Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Columns - Manual (Page 2)

[ Care and Use ManUal ]
2
I. GettInG Started
Each ACQUITY UPLC ® OST C
18
column comes with Certificate of
Analysis and a Performance Test Chromatogram embedded within
the eCord
™ intelligent chip. The Certificate of Analysis is specific to
the batch of packing material contained in the ACQUITY UPLC ® OST
C
18
column and includes the gel batch number, analysis of unbonded
particles, analysis of bonded particles, and chromatographic results
and conditions. The Performance Test Chromatogram is specific to
each individual column and contains such information as gel batch
number, column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data should
be stored for future reference.
a. Column Connectors
The ACQUITY UPLC
® system utilizes tubing and gold plated
compression screws that have been designed to meet stringent
tolerance levels and minimize extra column volumes. Optimized
column inlet tubing (part # 430001084) is supplied with the
ACQUITY UPLC
® system. The inject valve end of the tubing is
clearly marked with a blue shrink tube marker. Insert the opposite
end of the tubing into the ACQUITY UPLC
® column and tighten the
compression fitting using two 5/16-inch wrenches. For information
on the correct column outlet tubing, please refer to the relevant
detector section in the ACQUITY UPLC
® System Operator’s Guide
(part # 71500082502).
b. Column Installation
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the
injector outlet.
2. Flush column with 100% organic mobile phase
(acetonitrile with TEAA or methanol for TEA-HFIP ion-pairing
method) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline at 260 nm have been
achieved, proceed to the next section.
c. Column equilibration
ACQUITY UPLC
® OST C
18
columns are shipped in 100% acetonitrile.
It is important to ensure mobile phase compatibility before changing
to a different mobile phase system. Equilibrate the column with a
minimum of 10 column volumes of the mobile phase to be used for
the oligonucleotide separation.
Note: When mobile phase additives are present in low concentrations
(e.g., TEAA or TEA-HFIP ion-pairing reagents), 100 to 200 column
volumes may be required for complete ACQUITY UPLC
® OST C
18
column equilibration.
Table 1.
Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column Length (mm) Internal Diameter 2.1 mm
50 0.2
100 0.4
150 0.5
d. eCord
™ Installation
The eCord ™ button should be attached to the side of the column heater
module. The eCord ™ button is magnetized and does not require specific
orientation.
e. Initial Column efficiency determination
1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as found in
the “Performance Test Chromatogram”, to analyze the column
upon receipt.
2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different UPLC ® systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition, and operator technique.