Guard columns and their benefits in HPLC analysis - Lab Training
s. Precautionsin handling and use of HPLC mobile phase treated measures that should be adopted during preparation and use of mobile phase. Importanceof sample filtration discusses benefits of clean sample injections.
HPLC column
is a critical component of the HPLC system which requires careful handling and protection. It is
expensive to keep replacing columns frequently so your objective should be to
maximize the useful life of the column so that every time you get the desired
accuracy and consistency of results.
The
chromatographic behavior of the HPLC column begins to decline over use due to
gradual accumulation of impurities and suspensions. Particles larger than 2μm
present in mobile phase or sample start to deposit on the inlet frit of the
column thereby disturbing uniformity of flow. Smaller particles result in
increased backpressure so they begin to block the flow path in the stationary
phase.
Nature of
contaminants
- Highly retained compounds such
as fatty acids in reverse phase separations
- Irreversibly retained compounds
like residual proteins which were not removed completely at time of sample
extraction. Deprotenizatization
of proteins in biological fluids before HPLC analysis is strongly
recommended.
- Particulate impurities can
result from non filtration of samples, particulates released by wear of
system components such as seals in the pump or injector.
- Crystalline deposits resulting
from drying of residual buffers inside column. Washing of columns with
HPLC grade water after use or buffer solutions prevents such salt deposit
formation.
What is a
guard column?
A guard
column is a protective column or cartridge installed between the injector and
the analytical column. It serves to remove the impurities and suspended solids
from reaching the analytical column. Typically it has a length of about 2 cm
and internal diameter of 4.6 mm. Guard columns are packed with pelicullar
particles of around 40 μm size to offer negligible pressure drop.
Desirable
features of guard columns
- Guard column should have
preferably the same packing as the analytical column to eliminate
separation complications
- Internal ID of guard column
should be comparable to analytical column to minimize back-pressure.
Shorter guard column length is preferable but it should be long enough to
prevent strongly retained compounds from reaching the main column
- Frit facing the injector should
be removable for cleaning by removal of about 2 mm of material and filling
with fresh material
- Disposable cartridge type guard
columns are convenient and economical to use compared to refillable guard
columns.
Guard
columns need to be changed on regular basis but intermediate change becomes
necessary through observation of changes in chromatographic behaviour such as
increase in backpressure, peak broadening and, changes in retention time of
peaks. However, the frequency of change can be decided on the basis of chemical
composition of sample, presence of highly retained or irreversibly retained
components, injection volume or number of injections
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