Smart ideas for generating reliable HPLC results
research findings are based on decisions which can be arrived at only if high degree of reliability can be placed on the data generated by the HPLC system.
HPLC is a
multi component system so the accuracy and reliability of results is dependent
on the performance level of each component part. The present article offers
some suggestions which will contribute to greater authenticity of results. For
simplicity the contribution of each component part is discussed under a
separate heading.
Mobile phase
The
principal role of mobile phase is to carry the injected sample to the column
and after separation in the column to transfer the individual components to the
detector for their identification and quantification. Ideally a mobile phase
should be prepared fresh before start of analysis and HPLC grade solvents
should be used. The solvents should be free of any suspended particles.
Initially filter the solvents under vacuum using 0.45 μ filter. Further use of
online filters is recommended to remove any residual suspended particles. The
mobile phase solvents should be fully miscible with one another and the
injected sample should be miscible as well. When using buffers as mobile phase
components never allow the buffer to dry out inside the system as salts formed
on drying will deposit and cause hindrance to free flow of mobile phase in
addition to damage to pump components.
Sample
Like mobile
phase solvents it is a good practice to filter the sample before injection.
Large volume injections will cause peak shape distortions so columns should not
be overloaded by making large volume injections. Whenever a new method is to be
developed it is advised to check the compatibility of the sample with the
mobile phase. In case the solubility is low try a solvent or combination of
solvents matching the polarity of the mobile phase mixture.
Column
A column is a key component of HPLC system.
Its proper care and use will go a long way in providing high reliability of
results.
Operating
flow rates and pressures should be changed gradually so that such changes do
not disturb the stationary phase packing. Use of guard column will also prevent
impurities from gaining entry to the main column. As the flow of mobile phase
is dependent on temperature use of a column oven is strongly recommended so as
to maintain a constant column temperature
for consistency of flow rate.
Pump
The pump is
required to provide consistency of flow of mobile phase and maintain the
solvent composition throughout the analysis run. In case of gradient analysis a
pump is required to maintain the composition of mobile phase as per the
software program. Such conditions are
achieved by preventing damage to pump components and timely replacement of such
components on noticing any visible damage. Gradually flush the pump by allowing
rinsing intermediate polarity solvents or pure water as the case may be.
Injector/autosampler
Consistency
of injection volumes is a prime need for reproducibility of results. Manual
injectors comprise of fixed volume sample loops and in case of automated modes
the injection syringe maintains consistency of volume without operator
intervention.
Special care
needs to be taken to prevent contamination from previous analysis. In case of
manual injectors always flush the injection loop with sufficient sample and in
case of automated systems make sure that intermediate wash cycles are part of
the analysis program. A validation of the cleanliness of sample and standard
reusable vials is vital before commencement of analysis.
Light
sources
The majority
of HPLC analysis can be carried out using UV light source. Such light sources
have a useful life span. Lamp replacement as per recommendations of supplier
will contribute to a high degree of reproducibility of absorbance values.
Detectors
A detector
is the ultimate part of the HPLC system which confirms the presence of
different sample components as well helps in their quantification.
It is
recommended that the detector be cleaned periodically from time to time
off-line and flushed with water and mobile phase before being re- used. The
operating pressure range should never be exceeded to prevent possible damage to
detector cell walls.
The
suggestions offered in the article will certainly improve the quality of
results but it goes without saying that there is no substitute for periodic
calibration of the system against reference compounds
*Lab-Training*
*Lab-Training*
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