Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 5)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 5
III. MobIle phase and saMple guIdelInes
a. Buffers
Protein-Pak HR series columns and packings are stable in most
biologically-compatible buffering solutions. During analysis,
maintain buffers between pH 2 and 12. You may use buffers
containing halide ions (for example sodium chloride). However,
refer to the instrument manufacturer’s recommendation for system
compatibility and precautions.
Organic solvents such as short-chain alcohols (ethanol, isopropanol)
of up to 50% aqueous solution can be tolerated as mobile phases.
When switching from buffers to organic solvents, be careful not to
precipitate salts from solution or residual protein on the packing
material.
b. Sample Preparation and Filtration
Use HPLC-grade water to prepare buffers, samples, and reagents.
Filter all buffer solutions to remove microparticulate matter above
0.45 µm. This reduces the problem of plugged column filters and
preserves column life.
Employ sample clean-up such as centrifugation, filtration,
precipitation, and solid-phase extraction to prevent alteration of
the column chemistry. The Sep-Pak
® Cartridge Kit is available for
sample clean-up by solid-phase extraction. Filter all samples.
IV. operatIon
Liquid chromatography columns have a finite lifetime influenced
by their care and use, number of injections, sample and eluent
cleanliness, frequency of eluent changeover, and handling and
storage procedures.
a. Cautions
Note these cautions to avoid damage when using Protein-Pak HR
Ion-Exchange Glass series columns:
Column Hardware Pressure Limits
To avoid column hardware damage, never exceed the following
pressures inside the column:
Set the system high-pressure limit 0.7 MPa (100 psi or 7.0 atm)
below the maximum column pressure limit.
Operating Parameter Limits
To avoid bed damage to any of the Protein-Pak HR column products,
never exceed an operating backpressure of 3.5 MPa (500 psi or 35 atm).
The suggested flow rate range for each column size while maintaining
an operating backpressure of 3.5 MPa (500 psi or 35 atm) or less is:
b. General Guidelines
Refer to the cautions in Section II for additional cautionary
information.
•
When possible, dedicate columns to specific applications. Repeated
sample and solvent changeover accelerates column deterioration and
loss of resolution.
• DO NOT exceed 50% organic content in the mobile phase. Up to 50%
short-chain alcohols may be used for column storage or cleaning if the
column is free from salts and protein.
• DO NOT use the following solutions with the following columns:
• Protect the column from vibration, mechanical shock, and rapid
changes in pressure. Column packings are based on a highly-porous
and rigid resin. Any thermal, physical, or chemical shock (such as
changing eluents rapidly or at high flow rates) can cause the particles
to shift and may result in a loss of efficiency. If a void forms at the
column inlet, adjust the plunger (see next section for instructions on
eliminating voids).
• DO NOT expose columns to freezing temperatures (below 0 °C or
32 °F).
• To avoid precipitation of the sample and assure reproducible results,
dissolve sample in the initial buffer. Samples dissolved in high-ionic-
AP Mini 10 MPa (1500 psi or 100 atm)
AP-1 10 MPa (1500 psi or 100 atm)
AP-2 7.0 MPa (1000 psi or 70 atm)
AP-5 3.5 MPa (500 psi or 35 atm)
AP Mini 0.2-2 mL/min
AP-1 0.5-3 mL/min
AP-2 2-10 mL/min
AP-5 12-50 mL/min