Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns - Manual (Page 3)

[ Care and Use ManUal ]
Protein Separation Technology ACQUITY UPLC BEH300, C
4
, 1.7 �m
3
b. Column Connectors
The ACQUITY UPLC system utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to 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).
c. Column Installation
Note: The flow rates given in the procedure below are for a typical
1.7 µm packing in a 2.1 mm i.d. column.
1. Purge the solvent delivery system of any buffer-containing
or water-immiscible mobile phases and connect the inlet end
of the column to the injector outlet. An arrow on the column
identification label indicates the correct direction of solvent flow.
2. Flush column with 100% organic mobile phase (acetonitrile) by
setting the pump flow rate to 0.2 mL/min.
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 stable backpressure and baseline have been achieved,
proceed to the next section.
d. Column Equilibration
Protein Separation Technology 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 (refer to Table 1 for column volumes).
Table 1: Empty Column Volumes in mL
(multiply by 10 for flush solvent volumes)
To avoid precipitating mobile phase buffers on your column or in
your system, flush the column with five column volumes of a water/
organic solvent mixture, using the same or lower solvent content as in
the desired buffered mobile phase. For example, flush the column and
HPLC system with 50% acetonitrile in water prior to introducing 50%
acetonitrile/50% buffer mobile phase.
Column equilibration may be judged initially by stable pressure and
by a stable detector baseline. For a specific application, it is, however,
necessary to test the required duration of equilibration. The criteria
for adequate equilibration include reproducibility of retention time
for major and minor peaks, resolution for critical pairs, and consistent
baseline characteristics.
Note: Low concentration mobile phase additives, particularly those
with minimal buffering capacity may require extended equilibration
and re-equilibration between gradient analyses.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in
the desired application. Waters recommends using the solute
mixture and conditions described in the “Performance Test
Chromatogram” to test the column upon receipt.
2. Measure retention of the test compounds and the number of
theoretical plates (N).
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different HPLC systems due to the quality of the connections,
operating environment, system electronics, reagent quality,
condition of column, and operator technique.
Column Length Vol. mL (2.1 mm i.d.)
50 0.17
100 0.35
150 0.52