Retention studies of DNA on anion-exchange monolith chromatography binding site and elution behavior

Linear gradient elution experiments were carried out on monolithic anion-exchange chromatography (AEC) with oligo-DNAs of various sizes (4-50mer, molecular weight M-W = 1200-15,000) and compositions in order to investigate the retention mechanism. The binding site (B) values as well as the peak salt elution concentration IR values were determined. The B values determined for the monolithic AEC were similar to the values for non-porous AEC and porous AEC. The B value increased linearly with the number of charges (bases) of single-strand DNA when M-W is less than ca. 3600 (12mer). When M-W is greater than 6000, the slope of B versus M-W decreased, and became very small at M-W > 30,000. The IR value also increased linearly with M-W for M-W < 6000, and slightly with M-W for M-W > 10,000. It was shown that a very difficult separation of a single-strand 50mer poly(T) and a double-strand 50mer poly(A) and poly(T) was accomplished within 10 min by using a very shallow gradient at a high initial salt concentration (0.5 M) and a high flow-velocity (2.7 cm/min). (c) 2007 Elsevier B.V. All rights reserved.