Operating Instructions RF-2000
52
(2) Check the connection of the units. Refer to the instruction manuals for details
of the connection of each unit.
(3) Before installing the column, observe tubing connection of the LC system.
Use tubing with 0.3mm I.D. or less from the outlet of auto injector to the
column inlet and from the column outlet to the detector inlet. Length of the
tubing should be 30cm or less to minimize the extra-column band broadening.
(4) Flush the flow line with appropriate solvents dependent on the operation
status of the system. General guideline is shown below. To flush the flow line
with solvents, connect the inlet and outlet tubing of the column directly using a
proper union.
Column should be connected after flushing.
* Newly installed system
Flush the flow line with isopropyl alcohol, then with water at a flow rate of 2
ml/min for ten minutes.
* When low polarity solvent (such as hexane) is used as the mobile
phase in the system:
Flush the flow line with isopropyl alcohol, then with water at a flow rate of 2
ml/min for ten minutes.
* When mixture of water and organic solvent, or organic solvent
miscible with water (methanol, acetonitrile, etc.), or buffer solution is
used as the mobile phase in the system:
Flush the flow line with water at a flow rate of 2 ml/min for ten minutes.
(5) Set the mobile phase (b) and flush the flow line, then connect the column (c)
to the LC system.
6.2.3 Procedure
The outline of the procedure is described below:
(1) Set the flow rate of the pump to 1ml/min and column oven temperature to
40°C. Start pump flow and temperature control. Then, confirm that the solvent
comes out from the outlet tubing of the detector and no leak of solvent is
observed at the connection.
(2) Set the parameters of detector.
EX (nm) : 360 GAIN : 1
EM (nm) : 450 LAMP : 1
Response : 2 RATIO : 1
Sens : 2
(3) Monitor the baseline. When a stable baseline is obtained, press the zero point
adjustment key of the detector. Then, inject 10µl of mobile phase and confirm
no peak is observed.
(4) Next, inject 10µl of test sample solution. Repeat measurement at least five
times.
(5) Obtain the chromatographic data of retention time, peak area, and peak
height from five successive analyses.
(6) Calculate the average (X) of the data and %RSD using the equations as
shown below.
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