Method guidelines, Dc a b, Ca b – Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column User Manual

Page 5

[ method guidelines ]

enhanced component resolution. A suitable gradient separation method
can be approximated from the oligonucleotide base (C, G, A, and T)
composition. The initial gradient is typically adjusted while keeping the
gradient slope constant.

Figure 3: Impact of gradient slope on 15-60mer oligodeoxythymidine
ladder resolution and analysis time. Initial mobile phase strength is
kept constant, gradient slope for Panel A= 0.15% acetonitrile/min,
Panel B= 0.30% acetonitrile/min, and Panel C= 0.45% acetonitrile/min.

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 oligonucle-

otide

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 conditions: All start at 40%B (i.e., 8% Acetonitrile)
Detection:

260 nm, 20 scans per second

Figure 3 illustrates analysis time reduction when using steeper gradi-
ents. The disadvantage of using steeper gradients is that the resolution
of oligonucleotides is negatively affected. An alternative approach to
method development is to equilibrate the column and begin the separa-
tion at a low concentration of organic solvent. The column equilibrated
organic solvent concentration at sample loading has to be well chosen.

If the initial solvent strength is too high, then some desired small
oligonucleotide sequences may elute from the column unretained. At
the other extreme, when the gradient starts at too low an organic con-
centration, the analysis may be excessively long without the benefit of
enhanced component resolution. A suitable gradient separation method
can be approximated from the oligonucleotide base (C, G, A, and T)
composition (see Table 2). Figure 4 illustrates the impact of the initial
gradient strength on analysis time and oligonucleotide resolution
(gradient slope was kept constant). No resolution loss is observed.

Figure 4: Impact of initial mobile phase strength on 15-
60mer oligodeoxythymidine ladder analysis time. Gradi-
ent started at 7, 8, 9, or 10 % acetonitrile (A, B, C, or D
chromatograms, respectively). When keeping gradient slope con-
stant, the analysis time can be reduced without a loss of resolution.

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 slope: 0.15% Acetonitrile change per min for all runs
Detection:

260 nm, 20 scans per second

minutes

13.5

0.45% ACN per min change

0.15% ACN per min change

0.30% ACN per min change

minutes

Start at 50%B (10% ACN)

Start at 45%B (9% ACN)

Start at 40%B (8% ACN)

Start at 35%B (7% ACN)