Method guidelines, I. principles of oligonucleotide separations, Ost c – Waters Oligonucleotide Separation Technology XBridge OST C18 Columns User Manual

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[ method guidelines ]

i. prinCiples oF oligonuCleotide separations

Separations of detritylated synthetic oligonucleotides on an XBridge

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OST C

column are based on ion-pair, reversed-phase chromatographic

principles (IP-RP-LC). As shown in Figure 1, the ion-pairing additive in
the mobile phase is adsorbed on a hydrophobic sorbent and provides
for charge-to-charge interactions with negative charges contained on
the oligonucleotide backbone (e.g., phosphate groups).

Figure 1: Proposed Mechanism of IP-RP-LC for Synthetic Oligonucleotide
Separations

As a result, an efficient charge-based (length-based) oligonucleotide
separation is achieved (Figure 2). Gradient elution using an acetonitrile
or methanol eluent displaces both ion-pairing agent and the oligo-
nucleotides from the sorbent surface.

Separation selectivity and resolution decreases with increasing
oligonucleotide length (Figure 2) making the separation of long
oligonucleotides challenging. Modified oligonucleotides such as phos-
phorothioates and 2-O alkyl modified species are also more difficult
to analyze. Special mobile phase may be required (see Section III,
Recommended Mobile Phases).

Figure 2: Separation of a 15 - 60mer Deoxythymidine Ladder on
XBridge

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OST C

HPLC system:

Waters BioAlliance

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2796, PDA Detector with micro UV cell

Sample Injected:

Approximately 100 pmoles of a detritylated 15 – 60mer

oligonucleotide ladder diluted in

0.1 M TEAA

Column:

Waters XBridge

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OST C

, 2.5 µm (2.1 x 50 mm)

Mobile Phases:

A: 0.1 M TEAA,

B: Acetonitrile / 0.1M TEAA, 20/80, v/v

Flow rate:

0.2 mL/min

Column Temp.:

60 ˚C

Gradient delay:

0.45 mL

Gradient:

40 to 62.5% B in 30 minutes (8-12.5% acetonitrile, 0.15%

acetonitrile per minute)

Detection:

260 nm, 5 scans per second

Two commonly used ion-pairing agents for oligonucleotide applications
are triethyl ammonium and dimethylbutyl ammonium ions. The final pH of
these mobile phases containing either of these ion-pairing reagents is
adjusted by the addition of Acetic Acid, or in some cases, Hexafluo-
roisopropanol (HFIP). These mobile phases are volatile making them
suitable for LC-MS applications.

The ability to adequately resolve synthetic oligonucleotide mixtures
by ion-pair, reversed-phase chromatography is significantly affected
by the particle size of the material contained in an efficiently packed
column (see Figure 3). Consequently, XBridge

™

OST C

columns are

efficiently packed with 2.5 micron material to maximize detritylated
oligonucleotide component resolution. In order to improve oligonucleotide
separation efficiency and speed, elevated separation temperature (e.g.
60 ˚C) is recommended. Elevated temperature will also reduce operating
LC System back pressure.

T EA

XBridge

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OST C

chain

PO group on Oligo chain