Waters OST C18 User Manual

[ method guidelines ]

XBridge™ ost C18 Method guidelines

*ost: oligonuCleotide separation teChnology

XBridge™ OST C18 columns are based on Waters second generation of
hybrid-silica BEH Technology™ particles and can be effectively used
for the lab scale purification and analysis of detritylated synthetic
oligonucleotides using ion-pair, reversed-phase chromatography. This
document provides useful method guidelines for the effective use of
this column chemistry for this group of compounds.

Contents

I. PRINCIPLES OF OLIGONUCLEOTIDE SEPARATIONS

II. SAMPLE PREPARATION

III. RECOMMENDED MOBILE PHASES

IV. RECOMMENDED INJECTOR WASH SOLVENT

V. GENERAL CONSIDERATIONS IN DEVELOPING SEPARATIONS

VI. ANALYSIS OF MODIFIED OLIGONUCLEOTIDES

VII. PURIFICATION CONSIDERATIONS

[ method guidelines ]

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TEA

XBridge™ OST C

18

chain

PO group on Oligo chain

i. p rinCiples o F o l i gonu C l eotide s e parat ions

Separations of detritylated synthetic oligonucleotides on an XBridge™
O ST C18 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 oligo­nucleotides from the sorbent surface.

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

18

HPLC system: Waters BioAlliance™ 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 X Bridge™ OST C18, 2.5 µm (2.1 x 50 mm)
Mobile P hases: 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

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

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, Hexafluo­roisopropanol (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 C18 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.

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[ method guidelines ]

Figure 3: Effectiveness of Waters BEH Technology™ Hybrid-Silica C18
Particle Size on Deoxythymidine Ladder Separations

HPLC system: Waters BioAlliance™ 2796, PDA Detector with micro UV cell
Sample Injected: Approximately 100 pmoles of detritylated 15 – 60mer crude
oligonucleotide ladder diluted in 0.1 M TEAA
Column: Waters BEH Hybrid-Silica C18 particles (2.1 x 50 mm)
Mobile P hases: 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

In addition to ion-pairing, a hydrophobic reversed-phase mechanism
also takes place in the oligonucleotide separation. The residual interaction
of nucleobases has an impact on overall retention and separation
selectivity, especially when using Triethylammonium Acetate (TEAA)
ion-pairing mobile phases. Separation of N and N-1mers may be either
enhanced or suppressed by the sequence contribution. More potent
ion-pairing systems such as Triethylammonium ion with Hexafluo­roisopropanol counter ion provide for more regular “charge-based”
separations (Figure 4).

Figure 4: Impact of Ion-pairing System on Separation of a 10-30mer
Heterooligonucleotide Ladder

HPLC system: Waters BioAlliance™ 2796, PDA Detector with micro UV cell
Sample: 20 mer: TCC C TA GCG T TG AAT TGT CC
25 mer: TC C CTA GCG TTG AAT TGT C CC TTA G
30 mer: TCC CTA GC G TTG AAT TGT CCC TTA GCG GGT
Ladder was prepared by hydrolyzing detritylated
20, 25, and 30mer oligonucleotides with a
3’-exonuclease
Column: Waters X Bridge™ OST C18, 2.5 µm (4.6 x 50 mm)
Mobile p hases: Upper chromatogram: 0.1 M TEAA with acetonitrile gradient; Lower
chromatogram: 16.3 mM T EA - 400 mM HFIP with methanol gradient
Flow rate: 1.0 mL/min
Column Temp.: 60 ˚C
Gradient delay: 0.45 mL
Detection: 260 nm, 5 scans per second

ii. saMple preparation

1. Dissolve the detritylated synthetic oligonucleotide sample in Mobile
Phase A (e.g., 0.1 M TEAA). For example, a 0.05 - 0.2 µmole scale
synthesis can be prepared in 0.1 mL of 0.1 M TEAA. Proportionately
larger or smaller volumes of 0.1M TEAA are required when dissolving
samples from different scale syntheses. Due to the nature of gradient
separations, relatively large volumes of sample (in low organic
strength eluent) can be injected and concentrated onto the head of
the column before beginning the gradient elution program.

2. Samples must be completely in solution and free of particulates
before injecting onto the column. Remove all particles from the
sample (Controlled Pore Glass Synthesis Support, etc.), which may
block the inlet column frit, increase the operating pressure, and
shorten the column life time. Sample contamination with high con­centration of salts and/or detergents may also interfere with analysis.

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