Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns, Glycan Performance Test Standard - Manual (Page 3)

[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Glycan, 1.7 �m Columns 3
4. Gradually increase the flow rate from 0.25 to 0.5 mL/min
over 3 minutes.
5. Once a stable backpressure and baseline have been achieved,
proceed to the next section.
d. 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 Volume in mL (2.1 mm i.d.)
50 0.17
100 0.35
150 0.52
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
UPLC 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.
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. These conditions can be found on
the eCord attached to the column.
2. Measure the 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 UPLC systems due to the quality of the connections,
operating environment, system electronics, reagent quality,
condition of column, and operator technique.
f. Useful Functional Tests for Benchmarking a New Column
The performance test uses a Glycan Performance Test Standard
(Part No. 186006349). This test sample consists of 2-AB Human
IgG and is QC verified to contain the components needed to
benchmark and monitor a new column, as shown in Figure 1.
To prepare the standard, add 100 µL of 100mM ammonium formate
buffer pH 4.5 and 100 µL of acetonitrile directly to the vial for a
total volume of 200 µL.
Gently mix the sample by inversion.
Human IgG Glycans Performance Test
The conditions shown below are for use on an ACQUITY UPLC system.
If you are using an ACQUITY UPLC H-Class or ACQUITY UPLC H-Class
Bio system, use 50/50 acetonitrile/water for the purge and wash
solvents. All of the other conditions can remain the same. Note that the
ACQUITY UPLC H-Class and ACQUITY UPLC H-Class Bio systems do
not have a strong and weak needle wash. Instead, they have one
purge and one wash solvent, both of which should be in 50/50
acetonitrile/water.
It is also important to note the injection solvent and injection volume
for this application. Larger glycans have limited solubility in solutions
that contain more than 50% acetonitrile. Gradual precipitation and
loss of these larger glycans will be observed under these condtions.
However, also note that large injections of water-containing samples
will distort the peak shape in HILIC chromatography. The optimum
injection volume for these applications is < 3 µL.