Method guidelines, I. principles of oligonucleotide separations, Ii. sample preparation – Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column User Manual

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[ method guidelines ]

I. PrInCIPLes oF oLIgonUCLeotIde sePArAtIons

Separations of detritylated synthetic oligonucleotides on ACQUITY
UPLC

OST C

columns are based on ion-pair, reversed-phase chro-

matographic principles (IP-RP-LC). As shown in Figure 1, the ion-pair-
ing 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 (i.e., 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 aceto-
nitrile or methanol eluent displaces both ion-pairing agent and the
oligonucleotides 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
ACQUITY UPLC

OST C

Column

UPLC

System:

Waters ACQUITY UPLC

System with installed 425 µL mixer

PDA Detector with standard UV cell

Sample Injected:

Approximately 100 pmoles of a detritylated 15 – 60mer

oligonucleotide ladder diluted in 0.1 M TEAA

Column:

Waters ACQUITY UPLC

OST C

column, 1.7 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:

50 to 65% B in 20 minutes (10-13% acetonitrile,

0.15% acetonitrile per min)

Detection:

260 nm, 20 scans per second

II. sAMPLe PrePArAtIon

Follow these steps to prepare your sample:

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 0.1 M 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.

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.

T EA

ACQUITY UPLC

OST C

chain

PO group on Oligo chain

minutes