Waters XBridge BEH Glycan, 2.5 μm XP and 3.5 μm Columns and Standards - Manual (Page 2)

II. GET TING START ED
Each XBridge BEH Glycan Column has a Certificate of Analysis
and a Performance Test Chromatogram. The Certificate of Analysis
is specific to each batch of packing material and includes the batch
number and analyses of the physical and chemical properties of
the particle. Particle size and pore structure are analyzed prior
to bonding. The carbon and nitrogen content of the bonding are
measured to insure consistent coverage. The selectivity of each
batch is also assessed with the chromatographic separation of
Waters Glycan Performance Standard, a 2-AB labeled N-linked
glycan standard mix spiked with Man-5 and Man-6, derived
from human IgG. The complex mixture of IgG glycans includes
high mannose structures as well as neutral and acidic complex
structures. The retention times and retention time differences of
selected components are used as the quality control test for each
batch of packing material. The Performance Test Chromatogram
is specific to each individual column and contains the following
information: batch number, column serial number, backpressure,
USP plate count, reduced plate height (RPH), USP tailing factor,
retention factor (k’), peak width, and chromatographic conditions.
These data can be found in the documentation supplied for each
column and should be stored for future reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical
2.5 µm XP packing in a 2.1 mm i.d. column. It is also highly
recommended that a column heater be used with our XBridge
BEH Glycan Column to help ensure consistent and reproducible
separated component retention times.
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 the column with 100% organic mobile phase
(acetonitrile) by setting the pump flow to 0.1 mL/min and
increase the flow to 0.17 mL/min over 3 minutes. Increase
the aqueous phase to 90% over 15 minutes. Note the
backpressure. Decrease aqueous phase to starting conditions
(22% aqueous in the test chromatogram).
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 from 0.17 to 0.34 mL/min
over 3 minutes.
5. Once a stable backpressure and baseline have been achieved,
proceed to the next section.
b. Column Equilibration
Glycan 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)
Column Length
(mm)
Column Internal
Diameter (mm)
Empty Column
Volume (mLs)
50 2.1 0.17
100 2.1 0.35
150 2.1 0.52
50 4.6 0.83
100 4.6 1.66
150 4.6 2.49
250 4.6 4.15
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 higher
acetonitrile content as in the desired buffered mobile phase. For
example, flush the column and LC system with 50% acetonitrile in
water prior to introducing 50% acetonitrile/50% buffered 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.
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XBridge BEH Glycan, 2.5 µm XP and 3.5 µm Columns and Standards