Waters ACQUITY UPLC SEC Columns and Standards - Manual (Page 6)

6
[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH SEC Columns
 Volume Load: < 20 μL for 4.6 x 150 mm
 Recommended pH Range: 2 to 8. The column lifetime will vary
depending upon the operating temperature as well as the type
and concentration of buffer used.
 Recommended Salt Conc.: less than or equal to 0.5 M
 Recommended Organic Conc.: < 20% acetonitrile (Caution: Many
proteins are insoluble at elevated organic concentrations. Prior to
chromatography, test to ensure the sample does not precipitate at
the organic concentration to be used for the chromatography. Also,
if column is run under denaturing conditions (greater than 10%
organic), subsequent column performance under 100% aqueous
conditions may be affected.)
 Recommended Temperature: 4–60 °C. Reduce flow rate when
operating at low temperatures (e.g. 10 °C) to avoid excessive
column pressure.
 Recommended Storage:
For overnight storage, continuously flush the column with the mobile
phase at 10–20% of the maximum recommended flow rate. Store
the column in the HPLC grade water when it will be used within
24 hrs or in 10–20% methanol for long term storage.
Note: Working at extremes of pressure, pH and/or temperature may
result in shorter column lifetimes.
V. TROUBLESHOOTING
The first step in systematic troubleshooting is comparison of the column
performance in its current state to the performance when it was
functioning properly. The functional tests with the protein mixture may
reveal 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 decreased
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 monitoring
pressure of the system as each connection is broken from the outlet
end to the inlet. If the system is occluded, the blockage should be
identified and removed. If the pressure increase resides in 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 in Section VI.
If a single sample caused the pressure increase, it likely reflects
particulates or insoluble components, such as lipids or higher order
aggregates. Cleaning is still an option, but using the more
aggressive options. If samples appear cloudy or turbid, they should
not be injected, as this will lead to pressure increases. Sample
preparation such as filtration or centrifugation may be used, but one
should first check whether this impacts the results.
2. Loss of resolution and increased peak tailing can be caused by
microbial contamination. It is important to follow good standard
laboratory practices to prevent microbial contamination,
This includes changing buffer bottles frequently, using high purity
water, using a sterile filtration apparatus, and storing system and
column under recommended conditions. If microbial contamination
has occurred, cleaning the column will have no effect on
performance. When changing the flow rate, ramp it at a rate of
0.1 mL/min and avoid immediate flow-rate increases greater than
0.1 mL/min.
3. Increased peak tailing can be caused by failure of a tubing
connector or a build-up of material on the column inlet frit.
Before proceeding with diagnostic or corrective measures, check
all connections that the mobile phases have been correctly
prepared and the correct method has been selected. Then repeat
the protein standard test. If the proteins shows increased peak
tailing, it is likely that there is significant build-up of material
on the column inlet and the column will require replacement.
4. Carryover is defined as the appearance of the constituents of one
sample in the next analysis. In size-exclusion chromatography
carryover is typically due to system components or improper
wash solvents. Run a blank injection. If the protein peaks only
appear when an injection is made, they likely originate from
system component or inadequate wash solvents. Adsoprtion in
the system components most likely occurs in the loop or needle.
In these instances the component may need to be changed.
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 ), and the Waters website, www.waters.com