1, MW 1,460,000; 2, MW 288,000; 3, MW 92,000; 4, MW 19,000; 5, MW 2100.

was used as the mobile phase with a volumetric flow rate of 1 mL/min. The polystyrene samples were monitored with a UV-Vis detector at a wavelength of 254 nm.

The calibration curves obtained with the evaluation of these chromatograms are given in Fig. 15. Here the curves obtained with the columns including mono-disperse-macroporous poly(S-co-DVB) beads produced with two different cross-linking agent (i.e., DVB) feed concentrations were exemplified. The curves were almost linear for the entire range of MW tried. Thus, one concludes that the tried SEC columns can be utilized for the determination of average MW in the range of 2100-1,460,000. The variation of column back pressure with the mobile phase flow rate should be considered as another important parameter strongly affecting the flow regime in the chromatographic column. For this reason, the variation of column back pressure with the mobile-phase flow rate was investigated by selecting a commercial column containing monodisperse-macroporous particles in the polydispersed form as a reference material. The variation of column back pressure with the flow rate of mobile phase is comparatively shown for monodispersed and polydispersed poly(S-co-SVB) particles

10"

Log (molecular weight)

FIG. 15 Calibration curve indicating the variation of average MW of polystyrene standards with the retention time within the SEC column containing monodisperse-macropo-rous 7-|lm particles.

FIG. 16 Variation of column back pressure with the mobile phase flow rate. Mobile phase:THF.

in Fig. 16. In the studied range of flow rate, highly linear relations were obtained for both columns. The columns containing monodispersed particles provided lower back pressures at constant flow rate.

The chromatographic performance of the poly(S-co-DVB) particles was also investigated in the RPLC mode for the separation of proteins. Here a protein mixture that was very similar to those used by different researchers, including four proteins with different hydrophilicities (i.e., cytochrome c, lysozyme, albumin, and insulin), was used. Figure 17 exemplifies the liquid chromatograms of the protein mixture taken with acetonitrile/water gradient from 30% to 70% (v/ v) acetonitrile in 40 min. Here the 7-p.m poly(S-co-DVB) particles were used as the column-packing material. The chromatograms were taken with a UV-Vis detector at 280 nm. As seen here, the chromatograms with high resolutions could be achieved by using monodisperse and macroporous poly(S-co-DVB)

0 10 20 30 Time {minutes)

FIG. 17 Sample liquid chromatogram of a protein mixture including four different proteins. UV-Vis detector at 280 nm, Acetonitrile/water gradient from 30% to 70% ace-tonitrile in 40 min. Flow rate: 1 mL/min. Order of elution: l, Cytochrome c; 2, Lyso-zyme; 3, albumin; 4, insulin.

beads. It should also be noted that these high resolutions are valid for a broad range of mobile phase flow rate (i.e., 1-4 mL/min).

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