Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns, Glycan Performance Test Standard - Manual (Page 6)

[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Glycan, 1.7 �m Columns 6
IV. TROUBLESHOOTING
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 ladder or IgG
glycans 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 exacerbated by high flow rates. Reduce the flow rate by
one half while doubling the gradient time to maintain a constant
slope. Finally, apparent memory effects may actually reflect the
solubility of the sample in the mobile phase. Reducing the amount
injected may eliminate the effect.
Note: Useful general information on column troubleshooting problems
may be found in HPLC Columns Theory, Technology and Practice,
U.D. Neue, (Wiley-VCH, 1997), the Waters HPLC Troubleshooting
Guide (Literature code # 720000181EN), or visit www.waters.com.