Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase...

Improved resolution in slalom chromatography, a novel size-fractionation method discovered recently for relatively large DNA molecules (> 5 kpb), was obtained by using columns generally employed for reversed-phase chromatography: i.e., two types of Capcell-Pak (methyl or phenyl-derivatized 5-microns microbeads), and five types of Hypersil-3 packings (trimethylsilyl, dimethyloctyl, cyanopropyl, octadecyl or phenyl-derivatized 3-microns microbeads). The resolution of 5-15-kbp DNA was significantly improved by employing these columns, though the separation characteristics differed. When Capcell-Pak columns were used with a normal low-salt eluting solvent (10 mM sodium phosphate, pH 6.8, 1 mM EDTA), chromatograms were obtained for lambda/HindIII fragments (a mixture of 0.1, 0.5, 2.0, 2.3, 4.4, 6.6, 9.4 and 23.1-kbp fragments) similar to those obtained previously with Asahipak GS-310 5-microns size-exclusion packings. However, when up to 0.2 M NaCl was added to the solvent, the DNA was increasingly retarded, particularly the 4.4, 6.6 and 9.4-kbp fragments, resulting in improved resolution in the low to middle molecular-mass range. The effect of salt was more significant with Capcell-Pak Phe than C1, although various features characteristic of slalom chromatography were preserved with both columns; i.e., dependency on DNA size, flow-rate, and temperature. This suggests that a mixed mode of separation, that is, slalom mode and hydrophobic-interaction mode, was operating. Although all of the Hypersil-3 packings showed significant adsorption of lambda/HindIII fragments under low-salt conditions, the fragments could be eluted with satisfactory yield and resolution by adding acetonitrile (> 5%) to the solvent. Notably, these Hypersil-3 packings allowed resolution of a 4.4-kbp lambda/HindIII fragment from the flow-through fraction for the first time, possibly due to their small particle size. Thus, various packing materials developed for high-performance liquid chromatography proved to be applicable for slalom chromatography, though the eluting conditions still need to be refined. The results support the concept that slalom chromatography is based on a hydrodynamic phenomenon.