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Comparison of the effects of pore structure and bead size on the degree of plasma chemical amine functionalization of polystyrene-divinylbenzene permanently porous resins. Permanently porous polystyrene-divinylbenzene (PS/DVB) resins were invented in the 1950s and are widely used as ion exchangers, adsorbents, separation media for chromatographic analysis, and solid-phase carriers in organic synthesis. They are excellent selective gel carriers with multiple applications, possessing special solid-phase chemistry, whose advantages are primarily ensured by a large number of functional groups. The various applications of polymer carriers in organic synthesis require careful design and control of the carrier’s pore structure. Polymer microspheres with fixed pores can be obtained through suspension polymerization, and they are particularly suitable for producing large-sized microsphere products, with typical particle sizes ranging from 5 to 1000 μm. By carefully selecting the pore-forming agent (along with the type and concentration of the crosslinking agent), a wide range of porous microspheres can be produced (according to the IUPAC definition – micropores: 500Å). The conditions for obtaining a permanent pore structure have been reviewed in the literature. It is certain that the porosity and specific surface area of microspheres have a strong influence on the polymer synthesis conditions, including the pore-forming agent composition, pore-forming agent concentration (dilution), degree of crosslinking, and reaction temperature. To obtain permanent pores with a high specific surface area, a high concentration of crosslinking agent is necessary ； Materials produced with insufficient crosslinking agents are essentially non-porous, even in the presence of a pore-forming agent solvent. This requires the improvement of solid-phase support reagents with higher loading capacities, as well as extracts that can easily reach the reaction site for use in organic synthesis. One approach may involve plasma chemical modification by introducing functional groups onto the surface of permanent resin pores. Plasmas are generally divided into three categories: plasmas in thermodynamic equilibrium ； Plasma in local equilibrium ； Plasma in a non-equilibrium state, namely so-called low-temperature plasma. The latter is usually achieved through low-pressure unloading, with the electron temperature (104–105 K) being much higher than that of the heavy particles (the overall temperature of the gas can be as low as room temperature). Thermodynamic equilibrium is not even achieved on a local scale ； Moreover, in these non-equilibrium plasmas, physical and chemical reactions can be initiated at relatively low temperatures. In the current study, functional group-free porous PS/DVB microspheres were treated with acrylamine low-temperature plasma to load amine groups on their surfaces. Using small batches of commercial resin, it is already known that the pore structure and size (particle diameter) of the microspheres are important in determining the degree of functionalization. However, it is also impossible to separate out the individual contributions of each characteristic pair to the degree of functionalization from this limited sample. Further work confirmed that the microsphere size has a strong influence, but this involved only one type of resin, so the effect of the pore structure was not significant. Therefore, it was decided to prepare a batch of PS/DVB resins with varying characteristics in terms of specific surface area, pore size, and pore volume. Many studies on the effects of pore-forming agent concentration and type utilize different levels of crosslinking agents in PS/DVB resins to analyze the physical properties of porous microspheres (primarily specific surface area). Studies have found that thermodynamically good solvents used to grow polymer networks can produce a higher proportion of micropores, resulting in a higher specific surface area. However, to our knowledge, no one has yet conducted a comprehensive study on the simultaneous effects of synthesis conditions on the specific surface area, pore volume, and pore size of permanent resinous adsorbents. Furthermore, the DVB levels in previous studies were relatively low; therefore, we used a resin with a crosslinking density of 72% in our study. The primary objective of the study is to detail the control of the parameters of interest—specifically, specific surface area, pore size, and pore volume—by the synthesis conditions (amount of crosslinking agent, type of pore-forming agent, and amount of crosslinking agent). To obtain the maximum amount of information from the minimum number of experiments, we employ multivariate analysis. Using the obtained results, during the iterative process, each variable is maximized independently to yield a set of good materials. Appropriate samples from this batch were treated with acrylamide plasma, and the degree of modification was quantified through elemental analysis and chemical methods. Therefore, the effect of pore structure on surface modification was investigated using plasma chemistry methods. Furthermore, a simple fractionation of a group of samples by screening was performed to investigate the effect of microsphere size on the degree of modification. 1. Experiment 2.1 Materials Divinylbenzene (DVB, 80%, industrial grade, Aldrich) and styrene (Aldrich) were washed with a 1% NaOH (Aldrich) solution to remove the polymerization inhibitors, and then rinsed thoroughly with large amounts of deionized water. Toluene (Fisher Scientific), heptane (Aldrich), dodecane (Aldrich), azobisisobutyronitrile (AIBN, BDH), polyvinyl alcohol (Mw 85000–146000, degree of hydrolysis 87–89%, Aldrich), NaCl (Avocado); all other solvents are of standard grade. 2.2 Instruments Pore volume and specific surface area are determined by nitrogen adsorption/desorption and mercury pressure intrusion analysis. The instrument used for nitrogen adsorption/desorption is the Micromeritics TriStar 3000 specific surface area and porosity analyzer. Before each experiment, the sample (about 0.2 g) was degassed at 50°C for 12 hours first. The specific surface area of the dry resin was determined using the nitrogen adsorption BET method, while the pore size distribution was determined using the BJH model. Mercury pressure porosimetry was carried out using a Micromeritics AutoPore III 9420, while the analysis of elements C, H, and N was performed with a CE440 element analyzer (Exeter Analytical Inc). 2.3 Suspension polymerization: The pore-forming agent concentration is expressed as a percentage of the monomer relative to the total volume, with the water/organic phase ratio remaining constant at 4:1. In a typical experiment, styrene (5 ml, 43 mmol, 12 mol% of the total monomer amount) and divinylbenzene (45 ml, 253 mmol of actual DVB; the remainder being 62 mmol of ethystyrene, 71 mol% of actual DVB) were thoroughly mixed with a pore-forming agent (50 ml) and AIBN (0.5 g, 3.0 mmol, 0.8 mol% based on the total monomer amount). The stabilizers, including PVA (polyvinyl alcohol, 2 g) and NaCl (2 g, 34 mmol), were dissolved in water (400 ml) before being transferred to a 500 ml suspension polymerization reactor (four-ported and equipped with a metal stirrer having two propeller blades). Then the monomer, heptane, and AIBN were quickly added to the aqueous phase, and stirred at 800 rpm. Thereafter, nitrogen was introduced into the reaction vessel for 10 minutes, and the temperature of the water bath was raised to 80°C and maintained for 6 hours. After 6 hours, the microspheres were collected by filtration using a pump and washed with hot distilled water. The microspheres were then transferred to a Soxhlet extractor and extracted with acetone for 24 hours. It is then air-dried at room temperature, and subsequently vacuum-dried at 50°C until a constant weight is reached. For each batch of samples, the portions with different particle sizes were separated using Endecotts’ Minor Sieve Shaker to obtain microspheres with sizes ranging from 20 to 300 μm. 2.4 Experimental Design The experimental design involves conducting a series of experiments to prepare porous PS/DVB microspheres, in order to determine which factors affect the specific surface area, pore volume, or pore size of the resin. 23 experimental groups were set up for factorial analysis. The three variables examined in the experimental study are as follows: 1. The difference in solubility parameters between PS/DVB and the pore-forming agent (Δδ) (x1); 2. The amount of pore-forming agent used (x2); 3. The concentration of divinylbenzene (x3). The “high” and “low” values for each parameter used in the experiment are shown in Table 1. Eight batches of polystyrene microspheres were prepared under different conditions, and a 23-factor design was used to represent the experiments (Table 2). The specific surface area, pore volume, and pore size of each resin were determined using nitrogen adsorption and mercury pressure intrusion methods. As can be seen from the results reported in Table 3, the pore-forming agent has a crucial influence on the porosity of the resulting microspheres. When toluene is mixed with heptane in a 1:1 ratio as a pore-forming agent, the specific surface area is quite high (480–544 m2/g), whereas when heptane is used as the pore-forming agent, the specific surface area is lower (366–499 m2/g). In contrast, the microspheres prepared using a toluene/heptane mixture as the pore-forming agent had lower pore volume and pore size (0.53–0.83 ml/g and 48–82 Å, respectively), while those prepared using heptane as the pore-forming agent had higher pore volume and pore size (0.68–0.94 ml/g and 81–112 Å, respectively). The results of specific surface area, pore volume, and pore size were used as response variables for multivariate data analysis (Table 3). The regression coefficients of the experimental variables can be calculated using MATLAB 5.3 via multiple linear regression; the regression analysis of the experimental data is shown in Figures 1-3. As can be seen from Figure 1, when the response variable is specific surface area, the regression coefficient for the experimental variable x3 (DVB concentration) is positive. This indicates that the higher the DVB concentration used (72% mol), the greater the specific surface area of the microspheres will be. Meanwhile, the regression coefficients of the other two experimental variables, x1 (the difference between the PS/DVB and the solubility parameter of the pore-forming agent, Δδ) and x2 (the amount of pore-forming agent used), on the specific surface area of the microspheres are negative, with Δδ having a more pronounced effect. These conclusions indicate that, if toluene/heptane (1:1) is used as the solvent at 67% concentration along with 72% mol DVB as the synthesis conditions, the specific surface area will increase (Table 3, Resin 5 – 544 m2/g). The polynomial obtained from the regression coefficients is: y=467.6-41.6x1-13.6x2+20.1x3……(1). Figure 2 shows that, with pore volume as the response, the regression coefficients for the three experimental variables are all positive; however, the effect of DVB concentration (x3) is relatively small. If heptane is used as a pore-forming agent at 100% dilution, along with a crosslinking agent used in an amount of 72% mol, the pore volume of the resulting microspheres will increase (Table 3, Resin 8 – 0.94 ml/g). The dilution degree has a significant effect on pore volume. The polynomial obtained from the regression coefficients is: y = 0.7441 + 0.0751x1 + 0.1254x2 + 0.0028x3……(2). As can be observed from Figure 3, the regression coefficients for the experimental variables x1(Δδ) and x2(porogen dosage) are positive, while the regression coefficient for x3(DVB concentration) is negative. The explanation for this is that if heptane is used at 100% as the pore-forming agent along with 49% mol of DVB, the pore size increases (Table 3, Resin 4 – 112Å). The polynomial obtained from the regression coefficients is: y=82.6+17.4x1+11.4x2-5.1x3……(3). 2.5 Plasma chemical functionalization: Acrylamine was subjected to plasma chemical modification. 25 mg of acrylamine was placed in a rotary glass reactor, and under a pressure of 0.4 mbar (1 mbar = 10-3 bar = 10-3*105 Pa = 100 Pa; in other words, 1 mbar equals 100 Pascals), continuous plasma was used to initiate the reaction for 20 minutes at 20 W. The specific experimental details are provided in the literature. 2.6 Determination of active amine loading from the From value: The quantification of the number of amino groups introduced through plasma chemical functionalization can be determined using the From loading, as shown in Figure 1. This requires the processed microspheres (about 30 mg) to be washed in a calcination tube with methanol (10×2 ml), and then all impurities to be removed using diethyl ether (2×2 ml). 2 ml of a solution of 9-fluorenylmethyl chloroformate (Fmoc-Cl) was dissolved in **furane (THF, 10 equivalents)** and added to each tube containing diisopropylethylamine (DIPEA). These tubes are then placed on a vibratory screen (Vibrax VXR) and vibrated for one hour to ensure complete reaction of Fmoc-Cl. The remaining solvent was removed using a vacuum pump, and the residue was washed thoroughly with an adequate amount of methanol (10×2 ml) and diethyl ether (2×2 ml). Finally, the sample is dried in a vacuum chamber. For each dry polymer sample lacking Fmoc-amino groups, 10 mg was taken and placed in a 5 ml volumetric flask; subsequently, 200 μL of a 20% **/N,N-dimethylformamide (DMF) solution was added, and the sample was left in it for 30 minutes. Each solution was diluted with methanol to a total volume of 5 ml. The absorbance of the compound at 300 nm was determined using a UV/VIS spectrophotometer (Unicam), with a 0.8% (v/v) solution of 20% / DMF in methanol serving as the reference solution. The Fmoc loading for each sample was calculated using the Beer-Lambert law. 2.7 Raman microscopy technique: Details regarding sample preparation, Raman images of 4-cyanobenzoic acid, and data processing can be found elsewhere. 3. Results and Discussion The type and concentration of the pore-forming agent used to prepare permanent porous resins have a crucial impact on the porosity of the resulting products; although the total pore volume is primarily controlled by the amount of pore-forming agent used, it is also influenced to some extent by the properties of that agent. The pore-forming agents used in the suspension polymerization to produce porous polymers can be divided into three categories: good solvents, poor solvents, and linear polymers or their mixtures, which can yield different types of pores and pore distributions. During the polymerization reaction, phase separation occurs between the pore-forming agent and the growing polymer network. When a pore-forming agent with affinity for the polymer is used (such as toluene/n-heptane), the residual polymer network is completely filled with solvated monomers, resulting in a high conversion rate of these monomers into polymer. Finally, there was less phase separation in the resulting microgel particles, which contained only a small amount of residual monomer and crosslinking agent. The separated pore-forming agent phase contained lower concentrations of untreated monomers and crosslinking agents. Further polymerization reactions that occur in the pore-forming agent phase produce a small amount of additional polymer, which acts on the fused microgel particles. Therefore, the microgel particles tend to retain their respective properties as well as the microporous and mesoporous networks, which are formed during the first shaping process of the microgel particles and remain largely intact. Such resins have a large specific surface area. On the other hand, when a precipitation pore-forming agent (such as n-heptane) is used, phase separation occurs at low conversion rates. Microgel particles are formed by the swelling of high-concentration monomers and crosslinking agents, and the separated pore-forming agent phase contains large amounts of monomers and crosslinking agents. A large amount of copolymers are generated after the pore-forming agent separates from the small droplet/particle phase, which affects not only the fusing of microgel particles but also the formation of numerous filled pores. In extreme cases, the particles lose their properties and aggregate to form labyrinthine macropores. The resin formed in this way has a smaller specific surface area, with a wide pore size distribution in the macroporous region. The effect of pore-forming agents can be explained by their solubility parameter δ (Table 4). The smaller the difference in solubility parameters between the resin and the pore-forming agent, the greater their affinity. The solubility parameter of styrene-divinylbenzene copolymers ranges from 17 to 18.6 MPa1/2. In recent related studies, Errede found that the concentration parameter δ of PS/DVB resin increases as the degree of crosslinking increases; at a crosslinking degree of 20%, the concentration parameter δ was 17.8 MPa1/2. Therefore, we assume that the concentration parameter value for resins with a higher degree of crosslinking is 18 MPa1/2. As can be seen from the data in Table 4, by changing the solvent from n-heptane to a toluene/n-heptane mixture, the affinity of the polymer network for the diluent increases. The results of the regression analysis showed that the type and approximate amount of pore-forming agent had a greater impact on the response factors than the DVB concentration; moreover, the higher the DVB concentration, the larger the values of these two parameters (specific surface area and pore volume). Therefore, it was decided to maintain the crosslinking degree at 72% in order to study the further effects of pore-forming agent types and concentrations on the response factors. At this point, the specific surface area, pore size, and pore volume of the resin are all at their maximum values. To obtain a wide range of response values, a series of resins with lower response factor values were prepared by varying the conditions. The resins were sorted in ascending order according to each response factor value (see Figure 4, using specific surface area as an example), and the selected resins were subjected to plasma chemical modification. For each response factor, two sets of samples were selected—one with a high parameter value and one with a low value—to be examined. To avoid extreme values for the samples, only those with maximum values of 33% and 66% were considered. Table 5 shows the three categories of “low-value” and “high-value” resins, along with their preparation parameters and properties. It should be noted that in order to obtain as many \"high-value\" and \"low-value\" resins as possible, one parameter is changed while keeping the other parameters constant in order to study the individual effect of each parameter. Elemental analysis (CHN) and the determination of Fmoc content are usually carried out using plasma-chemically modified samples (Table 6). Elemental analysis revealed the total amount of nitrogen on the polymer surface and the Fomc amount determined through the amine-reactive groups. Elemental analysis indicated a fairly high level of modification; however, there was still a large amount of unreacted Fmoc, so further chemical modification was not possible. The results also showed that the resin with a high specific surface area and pore volume (ACD68) had the highest modification level. The importance of pore volume can be seen by comparing it with ACD29 resin; this resin has a slightly higher specific surface area, but much lower pore volume and a lower Fmoc loading capacity. Another parameter that can affect the degree of chemical modification of the plasma is the microsphere size. Therefore, it was decided to screen all resin samples with a particle size of less than 53 μm that had been treated with plasma, along with their sister samples; the results are shown in Table 7. Clearly, particle size has a greater impact on the degree of modification. The increased degree of plasma chemical modification in the samples selected across the entire sample range has a much greater impact compared to Table 4. Similar reports on the effect of particle size on the degree of modification have also been found in previous studies. However, current research shows that particle size is the main factor determining the plasma chemical modification of acrylamide. The above conclusions indicate that the modification of microspheres mainly occurs on their outer surface. The smaller the microspheres, the larger their surface area (plasma-solid interface), and therefore the greater their degree of modification. This conclusion was verified using Raman microscopy (Figure 5), which shows a high degree of functionalization at a depth of approximately 3 μm within the shell. It should be noted that plasma chemical amine functionalization can reach surfaces throughout the entire microsphere, as gases can easily penetrate the internal pore structure. Functionalization can also take place in the absence of a swelling agent (as in the case presented in this paper) – functionalization occurs only on the surface; however, due to the high porosity of the microspheres and the presence of permanent pores, the surface that can be accessed covers the entire microsphere. Therefore, it is not simple to observe the preference for amine functional groups around the microspheres, as the microspheres cannot swell in the reaction medium. It should be noted that the modification of 4-cyanobenzoic acid and the Raman imaging images only reveal the reacted amine functional groups, whose concentration is much lower than that of the total nitrogen content (Table 6). Therefore, it cannot be concluded that plasma penetrates only into a thin shell from the outer surface of the microsphere. The highly functionalized microsphere shell offers many advantages on its surface, such as the removal and purification of reactive reagents. The high functionalization density on the external surface results in purified microspheres having better kinetics than traditional gel-type or permanently porous carriers, allowing for the slowdown of reactions. We have reported elsewhere on the applications of these microspheres in solid-phase organic synthesis (SPOS) and purification applications. 4. Conclusion An experimental design was employed to simultaneously study the effects of crosslinking degree, type of pore-forming agent, and level of pore-forming agent on the surface area, pore volume, and pore size of polystyrene-divinylbenzene microspheres with permanent pores. Studies have found that the type and level of pore-forming agents have a greater impact on various aspects than the degree of crosslinking. Using this information, the performance to be evaluated will be maximized, and a batch of samples will be produced so that each performance parameter takes on a wide range of values. Six sets of samples were selected, with each property represented by either a \"high\" or \"low\" value; the degree of functionalization after treatment with acrylamide plasma was determined using elemental analysis and Fmoc quantification. The results show that pore volume has the greatest influence on the degree of plasma chemical modification of propylamine. Finally, the effect of bead size on functionalization was investigated by comparing the degree of functionalization of existing samples with that of microspheres selected to have a size of less than 53 μm. It was found that smaller microsphere sizes result in higher modification levels. The explanation for these results is that most of the acrylamide modification occurs on the external surface rather than inside the microspheres.