Particles For Affinity Chromatography

The affinity high-performance liquid chromatography (affinity HPLC) applications have been performed by using stationary phases based on macroporous particles functionalized with the ligands sensitive to the biomolecules. The mac-roporous particles carrying functional groups are particularly important for the synthesis of affinity HPLC packing materials since their functional groups can be utilized for the covalent attachment of various ligands with specific recognition abilities.

FIG. 2 Variation of equilibrium BSA adsorption capacity with the initial BSA concentration for the CBF3G-A attached latex particles PVA-coated latex particles, and plain polystyrene particles. (Reprinted with permission from Ref. 13, Copyright © 1992, Elsevier Science.)

The variation of BSA adsorption capacity of the macroporous particles having different surface chemistries with the initial BSA concentration is given in Fig. 3 [14]. As seen here, the BSA adsorption capacities of plain poly(S-DVB) and the particles modified with relatively hydrophobic acrylic monomers like epoxypropyl methacrylate (EPMA) and methyl methacrylate (MMA) provided high nonspecific albumin adsorption at the IEP of BSA [14]. The macroporous particles produced in the presence of relatively hydrophilic monomers like 2-hydroxyethylmethacrylate (HEMA) and methacrylic acid (MAAc) exhibited lower non-specific albumin adsorption capacities [14]. Therefore, the presence of hydrophilic units on the macroporous surface caused a decrease in the hydro-phobic interaction between BSA and the particle surface. However, butyl methacrylate (BMA)-modified particles exhibited surprisingly low BSA adsorption capacity [14]. The macroporous particles carrying strongly entrapped or cova-lently bonded PVA chains on their surface had nearly zero nonspecific albumin adsorption [14]. This result is particularly important for the development of specific affinity HPLC sorbents exhibiting no nonspecific protein adsorption behavior.

The monodisperse-macroporous particles produced by including MMA, BMA, HEMA, or MAAc in the repolymerization stage and the plain poly(S-DVB) parti-

Initial BSA concentration (mg/mL)

FIG. 3 Variation of BSA equilibrium adsorption capacity of uniform macroporous particles prepared with different surface chemistries with the initial BSA concentration. (Reprinted with permission from Ref. 14, Copyright © 2002, John Wiley and Sons, Inc.)

cles provided desorption ratios higher than 80% (w/w) [14]. These results indicated that albumin was physically adsorbed onto the surface of these particles. However, the low desorption ratio with the EPMA-functionalized particles (about 5%) was evaluated as an evidence of the irreversible chemical interaction between epoxypropyl groups of the particles and the primary amine groups of BSA [14].

The use of macroporous particles as affinity supports in the chromatographic separation of proteins has attracted significant attention. The interactions of PVA-coated macroporous poly(S-DVB) particles were investigated by Leonard et al. [10]. Procion yellow HE3G-carrying macroporous particles were tried as the stationary phase for lysozyme and human serum albumin (HSA) separation [11]. Triazinyl dye-carrying perfluoropolymer supports in the particulate form were used as sorbents for the specific isolation of different proteins [12]. However, all particle types tried for protein separation were produced in the poly-dispersed form. By considering the potential advantages of the macroporous particles in the monodispersed form as a column-packing material in the chro-

matographic applications, we attempted to develop an affinity HPLC support material by starting from the monodispersed poly(S-DVB) particles.

A triazinyl dye acting an affinity ligand for albumin, CBF3G-A was immobilized on the surface of the PVA-carrying monodisperse-macroporous particles [15]. PVA-functionalized monodisperse-macroporous poly(S-DVB) particles of 6.25 pm were obtained by a multistage dispersion polymerization by including PVA as the steric stabilizer [15]. The representative electron micrographs of the particles are given in Fig. 4 [15]. The median pore diameter and the specific surface area of the particles were 104 nm and 24.8 m2/g, respectively [15]. The presence of PVA chains on the surface was shown by the comparison of the FTIR-DRS (Fourier transform infrared-diffuse reflectance spectroscopy) spectrum of PVA-functionalized particles with that of the plain poly(S-DVB) parti-

FIG. 4 Representative electron micrographs showing the size distribution and the detailed surface morphology of PVA functionalized, monodisperse-macroporous particles. Magnification (A) x1000, (B) x4000. (Reprinted with permission from Ref. 15, Copyright © 1999, VSP International Science Publishers.)

FIG. 4 Representative electron micrographs showing the size distribution and the detailed surface morphology of PVA functionalized, monodisperse-macroporous particles. Magnification (A) x1000, (B) x4000. (Reprinted with permission from Ref. 15, Copyright © 1999, VSP International Science Publishers.)

cles [15]. For the covalent attachment of CBF3G-A onto the PVA chains, a route similar to that used for nonporous particle was followed [13,15].

For the monodisperse-macroporous particles with different surface chemistries, the variation of equilibrium BSA adsorption capacity with the initial BSA concentration is presented in Fig. 5 [15]. As seen in the figures, the plain poly(S-DVB) particles exhibited some nonspecific BSA adsorption probably due to the relatively hydrophobic character of the particle surface. However, no significant nonspecific BSA adsorption with the PVA-functionalized poly-(S-DVB) particles indicated that the presence of a PVA layer on the surface strongly inhibited the hydrophobic interaction [15]. Albumin adsorption capacities up to 100 mg BSA/g particles were achieved with the CBF3G-A-attached particles due to the pseudospecific interaction between BSA and immobilized dye molecules [15].

It should be noted that the equilibrium BSA adsorption capacities of 60 mg/g could be obtained with the CBF3G-A-attached uniform polystyrene particles in the nonporous form [13]. Larger specific surface area of the macroporous material was probably responsible for the higher BSA adsorption [15]. In the

FIG. 5 Variation of equilibrium BSA adsorption capacity with the initial BSA concentration for the dye attached, PVA functionalized and plain poly(S-DVB) particles. (Reprinted with permission from Ref. 15, Copyright © 1999, VSP International Science Publishers.)

desorption experiments performed at pH 8 in the presence of 1 M NaSCN, desorption yields of approximately 90% (w/w) of adsorbed BSA could be obtained [15].

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