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:
- 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.
- Degas
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 at first, let it drop to a waste
first).
- 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.
Introduction
1-3: Solvent delivery system
4. The column
5 - Detector
6- Injector
7-Gradient
and isocratic
8-Data
Processing
Copyright
(C) since 1997: Dr. Shulamit Levin