Waters XBridge BEH Glycan, 2.5 μm XP and 3.5 μm Columns and Standards - Manual (Page 5)

c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. If filtering, Acrodisc
® filters
are recommended. Solvents containing suspended particulate
materials can damage the fluidic components of the UPLC system
and will generally clog the inlet distribution frit of the column.
This will result in higher operating pressure and poor performance.
d. Pressure
The XBridge BEH Glycan Columns will exhibit increased
backpressure when operated in 80–100% aqueous mobile phases.
As shown in the gradient table for Figure 1, the flow rate needs to
be lowered when washing a 2.1 x 150 mm Glycan column. XBridge
BEH Glycan Columns can tolerate pressures of up to 5,000 psi
(345 bar or 34 Mpa), although pressures greater than 4,500
psi should be avoided in order to maximize column and system
lifetimes.
Note: Working at the extremes of pressure, pH and/or temperature
will result in shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C–90 ˚C are recommended for
operating XBridge BEH Glycan Columns in order to enhance
selectivity, lower solvent viscosity, and increase mass transfer
rates. However, higher temperature will have a negative effect
on lifetime that will vary depending on the pH and buffer
conditions used.
IV. T ROUBLESHOOTING
The first step in systematic troubleshooting is comparing the
column, in its current state, to the column when it was functioning
properly. The method suggested in Section II for measuring plate
count is an essential first step. This technique detects physical
changes to the packed bed and chemical changes in the bonded-
phase surface. The functional test with the 2-AB labeled Dextran
Calibration Ladder (P/N 186006841) or the Glycan Performance
Test Standard (P/N 186006349) may reveal more subtle changes
in surface chemistry that affect the application.
There are several common symptoms of change in the column.
1. An increase in pressure is often associated with lost
performance in the application. The first step in diagnosis is to
ensure that the elevated pressure resides in the column rather
than somewhere else in the system. This is determined by
measuring pressure with and without the column attached to
the instrument. If the system is occluded, the blockage should
be identified and removed. If the pressure increase originates
from the column, it is helpful to know whether the problem
was associated with a single injection or whether it occurred
over a series of injections. If the pressure gradually built up,
it is likely that the column can be cleaned as described below
(Section V). For future stability, it may be useful to incorporate
a stronger regeneration step in the method. If a single sample
caused the pressure increase, it likely reflects particulates
or insoluble components. Cleaning is still an option, but
using the more aggressive methods. The sudden pressure
increase suggests that the user should consider some sample
preparation, such as high speed centrifugation.
2. Loss of retention can reflect a change in the column surface
chemistry. Before proceeding with diagnostic or corrective
measures, check that the mobile phases have been correctly
prepared and the correct method has been selected. Then
repeat the plate count test and the glycan test standard. If both
the plate count and glycan test show loss of retention, it is
likely that a significant fraction of the bonded phase has been
lost, and the column will require replacement. If the changes
are small and reflected only for some glycans, one of the
cleaning procedures may be effective.
3. Change in peak shape, resolution, or relative retention of peaks.
Follow the same steps as for loss of retention (Section II).
4. Carryover and memory effects are defined as the appearance
of the constituents of one sample in the next gradient analysis.
First determine whether the column or the system is the
source of carryover. Define a gradient method that includes
an “internal gradient”. That is, the analytical gradient is
repeated within a single method. If the glycan peaks appear
in both gradients, at the same time after start, the carryover
came from the column in what is often described as a “memory
effect”. If the glycan peaks only appear when an injection is
made, they likely originated from adsorption to some system
component. In that case follow the instrument manufacturer’s
recommendations. Memory effects as a source of carryover
may be reduced or eliminated in several ways. First, raising
the temperature of the separation reduces the possibility of
non-specific adsorption. Second, memory effects may be more
pronounced with steep gradients. Keep the gradient slope at
1% per column volume or less. Third, memory effects may be
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XBridge BEH Glycan, 2.5 µm XP and 3.5 µm Columns and Standards