Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column - Manual (Page 3)

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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 centrifuga-
tion 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, 0.20 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 applica-
tions 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
1 L 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 ACQUITY UPLC ® system injector and seals can
and should be washed with two separate solvents. A 90% water/10%
acetonitrile mixture is the recommended strong solvent injector wash
solution for the TEAA ion-pairing based method. A 90% water/10%
methanol mixture is the recommended strong solvent injector wash
solution for the TEA-HFIP based method. 0.20 µm membrane filtered,
LC grade Water is the recommended weak wash solvent solution for all
ACQUITY
® OST separation methods.
V. soLV ent MIXIng oPtIons
The standard Waters ACQUITY UPLC ® system is equipped with
50 µL in-line mobile phase mixer. For demanding biopolymer separa-
tions (e.g., peptide mapping), use of a shallow gradients (e.g., 0.15%