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

[ Care and Use ManUal ]
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ACQUITY UPLC OST C18 column performance can be tested with
the MassPREP OST Standard (P/N 186004135), a quality controlled
synthetic oligonucleotide sample consisting of 15, 20, 25, 30 and
35 mer deoxythymidine. Approximately 0.1 nmol of each oligo-
nucleotide was injected onto the ACQUITY UPLC OST C18 column.
Refer to P/N 715001677 for more information on sample prep for
the MassPREP OST Standard. Smaller peaks eluting prior to the main
peaks are failure, by-product sequences from the synthesis.
Figure 1: Separation using the MassPREP OST Standard on ACQUITY
UPLC OST C18
UPLC ® System: Waters ACQUITY UPLC ® System with installed 425 µL mixer
(see Section II, g), PDA Detector with standard UV cell
Sample Injected: Approximately 0.1 nmol of MassPREP OST Standard (P/N 186004135)
diluted in 0.1 M TEAA
Column: Waters ACQUITY UPLC ® OST C
18
column, 1.7µm (2.1 x 50 mm)
Mobile Phases: A: 0.1 M TEAA,
B: Acetonitrile / 0.1M TEAA, 20/80, v/v
Flow rate: 0.2 mL/min
Column Temp.: 60 ˚C
Gradient delay: 0.45 mL
Gradient: 40 to 59.5% B in 26 minutes (8-11.9% acetonitrile,
0.15% acetonitrile per minute)
Detection: 260 nm, 5 scans per second
II. Column uSe
To ensure the continued high performance of your ACQUITY UPLC ®
OST 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 0.1M 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 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 oligonucle-
otide 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, 0.2 µm mem-
brane and contained in bottles that are clean and particulate free.
teaa
1 L of 0.1 M TEAA may be prepared as follows:
1) Perform work in a hood.
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