Bio-Rad Aminex Organic Acid and Alcohol Analysis Columns - Manual (Page 9)

compounds adsorbing to the column matrix over a period of many injections.
Occasionally, back pressure increases are caused by clogged frits. In any case,
following the cleaning procedures and/or reversing the column flow may help to
reduce the back pressure. If the guard system is in place and if the cartridge is
changed according to directions, fewer contaminants adsorb to the matrix and
higher column back pressures are delayed over the life of the column. Do not open
the column to resolve back pressure problems. The packing material of an opened
column may partially extrude from the opening and ruin the column.
When using an organic modifier, run column at 0.1 ml/min at 5% solution until a
stable baseline is obtained, then increase the organic modifier to the desired
concentration. Observe maximums for each column. A first step using a 5%
solution reduces immediate swelling of the column material and possible over
pressure.
Avoid sudden pressure surges on the column. The packing may compress, which
will result in tailing and decreased column efficiency.
5.4 Column Testing
All new columns should be tested to verify proper performance before they are
used for analysis. Column performance can change after extended use, and
retesting is useful to evaluate the changes. For successful analysis, the efficiency
(number of theoretical plates) and selectivity (resolution) must both meet minimum
requirements.
The performance of the HPLC system should be verified independent of the
column. Since each column is checked just prior to shipping, failure to reproduce
the supplied chromatogram may reflect problems with the HPLC hardware or the
preparation of the mobile phase. Be sure to keep all extra-column volumes to a
minimum. Loss of efficiency is often due to tubing or injector problems. If the peak
separation differs dramatically from the chromatogram supplied, the mobile phase
should be prepared again with careful consideration of each of its components.
If the peak elution order and peak shapes resemble the test chromatogram, but the
compounds are not fully resolved, minor changes in mobile phase and other
chromatographic variables may be made to achieve optimal separation or to
improve resolution. When all components of the standard are resolved sufficiently,
the analysis of samples may begin.
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