Before beginning to work
with any HPLC instrument one must understand
the basic principles of liquid chromatography
at its various modes.
chromatograph is very complex and it consists
of sophisticated technology and sometimes
delicate and total ignorance in the manner it
works might sabotage its parts which are very
expensive. In addition, there are
chemical processes of distribution between the
two phases, the mobile and stationary, while
the analysis goes on, and there is a need for
a minimum understanding of their theoretical
basis to operate the system correctly.
Also, a minimum understanding of the method,
in which the system performs data analysis, is
needed so that no false data or results will
be obtained in the analysis.
LISTS OF STEPS NEEDED BEFORE ANY RUN BY
HPLC רשימת צעדים מקדימים לפני הרצה :
- Filter the solvents with membranes with
cutoff of 0.22-0.45 mM
- Use clean and transparent reservoirs
through which precipitates and colloids
can be distinguished.
- Make sure that the solvents will be
easily mixed with the previous solvents in
the same inlets (A, B, C, D). For
example methanol or water should not be
placed instead of hexane directly, or any
organic solvent should not be placed
directly instead of a buffer reservoir.
- Degass the solvents and purge all the
tubing that lead to the pump.
- Connect the column carefully according
to the flow direction marked on it (do not
connect directly to the detector).
- Flow the
appropriate solvents through the column at
a low flow-rate (0.1-0.5 ml/min) or reach
the composition gradually using the
- Select the appropriate wavelength
(or other type of setting) in the detector
and wait for stable baseline
- Prepare the set of methods in the
workstation: Instrument method for the
control on the system, Processing method
for the data processing and the Report
method for the report of final results.
- When the system and the methods are
ready a blank run should be performed to
test the system and verify that it is
clean from interferences.
The rest of the
the rest of the article:
delivery system המשאבה
and isocratic גרדיינט או
Processing עיבוד נתונים