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POSTED ON AUGUST 23, 2016 BY DR. DEEPAK BHANOT LEAVE A COMMENT

Important suggestions for

generating reliable HPLC results
HPLC has a marked
presence in analytical
research and quality
control laboratories.
Important decisions
concerning both
manufacturing processes
as well as publication of
research ndings 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 Each instrument component contributes to high reliability of
results
system.

HPLC is a multi component

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system so the accuracy and reliability of results is dependent on the performance level of each
component part. The present article o ers some suggestions which will contribute to greater TODAY WEEK MONTH ALL
authenticity of results. For simplicity the contribution of each component part is discussed under
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Mobile phase

The principal role of mobile phase is to carry the injected sample to the column and after TOPICS
separation in the column to transfer the individual components to the detector for their
AAS (53)
identi cation and quanti cation. 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 Analytical Chemistry (3)

particles. Initially lter the solvents under vacuum using 0.45 μ lter. Further use of online lters Calculations (5)
is recommended to remove any residual suspended particles. The mobile phase solvents should
Chemical (2)
be fully miscible with one another and the injected sample should be miscible as well. When
using bu ers as mobile phase components never allow the bu er to dry out inside the system as Chromatography (9)
salts formed on drying will deposit and cause hindrance to free ow of mobile phase in addition
Environmental Analysis (2)
to damage to pump components.
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Gas Chromatography (55)

Like mobile phase solvents it is a good practice to lter the sample before injection. Large volume
injections will cause peak shape distortions so columns should not be overloaded by making General Topics (140)
large volume injections. Whenever a new method is to be developed it is advised to check the
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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. HPLC (60)

ICP Techniques (13)

Column
Jobs (1)

  A column is a key component of HPLC system. Its proper care and use will go a long way in Lab Bytes (17)
providing high reliability of results.
Lab Management (95)

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28/04/2019 Important suggestions for generating reliable HPLC results - Lab-Training.com

Operating ow rates and pressures should be changed gradually so that such changes do not Laboratory Insurance (8)
disturb the stationary phase packing. Use of guard column will also prevent impurities from
Laboratory Safety (13)
gaining entry to the main column. As the ow of mobile phase is dependent on temperature use
of a column oven is strongly recommended so as to maintain a constant column temperature  Method Validation (1)

for consistency of ow rate. Microbiology (14)

News (3)
Pump
Paper Chromatography (10)
The pump is required to provide consistency of ow of mobile phase and maintain the solvent
pH Measurement (3)
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 Preformulation Studies (3)

achieved by preventing damage to pump components and timely replacement of such Quality System (9)
components on noticing any visible damage. Gradually ush the pump by allowing rinsing
Separation Science (11)
intermediate polarity solvents or pure water as the case may be.
Spectroscopy (80)
Injector/autosampler
Stability studies (8)

Consistency of injection volumes is a prime need for reproducibility of results. Manual injectors Training (21)

comprise of xed volume sample loops and in case of automated modes the injection syringe Weighing (3)
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 ush the injection loop with su cient 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 con rms the presence of di erent
sample components as well helps in their quanti cation.

It is recommended that the detector be cleaned periodically from time to time o -line and
ushed 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 o ered 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.

Filed Under: HPLC Tagged With: Each instrument component contributes to high reliability of results,
Hplc

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise
beginning from sales and product support of analytical instruments. After
completing his graduation and post graduation from Delhi University and IIT
Delhi he went on to Loughborough University of Technology, UK for doctorate research in
analytical chemistry. His mission is to develop training programs on analytical techniques and
share his experiences with broad spectrum of users ranging from professionals engaged in
analytical development and research as well as young enthusiasts fresh from academics who
wish to embark upon a career in analytical industry.

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