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

[ Care and Use ManUal ]
Glyco-Pak N Columns 5
b. Operation Precautions
Refer to the following list for operation precautions:
• Stay within a pH range of 2 -12.
• Filter all solvents. Avoid using turbid or cloudy mobile phases. Be sure
that any solutions containing buffers, salts, and so on are compatible
with the wetted surfaces of the column and equipment.
• 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 0.3 ml/min increments.
• Refer to Table 1 for recommended initial mobile phase compositions for
various neutral oligosaccharides.
Table 1: Recommended Mobile Phase Compositions for Neutral
Oliogiosaccharides
The table above represents retention relative to N-acetylglucosamine calcu-
lated as follows:
The numbered structures refer to those found in Figure 4. Please note that the
relative retention of these structures represent only approximate values and
may be affected by variations in instrument configuration and mobile phase
composition. They are provided as an aid to mobile phase selection and not as
a diagnostic indicator of column performance.
c. Column Efficiency
Liquid 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.
Follow generally accepted procedures for quality control and methods devel-
opment when using these columns.
Waters columns are thoroughly tested in our quality control laboratories for
adherence to our specifications. Variations in your results may occur depend-
ing on the equipment used, test sample makeup, mobile phase and equipment
settings and conditions.
Note: Be sure to record results and instrument settings (and configurations)
to allow exact reproduction and comparison in the future.
d. 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 naturally occurring peak asymmetry.
Figure 6: 5-Sigma Test Method
Oligosaccharide* Retention Relative to N-Acetylglucosamine
% Acetonitrile
65 68 71 74
1 1.0 1.0 1.0 1.0
2 1.56 1.81 2.62 3.4
3 1.56 2.03 3.20 4.69
4 -- 2.40 3.87 6.13
5 2.05 3.00 5.48 9.87
6 2.83 4.73 8.97 11.48
Relative Retention = k’x
k’ N-acetyglucosamine