Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 5)

[ CARE AND USE MANUAL ]
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3. To remove particulates the sample may be filtered with a 0.2 μm
membrane. Be sure that the selected membrane is compatible and
does not dissolve with the selected Mobile Phase diluent. Contact
the membrane manufacturer with solvent compatibility questions.
An alternative method of particulate removal involves centrifugation
for 20 minutes at 8,000 rpm, followed by the transfer of the
supernatant liquid to an appropriate vial.
b. Recommended Mobile Phases
The most common ion-pair mobile phase for synthetic oligonucleotide
separations is based on of triethylammonium acetate (TEAA). This
mobile phase can be prepared by titrating glacial acetic acid aqueous
solution with triethylamine (TEA).
Note: To maximize column life, it is ESSENTIAL that all prepared OST
Mobile Phases be filtered through a solvent compatible, 0.45 µm membrane
and contained in bottles that are clean and particulate free.
TEAA
1L of 0.1 M TEAA may be prepared as follows:
1) Perform work in a hood.
2) Add 5.6 mL of glacial acetic acid into 950 mL of water and mix well.
3) Slowly add 13.86 mL of TEA.
4) The pH should be adjusted to pH 7.0 +/- 0.5 by careful addition
of acetic acid.
5) Adjust final volume to 1 L with water.
Alternatively, premixed TEAA can be used [e.g., Sigma 1 M TEAA
(part no. 90357)]. Mix 100 mL with 900 mL of water to prepare 1 L
of 0.1 M TEAA mobile phase
Alternative ion-pairing reagents are recommended for improved
separation of phosphorothioates or when performing LC-MS analyses.
An ion-pairing mobile phase based on Triethylamine (TEA) and
Hexafluoroisopropanol (HFIP) as the buffering acid produces an
efficient eluent system for improved separations involving these
application types. As indicated below, two ion-pairing systems
are useful. For routine detritylated oligonucleotide applications,
aqueous buffer consisting of 8.6 mM TEA and 100 mM HFIP is
effective. For applications such as those involving the separation of
G-rich oligonucleotides, it is advisable to use aqueous buffer consisting
of 15 mM TEA and 400 mM HFIP (pH approximately 7.9).
TEA-HFIP System 1
1L of 8.6 mM TEA / 100 mM HFIP is prepared as follows:
1) Perform work in a hood.
2) Add 10.4 mL of HIFP (16.8 g) into 988.4 g of water and mix well.
3) Slowly add 1.2 mL of TEA.
4) The pH is approximately 8.3 +/- 0.1.
TEA-HFIP System 2
1 L of 15 mM TEA / 400 mM HFIP is prepared as follows:
1) Perform work in a hood.
2) Add 41.56 mL (67.17 g) of HFIP into 956.36 g of water and mix well.
3) Slowly add 2.08 mL (1.52 g) of TEA.
4) The pH is approximately 7.9 +/- 0.1.
c. Recommended Injector Wash Solvents
Between analyses, the HPLC System Injector seals should be washed. A
90% Water / 10% Acetonitrile injector wash solvent is recommended.
d. pH Range
The recommended operating pH range for XBridge OST C
18
columns
is 1 to 12.
e. Pressure
XBridge OST C
18
columns can tolerate pressures of up to 6,000 psi
(400 bar or 40 Mpa).
f. Temperature
Temperatures between 20 ˚C – 65 ˚C are recommended for operating
XBridge OST C
18
columns in order to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. Note: Operating at
elevated pH, temperature, and/or pressure may potentially result in
shortened column life.
g. Flow Rate
The recommended flow rate for oligonucleotide separations performed on
a 2.1 x 50 mm XBridge OST C
18
column is 0.2 mL/minute. 1.0 mL/min is
the recommended flow when using a 4.6 x 50mm column.