Novel Strategy in Slalom Chromatography for Studying Both the Protein Reptation Mechanism and the Compacting Agent Effect to Improve Oligonucleotide Separation

Abstract

Slalom Chromatography (SC) is a chromatographic mode for separate flexible biological molecules. In this technique, the reptation of the DNA fragments through the column packing follows the flow direction and its like a snake edging is way into long grass. A novel equation (Guillaume Y.C. et al. (2002) Anal. Chem. 74(6): 1217–1222) was recently developed to model the retention variation of linear double stranded DNA molecules with the mobile phase velocity under SC. As well, in a previous paper (Guillaume Y.C. et al. (2001) J.Chromatogr.Sci. 39: 361–364) the SC mode was clearly visualized for the first time for specific values of the mobile phase viscosity (h) for proteins which are less flexible than DNA. In this manuscript, the effect of (i) the mobile phase viscosity (h) and (ii) three compacting agents (CAs) i.e. spermine, spermidine, hexamine cobalt on the constants of this new equation was analysed to (i) confirm that h plays a great role on the protein stretching and (ii) analyse the effect of CAs for linear DNA fragments and plasmids. Then, for the first time, it was demonstrated that CAs have different behaviour; for example, hexamine cobalt compacted only the linear DNA fragments. Thus this CA difference effect may be useful for separations of nucleic acids.