## 3 Results and Discussion
### Preparative Chromatography
The hydrophobic interactions are favored by the use of high (NH4)2 SO4 concentrations, since the kosmotropic ions influence the exposure of hydrophobic regions of molecules [26]. Considering the pDNA isoforms, sc pDNA has some hydrophobic regions, naturally exposed because of its conformational torsions [27], and this effect can be enhanced under high (NH4)2 SO4 concentrations, allowing the establishment of hydrophobic interactions between non-polar regions of the ligands and the sc pDNA. On the other hand, the oc isoform has the bases inward and they are less available to interact with histidine even in the concentration of 2.3 M (NH4)2 SO4, being immediately eluted in the first step. Concerning RNA, the bases are more exposed because of its main single-stranded nature, being in this way more available to interact with the matrix. The decrease of the (NH4)2 SO4 concentration increases the reorganization of water molecules, consequently minimizing the hydrophobic effects, and disfavoring these interactions. Despite the dominant effect of ring stacking/hydrophobic interactions, the water-mediated hydrogen bonds and van der Waals forces are also present between histidine-agarose matrix and pDNA [28; 29], evidencing the affinity character of this matrix. The results obtained in Figure 2A are in accordance with other works, made under the same conditions but using other plasmids [23; 24]. Figure 2B exhibits the chromatographic profile of the clarified lysate in arginine-macroporous support, as well as the respective agarose gel electrophoresis. Here, an increasing gradient of NaCl was used to elute the bound molecules. First, RNA was eluted in peak 1 at 124 mM NaCl in 10 mM Tris-HCl buffer (pH 8.0), as confirmed by the agarose gel electrophoresis (lane 1, Figure 2B). The sc pDNA isoform was then eluted in peak 2, at 500 mM NaCl, together with a small amount of oc pDNA (lane 2 Figure 2B). A final step of 2 M NaCl was used to confirm the elution of all retained species. The arginine ligands immobilized in the macroporous support, under the influence of the mobile phase, mainly promote electrostatic interactions, not disregarding the involvement of other non-covalent interactions occurring between the lysate molecules and the arginine ligand [21; 23]. The negatively charged groups in the pDNA backbone interact with the positive groups of the arginine support, and as expected these molecules bind more strongly than RNA, because pDNA has a double stranded negatively charged chain, while RNA is smaller and presents a single-stranded nature [28; 29]. The histidine-monolithic support was applied to purify the sc pDNA from the clarified lysate sample by using a decreasing stepwise gradient of ammonium sulfate (Figure 2C). The first step was established at 3 M (NH4)2 SO4 in 10 mM citrate-EDTA buffer, pH 5.0, to promote the retention of all biomolecules present on the monolithic column. The RNA and a small fraction of sc pDNA were eluted in the second gradient step (peak 2) at 2.52 M (NH4)2 SO4, as it can be observed in the agarose gel electrophoresis (lane 2, Figure 2C). Finally, the more tightly bound biomolecules were eluted in the third peak by a decrease in the ionic strength to 10 mM Tris-HCl (pH 8), which correspond to sc pDNA as visualized in lane 3 of the agarose gel electrophoresis (Figure 2C). In this particular case, due to the pH 5 of the mobile phase, the imidazole ring of histidine is protonated and hence positively charged, which favors the establishment of electrostatic interactions between nucleic acids and the stationary phase, in addition to the hydrophobic effects previously discussed. Actually, the difference on the chromatographic elution behavior found on the histidine-monolith and the histidine-agarose can be related to the additional electrostatic effect resultant from the pH 5.0 used in this study, as it was already observed by [PERSON] and co-workers to purify the HPV-16 E6/E7 pDNA [25].