Waters ACQUITY UPLC - Column Use; Sample Preparation

4ACQUITY UPLC and ACQUITY Premier Oligonucleotide BEH C
18
Columns
[ CARE AND USE MANUAL ]
III. COLUMN USE
To ensure the continued high performance of your
ACQUITY UPLC and ACQUITY Premier Oligonucleotide
BEH C
18
Columns, follow these guidelines:
a. Sample Preparation
1. Dissolve the detritylated synthetic oligonucleotide
sample in mobile phase A (e.g., 0.1 M TEAA). For example,
a 0.05–0.2 µmole scale synthesis can be prepared in
0.1 mL of 0.1 M TEAA. Proportionately larger or smaller
volumes of 0.1 M TEAA is required when dissolving
samples from different scale syntheses. Due to the nature
of gradient separations, relatively large volumes of
sample (in low organic strength eluent) can be injected
and concentrated onto the head of the column before
beginning the gradient elution program.
2. Samples must be completely in solution and free of
particulates. Remove all particles from the sample
(Controlled Pore Glass Synthesis Support, etc.), which
may block the inlet column frit, increase the operating
pressure, and shorten the column life time. Sample
contamination with high concentration of salts and/or
detergents may also interfere with analysis.
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 8000 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 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
oligonucleotide mobile phases be filtered through a solvent
compatible, 0.2 µm membrane and contained in bottles that are
clean and particulate free.
TEAA
One liter 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 [p/n: 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 7.9).
TEA-HFIP System 1
One liter 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
One liter 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.