Peculiarities of the Retention Mechanism of Circular and Linear Dna Fragments Using Non-Equilibrium Chromatography

Two different methods have been used to investigate the retention mechanism of a series of DNA fragments in non equilibrium chromatography (NEC) over a range of column temperatures (T) and with different mobile phase flow-rates (F). The first approach was the separate study of each factor affecting the retention mechanism; the second method was the simultaneous variation of all these factors. The apparent relative retention time of each DNA fragment was used as a retention marker. The data obtained showed that the retention mechanism of the DNA fragment was dependent on the F and T values, and also on the DNA form, i.e., linear or circular form. For low F values, the retention mechanism was based on the classical hydrodynamic regime (HDC) specific to the circular form. On the contrary, for the linear forms, their retention mechanism was based on a slalom chromatographic process (SC) specifically for high F values at low column temperature. This confirms that the SC and HDC modes are interconnected and that the HDC⇄SC transition exists and was clearly visualized. In addition, using a simplex process connected to the chemometric methodology, the optimal conditions for F and T were calculated to obtain the more efficient separation of the linear DNA forms in a minimum analysis time.