Waters Protein-Pak SEC Columns - Manual (Page 3)

[ CARE AND USE MANUAL ]
Protein-Pak SEC Columns 3
f. Equilibration
A necessary step to sucessful use of your new column is the inital
solvation (or wetting) of the packing. Purge the column with 5 - 10
column volumes of the stronger eluting component of the mobile phase
alone before a a final purge with the actual mobile phase. Equlibration
between the mobile phase and packing is established when a stable
baseline can be produced. If your result is unsatisfactory repeat the
equilibration process.
g. Column Purge Volumes
Column Size (mm x cm) Recommended Volumes (mL)
7.5 x 30 100
7.8 x 30 200
II. CARE AND USE
Liquid chromatography columns have a finite life influenced by their
care and use, number of injections, sample and solvent cleanliness,
frequency of solvent changeover and handling and storage procedures
among other factors. If a change is observed in the :
• Retention of a particular compound
• Resolution between two compounds
• Peak shape
Take immediate steps to determine the reason for the changes, and until
the determination is made, the results of any separations using the
column must not be relied upon. Follow generally accepted procedures
for quality control and methods development when using these columns.
Important Note: Before running the first analysis on your new column per-
form the test sample separation given in the test conditions section.
a. Sample Preparation and Filtration
Use HPLC grade solvents, filtered to remove microparticulate matter
above the 0.45 μm. This reduces the problem of plugged filters and
preserves column life. Vacuum filtration or sonification may be used to
remove dissolved gasses which could affect your solvent delivery system.
Always filter prepared samples to prevent excessive pressure buildup due
to particlate matter. Adequate sample cleanup (using Sep-Pak
® cartridges
designed for this purpose) prevents alteration of the column chemistry by
strongly adsorbing or precipitatin sample components.
b. Precautions
Normal recommended pressure should not exceed 3500 psi for the
Protein-Pak SEC steel columns and no greater than 300 psi for glass SEC
columns. Maximum flow rate should not exceed: 1 mL/min
– For all silica-based packing materials, stay within a pH range of 2-8 (i.e.
avoid using concentrated acids or bases). For maximum column life stay
within the pH range: 3.5 - 6.5.
– Use Sentry™ Universal Guard Column Holder (WAT046910) and Protein-
Pak 125 Sentry Guard 2/pkg. (186000926) to protect your column from
contaiminants and extend column life.
– Try to dedicate more columns to specific applications. Constant switching
of samples and solvents will cause a more rapid column contamination
and loss of resolution.
– Filter all aqueous buffers. Avoid using turbid or cloudy buffers. Be sure that
any solutions containing buffers, salts, etc. are compatible with the wetted
surfaces of the column and equipment.
– Protect column from vibration, mechanical shock, and rapid changes in
pressure. Column packings are based on a highly porous and delicate silica
gel alignment. Any thermal, physical or chemical shock (such as changing
solvents rapidly or at high flow rates) can cause the particles to shift and
may result in a loss of efficiency.
– When using water, distill or treat with a Milli-Q ® or equivalent system.
De-ionized water is not acceptable because it contains organi compounds
which alter column selectivity.