NB|Chem.de

Topic of the Month: October 2011

 

Impact of mobile phase pH on Ion-Exchange-Chromatography of Oligonucleotides

pH is a key factor in IEX chromatography, since it may have significant impact on analyte and resin stability. In addition, pH may have an impact on the retention time of the oligonucleotides during chromatographic separation, due to the associated changes in the charge state of analytes and resins. Due to the nature of the hydrogen bonds between the complementary strands that stabilize the Watson-Crick duplex, a double helix is not stable above a pH of 10.5. High pH values are therefore often used in chromatographic separations to disrupt otherwise stable secondary structures, especially in RNA or modified DNA, that stabilize structures. See below for examples:

Figure 1: Impact of pH on retention time (high pH). Retention time in IEX-HPLC shifts in high pH and neutral pH applications, depending on the G/U content and base position within the sequence. Chromatography was performed using a DNA-Pac200 column (2.1 x 100 mm) (Dionex) at a flow rate of 1 mL/min. Mobile Phase A was {20 mM Sodium Phosphate (pH 11.0), 10% ACN} and Mobile Phase B was {20 mM Na3PO4, 1.0 M NaBr (pH 11.0), 10% ACN}. Column temperature was 30°C. A gradient was run from 25-62% B in 12 minutes.

 

Figure 2: Impact of pH on retention time (neutral pH). Mobile Phase A was {10 mM Tris/HCl (pH 8), 10% ACN} and Mobile Phase B was {10 mM Tris/HCl (pH 8), 1.0 M NaBr (pH 11.0), 10% ACN}. Column temperature was 75°C. A gradient was run from 30-60% B in 12 minutes. Traces 1 to 6 represent RNA-1 to RNA-6 respectively (See also Table 1).

Table 1: Base composition of RNA single strands analyzed in Figures 1 & 2. Number of guanines and uridines in the sequence of six different RNA strands (RNA-1 to RNA-6) is indicated.

 

 

 

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