Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 6)

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[ method guidelines ]
Figure 5: Analysis of a Digested 25mer Phosphorothioate Oligonucleotide
HPLC system:
Waters BioAlliance ™ 2796, PDA Detector with micro UV cell
Sample: Detritylated 25mer phosphorothioate oligonucleotide mix
(CTC TCG CAC CCA TCT CTC TCC TTC T) digested with 3’ exonuclease
Column: XBridge ™ OST C
18
, 2.5 µm (2.1 x 50 mm)
Mobile phase: A: 15 mM TEA with 400 mM HFIP
B: methanol
Flow rate: 0.2 mL/min
Column Temp.: 60 ˚C
Gradient delay: 0 mL
Gradient: 15 to 20% B in 20 minutes (0.25% methanol per minute)
Detection: 260 nm, 2 scans per second
Peptide nucleic acids (PNA) can also be analyzed using XBridge ™ OST C
18
columns. The ion-pairing system recommended for analysis of PNA is
similar to those used for peptide analysis (0.1% Trifluoroacetic Acid
or Formic Acid).
UV detection of eluted oligonucleotide peaks is often performed at
260 nm. Injection of 50 pmol of detritylated oligonucleotide sample
on a 2.1 x 50mm XBridge
™ OST C
18
column yields relatively abundant
peaks. Limits of quantitation (LOQ) vary with the type of oligonucleotide,
LC system and detector; LOQ generated on Waters 2996 PDA detector
equipped with micro UV cell is approximately 1 pmol (2.1 x 50mm
XBridge
™ OST C
18
). The Limit of detection (LOD) estimate is shown in
Figure 6.
Figure 6: Analysis of a 20mer Oligodeoxythymidine Crude Synthesis Mixture
HPLC system: Waters BioAlliance ™ 2796, PDA Detector with micro UV cell
Sample: ~600 pmol of a detritylated 20mer, ~18 pmol of 19mer, ~4.5 pmol
of 17mer was injected on column.
Column: XBridge ™ OST C
18
, 2.5 µm (2.1 x 50mm)
Mobile phase: 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 mL
Gradient: 35 to 50% B in 30 minutes (7-10% acetonitrile)
Detection: 260 nm, 2 scans per second
Vii. puriFiCation Considerations
XBridge ™ OST C
18
columns are designed for laboratory scale oligo-
nucleotide purifications and analyses. Sufficient amount of isolated
material suitable for molecular biology and other experiments can
be prepared in a single injection. For example, a 4.6 X 50 mm XBridge
™
OST C
18
column can suitably purify approximately 20-200 nmoles of
sample in a single injection. It is important to understand that column
overloading results in a peak broadening and that some earlier eluting
impurities may co-elute with the component of interest. With a proper
heart-cutting technique, a good purity of the target oligonucleotide
can be obtained without significant yield sacrifice (Figure 7).
Chromatographers frequently develop a separation on the analytical
scale before moving to preparative work. The steps required to opti-
mize the analytical separation involve:
1) Selecting the appropriate column packing material and mobile phase.
2) Determining the optimal flow rate, gradient during and separation
temperature.
3) Determining the amount of material that can be satisfactorily
loaded and separated on the analytical scale column.