Waters Pico-Tag Columns for Free Amino Acids - Manual (Page 3)

[ Care and Use ManUal ]
The Pico•Tag Column for Free Amino Acids 3
3. Slide the compression fitting, followed by the ferrule (large end of the
taper first) over the tube. Be cer-tain to bottom the tube in the fitting
seat to assure a leak-free connection.
Note: Attach a union in place of the column and flush the lines free of previous
solvents before attaching the column.
Figure 2: Ferrule and Compression Assembly
d. Equilibration
A necessary step to successful use of your column is the initial solvation
(or wetting) of the packing. Purge the column in a column heater and bring
the temperature to 46 °C. Purge the column with 30 mls of Eluent 2, then
equilibrate with Eluent 1. Equilibration between the mobile phase and pack-
ing is established when a stable baseline can be produced. If your result is
unsatisfactory repeat the equilibration process.
III. operatIng tIps
• Normal recommended pressure should not exceed 3500 psi.
• For all silica based packing materials, stay within a pH range of 2-8
(avoid using concentrated acids or bases). Maximum column life (pH)
3.5 - 6.5.
• Filter all aqueous buffers (Pico•Tag eluents are prefiltered). 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 organic com-
pounds which alter column selectivity.
• Protect the column from rapid changes in solvent composition. DO NOT
change the flow rate faster than 0.5 ml/min increments.
IV. 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 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. 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.
Note: Before running the first analysis on your new column perform the test
sample separation given in the test conditions section.
a. Column Testing
The w5 sigma method shown in Figure 3 is used to measure column efficiency.
Unlike the tangent method used to determine system efficiency, this stringent
method considers naturally occurring peak asymmetry.
Figure 3. 5 Sigma Test Method
Columns are thoroughly tested in our quality control laboratories for adher-
ence to our specifications. Since slight variations in your results will occur
depending on the equipment used, test sample makeup and equipment set-
tings and conditions, perform the test sample run given here for your new
column and record the results (retention time and the settings used) before
attempting the first analysis. Use these results for comparison throughout the
life of your column.
Note: Be sure to record results and instrument settings (and configurations)
to allow exact reproduction and comparison in the future.