Waters Sugar-Pak I Column - Manual (Page 4)

[ Care and Use ManUal ]
Sugar-Pak I Columns 4
 The pressure limit setting on the solvent delivery system should
be set to a few hundred psi above the normal working pressure
to avoid any excessive pressure buildup while the instrument is
unattended.
 If sugars not prone to inversion are analyzed (e.g., hydrolyzates),
it is not essential to use calcium EDTA in the mobile phase. Pure
water can be used but it should be filtered and degassed as
recommended. Column regeneration and flow direction changes
should be carried out regularly for optimum column performance.
b. Storage Considerations (more than 72 hours without use)
 Turn off the column heater and stop the pump.
 When the column is cool, remove it from the system and replace the
endcaps originally supplied with the column. The column should not
be allowed to dry out during storage. The normal mobile phase is
suitable as a storage solvent in the column. Refrigerate the column
at 5 °C (not frozen) to minimize any microbial growth.
 The end fittings in the system should be joined by a union after the
column is removed. Flush the entire liquid chromatography system
first with water, then methanol, and store it in methanol.
c. Starting Up After a Shutdown
Note: Make sure all methanol is flushed from the system with water
before the column is reinstalled.
When starting up a system containing a cold column, turn on the
column heater and start the mobile phase flowing at no more than
0.2 mL/min. Allow the instrument to stabilize under these conditions
for at least 20 minutes after the column reaches working temperature
before running the system under usual working conditions.
d. Overnight Shutdown (less than 72 hours without use)
The column may be left in the system with the flow rate reduced to
0.2 mL/min. Recirculate the solvent by placing the effluent line in the
supply flask. If it is undesirable to leave the column heater on, shut off
the pump and the column heater and allow them to cool down.
e. Starting Up After an Overnight Shutdown
Turn on the column heater and start the mobile phase flowing at no
more than 0.1 to 0.2 mL/min. Continue at this flow rate for at least
20 minutes after the column has reached its working temperature
before setting normal operating conditions for analysis.
IV. Sample Preparation
a. Pre-Injection Filtration
Filter all samples through a 0.45 µm membrane filter just before
injection to remove particulates. Suitable replacement filters are
provided in the Waters Aqueous Sample Clarification Kit (P/N
WAT026865). Suitable disposable filters with a larger surface area
are also available (HV Filter Unit, P/N WAT085996).
b. In-Line Filtration
If a Guard-Pak ™ Pre-Column module is being used, disposable
pre-column area filter inserts (P/N WAT032472) for use instead of
Guard-Pak Pre-Column inserts. If guard columns are not being used,
it is advisable to use an in-line pre-column filter (P/N WAT084560)
immediately before the column to protect it from any particulate
matter in the samples or mobile phase.
c. Chemical Cleanup of Samples
Substances such as proteins, lipids, polysaccharides, salts and acids,
which could contaminate your column, should be removed from samples
using the suggested techniques outlined in sections d through h.
d. Pre-Injection Cleanup of Proteins and Lipids
Proteins, or lipids such as fats and oils, can contaminate the column
and cannot be removed by washing with organic solvents. If samples
are contaminated with large amounts of lipid, the excess can be
removed by liquid-liquid extraction into a solvent such as chloroform.
Residual lipophilic compounds can be removed using a Sep-Pak ® C
18
Cartridge (P/N WAT051910) as explained in the following procedure:
1. Fill a 10 mL syringe with 5 mL of methanol and slowly flush
the methanol through the cartridge to condition the
Sep-Pak C
18
packing.