Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns - Manual (Page 7)

[ Care and Use ManUal ]
Protein Separation Technology ACQUITY UPLC BEH300, C
4
, 1.7 �m
7
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior to
use. Pall Gelman Laboratory 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
ACQUITY UPLC BEH300, C
4
columns can tolerate pressures of
up to 15000 psi (1034 bar or 103 MPa). Under optimum use
conditions, however, the ACQUITY UPLC System will be configured
with Waters ACQUITY UPLC Binary Solvent Manager 425 μL Mixer
(P/N 205000403) that has a working pressure limit of 10000psi.
This does not limit the utility of the column because the low optimum
linear velocity for high molecular weight analytes results in system
pressures well below the limit of the mixer.
Note: The mixer has a pressure rating of 690 bar (10,000 psi).
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 oper-
ating ACQUITY UPLC BEH300, C
4
columns in order to enhance
selectivity, lower solvent viscosity and increase mass transfer rates.
However, any temperature above ambient will have a negative effect
on lifetime which will vary depending on the pH and buffer condi-
tions used.
V. trouBLesHootInG
The first step in systematic troubleshooting is comparison of the
column, in its current state, to the column when it was functioning
properly. The method suggested in Section I 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 two functional tests with the Peptide Standard and the
Protein Mixture 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 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 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, such as lipids. Cleaning is still an option, but using
the more aggressive options. The sudden pressure increase
suggests that the user should consider some sample preparation,
such as filtration or 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 functional peptide or protein test. If
both the small and large molecule 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 proteins, one of the cleaning
procedures may be effective.