Waters Glyco-Pak DEAE Columns - Manual (Page 5)

[ Care and Use ManUal ]
Glyco-Pak DEAE Columns 5
III. operatIon
a. Flow Rate
Maintain Glyco-Pak DEAE flow rate at less than 1.2 ml/min. In general, flow
rates of 0.5-1.0 ml/min are recommended.
a. Operating Precautions
Refer to the following list for operation precautions:
• Glyco-Pak DEAE columns are compatible with buffers in the pH range of
2 -12.
• Flush system after use with HPLC grade or Milli-Q ® water when using
buffers containing halide ions.
• Filter all aqueous buffers. Avoid using turbid or cloudy mobile phases.
Be sure that any solutions containing buffers, salts, etcetera are com-
patible with the wetted surfaces of the column and equipment.
• DO NOT exceed 20 % organic content in the mobile phase.
• DO NOT expose columns to freezing temperatures.
• Protect the column from vibration, mechanical shock and rapid changes
in pressure. Column packings are based on a porous rigid polymer
alignment. Any thermal, physical or chemical shock (such as changing
mobile phases rapidly or high flow rates) can cause the particles to shift
and may result in a loss of efficiency.
• Treat water with a Milli-Q or equivalent system. De-ionized water is not
acceptable because it contains organic compounds which alter column
selectivity.
• Protect the column from rapid changes in mobile phase composition. DO
NOT change the flow rate faster than 0.5 ml/min increments.
• Do not use sodium azide, sodium dodecylsulfate (SDS) or anionic
detergents.
c. Typical Operating Conditions
Mixtures of acidic oligosaccharides typically are separated with a linear
gradient from A to B over 30 minutes at a flow rate of 0.8 ml/min with the
following mobile phases:
A B
Water 100 mM NaCl
1.0 mM NaH
2
PO
4
100 mM NaH
2
PO
4
pH 5-6 with NaOH pH 5-6 with NaOH
Volatile buffers such as ammonium acetate may be used for preparative work.
Final conditions must be appropriately adjusted for the individual sample.
For sub-fractionation of peaks collected from a gradient run, phosphate buffers
of appropriate pH and molar strength can be used isocratically for enhanced
resolution.
d. Column Efficiency
L iquid chromatography columns have a finite life which is directly related
to the care and use they receive. Column life is influenced by the number of
injections, sample and solvent cleanliness, frequency of solvent changeover,
and handling and storage procedures.
If you observe a change in the (1) retention of a particular compound, (2)
resolution between two compounds, or (3) peak shape, take immediate steps
to determine the reason for the changes. Until the cause of the change is
determined, the results of any separation using the column must not be
relied upon.
Waters columns are thoroughly tested in our quality control laboratories for
adherence to our specifications. Variations in your results can occur depend-
ing on the equipment used, test sample makeup, mobile phase, equipment
settings and conditions.
Note: Be sure to record results and instrument settings (and configurations)
to allow exact reproduction and comparison in the future.
e. Column Testing
Waters uses the 5-sigma method shown in Figure 6 to measure column effi-
ciency. Unlike the tangent method used to determine system efficiency, this
stringent method considers natural peak asymmetry.
Figure 6: 5-Sigma Test Method