The efficient operation of HPLC system is dependent on freedom of mobile phase and sample from chemical impurities or solid suspensions. Precautions in handling and use of HPLC mobile phase discusses measures that should be adopted during preparation and use of mobile phase. Importance of 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.
Please share your experiences on use of guard columns and do leave your comments.