Chemistry Reference
In-Depth Information
conditions [16,17]. Extra column band broadening from improperly made fittings or
longer tubing lengths also can contribute variability.
8.6.2 c
olumn
c
onSIderAtIonS
Historically, LC columns have been a significant source of variability. However,
recently, manufacturers have vastly improved the reproducibility on both a column-
to-column and a batch-to-batch basis by having complete control over the manufac-
turing process and raw materials. It is now a recommended practice to specify the
brand and other details of the column used. If additional columns have been tested
and approved for the method, they also should be specified by name. Using the blan-
ket statement “or equivalent” should be avoided, given the variability of columns
between manufacturers, even if they are of the same type (e.g., C
18
). The United
States Pharmacopeia (USP) has also addressed this issue by creating a database that
uses chromatographic tests to classify columns according to various parameters [18].
When finalized, users will be able to cross-reference columns that can be equivalent
to the one currently in use.
Column temperature is another source of variability. The days of room temper-
ature or “controlled” room temperature separations are long past; to achieve the
kind of reproducibility demanded by today's separation columns and in some cases,
mobile phase temperature control is critical. Nowadays, columns are thermostated
at least a few degrees above the highest room temperature to compensate for both
intra- and interlab temperature variability, either with (preferred) or without solvent
preheating, and some column control modules are also capable of cooling to temper-
atures below room temperature. The accuracy and precision of the set temperature
between different column heaters (particularly between different manufacturers)
also can vary and should also be considered.
Proper column equilibration also should be observed, either before being used
for the first time (isocratic mode) or between runs (gradient mode). A common rule
of thumb is to equilibrate a column with ten column volumes of mobile phase, or a
total volume equivalent to five times the column volume and three times the system
volume.
8.6.3 g
enerAl
c
onSIderAtIonS
There are other sources of variability that can lead to pitfalls. Training can be
addressed at any time, but it is not uncommon to train new users of the method
before formal AMT. Sometimes despite all the upfront work, errors are still made;
either honest mistakes or errors in procedure that result from method ambiguities.
Procedures should be written so that there is only one possible interpretation of how
to perform the method, with enough detail so that nothing is left to chance.
8.7 conclusIon
The development and validation of robust methods and strict adherence to well-
documented SOPs is the best way to ensure the ultimate success of the AMT. A
