Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 4)

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[ method guidelines ]
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.
iii. reCoMMended MoBile phases
The most common ion-pair mobile phase for synthetic oligonucleotide
separations is based on 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 mem-
brane 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 +/- 0.5 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 sepa-
ration of phosphorothioates or when performing LC-MS analyses. An
ion-pairing mobile phase based on Triethylamine (TEA) and Hexafluo-
roisopropanol (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 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 HFIP (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 of final buffer is approximately 7.9 +/- 0.1.
iV. reCoMMended inJeCtor Wash solV ents
Between analyses, the HPLC system injector seals should be washed. A
90% Water / 10% Acetonitrile injector wash solvent is recommended.
V. general Consideration in deVeloping separations
Separation of detritylated synthetic oligonucleotides by ion-pair,
reversed-phase chromatography uses very shallow gradients. With
both TEAA and TEA-HFIP ion-pairing systems, a rate of strong eluent
change between 0.1-0.25 % Acetonitrile (or Methanol) per minute is
recommended. However, the formation of shallow gradients can place
performance demands on LC pumps and mixers that can compromise
the quality of the separation. Consequently, it is strongly advised
that Mobile Phase B formulation contain a premix blend of aqueous
and organic solvents (e.g., Mobile Phase A= 0.1 M TEAA and Mobile
Phase B = Acetonitrile / 0.1M TEAA, 20/80, v/v) to minimize poten-
tially inadequate solvent mixing that can compromise component
resolution.