Waters Oligonucleotide Separation Technology Standard - Manual (Page 2)

[ Care and Use ManUal ]
MassPREP Oligonucleotide Separation Technology Standard 2
Table 1: System Troubleshooting using MassPREP OST
Standard
III. PreParatIon ProCedure
The following procedure is provided as a general guideline for
MassPREP OST standard reconstitution. The described method
only serves as a starting point. Depending on the specific
application, one may consider using other solvents and/or dilutions.
Recommended chemicals to prepare sample diluent and LC mobile
phase are following: Acetic Acid (2M):Triethylamine (2M), Fluka,
P/N 09748; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), Fluka, P/N
52512; triethylamine (TEA), Sigma, P/N 417283.
1. Prepare 100 mM of triethylammonium acetate (TEAA) by diluting a
2M:2M stock solution of Acetic Acid:Triethylamine 20 fold in deionized
or HPLC grade water.
2. Add 0.5 mL of 100 mM TEAA in MassPREP OST standard vial. The
final concentration is ~2 pmole/µL for each oligonucleotide. Vortex the
vial briefly to dissolve and homogenize the sample.
3. For MassPREP OST standard analysis in UPLC mode, prepare the
following mobile phases:
a. Mobile phase A is 15 mM TEA, 400 mM HFIP in water. Add
8.31 mL (13.44 g) of HFIP into 191.3 g of water, add 416 µL of
TEA; the final volume is 200 mL, the buffer pH is ~7.9.
b. Mobile phase B is 50% A, 50% MeOH. Prepare TEA-HFIP buffer
as described above, and add 158.2 g of MeOH (200 mL).
c. The LC solvents are highly volatile; the mobile phase should
be used only 24 hours only before making a fresh one. Keep
the mobile phase containers sealed to minimize the buffer
evaporation.
4. Prime the ACQUITY UPLC system and connect a 2.1 x 50 mm
ACQUITY UPLC OST C18, 1.7 µm column (P/N 186003949). Setup
flow rate at 0.2 mL/min, and column temperature at 60 °C. Equilibrate
with initial mobile phase conditions (38% B) for ~20 minutes.
5. Inject 10 µL of the MassPREP OST standard. Run the gradient from 38
to 50% B in 12 minutes. The example of chromatogram is shown in
Figure 1.
6. The MassPREP OST sample can also be used to troubleshoot XBridge
OST C18, 2.5 µm columns configured to a HPLC system. The resolution
is not expected to match the performance obtained with a ACQUITY
UPLC OST C18, 1.7 µm column and UPLC System.
7. The alternative conditions for HPLC (UPLC) oligonucleotide analysis
use TEAA ion-pairing system:
a. Mobile phase A is 100 mM TEAA (measure 190 g of water,
add 10 mL of 2M:2M Acetic Acid:Triethylamine Stock Solution.
The pH adjustment is not necessary.
b. Mobile phase B: 20% acetonitrile, 80% TEAA. Prepare 200 mL
of TEAA as above, and add 50 mL (39.3 g) of acetonitrile.
c. The most useful analytical column for HPLC is a 2.1 x 50 mm,
XBridge OST C18, 2.5 µm column (P/N 186003952). Flow rate
is set to 0.2 mL/min, column temperature to 60 °C. Gradient
is 40-60% B in 25 minutes. Approximately 20-40 µL of the
MassPREP OST sample is typically injected on column (80 pmole
per peak). Example of chromatogram is not shown.
Chromatogram Appearance Potential problem
Peaks elute outside of
the expected retention
time window Incorrectly prepared mobile phase,
aged mobile phase, incorrect col-
umn temperature setting; excessive
gradient delay (method transfer
between different LC systems).
Tailing peaks Poor tubing connections (especially
between column and detector);
column bed deterioration, column
contamination.
Inconsistent peak width,
split peaks Inadequate gradient mixing,
incompatible sample solvent or
weak wash (purge solvent for
Alliance
® HT).
Lost resolution Aged or contaminated column.
(Has column backpressure
changed?)