TY - JOUR
AU1 - Gama, Mariana, R.
AU2 - Aggarwal,, Pankaj
AU3 - Liu,, Kun
AU4 - Lee, Milton, L.
AU5 - Bottoli, Carla, B.G.
AB - Abstract Capillary columns containing butyl or lauryl methacrylate monoliths were prepared using two different free-radical polymerization methods: conventional free-radical polymerization and controlled/living free-radical polymerization, both initiated thermally, and these methods were compared for the first time. Both monolith morphology and chromatographic efficiency were compared for the synthesized stationary phases using scanning electronic microscopy (SEM) and capillary liquid chromatography, respectively. Columns prepared using controlled method gave better chromatographic performance for both monomers tested. The lauryl-based monolith showed 7-fold improvement in chromatographic efficiency with a plate count of 42,000 plates/m (corrected for dead volume) for a non-retained compound. Columns fabricated using controlled polymerization appeared more homogenous radially with fused small globular morphologies, evaluated by SEM, and lower column permeability. The columns were compared with respect to resolving power of a series of alkylbenzenes under isocratic and gradient elution conditions. Introduction Monolithic (both silica and organic) columns were introduced as a low pressure alternative to particle packed columns for liquid chromatography (LC) separations in the late 1980s and early 1990s, especially for miniaturized separation instrumentation (1–5). Monolithic columns were seen as a major breakthrough in column technology for LC, providing performance comparable to classical high performance liquid chromatography (HPLC) columns packed with 3–5 µm diameter particles, at 5–10 times lower back pressure (6–8). However, a significant improvement in chromatographic efficiency of packed columns associated with development of sub-2 micron particles and modern core–shell technology quickly decreased enthusiasm for monolithic columns (9). The performance of silica monoliths improved significantly through understanding and optimizing the sol–gel synthesis method. Silica monoliths have been successfully used for both small and large molecule separations, and are commercially available. However, organic monoliths have lagged behind the other column technologies in chromatographic performance, and are still largely employed for large biomolecule separations. This is primarily due to their high gel porosity, low mesopore volume and inherent structural heterogeneity (10, 11) associated with the polymerization conditions (12–14). Organic monoliths have conventionally been fabricated using conventional free-radical polymerization, which is difficult to control with regard to molecular weight of the growing polymer chain. The rapid initiation and terminations reactions usually result in the formation of microgels with highly heterogeneous polymer network structures, providing mediocre separation performance (15–17). Major efforts are now being directed toward obtaining more homogenous monolith structures with well-defined skeleton and pore size dimensions, essential for obtaining excellent chromatographic efficiencies. Living free-radical polymerization, also known as controlled polymerization, has been employed for fabricating branched polymers and monoliths (18–21). In living radical polymerization, there exists an equilibrium between the growing radical chain and dormant species, slightly favoring the dormant species (22, 23). This reversible equilibrium increases the time of chain propagation, giving it sufficient time to relax and distribute homogenously (24, 25). Moreover, the reversibility provides much better control over the molecular weight distribution of the growing radical chain and on the final monolith morphology. There have been reports of using living radical polymerization for controlling monolith morphology using different initiation systems. Yu et al. used atom transfer free-radical polymerization to prepare a poly(ethylene glycol dimethacrylate-co-ethylene glycol methyl ether methacrylate) monolith (15). Nitroxide-mediated living radical polymerization was used for fabricating a poly(styrene-co-divinylbenzene) monolith (13). However, the complex reaction kinetics, the difficulty in optimizing the reaction conditions in atom transfer polymerization, and the high temperatures required for nitroxide reaction of styrene monomers prevent the using of this reaction for easy syntheses in capillary dimensions (12). Recently, organotellurium-mediated living radical polymerization (TERP), a new branch of living radical polymerization, was employed to fabricate poly(styrene-co-divinylbenzene) (26), poly(glycidyl methacylate) (27) and poly(N,N-methylenebiacrylamide) (28) monoliths. The reaction mechanism involved generation of carbon-centered radicals by thermolysis to initiate polymerization in the presence of a thermal initiator, azo-bis isobutyronitrile (29, 30). This polymerization method takes place under mild polymerization conditions, is compatible with different functional group monomers, and is relatively easy to optimize, thereby overcoming the limitations of atom transfer free-radical and nitroxide-mediated living radical polymerizations while still maintaining precise control over the monolith morphology. Therefore, there has been a growing interest in fabricating monolithic columns using organotellurium polymerization (31, 32). Although there are numerous reports of using TERP for monolith fabrication and comparing bulk morphologies, a direct comparison of capillary LC column performance for similar columns prepared by conventional and TERP methods has not been conducted. In this work, the morphologies and chromatographic performance of butyl methacrylate-co-ethylene glycol dimethacrylate (BMA-co-EGDMA) monoliths fabricated using conventional and TERP methods were investigated. The results were further confirmed by testing an additional monomer, i.e., lauryl methacrylate (LMA). The monolith morphologies were characterized using scanning electron microscopy (SEM), and the chromatographic performance was evaluated by measuring the chromatographic efficiencies for a series of alkylbenzenes. Experimental Chemicals and materials The chemicals 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), azobisisobutyronitrile (AIBN), LMA (96%), BMA (99%), ethylene glycol dimethacrylate (EGDMA, 98%), 3-(trimethoxysilyl)propyl methacrylate (TPM, 98%), toluene, 1,4-butanediol, uracil, propylbenzene, butylbenzene and pentylbenzene were purchased from Sigma-Aldrich (St. Louis, USA). Ethylbenzene was purchased from Fisher Chemicals (Fair Lawn, NJ, USA); water (HPLC grade) was purchased from Macron Fine Chemicals (Center Valley, PA, USA); and n-propanol was acquired from Mallinckrodt (Phillipsburg, USA). The TERP promoter, ethyl-2-methyl-2-butyltellanyl propionate (BTEE), was a gift from Otsuka Chemical Co. (Osaka, Japan). Since BTEE is oxygen sensitive, it was stored in vials that had been carefully cleaned and dried, and all transfers were conducted inside a nitrogen glove box. All reagents were analytical grade and were used as received. Preparation of polymeric monolithic columns Fused silica capillaries, 150 µm i.d., from Polymicro Technologies (Phoenix, AZ, USA) were treated using TPM to anchor the polymeric monolith on the capillary wall as described previously (33, 34). Monoliths were prepared by weighing the appropriate amounts of BMA or LMA as monomer, 1,4-butanediol and n-propanol as porogenic solvents, and DMPA or AIBN as initiator, for thermal polymerization (see Table I). To perform TERP, a fixed amount of BTEE was added to the polymerization mixture. Each solution was sonicated for 1 min and degassed using nitrogen flow for 3 min before it was introduced into a pre-treated capillary. Both column ends were sealed with rubber septa before continuing with thermal initiation. Thermal initiation was performed at 60°C for 3 h in an oil bath. For TERP, the column was placed in an oil bath at 60°C for 24 h. After preparation, each monolithic column was flushed with methanol and water for 2 h to remove unreacted monomer and residual porogens. Table I. Chemical Compositions of Polymeric Monolithic Columns Prepared Using Two Different Initiation Methods Monolitha . Polymerization method . Polymer chemistryb . Porogens (%) . Initiatorc (%) . n-propanol . 1,4-butanediol . 1 Conventional BMA-co-EGDMA 34 26 1.0 2 TERP BMA-co-EGDMA 40 20 0.1 3 Conventional LMA-co-EGDMA 34 26 1.0 4 TERP LMA-co-EGDMA 30 30 0.1 Monolitha . Polymerization method . Polymer chemistryb . Porogens (%) . Initiatorc (%) . n-propanol . 1,4-butanediol . 1 Conventional BMA-co-EGDMA 34 26 1.0 2 TERP BMA-co-EGDMA 40 20 0.1 3 Conventional LMA-co-EGDMA 34 26 1.0 4 TERP LMA-co-EGDMA 30 30 0.1 aPolymerization mixtures contained 40% (wt/wt) monomers, 60% (wt/wt) porogenic solvents and a variable amount of initiator, depending on the polymerization method. The reaction was carried out in 15 cm × 150 µm i.d. capillaries. bThe ratio of monomer and cross-linker was kept constant in all recipes (24:16 % wt/wt). cConventional polymerization was performed with 1.0% AIBN (wt% relative to total monomers) and controlled polymerization was performed with 0.1% AIBN (wt% relative to total monomers) and a fixed amount of BTEE (0.6 µL) as polymerization promoter. Table I. Chemical Compositions of Polymeric Monolithic Columns Prepared Using Two Different Initiation Methods Monolitha . Polymerization method . Polymer chemistryb . Porogens (%) . Initiatorc (%) . n-propanol . 1,4-butanediol . 1 Conventional BMA-co-EGDMA 34 26 1.0 2 TERP BMA-co-EGDMA 40 20 0.1 3 Conventional LMA-co-EGDMA 34 26 1.0 4 TERP LMA-co-EGDMA 30 30 0.1 Monolitha . Polymerization method . Polymer chemistryb . Porogens (%) . Initiatorc (%) . n-propanol . 1,4-butanediol . 1 Conventional BMA-co-EGDMA 34 26 1.0 2 TERP BMA-co-EGDMA 40 20 0.1 3 Conventional LMA-co-EGDMA 34 26 1.0 4 TERP LMA-co-EGDMA 30 30 0.1 aPolymerization mixtures contained 40% (wt/wt) monomers, 60% (wt/wt) porogenic solvents and a variable amount of initiator, depending on the polymerization method. The reaction was carried out in 15 cm × 150 µm i.d. capillaries. bThe ratio of monomer and cross-linker was kept constant in all recipes (24:16 % wt/wt). cConventional polymerization was performed with 1.0% AIBN (wt% relative to total monomers) and controlled polymerization was performed with 0.1% AIBN (wt% relative to total monomers) and a fixed amount of BTEE (0.6 µL) as polymerization promoter. Capillary LC The literature has described the use of alkylbenzenes as standards for the characterization of selectivity of stationary phases for reversed-phase separations (35, 36). A test mixture of alkylbenzenes (benzene, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene) was prepared in acetonitrile/water (70:30, v/v) to give a concentration of each analyte of 0.5% (v/v). A uracil solution was prepared in the same solvent composition at a concentration of 2 mg mL−1. Separations were performed using an Eksigent Nano 2D LC system (Dublin, CA, USA) with an injection volume of 30 nL. The mobile phase was a mixture of acetonitrile and water. On-column detection was performed at 214 nm using a Crystal 100 variable wavelength UV-detector from Thermo Scientific (San Jose, CA, USA), and data were processed using Chrom Perfect software (Mountain View, CA, USA). UV absorbance was monitored at 214 nm. Column permeability The chromatographic permeability (K) was determined using Darcy's law K=ηLuΔP, where η is the mobile phase viscosity, L is the column length, u is the mobile phase linear velocity and ΔP is the column back pressure (i.e., column pressure drop). The mobile phase used for calculating permeability values was HPLC grade water. Scanning electronic microscopy For analysis by SEM, fragments of the column (4.0 mm length) were cut and fixed to a gold metallic support using double-sided carbon tape. Then, a gold layer of 10 nm was formed on each sample by vapor deposition under vacuum. After that, the monoliths were characterized using a Jeol JMS 6360-LV (Tokyo, Japan) scanning electron microscope. Results Rigid macroporous monoliths were obtained in this study using two polymerization methods, i.e., conventional and TERP, with different functional monomers (i.e., BMA and LMA) and the same cross-linker (EGDMA). The effect of mixture composition (i.e., porogens, weight proportion of simple monomers and initiator/promoter constituents) and fabrication conditions were also investigated for each method. Figure 1 compares the SEM morphology obtained from BMA-co-EGDMA and LMA-co-EGDMA monoliths when prepared by conventional polymerization and TERP, respectively. Figure 1. Open in new tabDownload slide SEM images of (A) BMA-co-EGDMA and (B) LMA-co-EGDMA monoliths using (A1, B1) conventional free-radical polymerization and (A2, B2) TERP at 60°C. The images represent to ×1200 magnification. Figure 1. Open in new tabDownload slide SEM images of (A) BMA-co-EGDMA and (B) LMA-co-EGDMA monoliths using (A1, B1) conventional free-radical polymerization and (A2, B2) TERP at 60°C. The images represent to ×1200 magnification. The chromatographic parameters (chromatographic efficiency, in plates per meter) for the separation of alkylbenzenes—toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene—using BMA and LMA stationary phases are summarized in Tables II and III. Table II. Separation Efficiencies for Alkylbenzenes Measured Using BMA and LMA Monolithic Columns Monolith . Polymerization method . Chromatographic efficiency (plates/m) . Uracil . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6,400 10,000 8,000 6,100 5,000 4,500 TERP 18,200 20,800 22,000 24,000 23,000 18,000 LMA-co-EGDMA Conventional 4,600 3,600 2,800 2,600 3,800 TERP 28,000 35,000 33,000 31,000 28,000 28,000 Monolith . Polymerization method . Chromatographic efficiency (plates/m) . Uracil . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6,400 10,000 8,000 6,100 5,000 4,500 TERP 18,200 20,800 22,000 24,000 23,000 18,000 LMA-co-EGDMA Conventional 4,600 3,600 2,800 2,600 3,800 TERP 28,000 35,000 33,000 31,000 28,000 28,000 Table II. Separation Efficiencies for Alkylbenzenes Measured Using BMA and LMA Monolithic Columns Monolith . Polymerization method . Chromatographic efficiency (plates/m) . Uracil . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6,400 10,000 8,000 6,100 5,000 4,500 TERP 18,200 20,800 22,000 24,000 23,000 18,000 LMA-co-EGDMA Conventional 4,600 3,600 2,800 2,600 3,800 TERP 28,000 35,000 33,000 31,000 28,000 28,000 Monolith . Polymerization method . Chromatographic efficiency (plates/m) . Uracil . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6,400 10,000 8,000 6,100 5,000 4,500 TERP 18,200 20,800 22,000 24,000 23,000 18,000 LMA-co-EGDMA Conventional 4,600 3,600 2,800 2,600 3,800 TERP 28,000 35,000 33,000 31,000 28,000 28,000 Table III. Retention Factors for Alkylbenzenes Measured Using BMA and LMA Monolithic Columns Monolith . Polymerization method . Permeability (×10−14 m2) . Retention factor (k) . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6.01 0.88 1.11 1.42 1.87 2.43 TERP 0.08 1.17 1.52 2.01 2.72 3.64 LMA-co-EGDMA Conventional 4.44 0.96 1.36 1.82 2.65 TERP 2.27 0.93 1.20 1.69 2.44 3.49 Monolith . Polymerization method . Permeability (×10−14 m2) . Retention factor (k) . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6.01 0.88 1.11 1.42 1.87 2.43 TERP 0.08 1.17 1.52 2.01 2.72 3.64 LMA-co-EGDMA Conventional 4.44 0.96 1.36 1.82 2.65 TERP 2.27 0.93 1.20 1.69 2.44 3.49 Table III. Retention Factors for Alkylbenzenes Measured Using BMA and LMA Monolithic Columns Monolith . Polymerization method . Permeability (×10−14 m2) . Retention factor (k) . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6.01 0.88 1.11 1.42 1.87 2.43 TERP 0.08 1.17 1.52 2.01 2.72 3.64 LMA-co-EGDMA Conventional 4.44 0.96 1.36 1.82 2.65 TERP 2.27 0.93 1.20 1.69 2.44 3.49 Monolith . Polymerization method . Permeability (×10−14 m2) . Retention factor (k) . Toluene . Ethylbenzene . Propylbenzene . Butylbenzene . Pentylbenzene . BMA-co-EGDMA Conventional 6.01 0.88 1.11 1.42 1.87 2.43 TERP 0.08 1.17 1.52 2.01 2.72 3.64 LMA-co-EGDMA Conventional 4.44 0.96 1.36 1.82 2.65 TERP 2.27 0.93 1.20 1.69 2.44 3.49 The chromatograms for the separation of alkylbenzene series with BMA-co-EGDMA and LMA-co-EGDMA monolithic columns are shown in Figures 2 and 3, respectively. Figure 2. Open in new tabDownload slide Separations of alkylbenzenes on BMA-co-EGDMA monolith columns fabricated using (A) conventional free-radical polymerization and (B) TERP. Conditions: linear gradient from 70% to 100% acetonitrile in 15 min, then isocratic elution with 100% acetonitrile; mobile phase flow rate of 200 nL/min; on-column UV detection at 214 nm. Peak identifications in order of elution: uracil, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene. The chromatogram was baseline corrected using Excel. Figure 2. Open in new tabDownload slide Separations of alkylbenzenes on BMA-co-EGDMA monolith columns fabricated using (A) conventional free-radical polymerization and (B) TERP. Conditions: linear gradient from 70% to 100% acetonitrile in 15 min, then isocratic elution with 100% acetonitrile; mobile phase flow rate of 200 nL/min; on-column UV detection at 214 nm. Peak identifications in order of elution: uracil, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene. The chromatogram was baseline corrected using Excel. Figure 3. Open in new tabDownload slide Separations of alkylbenzenes on LMA-co-EGDMA monolith columns fabricated using (A) conventional free-radical polymerization and (B) TERP. Conditions: linear gradient from 70% to 100% acetonitrile in 15 min, then isocratic elution with 100% acetonitrile; mobile phase flow rate of 200 nL/min; on-column UV detection at 214 nm. Peak identifications in order of elution: uracil, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene. The chromatogram was baseline corrected using Excel. Figure 3. Open in new tabDownload slide Separations of alkylbenzenes on LMA-co-EGDMA monolith columns fabricated using (A) conventional free-radical polymerization and (B) TERP. Conditions: linear gradient from 70% to 100% acetonitrile in 15 min, then isocratic elution with 100% acetonitrile; mobile phase flow rate of 200 nL/min; on-column UV detection at 214 nm. Peak identifications in order of elution: uracil, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene. The chromatogram was baseline corrected using Excel. SEM morphologies and column efficiencies for uracil—the non-retained compound—measured using BMA and LMA monolithic columns prepared by TERP using three different porogen ratios, are compared in Figure 4 and Table IV, respectively. Figure 4. Open in new tabDownload slide SEM images of (A1, B1, C1) BMA-co-EGDMA and (A2, B2, C2) LMA-co-EGDMA monoliths prepared using TERP with (A) 0.30:0.30, (B) 0.34:0.26 and (C) 0.40:0.20 (wt/wt) ratios of n-propanol and 1,4-butanediol, respectively, of porogenic solvents. The images represent ×1200 magnification. Figure 4. Open in new tabDownload slide SEM images of (A1, B1, C1) BMA-co-EGDMA and (A2, B2, C2) LMA-co-EGDMA monoliths prepared using TERP with (A) 0.30:0.30, (B) 0.34:0.26 and (C) 0.40:0.20 (wt/wt) ratios of n-propanol and 1,4-butanediol, respectively, of porogenic solvents. The images represent ×1200 magnification. Table IV. Column Efficiencies for a Non-retained Compound, Uracil, Measured Using BMA and LMA Monolithic Columns Prepared by TERP Using Three Different Porogen Ratios Monolith . Chromatographic efficiency (plates/m) . 1.0a . 1.3a . 2.0a . BMA-co-EGDMA 15,000 9,700 18,000 LMA-co-EGDMA 23,000 28,000 7,300 Monolith . Chromatographic efficiency (plates/m) . 1.0a . 1.3a . 2.0a . BMA-co-EGDMA 15,000 9,700 18,000 LMA-co-EGDMA 23,000 28,000 7,300 aPorogen ratio. Table IV. Column Efficiencies for a Non-retained Compound, Uracil, Measured Using BMA and LMA Monolithic Columns Prepared by TERP Using Three Different Porogen Ratios Monolith . Chromatographic efficiency (plates/m) . 1.0a . 1.3a . 2.0a . BMA-co-EGDMA 15,000 9,700 18,000 LMA-co-EGDMA 23,000 28,000 7,300 Monolith . Chromatographic efficiency (plates/m) . 1.0a . 1.3a . 2.0a . BMA-co-EGDMA 15,000 9,700 18,000 LMA-co-EGDMA 23,000 28,000 7,300 aPorogen ratio. Discussion Selection of polymerization conditions The selection of organic solvent porogens is an important step in monolith fabrication. One of the monomers, BMA, was used to select the porogenic solvents for conventional initiation. Solvent polarity and solubility values were used as physical/chemical parameters to aid in porogen selection. A porogen with a solubility value similar to the monomer was considered to be a good porogen, while one with a large difference in solubility value was a bad porogen. The porogens in this study varied widely in terms of their polarity and solubility values. It was found that use of a long chain aliphatic alcohol, such as dodecanol, resulted in formation of a gel or a monolith with very low permeability. Monoliths fabricated using methanol as one of the porogens gave monoliths with very high permeability; however, the chromatographic performance was very poor for these columns. Therefore, to fabricate a monolith with intermediate permeability and reasonable chromatographic performance, solvents such as n-propanol, isobutanol, 1,4-butanediol and ethylene glycol were studied. Although BMA monoliths could be formed from various combinations of these four porogens, those prepared from n-propanol and 1,4-butanediol gave better chromatographic performance at reasonable column back pressure. Therefore, n-propanol and 1,4-butanediol were used as porogens for both BMA and LMA monoliths (Table I). Also, the same porogens were used irrespective of the initiation method so as to limit the number of variables and for understanding the influence of initiation method on column morphology and performance. The polymerization time for conventional method was selected to be 3 h. Any longer polymerization time was found to have no effect on chromatographic performance of methacrylate-based monolith (37). However, since the polymerization of TERP was slower, it was carried out for 24 h. The second important parameter in monolith fabrication is the polymerization temperature. The most commonly used polymerization temperature reported for conventional polymerization is 60°C. Therefore, the polymerization temperature was set at 60°C for both initiation methods to minimize the number of variables for comparison. Moreover, the polymerization was too slow at 50°C with AIBN as initiator, and a temperature above 60°C resulted in a monolith with very low permeability when polymerized using TERP. Monolith morphology and chromatographic performance The BMA-co-EGDMA monoliths were fabricated using the same porogens (Table I). Those fabricated using TERP showed a 3-fold improvement in chromatographic efficiency (Table II) for uracil, the non-retained analyte. Similar improvement in column performance was observed for alkylbenzenes, the retained analytes. This improvement in column efficiency can be ascribed to the difference in monolith morphology as seen in the SEM images in Figure 1. The monolith fabricated using TERP shows a more homogenous morphology with smaller globule in comparison to the monolith fabricated using conventional method. The smaller globule lessen the resistance to mass transfer of the analyte, while the improved homogeneity reduces the dispersion due to eddy diffusion. These changes in skeletal dimensions were further confirmed by the lower column permeability and increased retention values for the retained compounds when separated using column fabricated using TERP (Table III). Monoliths fabricated using TERP also showed significant improvement in resolution. A mixture of five alkylbenzenes was base-line resolved with good peak shapes, which was not the case using columns fabricated using conventional polymerization, and the Figure 2 shows gradient elution separations of a mixture of alkylbenzenes on BMA-co-EGDMA monoliths fabricated using the different initiation methods. The improvement in monolith morphology and accompanying improvement in chromatographic performance can be explained on the basis of the reaction kinetics of TERP. The reversible and slow polymerization rate in TERP provides better control over the molecular weight of the growing polymer chain, leading to a narrower distribution and a more homogeneous monolith morphology. LMA-co-EGDMA monoliths showed similar improvement in chromatographic performance and in monolith morphology. Monoliths fabricated using TERP showed a fused morphology that was more homogenous than that obtained by conventional method. The chromatographic efficiency was improved by a factor of 7 for a non-retained compound and by a factor of ~9 for a retained compound (toluene). The retention was found to be slightly less for monoliths fabricated using TERP in comparison to monoliths fabricated using conventional polymerization reaction. This lower retention could be due to the fused morphology rather than the globular. However, the improved chromatographic performance with change in morphology was clearly evident. The improved resolution in gradient elution of alkylbenzenes (Figure 3) and improved efficiency (Table II) when using LMA-co-EGDMA monoliths fabricated using TERP further verifies the improved performance observed when using a TERP. In a recent work, we found that the injection system often contributes significant extra-column volume, which adversely affects the measured column efficiencies for small-diameter columns (38). The extra-column volume of the injection valve for the capillary LC system used in this work was determined to be ~15 nL at flow rate of 200 nL/min. Correcting for this extra-column contribution, the column performance was found to improve by ~49% (e.g., 28,000–42,000 plates/m) for a non-retained compound (i.e., uracil) and ~13% for a retained compound (i.e., toluene, k = 0.93). All efficiency values reported in this manuscript represent the actual measured values (unless stated otherwise) and could be corrected to indicate the true column performance. The retention times observed when using the LMA monolith were found to be higher for all of the compounds in comparison to the BMA monolith for columns fabricated using conventional method. The increased hydrophobicity of the monolith because of the longer alkyl chain length (C12 vs C4) and the different monolith morphology well explain the observed trend. However, the retention times were lower for LMA monoliths fabricated using TERP. This difference could be assigned to differences in monolith morphologies of BMA and LMA columns fabricated using TERP. The LMA monoliths show more fused morphology in comparison to the tiny microglobules observed in the BMA monolith. The fused morphology displays better homogeneity, thereby leading to improved chromatographic efficiency. However, the fused skeletal mass would have lower surface area, thereby causing less retention. Morphology optimization for TERP The morphology and homogeneity of a fabricated column are determined by the processes occurring during monolith synthesis, i.e., gelation and phase separation. Fabricated monoliths have been shown to have both homogeneity and good performance if both gelation and phase separation occur at the same time. If gelation occurs earlier than phase separation, the resultant structure is a gel with no distinct macropores. Early or delayed phase separation of the growing polymer chain leads to a monolith with globular morphology and increased structural heterogeneity. Such columns show relatively poor chromatographic performance for small molecule separations. Therefore, preparation of BMA and LMA monoliths using TERP was further investigated using different weight ratios of porogens to provide improved chromatographic performance. Uracil was used as a non-retained analyte in the efficiency measurements. The chromatographic performance of BMA monoliths was found to first worsen and then improve (Table IV) with an increase in porogen ratio (i.e., ratio of the amount of n-propanol to 1,4-butanediol). This change in performance can be ascribed to a change in skeletal dimensions visible in the SEM images. The average globule size increased with an increase in porogen ratio from 1.0 to 1.3. However, further increase in porogen ratio to 2.0 caused a significant decrease in globule size (Figure 4). The change in porogen ratio changes the times that gelation and phase separation occur. For phase ratios of 1.0 and 1.3, phase separation appears to occur much later than gelation, leading to formation of large globular morphology. This lag in phase separation and gelation is reduced significantly at a porogen ratio of 2.0, leading to formation of very small globules. The behavior of BMA monolith formation was further verified by investigating the effect of porogen ratio on morphology of LMA monoliths. Similar trends were observed with improved chromatographic performance when using monoliths with fused morphology. A porogen ratio of 1.3 gave the best performance for LMA monoliths. At higher porogen ratio (i.e., 2.0) the morphology became globular, indicating an occurrence of delayed phase separation. Conclusion The chromatographic efficiency of monolithic columns was improved by adjusting the monolith morphology. The inherent structural heterogeneity associated with free-radical polymerization was significantly reduced by using TERP. The differences in chromatographic performance resulting from different polymerization methods were verified using two different monomer systems. BMA and LMA monolithic columns fabricated using TERP showed >3-fold and 7-fold improvement in column performance, respectively. The LMA-co-EGDMA monolith fabricated using TERP showed a chromatographic efficiency of 42,000 plates/m (when corrected for dead volume) for a non-retained compound. The observed trend in chromatographic efficiency was correlated to the structural morphology of the monolith. 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TI - Improvement in Liquid Chromatographic Performance of Organic Polymer Monolithic Capillary Columns with Controlled Free-Radical Polymerization
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmw193
DA - 2017-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/improvement-in-liquid-chromatographic-performance-of-organic-polymer-rOBMOL2fK8
SP - 398
VL - 55
IS - 4
DP - DeepDyve
ER -