## 3 Results and Discussion
### Structural Analysis of pDNA  
#### 3.2.1 Circular Dichroism  
The structural stability of purified sc pDNA was assessed by circular dichroism (CD), to evaluate if the distinct chromatographic strategies induce any changes in the topology of purified sc pDNA. Besides the information about the structural conformation of pDNA, this technique also shows the stability of the target molecule. Figure 3 shows the CD spectra of the sc pDNA samples recovered from the three different purification strategies, and for comparison purposes, a sample containing oc and sc pDNA obtained with a commercial kit (oc + sc) was also analyzed. The CD spectra of the pDNA (oc + sc) samples obtained using the commercial kit were very similar for both buffers used (Figure 3A,B), and it was possible to identify two positive ellipticity peaks at 220 nm and 275 nm and a negative at 245 nm, which are indicative of the typical topology of B-DNA [35; 36]. This result demonstrates that these buffers did not induce significant changes to pDNA structure. When comparing these spectra with the ones obtained for the pDNA samples purified by our chromatographic methodologies, some minor alterations to the signals can be observed (Figure 3). These slight changes in the bands' magnitude or deviations of the maximum can be associated with little changes in the pDNA molecules caused by the different chromatographic conditions used during the purification process, namely different temperature, ionic strength, and pH. These differences are most evidenced in the samples purified by the supports modified with the histidine ligand, where we can note less intense bands. As reported in the literature, during the purification procedure, several parameters should be considered and modulated, such as the mobile phase conditions, pH, or temperature, in order to reach a better interaction between the ligands and the target. However, these parameters may also cause conformational changes in the molecule, which should be avoided [36; 37]. In the present study, the different conditions and interaction profiles occurring between the target molecule and the supports can explain the small differences achieved on the CD spectra of samples purified with the histidine matrices. Both strategies were based on the use of a decreasing ammonium sulfate gradient, but they differ in the pH used. Thus, some structural changes can occur on pDNA due to the acidic conditions, as the low pH can lead to subtle depurination of pDNA, which can be detected by CD. On the other hand, it is feasible to relate some CD differences to the ionic strength used, as the salt concentration can modify the double-helix condensation, depending on the amount of available ions, especially cations [9], and in some extreme cases, it can undergo transition to A or Z-DNA conformation [38]. It is noteworthy that the signal of the sc pDNA purified by arginine-macroporous present a small deviation referring to the positive region for approximately 270 nm, while those purified by histidine point to 275 nm. This detail may also be a reflection of the binding conditions of the target molecule to the matrix promoted by the mobile phase buffer, since CD is known to reflect little conformational DNA changes [39], or just a reflection of the presence of a small amount of oc pDNA isoform (Figure 3B).