Control of oligonucleotide retention on a pH-stabilized strong anion exchange column

New monolith technology for automated anion-exchange purification of nucleic acids

“…In general, gradient elution of ON samples is employed, and we used a previously described eluent system [9,25]. In brief, this system consists of 4 eluents: (A) deionized H 2 O, (B) 0.2 M NaOH, (C) 0.2 M Tris, 0.2 M AMP and 0.2 M DIPA adjusted to pH 7.2 with MSA, and (D) either 0.33 M NaClO 4 or 1.25 M NaCl.…”

“…on the length and composition of the oligonucleotides. Purified fractions were analyzed on a 2 mm × 250 mm DNAPac PA200 at various temperatures and pH values (as indicated), using linear or curved gradients [9,36] at 300 L/min. Using a Dionex WPS-3000 TBFC fraction collecting autosampler, we configured a system for automatic purification and desalting (Scheme 1).…”

“…The gradient for the DNAPac analysis was 50-150 mM NaClO 4 in 12 min using curved gradient 4 (see Ref. [9] for the gradient number equation). The eluent was buffered to pH 7 with 20 mM Tris-HCl, flow was 300 L/min, and the temperature was 60 • C. The yield of collected ON was calculated based on the known dilutions, injection volumes, fraction volumes and peak areas, as compared to the known amount loaded (8.25 mg) by full loop injection onto each column.…”

“…While improvements to ON synthesis can often provide sufficient purity for use as amplification probes, ONs intended for diagnostic and therapeutic applications must be purified and thoroughly characterized after synthesis [8]. Such purifications often employ anion-exchange (AE) chromatography, because unlike gel electrophoretic and reversed-phase separations, the AE approach offers several powerful mechanisms for control of retention and selectivity [8][9][10]. In the initial stages of development, smallscale samples up to 200 g may be purified for early screenings, but milligram quantities are needed for bioanalysis of promising candidates.…”

Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography

“…The employed techniques include denaturing anion exchange (AEX) HPLC [22][23][24][25], denaturing ion-pairing reversed-phase (IP RP) HPLC [17,[26][27][28][29], non-denaturing AEX HPLC [30][31][32]; and non-denaturing IP RP-HPLC [33][34][35][36][37].…”