Waters Protein-Pak Hi Res IEX Columns - Manual (Page 6)

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[ CARE AND USE MANUAL ]
Protein-Pak Hi Res IEX Columns and Standards
d. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
to use. Solvents containing suspended particulate materials can
damage the fluidic components of the UPLC system and may clog
the inlet distribution frit of the column. This will result in higher
operating pressure and poor performance.
The maximum organic concentration must be <50%. When the
solvent is replaced by distilled or ion exchanged water, the flow
rate must be less than 0.25 mL/min for Protein-Pak Hi Res Q, 5
µm column or less than 0.5mL/min for Protein-Pak Hi Res CM or
SP, 7 µm columns.
e. Pressure
Protein-Pak Hi Res IEX columns can tolerate pressures across the
column of up to 2175 psi for Protein-Pak Hi Res Q, 5 µm column
and up to 1450 PSI for Protein-Pak Hi Res SP, 7 µm and Protein-
Pak Hi Res CM, 7 µm columns. These suggested limits are for the
pressure drop across the column. The actual recorded pressure on
an HPLC or UPLC system is a sum of the pressure generated from
solvent flow through the LC tubing and detector cell combined
to the pressure generated from solvent flow through the packed
IEX column. To determine the actual pressure drop across the
IEX column, subtract the above value from the system pressure
obtained (using the same eluent, flow, and temperature selected
for the IEX separation method) when a union is substituted in place
of the column on the LC System.
Note: Working at the extremes of pressure, pH and/or temperature
may result in shorter column lifetimes.
f. Flow Rate
Standard flow rate:
0.3–0.6 mL/min: Protein-Pak Hi Res Q, 5 µm column
0.5–1.4 mL/min: Protein-Pak Hi Res CM and SP, 7 µm columns
Under certain conditions (viscous buffers, low temperatures) the
maximum flow rate may have to be reduced so as to not exceed
the maximum column pressure limits.
g. Temperature
Temperatures between 10 ˚C–60 ˚C are recommended for
operating Protein-Pak Hi Res IEX columns.
V. T ROUBLESHOOTING
The first step in systematic troubleshooting is comparison of the
column, in its current state, to the column 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 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 possible that
the column can be cleaned as described in Section VI. 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 functional or protein test. If the proteins show
loss of retention, the column may require replacement. If the
changes are small and reflected only for some proteins, 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 (Symptom 2).
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 protein peaks appear
in both gradients, at the same time after start, the protein
came from the column in what is often described as a “memory
effect”. If the protein peaks only appear when an injection is