Waters XBridge Protein BEH SEC Columns and Standards - Manual (Page 5)

5
[ CARE AND USE MANUAL ]
XBridge Protein BEH SEC Columns and Standards
■ ■
Mass load: < 300 μg for a 7.8 x 150 mm
■ ■
Volume load: < 60 μL for 7.8 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 20%
methanol for long term storage.
Note: Working at extremes of pressure, pH and/or temperature may
result in shorter column lifetimes.
IV. T ROUBLESHOOTING
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 .