2ACQUITY UPLC and ACQUITY Premier Oligonucleotide BEH C
18
Columns
[ CARE AND USE MANUAL ]
II. GETTING STARTED
Each ACQUITY UPLC and ACQUITY Premier Oligonucleotide
BEH C
18
Column comes with a 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
and ACQUITY Premier Oligonucleotide BEH 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 (p/n: 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 (p/n: 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 five 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 and ACQUITY Premier Oligonucleotide BEH
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 and ACQUITY Premier Oligonucleotide BEH
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. Procedure for Using New, Out-of-Box Columns
Prior to using a new column, it is important to confirm that
it produces reproducible chromatography and the desired
level of chromatographic resolution. To this end, it is useful
to benchmark column performance with a sample that is
representative of the intended application. The number of
injections necessary to achieve reproducible performance
may be dependent on sample characteristics and system
type. Method variables like pH, mass load, ionic strength,
and ion pairing could also have impact. ACQUITY Premier
Columns have MaxPeak™ High Performance Surfaces that
reduce the number of injections necessary to achieve desired
performance due to the improved hardware inertness.
e. 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.
f. 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.
To Next Page
Loading...
