## 2 Results and Discussion
### Chromatographic Performance  
To investigate their chromatographic performance, the porous P(S-DVB) microparticles were packed into stainless-steel chromatographic columns. Figure 9 shows the back pressure of columns packed with P(S-DVB)-3 and P(S-DVB)-4 at different flow rates. The back pressure demonstrates a decent linearity with the stream flow rate in the acetonitrile (ACN)-water mixture (\(v/v\) = 7:3). The back pressure of the column packed with P(S-DVB)-4 is lower than that of P(S-DVB)-3, because the average pore size of P(S-DVB)-4 (~18 nm) is bigger than that of P(S-DVB)-3 (~10 nm). The mixture of uracil, toluene, naphthalene and fluorene were separated by the packed column with various permeable polymer microparticles. As shown in Figure 10, the column packed with P(S-DVB)-4 isolates the mixture faster than the column packed with P(S-DVB)-3 due to the higher specific surface area of the former. Because the P(S-DVB)-4 microparticle performed better than the P(S-DVB)-3 microparticle, it was selected for the DR surface modification and the following chromatographic tests. Figure 8: Fourier Transform Infrared(FT-IR) spectra of porous P(S-DVB) microparticles (**a**); sulfonated P(S-DVB) microparticles (**b**); and DR-modified P(S-DVB) microparticles (**c**). Figure 9: Back pressure of columns packed with different porous polymer microparticles: column, 75 mm \(\times\) 4.6 mm Inner Diameter(I.D. ); mobile phase, ACN–water mixture (\(v/v\) = 7:3). Depending on the chemical structure of DR and mobile phase composition, the DR-modified porous P(S-DVB)-4 stationary phase performed different separation mechanisms, including reversed phase (RP) and hydrophilic interactions. As shown in Figure 11, the benzene analogues were also successfully separated by porous P(S-DVB)-DR microparticles. The affinity interaction between benzene rings of DR and the benzene analogues make the separation practical. Compared with Figure S2, the separation performance of the P(S-DVB)-DR column is better than a commercial C18 column (Agela Technologies, S185006, Wilmington, DE, USA) at the same 75 mm column length. Table 2 shows that the run-to-run (\(n=5\)) relative standard deviation(RSD) of retention time for the benzene analogues was less than 1%, day-to-day (\(n=7\)) RSD was less than 2.5%, and column-to-column (\(n=5\)) RSD was less than 3.5%. After a continuous 100 times running in one column, the RSDs of retention time for the benzene analogues were all less than 2.5%, and the separation performance of the P(S-DVB)-DR column was not deteriorated. Therefore, the P(S-DVB)-DR columns demonstrated very good stability and repeatability. Figure 11: Chromatograms for the separation of benzene analogues on the porous P(S-DVB)-DR column: column, 75 mm \(\times\) 4.6 mm I.D. ; injection size, 3 \(\mu\)L; flow rate: 1.0 mL/min; mobile phase, ACN–water mixture (\(v/v=7\):3); elution order: (1) uracil, (2) toluene, (3) naphthalene, (4) fluorene. Figure 10: Chromatograms for the separation of benzene analogues on the porous P(S-DVB) column: column, 75 mm \(\times\) 4.6 mm I.D. ; injection size, 3 \(\mu\)L; flow rate: 0.5 mL/min; mobile phase, ACN–water mixture (\(v/v=7\):3); elution order: (1) uracil, (2) toluene, (3) naphthalene, (4) fluorene. With the introduction of hydrophilic secondary amine groups on the surface of the P(S-DVB)-4 particles, a series of organic acids were baseline separated on the P(S-DVB)-DR column (Figure 12b). The bare P(S-DVB)-4 column (Figure 12a) was used to compare with the P(S-DVB)-DR column. As shown in Figure 12b, the P(S-DVB)-DR column could effectively separate four kinds of organic acids. However, for the bare P(S-DVB)-4 column, the resolution decreased significantly (Figure 12a). The chemical structure of DR on the surface of P(S-DVB)-4 particles enhanced the interactions between organic acids and the stationary phase so that the separation was much more efficient than bare P(S-DVB)-4 particles.