Operating Instructions RF-2000
54
[Method for sample preparation]
Place 10mg of acridine and 10mg of anthracene in the volumetric flask of
100ml capacity. Add 100ml acetonitrile and shake it to dissolve. Transfer 1ml
portion of the sample solution into the 10ml volumetric flask and add
acetonitrile, resulting in a total volume of 10ml.
(e) Water (HPLC grade or equivalent)
(f) Isopropyl alcohol
(2) Check connection of the units. Refer to the instruction manuals for details of
connection of each unit.
(3) Before installing the column, check 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.
The 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.
6.3.3 Procedure
Outline of procedure is described below:
(1) Set the flow rate of the pump to 1ml/min, initial concentration of solvent B to
50%, and column oven temperature to 40°C. Then 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 RF-2000.
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.
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