Before beginning to work with any
HPLC instrument one must understand the basic principles
of liquid chromatography at its various modes.
The liquid 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
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
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 appropriate gradient.
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.