In Pharmaceutical Industries Related
Substances test analysis is very critical, particularly when the HPLC method
analysis is gradient method . To get smooth baseline, no additional peak is
always challenge in gradient method analysis.
In such cases Guard column is useful to get smooth baseline and
exclusion of any additional peak due to impurity available in solvent which is
used in mobile phase and diluents preparation.
"If you have already validated method, then method Equivalency data is required before routine use of Guard column".
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.
The proficient operation of HPLC instrument is dependent on freedom of mobile phase and sample from chemical impurities or solid suspensions. Precaution and handling of use of mobile phase discusses measures that should be adopted during preparation and use of mobile phase. Importance of cleaned sample (Centrifuge or filter with syringe filter) injection is always benefits.
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.
- 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.
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.
