Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 2)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH C
4
, 300Å, 3.5, 5, 10 �m
II. GET TING START ED
Each XBridge Protein BEH C
4
Column has a Certificate of
Acceptance and a Performance Test Chromatogram. The Certificate
of Acceptance is specific to each batch of packing material and
includes the 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 the information: batch number,
column serial number, USP plate count, USP tailing factor,
retention factor, and chromatographic conditions. These data
should be stored for future reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 3.5 μm packing
in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon
the column i.d of the column being installed. See “Scaling” section for calculating
flow rates when changing column i.d and/or length. See “Connecting the Column
to the HPLC” for a more detailed discussion on HPLC connections.
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 0.1 mL/min. and increase the
flow rate to 1 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 stable backpressure and baseline have been achieved,
proceed to the next section.
b. Column Equilibration
Your XBridge Protein BEH C
4
, 300Å Column is 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)
Column Internal Diameter
Column
Length (mm)
2.1 4.6 10 19 30
50 0.17 0.83 3.9 14.2 35.3
75 – – – – 53
100 0.35 1.7 7.9 28.4 70.7
150 0.52 2.5 11.8 42.5 106
250 0.87 4.2 19.6 70.9 176.7
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.
c. 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.